TRENDS IN BIOSCIENCES JOURNAL 5-3, 2012 EDITION

Trends in Biosciences Volume 5 Number 3 October, 2012 

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Trends in Biosciences 

A Quaterly International Scientific Journal 


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Trends in Biosciences 


CONTENTS 

MINI REVIEW 

1. Sustainable Crop Production Using Natural Resources 161 

Priyanka Bhareti and R.K. Panwar 

2. Mechanized Harvesting of Cereal Crops - An Overview of the Problems and Factors Affecting 163 

H. G. Ashoka and G. M. Prashantha 

RESEARCH PAPERS 

3. Record of the Genus Homolobus Foerster (Hymenoptera: Braconidae), with Description of a New Species 166 

from Saudi Arabia 

Hamid A. Ghrmah 

4. High Frequency Plant Regeneration through In Vitro Cormel Formation using In Vitro Root as 168 

Explant of Gladiolus sp. 

Arvind Arya and Sandeep Kumar 

5. Establishing Callus Formation of Swarna and IR 64 and Sub-culturing of Callus and Monitoring the Stages 171 

of Regeneration 

Kshitij Kumar and K.N. Singh 

6. In Vitro Regeneration of Banana Variety Grand Naine (G 9) 176 

Manju Rai, Pallavi Mittal, Amandeep Kaur, Gurpreet Kaur, Isha Gaur and Charandeep Singh 

7. A Rapid and Simplex Methodology for Development of Platform Bioprocess Technology for Manufacturing 180 

of Recombinant Therapeutic Proteins in Serum Free Medium at Small Scale 

Praveen Gupta 

8. Inheritance of Resistance to Yellow Mosaic Virus in Moth Bean 184 

L. N. Yogeesh, R. Madhusudhan, B.A. Ravi and S. Gangaprasad 

9. Efficacy of IPM Modules against Tomato Leaf Miner, Liriomyza trifolii (Burgess) 188 

Wagh S.S. and Patil P.D. 

10. Association of ABO Blood Groups with Lipid Profile and the Level of Oxidative Stress in Hypertensive Patients 191 

Shalini Kapoor, Rajesh Kumar and Neeraj Arora 

11. Effect of Feeding Mixed Leaves of Different Mulberry Varieties on Economic Traits of Silk Worm Bombyx mori L. 196 

G.B. Durande, R.S. Gade, R.N. Jagtap and D.S. Tayade 

12. Ecofriendly Approach for the Management of Shoot and Fruit Borer, Earias vittella (Fab.) on Okra 199 

in Allahabad (UP) 

K.P. Tulankar, A.D. Gonde, R.K. Wargantiwar, A. Kumar and P.S. Burange 

13. Diversity and Community Structure of Phytonematodes Associated with Guava in and Around Aligarh, 202 

Uttar Pradesh, India 

Rizwan Ali Ansari and Tabreiz Ahmad Khan

14. Anti Fungicidal Activity of Secretion from Scent Glands of Heteropteran Bugs 205 

Ch. Srinivasulu, V. Karunakar, S.M. Reddy and C. Janaiah 

15. Effect of Different Concentrations of SA on the Morphological and Agronomical Characteristics of Wheat 208 

(Triticum aestivum L. em. Thell.) 

Jyotsna Subha, S. Marker, A. Krupakar and Atul Tripathi 

16. Seroprevalence of Brucellosis in Cattle and Bufallo in Some Districts of South Bengal 212 

Prabir Kumar Karmakar, Bikash Kanti Biswas, Sourav Chandra and Mrs. R. Das 

17. Production of Cellulase and Bioethanol from Lignocellulosic Materials using Aspergillus niger and 214 

Sacchromyces cerevisiae 

Akhilesh Bind, Budh Prakash Kanoujia, Nabeel Ahmad, Sama. Masih and Abhishek Sharan 

18. Lipid Peroxidation as Biomarker for Evaluating the Level of Toxicity in Nostoc muscorum under Multiple Stress 218 

Khan Uzma Aftab, Rajesh Chaturvedi and Iffat Zareen Ahmad 

19. Isolation and Identification of Algae from Dhemaji District of Assam, India 220 

Jyoti Prasad Lahan, Rituparna Kalita, Sudipta Sankar Bora, Archana Deka, Robin Ch. Boro 

and Madhumita Barooah 

20. A Study on Impact of Watershed Development Programme in Uttar Pradesh 222 

Pushpendra Saroj, Basvaprabhu Jirli and Vinod Kumar 

21. Combining Ability Analysis for Yield and its Components in Indian Mustard (Brassica juncea L. Czern and Coss) 225 

Kanhaiya Lal, Ram Krishna, Ranjeet, Hasmat Ali and Rama Kant 

22. A Key to the Indian Species of Schizoprymnus Foerster (Hymenoptera: Braconidae: Brachistinae) with 231 

Description of a New Species 

Mohammad Shamim 

23. Effect of Organic and Inorganic Sources of Nitrogen on N, P, K and S Content of Rice Grain at Harvest 234 

and Straw at Different Stages of Rice (Oryza sativa) Crop Growth 

Debiprasad Dash and Hrusikesh Patro 

24. Incidence of Pod Borer, Helicoverpa armigera Hub. and Their Management Through Newer 240 

Insecticides in Chickpea 

Jeewesh Kumar, D. C. Singh and A. P. Singh 

25. Efficacy of Some Triazole and Strobilurin Fungicides against Rust Disease of Field Peas 244 

R S Bal and A Kumar 

SHORT COMMUNICATIONS 

26. Pseudomonas aeruginosa: of Course I am an Arsenic Eater 246 

Poonam Gusain, Rajesh Kumar and Vir Singh 

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Trends in Biosciences 5 (3): 161-162, 2012 

MINI REVIEW 

Sustainable Crop Production Using Natural Resources 

PRIYANKA BHARETI* AND R.K. PANWAR 

Department of Genetics and Plant Breeding, GBPUA&T Pantnagar, US Nagar, Uttarakhand 263 145 

e-mail: bhareti.priyanka9@gmail.com 

ABSTRACT 

Sustainable crop production using natural resources increase 

agricultural production and productivity by utilizing ecological 

and biological integrity of natural resources. It includes a 

various diverse approaches like hybridization and selection of 

intervarietal and interspecific species and different landraces 

and varieties that are well suited to the climatic conditions of 

the farm: mixed farming and cultural practices to enhance the 

biological and economic productivity and stability of the land; 

proper management of the soil to enhance and protect soil 

structure, soil texture, soil micro fauna and quality: growing 

of legume crops for soil nitrogen fixation; recycling crop waste 

and livestock. Sustainable crop production is a method of 

cultivation using principles of ecology and the study of 

relationships between organisms and their environment. It is 

an integrated system of crops and livestock production practices 

which is having a site-specific application which gives the long 

term benefits for our future needs. It depends on replenishing 

the soil while minimizing the use of non-renewable resources. 

Natural resources are an important source of nutrients and 

high yielding crop production. Balanced use of natural resources 

increased nutrient availability, nutrient and water use 

efficiency, improves soil fertility, enhances crop production and 

productivity, generate farmers income and create a beneficial 

interaction between crop and their surrounding environmental 

conditions. The chemical fertilizers should be used judiciously 

and proper utilization of natural resources along with chemical 

fertilizers for increasing demand of food, enhancing crop 

production and soil productivity in a sustainable way, all these 

efforts would lead to desired awareness and as a result proper 

utilization of natural resource would become a cause of a boom 

in agriculture sector. 

Key words 

sustainable, soil nitrogen fixation, livestock, natural 

resources, soil, productivity 

The World Commission on Environment and 

Development (Brundtland Report, 1987) defined sustainability 

as “ensuring that development meets the needs of the present 

without compromising the ability of future generations to meet 

their own needs”. This can be added the need to utilize the 

natural resources as biosafety to feed the world population. 

The concept of sustainable agriculture has come up because 

yields from modern farming technique reaching a plateau and 

the environmental problems due to excessive use of chemicals 

and fertilizers residue in food chain. Sustainable agriculture is 

that form of farming which produces sufficient food to meet 

the needs of the present generations without eroding the 

Dr. R.K. Panwar is a Professor of Genetics 

and Plant Breeding at G. B. Pant University 

of Agriculture and Technology Pantnagar. 

He has two decades experience of teaching, 

research, extension and guiding students. 

His research specialization is on Pulse crops 

including both kharif and rabi seasons. 

e-mail:panwarrk@rediffmail.com 

Priyanka Bhareti is currently associated 

with G. B. Pant University of Agriculture 

and Technology Pantnagar as a research 

scholar in Genetics and Plant Breeding. She 

has actively participated in many 

conferences and workshops. Presently she 

is working on morphological and molecular characterization 

of some Vigna species. 

ecological assets and productivity of life supporting systems 

of future generations. Natural farming is an excellent illustration 

of sustainable agriculture. It is also known as ecological 

farming/eco-farming or Organic farming or Permaculture. It is 

called eco-farming because ecological balance is given 

importance and organic farming because organic matter is the 

main source for nutrient management. 

An increment in agricultural production and productivity 

under normal environmental conditions is a challenge with 

increasing population and climate change. It is very important 

for ensuring security of food, reducing poverty, conserving 

and utilizing the vast natural resources to fulfill the demand of 

whole world’s population and future generations. In the 

process of attaining the higher level of crop production, 

emphasis should be laid on natural resources. Short term 

increase in crop yields from modern farming techniques in 

most of the countries and environmental degradation due to 

excess use of pesticides, insecticides, herbicides and chemical 

fertilizers, becoming a matter of concern. So the need of 

sustainable crop production using natural resources is 

increasingly being felt, across the world. In this way the 

recommendation of the Atlanta Conference on “Organic 

Farming” have a catalyst in creating interest in the organic 

agricultural practices all over the world (Dahama, 1997).

162 Trends in Biosciences 5 (3), 2012 

There are three types of reserves for natural resources 

can be identified (Reijnders, 1999, Chapman, and Roberts, 

1983). First one are continuous resources like wind and 

sunlight, which can not be reduced in size by their utilization, 

second are renewable resources like wood and crops, which 

we can harvest, but not at the very fast rate than their rate of 

replenishment and last is non-renewable resources like fuels, 

fossils and minerals, developed by a very slow geological 

processes. Agro-biodiversity, fertile soil, clean water can also 

be considered as non-renewable resources, which requires 

certain period for their recovery. 

Sustainable crop production using natural resources 

encourage the use of manures, crop rotation and minimal 

tillage. Landraces, hybridization practices involving 

interspecific, intervarietal, intergeneric crosses can also 

restores resources and contributing in various quality traits, 

insect-pest and disease resistance. It also involves agroforestry, 

multi-level cultivation and integrated animal 

husbandry. It discourages the use of synthetically produced 

agro-products for standing against biotic and abiotic stress. 

Sustainability refers the characteristics of a process that can 

be maintained in long term and sustainable use of the agroeco 

system denotes a system without impairing its capacity 

for renewal or regeneration. The excessive accumulation of 

chemicals and dissolve nutrients in the soil like nitrogen and 

phosphorous leads to increased algal biomass and eventually 

an anaerobic condition may prevail. This may leads to a serious 

loss of marine life and major reduction in real estate value of 

surrounding areas. The adequate N:P ratio in water around 

20:1 and the algal growth will increased when the ratio goes 

around 7:1 (Asmed, 1993). 

The continuous losses of nitrogen from the atmosphere 

due to dinitrification contribute into “green house gases” and 

increasing the breakage of ozone layer. Nitrogen losses can 

be high in heavily fertilized soil and intensively cultivated 

land, whether the fertilizer is from organic or inorganic (Aluned, 

1993). Excessive biotic and abiotic interferences have caused 

considerable degradation of our natural resources. Various 

types of chemicals like insecticide, pesticide and herbicides 

are extensively used to control these calamities. Most of the 

chemicals are chlorinated, non-biodegradable and leaves 

residues; which are hazardous to the environment, livestock’s 

and human beings. Even small quantity of the residues intake 

daily along with food can lead to high body fat (Dhaliwal and 

Singh, 1992), this may lead to increase the need for sustainable 

crop production. 

Adoption of early maturing varieties with tolerance to 

biotic and abiotic stresses can enable farmers to stand against 

draught, salinity, damage and insect-pest and still produce a 

crop to generate farmer’s income. Adequate management of 

soil with a high production of humus gives necessary benefits 

with respect to soil erosion, soil water retention capacity and 

micro and macro fauna in the soil. The integration of livestock 

with crops provides organic matter for soil microorganism’s 

as a source of energy to enhance soil fertility and nutrients 

for the crop. Cultivation of multi-purpose nitrogen-fixation 

trees with cash crops as in “Alley farming” system is also 

very useful and attractive to the farmers if some of the foliage 

can be utilized to give added value to livestock (Atta-Krah, 

1991). 

Green manuring and inter-cropping of leguminous crops 

is also very important to control the weeds and also for 

reducing the leaching of nutrients and reducing the soil erosion 

losses. It will increased soil organic matter and soil nitrogen 

as well as other essential nutrients. Mulching by using 

manures spreaders may also useful to control weeds. 

Sustainability of natural resources is under serious threat 

due to discriminate cutting of trees, conversion of forest land 

into agriculture, shifting cultivation, exploitation of fragile and 

marginal lands, faulty management practices and reduction in 

frequency of fallowing and excessive use of chemicals. The 

problem is further compounded with over-exploitation of sweet 

groundwater aquifers, ingress of sea water in the coastal 

ecosystem, floods and draughts. Thus the sustainability in 

crop production in which livestock play important role can 

also give solution to the domestic energy crisis, the promotion 

of multipurpose crops and trees, and the recycling of livestock 

excreta will provide the domestic fuels and also useful to 

control soil erosion and a source of fertilizer. 

LITERATURE CITED 

Asmed, S. 1993. Agriculture-Fertilizer Interference in Asia. Issue of 

Growth and Sustainability, Oxford and IBH Publishers, New Delhi, 

India. 

Atta-Krah, A. N. 1991. Fodder trees and shrubs in tropical Africa: 

importance, availability and patterns of utilization. In: Integration 

of livestock with crops in response to increasing population pressure 

on available resources (eds. T R Preston, M Rosales and H Osorio). 

CTA: Ede, Netherlands. 

Chapman, P.F. and Roberts, F. 1983. Metal resources and energy. 

Butterworths Monographs in Materials. 

Dahama, A.K. 1997. Organic Farming for Sustainable Agriculture, Ashila 

Offset Printers, Daruagung, New Delhi, India. 

Reijnders, L. 1999. A normative strategy for sustainable resource choice 

and recycling. Resources Conservation and Recycling, 28: 121- 

133. 

Recieved on 22-02-2012 Accepted on 01-09-2012


Mechanized Harvesting of Cereal Crops - An Overview of the Problems and Factors 

Affecting 

H. G. ASHOKA* AND G. M. PRASHANTHA 

Division of Agricultural Engineering, University of Agricultural sciences, G.K.V.K., Bangalore-560065 

(Karnataka), India 

*e-mail: agrilengineeruasb@gmail.com 

ABSTRACT 

Harvesting is one of the most labor intensive operations in the 

production of crops. Development of mechanical harvesters / 

reapers operated by self mounted engines, power tillers, tractors 

and combined harvesters for harvesting crops like paddy, wheat 

and other cereal crops are gaining the popularity. The efficiency 

of harvesting operation depends on the shear stress and the 

modulus of elasticity of the crop. The factors affecting the 

shearing principles are the moisture content, height and the 

diameters of cutting stem. The shearing stress of wheat stalk 

given the direct relation to wheat stems size and the moisture 

content, but has inverse relation with the cutting height of 

stalk because of a reduction in stalk diameter. The bending 

stress and modulus of elasticity has got inverse relation with 

moisture content (Esehaghbeygi et al., 2009). However the 

findings with the cotton crop contradicting with the shearing 

principle of wheat. The shearing stress and moisture content 

of cotton stalks leads to increasing the cutting efficiency, which 

means decreasing the shear stress (Metwalli et al. 1995). Also 

the reapers developed for harvesting crops like wheat and paddy 

could not become popular for harvesting finger millet and other 

rain fed crops because of varied crop materials. Several 

commercial reapers developed has got vide range of field 

capacities for wheat and paddy crops. 

Key words 

Harvesting, reapers, shear stress, modulus of 

elasticity, efficiency, field capacity 

Harvesting is one of the most labor intensive operations 

in the production of crops. Traditionally crops are harvested 

manually using locally made sickle which is time and labor 

consuming operation and both are scares during peak 

harvesting season. The harvesting period is very short and 

delay in harvesting reduces both quality and quantity of grain. 

Further, due to the rapid industrialization and large scale 

migration to urban areas, labor is becoming increasingly 

scarce and also proving costly. This labor shortage during 

harvesting resulted in delayed harvest and consequent field 

grain losses. Labor requirement for harvesting of cereal crops 

with traditional manual harvesting varies greatly depending 

on the crops, crop conditions, type of tools and the quality of 

man power used. Mechanization of harvesting is an 

alternative solution will also result in lesser cost of operation. 

Where farmers have adopted combines for harvesting, 

alternative straw handling and disposal technology may have 

to be developed and promoted, as burning of straw is creating 

environmental pollution and farmers are losing valuable animal 

Dr. H. G. Ashoka, Agril. Engineer (Res.), 

Division of Agril. Engineering, UAS, 

GKVK, Bangalore – 560065. He obtained 

post graduation in Agricultural 

Engineering from University of 

Agricultural Sciences Bangalore and Ph.D 

from Kuvempu University, During his 

university career, he has been working at 

different places in different capacity as a scientist and a 

university Post Graduate Teacher since 1990. He has 

undergone International Post Graduate training on 

protected agriculture at the Hebrew University Jerusalem, 

Israel, as well visited McGill University, Canada. He has 

been involved in several research projects on 

Mechanization and developed improved hand operated 

Maize Sheller and Seed Cotton Baler, he is closely working 

with the State Agricultural and Watershed Development 

Department since 2003. At present working as Agricultural 

Engineer at the Division of Agricultural Engineering, UAS, 

GKVK, Bangalore and involved in evaluation of variety of 

Agriculture Machineries. To his credit, he has got few 

National and International publications. 

feed material (Gyanendra Singh, 2002). The efforts of the 

research work in various research organizations in India and 

abroad resulted in developing the mechanical harvesters / 

reapers operated by self mounted engines, power tillers, 

tractors and combined harvesters for harvesting crops like 

paddy, wheat and other cereal crops (Pndey and 

Devanani,1987 and Mohammad Raza Alizadeh et al., 2007). 

Based on the power operated paddy reaper designed by IRRI, 

Philippines, power tiller operated reapers were developed and 

evaluated for harvesting wheat (Garg et al. 1984). However 

the reapers developed for harvesting crops like wheat and 

paddy could not become popular for harvesting finger millet 

and other rain fed crops because of various reasons. One of 

the main reasons attributed to the blade design and metallurgy. 

Cutting blades used for harvesting soft and non fibrous stem 

portion of the crops like wheat and paddy were not very 

effective in harvesting crops like finger millet, since the stem 

portion of the finger millet crop is hard and fibrous. Further 

the height, ear-head size and the size of the stem portion of 

wheat and paddy crop were small as compared to the height, 

ear-head size and the size of stem portion of finger millet. The


cutting blades fitted in the existing reapers used for harvesting 

wheat and paddy becomes wear and tear and requires very 

frequent replacement when they are used for harvesting crop 

like finger millet, jawar, bajra etc. due to hard and fibrous stem 

and needs frequent replacement. This necessitates the design 

and development of suitable blades material which can 

effectively used for harvesting hard stem cereal crops. 

HARVESTING 

Harvesting of crops is an important field operation and 

it is one of the most labor intensive operations in the 

production of crops. Mechanized harvesting is done under 

varied situation by several commercial reapers and their 

capacity varies greatly. The studies on cutting of the crop 

stalk with traditional sickles revealed that it requires 64-128 

man-h / ha (Fleurdeliz et al.1989). Nadeem (1983) has reported 

that the paddy harvesting by manual method require about 25 

% of the total labor requirement of the crop. Depending upon 

the crop yield it demands 120 to 250 man-hr/ha for cutting, 

bundling and on-field stacking of rice field by using traditional 

sickle. Feasibility study conducted by Manjunath et al.,(2009) 

during the period 2002 to 2005 showed the reduced cost of 

cultivation in paddy through mechanized harvesting at the 

Agricultural Research Station, as well in the farmer’s field of 

Gangavathi in the state of Karnataka. The studies revealed 

that the vertical conveyor power reaper (KAMCO Model KR 

120) was having the field capacity of 0.3 ha/hr with the field 

efficiency of 73 % at an average operating speed of 3.2 kilometer 

per hour. Gajendra Singh et al. (1988) reported that the locally 

manufactured and commercially available tractor front 

mounted reapers can harvest 0.284 ha / h and saves 129 manh/ha 

compared to traditional manual harvesting with sickle 

for wheat crop. It was also observed that the average field 

capacity of 0.4 ha/h with 4 per cent grain loss while harvesting 

by using reapers. Garg et al. (1984) reported the labour input 

for mechanical reaping was about 5 man-h / ha compared to 84 

man-h / ha in manual harvesting of wheat crop. Devani and 

Pandey (1985) obtained the wheat crop field capacity of 

0.269 ha/h with 1.6 m wide reaper, while for 2.08 m wide unit it 

was observed to be 0.337 ha/h. Also they observed the total 

harvesting losses in the range of 4 to 6 % of grain yield when 

grain moisture content was 7 to 11 %. The studies on cutting 

of the crop stalk with traditional sickles revealed that it requires 

64-128 man-h / ha (Fleurdeliz et al.1989). Kumar et al. (2006) 

reported the harvesting of finger millet with sickles requiring 

about 150-200 man-h / ha. 

APPLICATION OF SHEAR PRINCIPLE 

Shearing stress of wheat stalk was measured for four 

moisture content levels (15, 25, 35 and 45 %(w.b), three cutting 

heights (100, 200 and 300 mm), two types of cutting knives 

(smooth and serrated edge) with three blades oblique angle 

(0, 15 and 30 deg.). The results showed that the shearing 

stress of wheat stems decreased as the moisture content 

decreased. The shearing force of stems decreased as the 

cutting height of stalk increased because of a reduction in 

stalk diameter. Reduced in shearing stress was observed by 

using smooth edge knife because of less friction than serrated 

one. The blade oblique angle of 30 degree showed the least 

shearing stress. The average of shearing stress varied between 

3.25 and 3.86 Mpa. It was reported that the increased bending 

stress and modulus of elasticity with the decreased moisture 

content and an increased cutting height of stem. The average 

of bending stress observed to be between 17.74 - 26.77 Mpa 

and modulus of elasticity varied between 3.13 - 3.75 Gpa 

(Esehaghbeygi et al., 2009). 

FACTORS AFFECTING 

According to 1nce et.al.(2005) the value of shearing 

stress at low moisture content was approximately 19 % lower 

than at high moisture contents. This result was also reported 

for wheat straw, sunflower stalk and alfalfa stem (Dogherty et 

al. 1995, Crook and Ennos, 1994). The average shearing 

stresses were found to be 3.25, 3.57, 3.69 and 3.86 MPa for 

moisture contents 15%, 25%, 35% and 45% respectively. The 

moisture content had a significant effect on the shearing stress 

at 1% probability level. The studies of Nazari, et al.,(2008) are 

in line with the findings of 1nce et.al.(2005). They observed 

the decreased in shearing stress with increase in cutting height 

from ground level. Increasing the cutting height from 100 to 

300 mm, resulted in reduced shearing stress by 13%. Also 

they observed an increasing the stem height, resulted in 

decreased stem diameter in the range of 5.1 to 7.5 mm. 

The moisture content had a significant effect on bending 

stress (Annoussamy et al. [2000]). Bending stress decreased 

with increasing moisture content. Also it was reported that 

the moisture content and height had significant effects on 

bending stress and the value of bending stress at low moisture 

content was approximately 1.5 times higher than at high 

moisture content. However, the reports on moisture content 

and cutting efficiency for cotton crop are contradicting the 

findings of wheat (Metwalli et al., 1995). Increasing moisture 

content of cotton stalks leads to increasing the cutting 

efficiency, which means decreasing the power requirement. 

Hoseinzadeh et al.(2009), conducted experiment on three 

varieties of wheat and knife bevel angles, four levels of 

moisture content and three shearing speeds of pendulum to 

determine their effects on the shearing energy of wheat straw. 

Results showed that the effects of variety, knife bevel angle, 

moisture content and shearing speed on shearing energy were 

significant at 1%. Shearing energy decreased with decreasing 

moisture content and bevel angle and with increasing shearing 

speed. Minimum shearing energy was obtained at knife bevel 

angles of 25° and 30°, while its mean value of 35° showed a 

significant difference at 5%. 

Hadidi (1984) stated that, the height of crop stubbles 

increasing as stalk moisture content increased and decreased

ASHOKA AND PRASHANTHA, Mechanized Harvesting of Cereal Crops - An Overview of the Problems 165 

with increasing of knife velocity. He added that the percentage 

of wheat and rice grin losses increasing as the machine forward 

speed increased. Increasing cutter bar speed leads to decrease 

in percentage of grain loss. Also, increasing forward speed 

leads to increase in number of uncut stalks. 

Habib, et al., 2002 reported the parameters affecting 

cutting process are related to the cutting tool, machine 

specifications and plant material properties. Further, they have 

observed the cutting energy consumed in harvesting process 

is much lower than the energy consumed in crushing process 

due to the effect of moisture content. Further, Habib, et al., 

2001 reported that increasing plant diameter needs higher knife 

velocity for performing the free cutting operation. Whereas, 

increasing mass of plant stalks need low critical speed. Further, 

they have found that the critical knife velocity affected by 

both height of knife from ground and the plant overall length. 

Also, moisture content of plants materials affecting on the 

critical knife velocity throwing by the cutting force, where 

cutting force variation with the moisture content. 

Labor is becoming increasingly scarce and also proving 

costly and the mechanized harvesting is the need of the hour. 

The efficiency of harvesting operation mainly depends on 

the shear resistance offered by the crop for harvesting. For 

the application of shear principles for harvesting the crop, the 

factors influencing are, moisture content, cutting heights, 

cutting knives and the blades oblique angle. The observation 

of shearing stress on wheat crop revealed the decreased in 

shear stress with the decrease in moisture content. The 

reduction of shearing force was observed due to increased in 

cutting height of stalk because of a reduced stalk diameter. 

Also the reduced in shearing stress was observed by using 

smooth edge knife, because of less friction than serrated one. 

The blade oblique angle of 30 degree showed the least 

shearing stress. Bending stress and modulus of elasticity 

increased as the moisture content decreased with increased 

in cutting height of the stem. However, the findings are not 

true for the entire range of field crops. An increasing moisture 

content of cotton stalks leads to increasing the cutting 

efficiency, which means decreasing the power requirement. 

Similarly, use of reapers for mechanized harvesting of rain fed 

crop like Finger millet reported with frequent break down and 

repairs due to the hardy fibrous stem nature. There are no 

reports available for mechanized harvesting of crop like Jowar, 

Bajra etc. Hence there is need to improve the metallurgy of 

blade materials used for harvesting different crops. 

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parameters affecting the cutting process performance of agricultural 

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Moisture Content, Bevel Angle and Cutting Speed on Shearing 

Energy of Three Wheat Varieties. World Applied 

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performance of mechanical reapers for harvesting rainfed and 

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performance of mechanical reapers for harvesting rainfed and 

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and low cost of operation. J. Agric, 36:261-274. 



Record of the Genus Homolobus Foerster (Hymenoptera: Braconidae), with 

Description of A New Species from Saudi Arabia 

HAMID A. GHRMAH 

Department of Biology, King Khalid University, P.O. Box-9004, ABHA-61413. Kingdom of Saudi Arabia 

e-mail: hamidkku@gmail.com 

ABSTRACT 

Homolobus (Apatia) arabicus sp. nov., (Hym., Braconidae) is 

described from Saudi Arabia. Morphological diagnostic 

characters of the new species are figured, and they are compared 

with those of the related species. The genus Homolobus Foerster 

is reported for the first time from Saudi Arabia. 

Key words 

Homolobus, Braconidae, new species, Saudi Arabia. 

The genus Homolobus Foerster belongs to subfamily 

Homolobinae of Braconidae. Members of the genus 

Homolobus are koinobiont endoparasitoids of the Lepidoptera 

larvae, mainly in Geometridae and Noctuidae (van Achterberg, 

1979; Shaw and Huddleston, 1991; Shaw, 2006). The genus 

has been revised by van Achterberg, 1979, however, it is yet 

to be recorded from arabian region. The genus contains 45 

species and spread over four subgenra viz., Apatia Enderlein, 

1920; Chartolobus van Achterberg, 1979; Oulophus van 

Achterberg, 1979; Phylacter Reinhard, 1863 (Yu., et al., 2005). 

In this paper one new species viz., Homolobus (Apatia) 

arabicus, sp.nov, is described and illustrated with the help of 

photographs. 

MATERIALS AND METHODS 

Specimens were collected by sweeping net and malaise 

traps from different regions of Khamis Mushait, Saudi Arabia. 

The samplings were conducted between 2011 in the Asir 

province. The collected specimens were killed with ethyl 

acetate and mounted on triangular labels and were examined 

with a stereoscopic binocular microscope Nikon SMZ1200. 

Classification, nomenclature and distributional data of 

Braconidae suggested by Yu, et al., 2006 have been followed. 

The terminology for morphology follows that used by 

Achterberg, 1993. 

Abbreviations used in the text are: AOL= Anterior ocellar 

line (distance between the inner edges of an anterior ocellus 

and lateral ocellus); POL= Posterior ocellar line (distance 

between the inner edges of lateral ocelli); OOL= Ocello-ocular 

line (distance from the outer edge of a lateral ocellus to the 

compound eye); OD= Ocellus diameter; F= Flagellomere. 

The types of new species are deposited in Department 

of Zoology, King Khalid University, Abha, KSA. 

RESULTS AND DISCUSSION 

Description 

Homolobus (Apatia) arabicus sp. nov. 

Female: 6.8 mm, of fore wing 6.5 mm. 

Colour: Yellow-brownish; stemmaticum black; antenna brown, 

with outer side of scapus light brown; veins of fore wing 

largely dark brown, of hind wing only veins SR and 2-M, 

remainder of veins yellowish-brown 

Head: Antennal segments 48, length of third segment 1.2 times 

fourth segment, length of third, fourth and penultimate 

segments 3.5, 2.8 and 1.8 times their width, respectively; length 

of maxillary palp 1.5 times height of head; length of fourth 

segment of labial palp 2.3times length of third segment; in 

dorsal view length of eye 2.5 times temple; OOL:diameter of 

ocellus:POL = 2:8:3; frons smooth and shiny; vertex sparsley 

punctulate; face rather flat, sparsely and finely punctulate; 

clypeus sparsely punctate with long setae; length of malar 

space 0.45 times basal width of mandible. 

Mesosoma: Length of mesosoma 1.3 times its height; side of 

pronotum largely smooth, with some crenulae medio-anteriorly 

and few crenulate posteriorly; epicnemial area smooth; 

mesopleuron punctate-rugose dorsally; precoxal sulcus 

distinctly punctate-rugose, and remainder punctulate; 

metapleural flange smooth; notauli crenulate, narrow anteriorly 

and widened posteriorly; mesoscutum largely smooth, only 

near notauli punctate; surface of propodeum smooth anteriorly 

and remainder reticulate-rugose. 

Wings: Fore wing: SRI distinctly curved; r:3-SR:SRl = 7:9:33; 

cu-a distinctly inclivous, slightly curved basaad apically; 2- 

SR:3-SR:r-m = 7:11:5; r-m distinctly curved basad;. Hind wing: 

r present, dividing marginal cell into a long basal part (about 

1.3 times length of apical part) and a much shorter apical part; 

SC+R1 evenly curved. 

Legs: Hind claws without small sub-apical tooth, spiny setose; 

inner claw of hind tarsus equal to its outer claw; length of 

femur, tibia and basitarsus of hind leg 6.1, 9.6, and 9.4 times 

their width, respectively; length of hind tibial spurs 0.40 and 

0.70 times hind basitarsus 

Metasoma: Length of first tergite 3.2 times its apical width, 

hind wing vein r present; its surface distinctly rugose, its

HAMID, Record of the Genus Homolobus Foerster (Hymenoptera: Braconidae) 167 

Homolobus (Apatia) arabicus sp. nov., (holotype, female). 

Fig. 1. Female fore wing 

dorsal carinae absent; second tergite punctate medio-basally, 

its remainder largely smooth; length of ovipositor sheath 0.06 

times hind basitarsus. 

Male: Unknown. 

Distribution: Saudi Arabia: Asir region, Khamis Mushait. 

Host: Unknown 

Material examined. Holotype: Female (on card), 9.iii.2011, 

Khamis Mushyat, Asir region, Saudi Arabia, coll. Hamed, 

(Malaise trap). Paratypes: 4 females (on card) with same data 

as holotype. All the material is housed in Museum collection, 

Department of Biology, King Khalid University, Abha). (All 

the type material presently in the personal collection of author 

and will be deposited in King Saud university museum, Riadh, 

K.S.A.). 

Etymology: The species name is derived from its type locality. 

The new species Homolobus (apatia) arabicus sp. nov. 

resembles with H. (A.)elagabalus (Nixon, 1938). However, it 

differs from H.(A.) elagabalus in having length of malar space 

0.45 times basal width of mandible; basal part of vein SR of 

hind wing 1.2 times longer than second part of vein; veins SR 

and 2-M of hind wing similarly yellowish as veins of basal 

part of wing; first tergite finely sculptured. 

Fig. 2. Female habitus 

ACKNOWLEDGEMENT 

The authors are indebted to Dr. Sulaiman Al-ruman 

(Head, department of Biology, King Khalid University, Abha) 

for providing necessory research facilities. 


Achterberg, C.V. 1979. A revision of the subfamily Zelinae auct. 

(Hymenoptera, Braconidae). Tijdschrift voor Entomologie, 122: 

241-479. 

Achterberg, C.V. 1993. Illustrated key to the subfamilies of the 

Braconidae (Hymenoptera: Ichneumonoidea). Zoologische 

Verhandelingen Leiden, 288: 1-189. 

Shaw, M.R., Huddleston, T. 1991. Classification and biology of braconid 

wasps. Handbooks for the identification of British insects, 7: 1- 

126. 

Shaw, S.R. 2006. Chapter 12.2, Familia Braconidae: 487-525. In: 

Hymenoptera de la Región Neotropical. Memoirs of the American 

Entomological Institute, (eds. Hanson, P.E. and I.D. Gauld), 77: 1- 

994. 

Yu, D.S., Achterberg, C.V. and Horstmann, K. 2006. World 

Ichneumonoidea 2005. Taxonomy, biology, morphology and 

distribution [Braconidae]. Taxapad 2006 (Scientific names for 

information management); Interactive electronical catalogue on 

DVD/CD-ROM. Vancouver. 



High Frequency Plant Regeneration Through In Vitro Cormel Formation Using In 

Vitro Root as Explant of Gladiolus sp. 

ARVIND ARYA 1* AND SANDEEP KUMAR 2 

1 

Department of Biotechnology, Meerut Institute of Engineering and Technology, Meerut. U.P. 

2 

National Institute of Engineering and Technology, NIMS University, Jaipur 

*e-mail: arvindarya@hotmail.com 

ABSTRACT 

The direct organogenesis was derived from roots of in vitro 

plantlets as an explant in Gladiolus sp. (Iridaceae), a highly 

valuable ornamental variety. Direct shoot buds were initiated 

at a higher frequency from in vitro roots of Gladiolus sp. used 

as explant. Different basal medium viz., MS, B5 and 2iP were 

tested and found MS medium best for initiation of shoots. 

Highest number of shoots regenerated in 1x MS with 2.0 mg/l 

BAP. The shoots produced roots in MS with 1.0 mg/l IBA and 

6% sucrose. After five weeks, rooted plantlets were transferred 

for initiation of cormels. The protocol enabled to harvest more 

than 50,000 cormels (cormlets) within 150 days starting from a 

single in vitro root explant. 

Key words 

ABBREVIATIONS 

Cormel, Gladiolus, Ornamental, Regeneration. Root 

explant 

(2iP: 6-ã-ã-dimethylaminopurine, B5: Gamborg’s B5 

Medium, BAP: 6-benzylaminopurine, IAA: Indole acetic acid, 

IBA: Indole-3-butyric acid, Kn: N-(2-furanylmethyl)-1Hpuring-6-amine 

(kinetin), MS: Murashige and Skoog Medium, 

NAA: á-Naphthaleneacetic acid, SD: Standard Deviation) 

The propagation of Gladiolus is through seeds, corm 

formation or by cormel differentiation. Although, seeds are an 

effective means of Gladiolus propagation but seed-raised 

plants may not produce true-to-type population (Kumar, et 

al., 2011). 

Micropropagation offer promises of mass multiplication 

of bulbous and cormous plants(Novak and Petru, 1981; 

Takayama and Misawa, 1982; Takayama and Misawa, 1983). It 

also provide the materials free from viruses and other 

pathogens. Few reports are available pertaining to in vitro 

propagation of Gladiolus from axillary shoot formation (Logan 

and Zettler, 1984), through in vitro corm production (Bruyn 

and Ferreira, 1992) or from callus culture (Simonsen and 

Hildebrandt, 1971). However, in Gladiolus there is a clear scope 

for further refinement of in vitro culture methodology to 

acquire a higher number of shoots to complement traditional 

nursery methods (Kumar, et al., 2011). 

The present study was an attempt to produce multiple 

shoots by using roots of in vitro plantlets as explants source 

since propagation by corm and cormel formation may transmit 

several diseases thus causing a heavy loss. This report might 

be helpful for improvement of Gladiolus by mutation breeding, 

somaclonal variation and genetic engineering. 


The explant employed were roots of Gladiolus sp. of 

about 8-10 mm in length preserved from previous in vitro 

rooted plantlets culture and maintained in growth room (details 

not given). Roots of Gladiolus sp. were transferred to 25x100 

mm tubes containing 20 ml in MS media with (1-3 mg/l) 

concentrations of different cytokinins viz. BAP, Kn and 2iP. 

The pH of the medium was adjusted to 5.8 before autoclaving 

at 121 ºC for 15 min. Cultures were illuminated by cool-white 

florescent light (50 µMol m -2 .s -1 per 16h photoperiod) at 25 ± 

1ºC. 

Different cytokinins viz., BAP, Kn and 2iP were used to 

see their effect on shoot induction and proliferation. Basal 

MS medium solidified by agar was supplemented with different 

concentrations of cytokinins and root explant was aseptically 

inoculated on them. 

Different strength of basal medium viz., MS (1x, ½ x and 

¼ x) and B5 (1x and 1/2x) along with 2.0 mg/l BAP were 

prepared. 

Healthy in vitro shoots were transferred to jam bottles 

containing 30 ml of basal liquid MS media supplemented with 

different auxins. IAA, IBA and á-NAA alone as well as in 

combinations were added in basal liquid MS medium. The 

healthy rooted plantlets were transferred on low concentration 

of auxins, after five weeks initiation of cormlets. 


Four weeks after culture, the root segments with initiated 

shoot buds were transferred to MS media (data not given). 

The percentage of shoot formation was affected by the 

concentration of various cytokinins on MS media. Among 

the various cytokinins tested, BAP induced the maximum 

response in root explants of in vitro regenerated plantlets 

(Fig. 4). The shoot multiplication and shoot length on BAP 

(2.0 mg/l) showed better response (Fig. 4) than other 

concentration of cytokinins. Kn and 2iP failed to induce 

organogenesis and formation of shoot and produced groups 

of shoots of varying lengths depending upon the 

concentration of the cytokinins added to the medium.

ARYA AND KUMAR, High Frequency Plant Regeneration through In Vitro Cormel Formation using In Vitro Root 169 

Fig. 1. 

Fig. 2. 

Fig. 3. 

Fig. 1 

Fig. 2 

Fig. 3 

Effect of different concentrations of various cytokinins 

on shoot multiplication of Galdiolus spp. (MS medium 

after 8 weeks) 

Effect of different basal media containing 2.0 mg/l BAP 

on in vitro s hoot multiplic ation from in vitro 

regenerated root explants of Galdiolus spp. (After 6 

weeks) 

Root induction and cormlets formation in various 

permutations of auxins on MS med ia (20 sho ots 

pretreatment – 6 week old culture) 

rooting (84%), the maximum number of roots and root length 

(25.38 and 8.82 cm, respectively) were observed in 1 mg/l IBA. 

(Fig. 3, Fig. 7). IBA was superior to IAA and á-NAA for 

induction of root within seven days. The in vitro regenerated 

micro-corms were stored in a cool and dry place (Fig. 8). 

Rapid in vitro propagation techniques have been 

reported for some Gladiolus cultivars (Dantu and Bhojwani, 

1995; Ruffoni et al., 2008). Although, the in vitro multiplication 

of Gladiolus has been achieved by using axillary buds (Begum 

and Haddiuzaman, 1995), shoot tip and inflorescence axes 

(Ziv and Lilien-Kipnis, 2000) as explants but root was not 

used as explant for in vitro regeneration and micro-corm 

formation in Gladiolus spp. This is the first report describing 

the roots of Gladiolus sp. as explant source and its effect on 

micropropagation. In the present work, while searching for 

optimal conditions for in vitro corm development, we 

determined how the plant growth regulators, the types of basal 

media and their strength affect the in vitro regeneration of 

plants. 

Cytokinins especially BAP produced overall best 

response viz., induction and multiplication of shoot. Earlier 

reports also evident the participation of cytokinins in the in 

vitro stimulation of shoot induction and tuberization (Koda 

and Okazawa, 1983). However, there is apparent ambiguity 

about the role of cytokinins in the regulation of Gladiolus 

corm formation. Contrary to our findings kinetin is reported to 

induces cormel differentiation on excised stolon tips (Ginzburg, 

1973) and also that BA adversely affects corm formation at 

the shoot base (Steinitz and Lilien-Kipnis, 1989). 

In present study, it was found that high concentration 

of BAP suppress the shoot bud induction and further shoot 

growth. The shoot length was found to be significantly higher 

in MS medium with 1.0 mg/l 2iP compared to other cytokinins. 

In order to improve the shoot multiplication rate from in 

vitro root explant, they were cultured on various basal media 

and showed the maximum percentage of multiplication rate on 

full strength MS medium containing 2 mg/l BAP (Fig. 2, Figs, 

5 and 6). Even though the number of shoots was lower in B5 

½ strength medium when compared to other media. It was 

observed that the shoot induced on B5 medium exhibited scale 

like leaves. However, it has also been found that the quality of 

shoots was inferior as the regenerated shoots showed 

yellowish green leaves in B5 medium. 

Rooting and formation of cormlets from multiple shoots 

of Gladiolus sp. were achieved on different concentrations 

of auxins on liquid MS media. The maximum percentage of 

Fig. 4. In vitro root explants grown in MS medium with 1.0 

mg/l BAP (4-5 weeks old) 

Fig. 5. Initiation of organogenesis from in vitro rooted explants 

on 1X MS medium with 2.0 mg/l BAP 

Fig. 6. Enhanced shoot development during second sub-culture 

on MS medium containing with 2.0 mg/l BAP 

Fig. 7. Induction of corms with rooted shoots on 1.0 mg/l IBA 

with agar medium 

Fig. 8. In vitro regenerated micro-corms of Gladiolus


In present research the full strength MS medium was 

found best over other strengths of MS and B5 medium for 

further multiplication of shoots. This result is in line with many 

findings in which MS medium is reported best for shoot 

induction and multiplication (Bruyn and Ferreira, 1992; Kumar, 

et al., 1999). Rooting of shoots was achieved on auxins 

supplemented liquid MS medium. The transfer of in vitro 

produced plantlets and cormlets to soil and the direct transfer 

of in vitro plantlets to non-aseptic conditions resulted in a 

very low percentage of re-established plants. To overcome 

this problem the plantlets were thus left to dry in culture 

bottles, with the subsequent production of small, dormant 

cormlets. These cormlets had to be given low temperature 

treatment to break their dormancy before further planting. 


We express our heartily gratitude to Chairman, MIET, 

Meerut for providing funds and lab facility for the study. 


Begum, S. and Haddiuzaman, S. 1995. In vitro rapid shoot proliferation 

and corm development in Gladiolus grandiflorus cv. Redbrand. 

Plant Tissue Cult., 5: 7-12. 

Bruyn, M.H. and Ferreira, D.I. 1992. In vitro corm production of 

Gladiolus dalenii and G. tristis. Plant cell, tissue and organ culture 

31: 123-128. 

Ginzburg, C. 1973. Hormonal regulation of cormel dormancy in 

Gladiolus grandiflorus. Journal of Experimental Botany, 24: 558- 

566. 

Koda, Y. and Okazawa, Y. 1983. Influences of environmental, hormonal 

and nutritional factors on potato tuberization in vitro. Japanese 

Journal of Crop Science, 52: 582-591. 

Kumar, A., Sood, A., Palni, L.M.S. and Gupta, A.K. 1999. In vitro 

propagation of Gladiolus hybridus Hort.: Synergistic effect of heat 

shock and sucrose on morphogenesis. Plant cell, tissue and organ 

culture, 57: 105-112. 

Kumar, A., Palni, L.M. and Sood, A. 2011. Factors affecting in vitro 

formation of cormlets in Gladiolus hybridus Hort. and their field 

performance. Acta Physiologiae Plantarum, 33: 509-515. 

Logan, A.E. and Zettler, F.W. 1984. Rapid in vitro propagation of 

virus-indexed Gladioli. In: VI International Symposium on Virus 

Diseases of Ornamental Plants 164, pp.169-180. 

Novak, F.J. and Petru, E.A. 1981. Tissue culture propagation of Lilium 

hybrids. Scientia Horticulturae, 14: 191-199. 

Ruffoni, B., Pamato, M., Giovannini, A. and Brea, M. 2008. Gladiolus 

micropropagation in temporary immersion system. Propagation 

of Ornamental Plants, 8: 102-104. 

Simonsen, J. and Hildebrandt, A.C. 1971. In vitro growth and 

differentiation of Gladiolus plants from callus cultures. Canadian 

Journal of Botany, 49: 1817-1819. 

Steinitz, B. and Lilien-Kipnis, H. 1989. Control of precocious gladiolus 

corm and cormel formation in tissue culture. Journal of plant 

physiology, 135: 495-500. 

Takatsu, Y., Miyamoto, M., Inoue, E., Yamada, T., Manabe, T., Kasumi, 

M., Hayashi, M., Sakuma, F., Marubashi, W. and Niwa, M. 2001. 

Interspecific hybridization among wild Gladiolus species of southern 

Africa based on randomly amplified polymorphic DNA markers. 

Scientia horticulturae, 91: 339-348. 

Takayama, S. and Misawa, M. 1982. Regulation of organ formation by 

cytokinin and auxin in Lilium bulbscales grown in vitro. Plant and 

Cell Physiology, 23: 67-74. 

Ziv, M. and Lilien-Kipnis, H. 2000. Bud regeneration from inflorescence 

explants for rapid propagation of geophytes in vitro. Plant Cell 

Reports, 19: 845-850. 



Establishing Callus Formation of Swarna and IR 64 and Sub-culturing of Callus 

and Monitoring the Stages of Regeneration 

KSHITIJ KUMAR AND K.N SINGH 

Department of Plant Molecular Biology & Genetic Engineering 

Narendra Deva University of Agriculture & Technology Narendra Nagar (Kumarganj), Faizabad (U.P) 

e-mail: patelbiotech@gmail.com 

ABSTRACT 

The present regeneration from the target tissue (explant) of 

the genotype (s) is concerned. The present study was conducted 

with an objective to standardize efficient regeneration system 

for IR64 and Swarna at N.D.U.A.T (U.P.) India. MS medium 

supplemented with 2,4-D, (2mg/l) kinetin, (0.4mg/l). L. Proline, 

(500mg/L) Casein Hydrolysate, (300mg/L) agar-agar (0.8%w/v) 

was found best callus medium for Swarna. MS medium 

supplemented with 2,4-D, (2mg/L) L-Proline (500mg/L) maltose 

(30g/L) agar agar (0.8%w/v) was found to be best callus medium 

for IR64. 

Key words 

indica rice, IR64, regeneration, rice, 2, 4-D, Swarna 

Rice is stable food for more than 70% of the population 

(Prasad and Pandey, 2004). It is the source of livelihood for 

millions rural households and is the backbone of Indian 

agriculture. Swarna is mainly grown in Andhra Pradesh and it 

has a rainfed shallow land ecosystem (Singh and Singh, 2003). 

Swarna is indica type of rice variety released in 1982. Its 

parentage is vasistha x mahsuri with 155 days of maturity 

duration. Its grain type is medium slender. Special features of 

this variety are : good grain quality and higher yield under 

low nitrogen level. 

IR 64 is also indica type variety with long slender grain 

type. It has adaptability to irrigated ecosystem. It is of 130 

days maturity duration. Its parentage are IR 5657-33-2-1 X IR 

2061-465-1-5-5. Its special feature is : fine grain quality, high 

harvest index, resistance to blast and bacterial leaf blight. IR 

64 is released by IRRI and it grown across the world (Singh 

and Singh, 2003) but both the rice verities are susceptible to 

many abiotic and biotic factors. 


The present study was conducted establish callus 

formation of Swarna and IR 64 and Sub-culturing of callus 

and monitoring the stages of regeneration at N.D.U.A.T (U.P.) 

India for the objectives were to develop efficient cell culture 

two popular indica cultivars (IR 64 and Swarna) were 

subjected to somatic cell culture. 

The explants used for in vitro culture in the present 

study were mature seeds of the select rice cultivars. Fresh 

seeds obtained from crops raised by sowing the source seeds 

were dried well in sunlight and were stored at 4 0 C before use. 

The cultures were maintained at 2000 lux light intensity and a 

temperature of 25 0 C and 60% RH under 16 hour light and 8 

hour dark cycles. Growth regulators (2-4D, BAP) stock 

solutions were made with concentration of 10mg/10ml 

(Murashige and Skacethog). Ms media was used in the present 

study with modification (Murashige and Skoog, 1962) for 

callus culture. The basel medium comprised of the stock 

solution macronutrients, micronutrients, minornutrients, iron 

stock, vitamins and myo-inositol were mixed in known quantity 

in doubled distilled water accordance with composition of the 

basel medium requirement. Stock solution were stored at 4 0 C 

temperature in glass bottle whereas the iron stock solution in 

coloured bottle. Maltose was used as a carbohydrate source 

which was added and mixed well. Desired growth hormones 

were added to the media as per medium composition. For making 

one liter of culture media was prepared in 950 ml and pH was 

adjusted to 5.6 to 5.8 with 0.1 N HCl or 0.1N NaOH and volume 

was made upto 1 liter. After pH adjustment bacteriological 

grade agar-agar was added melted and added as per 

requirement to the boiling media and constantly stirred. Ten 

ml of media was dispensed in each test tube of dimension (250 

x150 mm) or transferred to 250 ml/ 500 ml/1lit.conical flask and 

plugged with non-absorbent cotton plugs. Media was 

autoclaved at a temperature of 121 0 C and at a pressure of 1.01 

kg/cm 2 for 20 minutes. Media prepared in conical flask were 

later melted and poured into sterile petriplates (25-30 ml per 

plate) under aseptic condition. Petriplates test tubes used for 

inoculation were sterilized in autoclave at a temperature of 

121 0 C and at a pressure of 1.01 kg/cm 2 for 20 minutes and 

equipments (like sterile blades and forceps) were presterilized 

and placed in glass bead sterilizer and cooled of before 

inoculation. Dehisced seeds were washed with sterilized 

double distilled water twice. Dehusked seeds were then 

soaked in 70% ethanol for 2 minutes. Dehusked seeds were 

washed twice with sterilized double distilled water. Dehusked 

seeds were surface sterilized using sodium hypochlorite (4% 

active chlorine and two drop of tween-20 to the seeds for 15 

minutes with continuous shaking. Dehusked seed were 

washed with sterilized double distilled water for two to three 

times. Dehusked seeds were re-exposed to 0.1% mercuric 

choloride and then kept for 5 minutes. The sterilized seeds 

were washed well with sterilized double distilled water for 5 to 

6 times to remove traces of sterilants. The seeds then


transferred to sterilized filter paper to remove excess moisture 

and was then inoculated to respective media and kept in 

culture room for in vitro germination. 

Mature seeds were dehusked manually prior to surface 

sterilization, placed in 70 per cent ethanol for 3 min, followed 

by treatment with 0.1 per cent mercuric chloride and a drop of 

tween-20 for 10 min with occasional stirring. The seeds were 

then rinsed thoroughly 4-5 times with sterile distilled water to 

remove any traces of mercuric chloride and were plated onto 

respective callus induction medium. The plates were incubated 

in the dark at 25±2 0 C. Calli obtained after 21-35 days were 

subculture onto fresh medium or used to initiate suspension 

culture. The callus was maintained by sub culturing onto the 

fresh but the same medium at 2-3 week intervals. 

Maltose (as a carbohydrate source) was used in 

concentration (30 g/l).media code Ms 9 

and Ms 7 

the 

carbohydrate source was changed from maltose to sucrose 

with the concentration (30 g/l). Organic supplements as L- 

Proline and Casine hydrolysate were added as described .pH 

of media was adjusted between 5.6-5.8 and agar agar of 

bacteriological grade was added with concentration with 0.8 

% w/v. Volume of the media was made 1 liter with 10 ml in each 

test tube and was autoclaved. One seed per test tube was 

inoculated for callus induction in dark condition for 21 days. 

Per treatment 50 test tubes was inoculated. Compact and 

nodular callus developed with in three weeks of culture, which 

could be subcultured to the same fresh media in order to 

maintain the culture. Callus of both the indica varieties (IR-64 

and Swarna) were put on the respective callus media as shown 

in (Table 1) for four week. 

Concentration of agar-agar and pH range was kept same 

as in callus induction media. Regeneration media contained 

the same basal media as described including the 

corresponding sugar supplements. The growth regulators 

supplements however, was changed as shown in Table 2. Agar 

agar of bacteriological grade was used at a concentration 0.6 

% w/v pH was adjusted to 5.6-5.8 with the help of 0.1N HCl or 

0.1N NaOH. Tubes were inoculated with callus and kept in 

dark for two weeks and then they were transferred to the light 

condition of 16 hours photoperiod and 8 hours dark period 

for another three weeks per-treatment 10 test tubes /callus 

were inoculated in both indica cultivars. 


The reason behind using the mature seed as an explant 

was that the explant should be readily available in large 

quantity so that the adds of obtaining the desired no. of 

regenerated plants can be improved .There had been reports 

on plants regeneration from mature embryo derived callus of 

japonica varieties (Fujimura, et al., 1985) but indica cultivars 

were found to be recalcitrant nature of tissue culture Koetji, 

et al., 1989 described callus induction in general, rice, Oryza 

sativa L. cultivars belonging to subspecies indica were known 

to be less responsive for callus induction as well as 

regeneration, however embryogenic callus induction which 

was dependent on the interacting between the genotype and 

culture condition and regeneration , rather the callus induction 

is the limiting factor of the indica rice cultivars. From the 

present study, it was clear from interaction between genotype 

and composition me not need the nutritional requirements 

may vary genotype to genotype. More ever, this differential 

requirement was also found to be significantly interaction 

between genotype and media as well as phytohormonal 

combination (Table 3) and carbohydrate source (Table 5 and 

6) as seen from result Swarna has the maximum response of 

MS media supplemented with a combination of 2,4-D (2 mg/ 

l) + kinetin (0.4 mg/l) + L-Proline (500 mg/l) casein hydrolysate 

(300 mg/l) resulting in 80% callus formation. While IR64 has 

shown good response on MS media supplemented with 

combination of 2,4-D (2mg/l) + L-Proline (500 mg/l) + casein 

hydrolysate (300 mg/l) with 70% callus formation (Table 6) 

callus of both the varities were creamy in colour. Genotypic 

difference for culture response in rice were also reported by 

several authors (Visarda, et al., 2002) earlier and present result 

are in agreement with earlier finding. 

The improve somatic cell culture response by addition 

of maltose might be due to reduce rate of hydrolysis of 

disaccharide to glucose -1 –phosphate as reported by Sopary, 

1979, same result was later reported by last and Bretlel, 1990, 

Maltose as carbohydrate source in callus induction is superior 

to sucrose in callus induction is superior to sucrose in callus 

quantity (Zhang, 1995) the present result (Table 5 and 6) are in 

agreement of earlier finding. In the present study to organic 

supplements were used i.e, L-Proline (500 mg/l) and casein 

hydrolysate (300 mg/l) (Sivamani, et al., 1996). 

It could be seen from the result that IR64 responded 

well on MS BAP (2 mg/l) + NAA (0.8 mg/l) + Kinetin (0.8 mg/ 

l) with mean number of shoot were as Swarna responded well 

as MS + BAP (1.5 mg/l ) + NAA (0.6 kinetin (0.6 mg/l ) with 14 

mean number of shoot (Table 9) Incubation of tube in dark 

condition is well documented by (Zhang, 1995). The addition 

of NAA alone in regeneration media was found to be inhibit 

regeneration of rice is well documented (Venkatachalam, et 

al., 2000). 

Explant source 

Mature seeds used as explants. The seeds were procured 

from Crop Research Station, Masodha, Faizabad. Main 

advantage of using seed as explant is that it is available year 

round. 

Callus induction 

Germination in the both indica cultivars seeds were 

noticed from the third day onwards. Swelling of the mesocotyl 

and radical was observed after 5-6 days and this was followed 

by the induction of callus from the scutellar region on 8-10

KUMAR AND SINGH, Establishing callus formation of Swarna and IR-64 and Sub-culturing of callus and monitoring 173 

days. The hypocotyls region was swollen and later 

appearance of callus was observed. 

Effect of hormones for callus induction in Swarna 

MS basal media different combination of 2, 4-D (0.5, 1.0, 

1.5, 2.0, 2.5 and 3.0 mg/l), Kinetin (0.2, 0.4 and 0.6 mg/l), L- 

proline (500 mg/l) and casine hydrolysate (300 mg/l) were used. 

All the combination responded differently. The culture media 

MS 1 

and MS 2 

did not formed callus, but resulted in plumule 

and radical formation. All other combinations produced callus. 

The percentage of callus was different from one treatment to 

another. Callus formed in medium with percentage 25, 54, 40, 

35, 45, 64, 80 and 48 in MS 3 

, MS 4 

, MS 5 

, MS 6, 

MS 7 

, MS 8 

, MS 9 

and MS 10 

were respectively (Table 3). The MS 9 

media callusing 

percentage was the highest (80%) and was selected as best 

callus forming media for Swarna. The callus were yellowish 

colour and globular in shaped (Fig. 1). 

Effect of hormone on callus induction in IR 64 

MS basal media with different concentration of 2, 4-D 

(0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l), Kinetin (0.2, 0.4 and 0.6 mg/ 

l), L-proline (500 mg/l) and casine hydrolysate (300 mg/l) were 

used for callus induction. The media combination MS 1 

and 

MS 2 

did not produce callus but resulted in plumule and radical 

formation after 21 days dark incubation. All other combination 

used in the present study resulted in callus formation in 

different percentage. The medium combination MS 3 

, MS 4 

, MS 5 

, 

MS 6, 

MS 7 

, MS 8 

, MS 9 

and MS 10 

formed callus with percentage 

of callus induction was 30, 55, 44, 34, 70, 56, 50 and 40 

respectively (Table 5). In MS 7 

media callus forming capacity 

was highest (70%) and was selected as the most suitable 

medium concentration for callus induction in IR 64 (Fig.1). 

Comparing callus induction percentage in IR 64 and 

Swarna 

Comparing callus induction percentage in both indica 

cultivar best callus induction media of both the variety were 

identified. The seeds were inoculated in respective media and 

kept in dark condition for 21 days at a temperature 23 ± 2 0 and 

Table 1. Sub culturing media of both the indica varities 

S.No. Variety Sub culturing media 

1 Swarna MS+2, 4-D (2.0mg/l) + L - Proline (500mg/l) 

+Casine hydrolysate (300mg/l+Kinitin(0.4mg/l) 

2. IR-64 MS+2, 4-D (2.0mg/l) + L - Proline (500mg/l) + 

Casine hydrolysate (300mg/l) 

Concentration of agar-agar and pH range was kept same as in callus 

induction media 

Table 2. 

Composition of shoot regeneration media 

S. 

No. 

Medium 

code 

Basal 

media 

BAP 

(mg/l) 

NAA 

(mg/l) 

Kinitin 

(mg/l) 

1. MSR 1 MS 0.5 0.2 0.2 

2. MSR 2 MS 1.0 0.4 0.4 

3. MSR 3 MS 0.5 0.6 0.6 

4. MSR 4 MS 2.0 0.8 0.8 

5. MSR 5 MS 2.5 1.0 0.8 

RH 60%. After expiry of period number of callus produced 

were counted. The percentage of callus formation of both 

indica cultivar if given in Table 5. 

Callus induction percentage of swarna was 80% whereas 

is IR 64 it was 70% which was lesser by 10% in IR 64. 

Effect of different carbohydrate source on callus by the 

indica test genotype 

When carbon source replaced maltose in the MS 0 

media 

with concentration 10 g/l for callus induction in Swarna a 

significant decrease in callusing percentage was recorded as 

detailed in Table 7. 

In Swarna MS 9 

media fortified with maltose had higher 

percentage of callus induction then MS 9 

media fortified with 

Sucrose by 32 per cent. Seeds were inoculated in dark 

condition for 21 days at a temperature of 23 ± 2 0 C and 60% 

RH. Under similar condition of incubation for IR 64 with media 

MS 7 

fortified with maltose (30g/l) and MS 7 

media fortified with 

sucrose (30g/l). The result of this is shown in Table 7. 

These experiments were not repeated. 

Fig. 1. Callus forming media 

Table 3. Percentage of callus induction in different 

combination in Swarna 

Medium Media composition 

Percentage 

of callus 

induction 

MS 1 MS +2,4-D (0.5 mg/l) - 

MS 2 MS +2,4-D (1.0 mg/l) - 

MS 3 MS +2,4-D (1.5 mg/l) 25 

MS 4 MS +2,4-D (2.0 mg/l) 54 

MS 5 MS +2,4-D (2.5 mg/l) 40 

MS 6 MS +2,4-D (3.0mg/l) 35 

MS 7 MS +2,4-D (2.0 mg/l) + L-proline (500 mg/l)+ 45 

Casine hydrolysate (300 mg/l) 

MS 8 MS +2,4-D (2.0 mg/l) + L-proline (500 mg/l) + 64 

Casine hydrolysate (300 mg/l)+ Kinetin (0.2 mg/l) 

MS 9 MS +2,4-D (2.0 mg/l) + L-proline (500 mg/l)+ 80 


MS 10 MS +2,4-D (2.0 mg/l) + L-proline (500 mg/l) + 


48


Different stages of embryo 

Different stages of embryo of both the indica cultivars 

are shown in Fig. 1. Different stages of embryo were compared 

at the intervals of 7, 14, 24, and 30 days. Both the varieties 

callus were globular and having knobby any structure. The 

callus formed shoots only directly on callus without any 

formation of embryo or embryo like structure. In some medium, 

callus formed nodular structure without any further 

differentiation. 

Regeneration 

Different media compositions were used for regeneration 

in IR 64 and Swarna as shown in (Table 2) callus of both the 

indica cultivars were put on the regeneration media and 

incubated in dark condition for two weeks and replaced to 

16hr light periods for three weeks with temperature 23 ± 2 0 C 

and 60% RH. Greening in callus started in third week onward. 

In subsequently formation of shoot resulted in fifth weeks. 

Effect of hormones used in regeneration of Swarna 

A combination of MS basal medium in different 

concentration of BAP (0.5, 1.0, 1.5, 2.0 and 2.5 mg/l) along 

NAA (0.2, 0.4, 0.6, 0.8 and 1.0 mg/l) and kinetin (0.2, 0.4, 0.6, 0.8 

and 1.0 mg/l) was used. None of the media produced shoots 

upto two weeks as the culture was maintained in dark. However, 

greening at different location observed on third weeks onward 

on transfer to illuminated racks. The media MSR 1 

did not 

responded to regeneration from callus culture and no shoot 

formation resulted. All other media resulted in shoot formation. 

However, frequency of regeneration differed in different 

hormone concentration used with MS basal media. Mean 

number of shoots formation was recorded in MSR 2 

, MSR 3 

, 

MSR 4 

and MSR 5 

with shoots 2, 10, 6 and 8 respectively. The 

best regeneration media for swarna is MSR 3 

with 10 mean 

Table 4. 

Percentage of callus induction in different 

combinations in IR64 

Medium Media composition Percentage of callus 

induction 

MS 1 MS +2,4-D (0.5 mg/l) - 

MS 2 MS +2,4-D (1.0 mg/l) - 

MS 3 MS +2,4-D (1.5 mg/l) 30 

MS 4 MS +2,4-D (2.0 mg/l) 55 

MS 5 MS +2,4-D (2.5 mg/l) 40 

MS 6 MS +2,4-D (3.0mg/l) 34 

MS 7 MS +2,4-D (2.0 mg/l) + L-proline 70 

(500 mg/l)+ Casine hydrolysate (300 

mg/l) 



mg/l)+ Kinetin (0.2 mg/l) 




MS 10 MS +2,4-D (2.0 mg/l) + L-proline 



40 

numbers of shoots (Fig.1). 

Effect of hormone on regeneration of IR 64 

A combination of MS basal medium different 

concentration of BAP (0.5, 1.0, 1.5, 2.0, and 2.5 mg/lk) along 

with NAA (0.2, 0.4, 0.6, 0.8 and 1.0 g/l) and kinetin (0.2, 0.4, 0.6, 

0.8 and 1.0 mg/l) was used. None of the media produced shoots 

upto weeks as the culture were maintained in dark. However, 

greening at different location observed on third weeks onward 

on transfer to illuminated racks. The media MSR 1 

did not 

responded to the regeneration from callus culture and no 

shoot formation resulted. The frequency of regeneration was 

different in different hormonal combination. The mean number 

of shoots in media combination MSR 2 

, MSR 3 

, MSR 4 

and MSR 5 

were 1, 2, 6 and 4 respectively. The best regeneration media IR 

64 was MSR 4 

on the basis of highest number mean shoot 

formation (8) (Table 8) (Fig. 1). 

Comparing regeneration percentage between IR 64 and 

Swarna 

For comparing the regeneration percentage both indica 

cultivars, media selected was on which maximum number of 

regeneration was recorded. Calli of the indica cultivars were 

inoculated on respective regeneration media for two weeks 

dark condition and were transferred to light condition of 16 

hrs light period and 8 hrs dark period with 60% RH and 23 ± 

2 0 C temperature for three weeks. Comparison of both indica 

cultivars is presented in (Table 9). Fig. 1. Mean number of 

shoots regenerated in swarna were 10 whereas in IR were 6, 

which was lesser by 4. 

Quality of callus induced on mature seeds 

Callus obtained by culturing of mature seed explants on 

callusing medium could be clearly distinguished into two type 

namely, embryogenic and non embryogenic calli. Embryogenic 

Table 5. 

Table 6. 

Comparison of callus induction percentage of both 

indica cultivars 

S. Variety Media composition 

No. 

1. Swarna MS +2,4-D (2.0 mg/l) + L-proline 



2. IR64 MS +2,4-D (2.0 mg/l) + L-proline 


mg/l) 

Percentage of 

callus induction 

Effect of different carbohydrate source on callus 

induction of IR64 

Medium Media composition Percentage of callus 

induction 

1 MS +2,4-D (2.0 mg/l) + L-proline 

(500 mg/l)+ Casine hydrolysate (300 75 

mg/l)+ Maltose (30g/l) 

2 MS +2,4-D (2.0 Mg/L) + L-Proline 

(500 Mg/L)+ Casine Hydrolysate 

(300 Mg/L) + Sucrose (30g/l) 

63 

80 

70

KUMAR AND SINGH, Establishing callus formation of Swarna and IR-64 and Sub-culturing of callus and monitoring 175 

Table 7. 

Mean number of shoot induction in Swarna 

Medium Media composition 

code 

MSR 1 MS+ BAP (0.5mg/l) + NAA (0.2 










Mean number of 

shoots formed 

- 

Table 8. Mean number of shoot induction in IR 64 

Medium 

code 

Media composition 


shoots formed 

MSR 1 MS+ BAP (0.5mg/l) + NAA (0.2 mg/l)+ - 

Kinetin (0.2 mg/l) 

MSR 2 MS+ BAP (1.0mg/l) + NAA (0.4 mg/l)+ 1 






MSR 5 MS+ BAP (2.5mg/l) + NAA (1.0 mg/l)+ 


4 

calli were composed of compact cell with dense cytoplasm 

and were darkly stained. In contrast non embryogenic calli 

were loosely packed with sparse cytoplasm were lightly 

stained. A number of nodulations could be seen on the surface 

of 30 days old embryogenic calli of swarna. However, such 

protuberances were not associated with IR 64. 

Regenerating calli 

A weeks after transfer of embryigenic calli onto the 

regeneration medium formation of globular embryoid like 

structure could be observed in case of IR 64, ten to twelve 

days later, development of heart shaped embryoid like 

structure could be observed. 

In Swarna, MS media supplemented with 2,4-D (2 mg/l) 

+Kinetin (0.4 mg/l ) + L-Proline (500 mg /l ) + Casein hydrolysate 

300 mg/ l) + Maltose (30 g/l ) + Agar agar (0.8% w/v ) was 

found best callus induction media with 80% callus induction 

percentage. 

IR 64 produced best callus in Ms media supplemented 

2 

10 

6 

8 

Table 9. 

Comparison of mean under of shoots in both indica 

cultivars 

S.No. Media composition 


shoots formed 

1. MS+ BAP (1.5mg/l) + NAA (0.6 mg/l)+ 10 


2. MS+ BAP (2.0mg/l) + NAA (0.8mg/l)+ 


6 

with 2, 4-D (2 mg/l) + L-proline (500 mg/l) + Casein hydrolysate 

(300 mg/l) +Maltose (30 g/l ) + Agar–agar (0.8 % w/v ) was 

found best callus induction media with 70 % callus induction 

percentage. 

The shoot induction media was different for both the 

indica cultivars IR 64. produced maximum number of healthy 

green shoot in MS media containing BAP (2.0 mg/l ) + NAA 

(0.8 mg/l ) + Kinetin (0.8 mg/ l) + maltose (30 g/l) + Agar-agar 

(0.6% w/v ). The mean numbers of shoots induced were 6. 

Whereas Swarna produced maximum shoot in MS 

containing BAP (1.5 mg/l) + NAA (0.6 mg/l) + Kinetin (0.6 mg/ 

l) + Maltose (30 g/l ) +Agar agar (0.6% w/v ) The mean of 

shoots induced were 10. 


Fujimura, T., Sakurai, M., Akagi H., Negishi, T. and Hirose, A. 1985. 

Regeneration of rice plants from protoplasts. Plants Tissue. Cult. 

Lett., 2: 74-75. 

Koetija, D.S., Grines, H.D., Wang, Y.C. and Hodges, T.K. 1989. 

Regeneration of indica rice (Oryza sativa L.) from primary callus 

derived from immature embryos. J. Plant Physiol., 135: 184-190. 

Prasad, R. and Pandey, 2004. Text book of field crop production, 

Directorate of Information and Publication of Agri., pp.1-54. 

Singh, C.P. and Singh, R. 2003. Modern Technique of Raising field 

crop, pp.1-54. 

Venkatachlam, P., Geetha, N., Priya, P., Rajasegar, G. and Jayabalan, N. 

2000. Efficient callus induction and plant regeneration from 

immature anther of Rice (Oryza sativa L.) via Somatic 

Embryogenesis. Plant Cell Biotech Mol. Biol., 1(1&2): 55-62. 

Zhang, S. 1995. Efficient plant regeneration from Indica (group I) rice 

protoplast of one advanced breeding line and three varieties. Plant 

Cell Reports, 15: 68-71. 



In Vitro Regeneration of Banana Variety Grand Naine (G 9) 

MANJU RAI*¹, PALLAVI MITTAL², AMANDEEP KAUR³, GURPREET KAUR³, ISHA GAUR³ AND 

CHARANDEEP SINGH 4 

1 

Raizo Biotec Labs, Ludhiana, Punjab 

2 

I.T.S Para Medical College, Muradnagar, Gaziabad 

3 

Chandigarh Group of Colleges, Landran, Mohali, Punjab 

4 

B.I.S Institute of Science and Technology, Moga, Punjab 

1 

e-mail: raizobiotec@raizo.com 

ABSTRACT 

This research was planned to study the effect of various 

combinations of auxins and cytokinins on micropropagation of 

“Grand Naine” cultivar of Banana (Musa). Rhizomes bearing 

the meristematic shoot tips were taken as explants from 

greenhouse maintained plants. These were surface sterilized 

with different concentrations of bavistin and HgCl 2 

for different 

intervals of time and cultured on MS media supplemented 

with different concentrations of BAP (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 

3.0 mg/l) and NAA (0.25 and 0.5 mg/l). BAP at 2.0 mg/l along 

with NAA at 0.5 mg/l proved to be the best combination and 

showed optimum shoot growth. Multiplicated shoots were 

inoculated on rooting media incorporated with either IBA or 

NAA (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and Charcoal (2 

gm/l) for root induction. IBA (2mg/l) and Charcoal (2 gm/l) 

produced maximum number of roots with a lot of root hairs. 

Shoots obtained in rooting media were hardened in portrays 

containing different potting mixtures, of which the mixture of 

Cocopeat and sand (2:1) showed maximum (96%) survival of 

plantlets. 

Key words 

Surface sterilization, multiplication, root induction, 

micropropagation, microshoots, hardening. 

Grand Naine are the cultivars of Musa acuminate and 

have become one of the most popular varieties for cultivation 

in Punjab under the crop diversification scheme. Punjab has 

suitable climate (sub-tropical) for banana cultivation as this 

particular cultivar is able to face climatic extrimities quite boldly. 

The main characteristics are its medium height about 6 ft and 

large fruit yields (20-25 kg/plant in Punjab). The moderate 

height allows easy harvesting and some resistance to 

windthrow. Micropropagation is an effective method for mass 

scale multiplication of disease free planting material in a short 

duration of time . Using vegetative parts such as suckers for 

propagation, there is a risk of transmitting the serious diseases 

like Banana Bunchy Top Virus (BBTV) which tremendously 

reduces the yield. As a non-seasonal crop, bananas are 

available fresh-year-round. 


Suckers of cultivar “Grand Naine” were collected from 

the green house growing plants of Raizo Biotec Labs. 

Meristematic shoot tips along with the underground rhizome 

(Fig.1a) were rinsed thoroughly with Teepol solution (2%) 

and then dipped in 1% Bavistin solution for 3 to 4 hours for 

surface decontamination. The explants were then trimmed off 

with the help of scalpel blade and converted to a suitable size 

(1-2 cm) for culturing (Fig. 1b). The rhizome bearing the shoot 

tip were further surface sterilized with 0.5% Bavistin for 12 

minutes followed by 0.1% HgCl 2 

for 7 minutes in the laminar 

air flow hood. Finally, explants were washed thrice with double 

distilled autoclaved water and cultured (Fig. 1c) on MS basal 

medium (Murashige and Skoog , 1962). The pH of the medium 

was adjusted to 5.8 and autoclaved at 1.04kg/cm² pressure 

and 121°C temperature for 15-20 minutes after dispensing 30 

ml each in jam jars. The cultures were incubated in the culture 

room maintained at standard conditions of temperature and 

light i.e, 25±2? under 3000 lux of light for 16 h light per day . 

Observations were recorded after one week. The shoots so 

induced on the basal media were subcultured on MS media 

supplemented with different growth regulators viz., BAP (6 

benzyl-amino purine) and NAA (á-naphthalene acetic acid). 

BAP at different concentrations (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 

3.0 mg/l) and NAA (0.25 and 0.5 mg/l) either individually or in 

combination and incubated for four weeks under the above 

mentioned culture conditions. After four weeks growth 

analysis, expressed as the number of shoots,length of shoots 

and type of shoots were recorded. Shoots grown on 

multiplication media for 7 cycles are further inoculated on 

rooting media for root induction. The rooting media is 

supplemented either with NAA or IBA (indole butyric acid) 

of different concentration (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l) 

along with charcoal. Observations were recorded after 14 days 

of incubation. The rooted plants were removed from the jars 

after 21 days on rooting media and were washed under running 

water for removal of agar and then were subjected to primary 

hardening in trays on moist cotton for 2 days (Fig.). Finally, 

the in vitro rooted plants were acclimatized on different potting 

mixtures Cocopeat and sand (2:1), farmyard manure and sand 

(1:1:1) and farmyard manure, sand and soil (1:1:1) to record 

the observations for the best potting mixtures for the survival 

of plants after 21 days of hardening. 

For each treatment, 12 replicates were used and each 

experiment was repeated thrice. The experimental data were

RAI et al., In vitro Regeneration of Banana Variety “Grand Naine” (G-9) 177 

analysed statistically by one way analysis of variance 

(ANOVA) to determine the variation between and within the 

treatments with respect to number of shoots and roots. 


The response of Musa spp. cv. Grand Naine rhizomes 

with apical buds as explants cultured on different proliferation 

media over a period of six weeks are presented in Table 1. For 

Multiplication of shoots cytokinin and auxin are the combined 

factors which influence the growth and multiplicity of the 

shoots. Culture medium devoid of growth regulators (control) 

failed to stimulate the bud break response in the explants 

even when cultures were maintained beyond the normal 

observation period of four weeks. MS medium with growth 

regulator supplements produced better results in terms of 

percentage explants response,shoots/explants and average 

shoot length . Of the combinations tested MS + BAP ( 2 mg/ 

l ) + NAA ( 0.5 mg/l ) elicited optimal response in which the 

average of 7.5 ± 0.45 shootlets (Fig. 2a , Table 1) with a mean 

shoot length of 6.2 ± 0.37 cm/explants was recorded. The 

second best shoot multiplication was obtained in the medium 

MS + BAP ( 1.5 mg/l ) + NAA ( 0.5 mg/l ) with a mean shoot 

length of 5.2 ± 0.43 cm (Fig. 2b). Higher concentration of BAP 

( 2.5 and 3.0 mg/l ) with NAA (0.5 mg/l) showed nodulation of 

the explants with fewer number of shoots. In such cultures 

shoots were stunted with minimum shoot length of 2.5 ± 0.43 

cm. significantly higher response in medium with combination 

of 2.0 mg/l BAP and 0.5 mg/l NAA has been recorded in the 

present study. The quality of shoots and the overall growth 

response in terms of average shoot length was shown better 

in this growth regulator combination. A comparatively low 

response was observed when BAP alone was added in the 

medium. This indicates that the addition of NAA , IBA or IAA 

to the medium improved the response of shoot growth in a 

number of species. Strosse, et al., 2008 reported that cytokinins 

NAA and BAP (5 mg/l) were used for the highest multiplication 

rate in banana. Height of the shoots was also maximum at this 

ncentration. Oliveira, et al., 2000 carried out the bud 

multiplication with 4.0 mg/l BAP for 3 subcultures. The 

multiplication rate was highest, without any contamination. 

Liang Jing Xan and Hu Yu Lin, 2011 used different hormone 

combinations of 0.4 – 0.6 mg/l BAP and 0.1 – 0.2 mg/l NAA to 

induce the adventitious buds. The highest multiplication rate 

was found in MS medium for subculturing containing 0.6 mg/ 

l BAP and 0.2 mg/l NAA, give rise to strong buds and normal 

leaf color were realized under these conditions. Muhammad, 

2004 studied the effect of BAP in combination with IAA (indole 

-3- acetic acid) on shoot proliferation. Concentration of BAP 

and IAA ranged from 0.1 to 0.4 mg/l each on solid or liquid 

medium. The minimum number of shoots regenerated from a 

single shoot tip was achieved in liquid MS media containing 

4.0 mg/l BAP and 0.1mg/l NAA. The best result demonstrated 

that 4.0 mg/l BAP and 1.0 mg/l NAA. Olivia, et al., 1990 carried 

out the culturing on ½ strength MS media with 5 mg/l BAP 

and 100 ml coconut water for shoot multiplication. Banerjee 

and Langhe, 1985 carried out the multiplication on the MS 

media supplemented with BAP 2.30 mg/l and IAA 1.80 mg/l. 

This concentration was used for rapid clonal propagation and 

stored under minimal growth conditions. 

Well developed elongated shoots measuring 4 – 5 cm in 

length were excised from shoot clump and transferred to MS 

medium containing IBA or NAA and charcoal (2 g/l). The 

rooting responses of excised shoots on different media , which 

includes rooting percentage, days required for root initiation, 

mean number of roots/shoot and mean root growth over a 

period of four weeks were recorded (Fig. 3, Table 2). No rooting 

was observed in auxin free basal medium (control). Similarly 

at lower level of IBA (0.25 mg/l) treatments, there was hardly 

any root emergence within four weeks of observation period. 

However on all the higher concentrations of IBA so tested 

(1.5 and 2.0 mg/l) good rooting response was noted. MS with 

2.0 mg/l IBA showed 95 % cultures responding to rooting 

with an average of 8.5 ± 0.33 roots per microshoot and an 

average shoot length of 3.5 ± 0.38 cm was recorded (Fig. 3a , 

Table 2). There was a remarkable increase in both shoot and 

root length after six weeks of culture (Fig. 3b). The second 

highest response (70%) was recorded at 1.5mg/l of IBA with 

an average root number of 6.0 ± 0.18 roots/plantlet with an 

average root length of 3.0 ±0.09 cm. In the present 

investigations it was observed that root primordial emerged 

from the base of the microshoots within 6 – 8 days after shoot 

inoculation and soon after that the growth was rapid. IBA 

was found to be more effective than NAA in induction of 

rooting as days required were only 6 – 8 as against 10 – 16 

days for similar response with IBA . Production of plantlets 

with profuse rooting in vitro is important for successful 

establishment of regenerated plants in soil (Sharma and Singh, 

1997). Similar findings were reported by Senthilkumar and 

Ramsundar, 2009 while carrying out the root induction with 

the medium supplemented with charcoal 1 gm/l and IBA 0.1 

mg/l. Yung I- Lee, et al., 2001 carried out the root induction 

with IBA 0.02 mg/l and charcoal 1gm/l resulting in long and 

hairy roots. Olivia, et al., 1990 added different components 

into the MS media. These components are micronutrient, 

macronutrient, vitamins, Fe-EDTA and coconut water (100 ml). 

When these plantlets are transferred to protreys, the leaves 

obtained are expanded and were also well rooted. 

Well rooted plants were removed from the jars and rinsed 

thoroughly before keeping on moist cotton for 48 hours in 

treys or jars in the incubation room (Fig. 4a) partly covered to 

prevent loss of moisture. The rooted shoots were then 

transferred to multiwell protreys containing different growth 

media viz., soil, sand and cocopeat (1:1:1), soil sand and 

farmyard manure (1:1:1) and mixture of cocopeat and sand 

(2:1). Plants were maintained at 28°C of temperature and 70 – 

80% relative humidity in greenhouse. Observations for the 

best growth media were recorded after three weeks. It was 

found that maximum survival rate (96%) was on the medium


Table 1. 

Shoot formation in rhizome sprouting of Grand Naine variety of banana cultured on semi solid MS medium 

supplemented with various concentrations of BAP and NAA (12 explants per treatment, data recorded at the end of 6 

weeks). 

Tre at men t 

Hor mo na l su pp le men ts 

mg/ l 

% o f e xpl an ts 

re s pon se 

M e an no . o f sh oo ts / 

e xpl a nt± S .D 

M e an sh oo t l e ngt h 

(c m.) ±S. D 

BAP 

NAA 

T1 0 0 - - - 

T2 0 .25 0 20 1.6 ±0 .30 1. 0±0 .09 

T3 0 .5 0 30 2.5 ±0 .12 1. 5±0 .04 

T4 1 .0 0 35 2.5 ±0 .38 2. 0±0 .14 

T5 1 .5 0 40 2.6 ±0 .35 2. 5±0 .26 

T6 2 .0 0 50 2.8 ±0 .14 2. 6±0 .23 

T7 2 .5 0 45 2.0 ±0 .33 2. 8±0 .21 

T8 3 .0 0 40 1.8 ±0 .49 2. 2±0 .18 

T9 0 .25 0. 25 30 2.1 ±0 .12 2. 4±0 .16 

T10 0 .5 0. 25 35 2.5 ±0 .14 2. 8±0 .04 

T11 1 .0 0. 25 40 3.0 ±0 .37 3. 0±0 .41 

T12 1 .5 0. 25 60 3.2 ±0 .25 3. 2±0 .28 

T13 2 .0 0. 25 60 3.2 ±0 .33 2. 0±0 .26 

T14 2 .5 0. 25 50 2.2 ±0 .24 1. 8±0 .12 

T15 3 .0 0. 25 45 2.0 ±0 .31 2. 0±0 .28 

T16 0 .25 0 .5 40 2.2 ±0 .28 2. 2±0 .30 

T17 0 .5 0 .5 50 2.3 ±0 .04 3. 0±0 .32 

T18 1 .0 0 .5 60 3.5 ±0 .42 5. 2±0 .43 

T19 1 .5 0 .5 75 4.5 ±0 .41 6. 2±0 .37 

T20 2 .0 0 .5 90 7.5 ±0 .45 3. 2±0 .18 

T21 2 .5 0 .5 60 3.5 ±0 .08 2. 3±0 .18 

T22 3 .0 0 .5 50 2.5 ±0 .43 3. 0±0 .36 

P< 0.05 level of significance S.D : Standard Deviation 

containing cocopeat and sand in the ratio of 2:1 (Fig. 4b). 

After three weeks these plants acquire well established root 

system and are transplanted to polybags containing a mixture 

of soil, sand and FYM in the ratio of 1:1:1 and kept in shade 

net house for further acclimatization (Fig. 5, 6). Upon transfer 

to shade net house, the plants started producing new leaves 

after two weeks of transplantation. Later, these were 

transferred to field conditions and the survival rate was 98%. 

Our findings are in accordance with Lima, et al., 2007 who 

used the different mixture in the portrays for acclimatization. 

The mixture contained subsoil land + carbonized rice hull + 

residue of tea decomposed in proportion 1:1:1. 95% survival 

of the plantlets was observed. Olivia, et al., 1990 used the 

mixture of coirdust and garden soil (1:1), decomposed saw 

Table 2. Effect of different levels of NAA and IBA on rooting response of microshoots of Grand Naine (12 replicates / 

treatment, data recorded after 4 weeks) 

Tre at men t 

MS me d iu m + % 

c ha rc oa l( 2 g/ l )+ gr owt h exp l ant s 

re gu la to r r es po nse 

IBA NAA 

P< 0.05 level of significance S.D : Standard Deviation 

Day s to ro ot 

i nd uc ti o n 

M e an ro ot no . 

± S. D 

Me a n r oo t l e ng th 

± S. D 

T1 0 0 - - - - 

T2 0.2 5 0 2 0 12 - 14 1 .0± 0.3 0 1 .0± 0.1 4 

T3 0. 5 0 4 0 10 - 12 2 .0± 0.2 4 2 .0± 0.1 2 

T4 1. 0 0 5 0 10 - 12 2 .5± 0.0 4 2 .2± 0.1 6 

T5 1. 5 0 7 0 6 - 9 6 .0± 0.1 8 3 .0± 0.0 9 

T6 2. 0 0 9 5 6 - 8 8 .5± 0.3 3 3 .5± 0.3 8 

T7 2. 5 0 5 0 10 - 12 2 .2± 0.3 2 2 .3± 0.1 4 

T8 3. 0 0 4 0 10 - 12 1 .4± 0.2 4 1 .7± 0.2 8 

T9 0 0. 25 3 0 12 - 16 1 .0± 0.1 4 1 .5± 0.1 2 

T10 0 0 .5 4 0 12 - 16 2 .2± 0.1 6 2 .4± 0.1 6 

T11 0 1 .0 6 0 10 - 12 2 .5± 0.0 4 2 .5± 0.1 2 

T12 0 1 .5 5 5 10 - 12 2 .6± 0.0 4 2 .2± 0.0 8 

T13 0 2 .0 7 0 10 - 12 2 .8± 0.1 2 2 .6± 0.0 4 

T14 0 2 .5 5 0 10 - 16 1 .8± 0.3 8 2 .2± 0.1 4 

T15 0 3 .0 4 0 10 - 16 1 .6± 0.1 4 1 .5± 0.1 4

RAI et al., In vitro Regeneration of Banana Variety “Grand Naine” (G-9) 179 

plantlets to polythene bags containing garden soil and humus 

(1:1). After 2 weeks 98% planlets survived and flushed new 

leaves. 

The efficient micropropagation technique described may 

be appropriated as a highly useful one for raising disease 

free quality planting propagules of Grand Naine variety of 

banana for commercial cultivation which will not only aid in 

socioeconomic development of the farmers but also fulfill the 

market demand. 


Fig. 1. 

Fig. 2. 

Fig. 3. 

Fig. 4. 

Fig. 5. 

Fig. 6. 

Preparation, inoculation and induction of shoot bud on 

MS medium (a) Explant taken from field (b) Explant 

ready for inoculation (c) Inoculation of explant into 

MS medium (d) Emergence of shoot bud after 3 weeks 

of inoculation (e) Shoot growth on MS basal media 

After 5 weeks of inoculation 

Shoot multiplication (a) Multiplication on MS media 

with 2 mg/l BAP after 3 weeks (b) Growth after 4 

weeks on 2 mg/l BAP 

Rooting of shoots on MS media with 2 mg/l IBA 

(a) Rooted plantlets after 3 weeks (b) Rooted plantlets 

after 6 weeks 

(a) In vitro hardening (b) Primary hardening in protreys 

90 days old plant ready for transplanting into the field. 

110 days old banana plants in the nursery 

dust and garden soil (1:1), decomposed animal manure and 

garden soil (1:1), sand and garden soil (1:1). The best survival 

rate of plantlets were obtained on sand and garden soil in 

equal proportion. Islam, et al., 2010 transferred the rooted 

Banerjee, N., De Langhe, E. 1985. A tissue culture technique for rapid 

clonal propagation and storage under minimal growth conditions 

of Musa (banana and plantain). Plant Cell Reports, 4:351-154. 

Islam, R., Rahman, M.H.I., Ahmad, M.A., Bari, M.Z. Rahman and M., 

Hassain. 2010. Micropropagation of Banana Cv. Ranginsagar (AAA 

Group), A Commercial Cultivar of Northern Region of Bangladesh. 

SUST Studies. 7(1):19-24. Bangladesh 

Lima, J.D., Silva, S.H.M.G. da, Santos, E.M.H. dos, Lima, A.P. de S., 

Hirata, D.M., Santos, F. dos 2009. Crescimento e nutrição de 

mudas de bananeira em substrato contendo resíduos da agroindústria 

de chá preto durante a aclimatização. Scientia Agraria, 10(1): 37- 

42. 

Muhammad Aish. 2004. In vitro multiplication of banana (Musa 

spp.). HortScience, 42: 1253-1255. 

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth 

and bioassays with tobacco tissue cultures. Physiologia Planta., 15: 

473-497. 

Oliveira, R.P., de, Silveira, D.G., Oliveirad e Silva, S. de 2000. Effect of 

disinfection and use of contamination indicators in banana 

micropropagation. Revista Brasileira de Fruticultura, 22(1): 57- 

61. 

Olivia, P.D., Glenn, C.G., Robert, J.H., Steve, W.A., Mike, K.S., Ian, 

D.G. 1990. Random amplified polymorphic DNA (RAPD) detection 

of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) 

bananas. Plant Cell Reports, 16(1): 118-123. 

Senthilkumar, M., Ramsundar, V. 2009. Micropropagation of banana 

Musa spp. cv. Robusta (AAA). Plant Cell Biotechnology and 

Molecular Biology, 10(1/2):19-24. 

Sharma, T.R., Singh, B.M. 1997. High frequency in vitro multiplication 

of disease free Zingiber officinale Rosc. Plant Cell Reports, 17: 68- 

72. 

Strosse, H., André, E., Sági, L., Swennen, R., Panis, B. 2008. Adventitious 

shoot formation is not inherent to micropropagation of banana as 

it is in maize. Plant Cell Tissue and Organ Culture, 95:321-332. 

Young I. Lee. 2001. In vitro culture and germination of terrestrial asian 

orchid seeds. Methods in Molecular Biology, 710:53-62 



A Rapid and Simplex Methodology for Development of Platform Bioprocess 

Technology for Manufacturing of Recombinant Therapeutic Proteins in Serum Free 

Medium at Small Scale 

PRAVEEN GUPTA 

Apcegen Technologies Private Limited, Indian Institute of Technology, Kanpur, U.P. 208 016 

e-mail: praveenshrihari@rediffmail.com 

ABSTRACT 

One of the key dynamics in development of mammalian cell 

culture process development is the identification, selection and 

optimization of a suitable cell culture production medium. 

There are several basal se rum free medium available 

commercially in the market, which are still not meeting the 

demand of particular mammalian cell line with respect to 

maximum cell density, maximum productivity and quality of 

recombinant monoclonal antibodies. In order to necessitate 

these needs, a rapid and simplex methodology was developed 

fo r de velo pment of bio proc ess technolo gy for GMP 

manufacturing of recombinant monoclonal antibodies at shale 

flask level. 

Key words 

Cell Culture, Medium Optimization, DOE, Bioprocess, 

Monoclonal Antibodies 

The class of recombinant therapeutic proteins is 

growing rapidly due their ability to address human health 

needs. Globally, the market for these drugs is valued about $ 

100 billion – 15% to 20% of the total pharmaceutical market. 

The race has begun to make biosimilar versions as several of 

these biomolecules Patent protections are going off by 2015 

naturally; this offers a huge commercial opportunity to the 

generic drug producers. Recently, USFDA has also put their 

effort to promote this business segment area by releasing of 

draft guidelines for manufacturing of biosimilars which will 

help manufacturer to get a clear overview to launch a biosimilars 

product in the US market. Indian biopharmaceuticals market 

has also rapidly grown to US $2000 million by 2010. This 

success has placed the Indian biopharmaceuticals 

manufacturers in the forefront in the global biosimilars 

manufacturers. Selection of commercially available medium 

and development of feed strategy for bioproduction of 

recombinant therapeutics is traditionally a difficult and time 

consuming process. Earlier work showed simple strategies 

based on cell and spent media analysis (McKeehan, et al., 

1981) and highly complex factorial designs requiring multiple 

iterations for data analysis (Delacruz, et al., 2001). These 

methodologies typically results in optimization that is highly 

cloned specific as nutritional requirement of different cell clone 

is unique. Using multiple approaches a well balanced cell 

culture bioprocess platform developed consisting of basal 

medium and concentrated feed formulation with sufficient 

diversity to support process optimization. This approach will 

result in development of cost effective and robust platform 

bioprocess technology to meet all current demands for 

production of human recombinant therapeutic proteins such 

as high cell density growth coupled with productivity. 


Cell culture 

The CHO cell line CRL-9606 was obtained from ATCC. 

This particular cell line was designed to produce IgG using a 

DHFR - expression system with methotrexate (MTX) for 

selection. The culture was adapted to CD-CHO protein-free 

media (Invitrogen, Carlsbad, CA) deficient in hypoxanthine 

and thymidine. The culture medium was supplemented with 

L-glutamine (Gibco) to a final concentration of 3.4 mM and 

MTX (SIGMA) to a final concentration of 500 nM. A stable 

cell pool generated by transfection the plasmid containing 

IgG gene with DHFR as a selection marker. The cells were 

maintained and grown in DMEM with 10% FBS in tissue culture 

flasks in a CO 2 

incubator containing a humidified atmosphere 

of 8% CO 2 

in air at 37 0 C. 

Adaptation of adherent to serum free suspension cells 

Clone was adapted in serum free medium in shake flasks 

by sequential adaptation method. In adaptation, two passages 

were given in each reduction of FBS concentration in order to 

maintain viability more than 85%. Seeding Cell count was 

maintained ~ 0.5 million cells/mL for each passage level during 

entire adaptation process. In final passage with 1% FBS cells 

were directly taken up in shake flasks and incubated in a CO 2 

incubator containing a humidified atmosphere of 8% CO 2 

in 

air on an orbital shaker (Sartorius) rotating 130-150 rpm at 

37 0 C. 

Cell counting and viability measurement 

Cell counting and viability of culture was determined 

based on Trypan blue exclusion method (Patterson, 1979) by 

using Beckman Vi-CELL coulter. 

Expression determination of IgG 

Yield of IgG production was measured by an ELISA 

assay (Veronica, et al., 2006). Briefly, micro platyes (96-well, 

Nalge-Nunc) were coated with 100 uL/ well of polyclonal antihuman 

Antibody in 0.1 M sodium bicarbonate buffer (pH8.3)

PRAVEEN GUPTA, A rapid and simplex methodology for development of platform bioprocess technology 181 

at 4 0 c overnight. Each plate was blocked with 150 uL of 1% 

BSA in PBS fro 2 hrs at room temperature. Rest procedure is 

followed from manufacturer of kit (R&D Systems Inc, MN). 


Optimization of Production Medium 

A simple and rapid design of experiment for optimization 

of medium was developed which was divided in three major 

categories; 

1. Selection of basal medium 

2. Development of feed formulation 

3. Development of feed strategy 

1. Selection of basal medium 

In order to selection of basal medium; five serum free 

commercially available medium were identified based on 

concentration of D-Glucose, L-Glutamine, Trace Elements and 

Amino Acids. They were CD Forti CHO (Life technologies), 

Opti CHO (Life technologies), CD-CHO Excell (Sigma), 

CD4Mab (Hyclone), CD4CHO (Hyclone)). A selection criterion 

was defined by maximum viable cell count and productivity. 

All the experiment was performed in Erlenmeyer 125 mL shake 

flaks in duplicates with a working volume of 30 mL and average 

data was plotted in Fig. 1 and 2, where maximum viable cell 

count was found in CD Forti CHO media whereas maximum 

productivity was found in CD4CHO (Hyclone). Hyclone 

CD4CHO media was selected for further optimization of 

medium. 

2. Development of Feed Formulation 

A Design of experiments was development based on 

Plackett Burman Methodology. A statistical software Minitab 

was used for creating the factorial design (Table 1). Full 

factorial (2k) includes all possible combinations of ê 

components, each tested at two levels. In this study 10 

potential additives was selected which have shown the effect 

on cellular growth of various cell lines and the basis of selection 

is described below: 

Fig. 1. 

Basal medium selection, criteria maximum viable cell 

count 

Fig. 2. 

Basal medium selection, criteria productivity 

Table 1. 

Factorial design for development of feed formulation 

Flask ID Group D-Fructose Lipid BFGF ITS ZAP L-Glycine L-Aaparagine Sodium h-EGF h-IGF 

CHO 

Pyruvate 

6 1 + + - + + + - - - + 

7 2 - + + - + + - - - - 

8 3 + - - + - + + + - - 

9 4 - + - + + - + + + - 

10 5 - - + - + + + + + + 

11 6 - - - + - + - - + + 

12 7 + - - - + - + + - + 

13 8 + + - - - + + + + - 

14 9 + + + - - - - - + + 

15 10 - + + + - - + + - + 

16 11 + - + + + - - - + - 

17 12 - - - - - - - - - - 

18 13 + + + + + + + + + + 

Final Concentration 

used 

Fructose Lipid BFGF ITS ZAP L-Glycine L-Asparagine Sodium h-EGF h-IGF 

CHO 

Pyruvate 

10mM 1X 4 ug/L 1X 1X 1g/L 28.4 ug/L 110 mg/L 5 ug/mL 2 ug/mL


A) D-Fructose (Sigma) in the medium helps to maintain 

the pH of the medium by controlling the rate of 

production of lactic acid and ammonia (Barngrover, et 

al., 1985). 

B) Insulin Transferrin Selenium (ITS) (Gibco) have been 

shown to be components which are required for optimal 

performance of serum-free media (Bottenstein, et al., 

1979). 

C) L-Glycine (Sigma) simplest amino acid playing various 

role in cellular mechanisum ( Narhi, et al., 1997). 

D) L-Asparagine (Sigma) has been shown to enhance 

ornithine decarboxylase activity in cultured human colon 

adeno carcinoma Caco-2 cells (Chabanon, et al., 2000). 

E) ZAP-CHO improves CHO growth and productivity. 

F) basic Fibroblast Growth Factor (B-FGF) (Sigma) potent 

regulators of cell proliferation, differentiation and 

function (Galzie, et al., 1997). 

G) human Epidermal Growth Factor (h-EGF) (Sigma), a 

mitogenic for variety of epidermal and epithelial cells, 

including fibroblasts, glial cells, mammary epithelial cells, 

vascular and corneal endothelial cells, bovine granulosa, 

rabbit chondrocytes, HeLa cells, and SV40-3T3 cells 

(Carpenter and Cohen, 1979). 

H) human Insulin Linked Growth Factor (h-IGF) (Sigma), 

a mitogenic in nature and playing a role in cellular 

metabolism (Eckhard, et al., 1996). 

I) Sodium pyruvate (Gibco) an intermediary organic acid 

metabolite in glycolysis and the first of the Embden 

Myerhoff pathway that can pass readily into or out of 

the cell. Thus, its addition to tissue culture medium 

provides both an energy source and a carbon skeleton 

for anabolic processes. 

J) Lipid supplement (Gibco) chemically defined lipid 

concentrate is a concentrated lipid emulsion designed 

to reduce or replace fetal bovine serum in cell culture 

media for a wide variety of applications, including growth 

and maintenance of CHO, hybridoma, and insect cells in 

culture; monoclonal antibody production by 

hybridomas; and viral expression in insect cells. 

Fig. 3, showed that Feed Group-3 in combination of D- 

Fructose, ITS, L-Glycine, L-Asparagine and Sodium Pyruvate 

was helping to increase productivity by about 60% as 

compare to basal medium CD4CHO whereas Feed Group-7 D- 

Fructose, ZAP-CHO, L-Asparagine, Sodium Pyruvate and h- 

IGF increased productivity by about 40% as compare to basal 

medium CD4CHO. These two feed formulation were selected 

for further experiments. 

3. Development of Feed Strategy 

From selection of basal medium experiment next 

Fig. 3. 

Development of feed formulation, criteria productivity. 

promising basal media was selected based on the productivity 

and designed an experiment for feed strategy. Table 2 showed 

feeding of cells at different time feed intervals with 

combination of Feed Group 3 and Feed Group 7 with a basal 

medium Hyclone CD4 CHO medium and Opti-CHO medium. 

Graph 4; showed that when Opti-CHO : Hyclone CD CHO 

medium (1:1) ratio was used with daily feeding of Feed Group- 

3 in 5% V/V, productivity was found 3X of productivity in 

CD4CHO medium. 

In this study, we developed a bioprocess platform 

technology which may be used to develop human recombinant 

therapeutic such as monoclonal antibodies, hormones, fusion 

Table 2. 

Hyclone CD4 

CHO medium 

Fig. 4. 

Feed strategy development 

Shake flask - 

ID 

Feed 

group 

Feed 

time 

19 F-3 24 hrs 5 

20 F-7 24 hrs 5 

21 F-3 48 hrs 5 

Development of feed strategy 

Feed % 

V/V 

22 F-7 48 hrs 5 

23 F-3 24 hrs 5 

Opti-CHO 

24 F-7 24 hrs 5 

25 F-3 48 hrs 5 

26 F-7 48 hrs 5 

Opti-CHO : 27 F-3 24 hrs 5 

Hyclone CD 28 F-7 24 hrs 5 

CHO medium 29 F-3 48 hrs 5 

(1:1) 30 F-7 48 hrs 5

PRAVEEN GUPTA, A rapid and simplex methodology for development of platform bioprocess technology 183 

proteins and vaccines. We developed multi-approaches 

coupled with Design of Experiments (DOE) methodology for 

this study to keep in mind simplicity and rapidness of 

technology. In this work, its defined the strategy into three 

category; Selection of commercial available Basal Medium, 

Development of Feed Formulation and Development of Feed 

Strategy. By defining of criteria for each sub category helps 

to eliminate rest un-potential variables from the exercise. In 

first step, we eliminated three un-potential commercially 

available medium and selected two potential commercially 

available medium. After this step we indentified two potential 

additives sub-sets to develop a feed formulation and finally 

playing with feed development strategy, we defined that 50% 

mixture of Opti-CHO and Hyclone CD CHO medium each with 

daily feeding of Feed Group 3 (Fructose, ITS, L-Glycine, L- 

Asparagine and Transferring) in 5% V/V increased the 

productivity 3X of productivity in basal medium. This clearly 

shows a potential methodology to adopt in biomanuafcturing 

of Biotherapeutic products as a platform bioprocess 

technology. 


Barngrover, D., Thomas, J. and Thilly, W.G. 1985. High Density 

Mammalian Cell Growth in Leibovitz Bicarbonate-Free Medium: 

Effects of Fructose and Galactose on Culture Biochemistry; J. Cell 

Set., 78: 173-189. 

Bottenstein, J., Hayashi, I., Hutchings, S., Masui, H., Mather, J., 

McClure, D.B., Ohasa, S., Rizzino, A., Sato, G., Serrero, G., Wolfe, 

R. and Wu, R. 1979. Methods in Enzymology, vol.LVIII pp. 94- 

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Inheritance of Resistance to Yellow Mosaic Virus in Moth Bean 

L. N. YOGEESH 1 , R. MADHUSUDHAN 2 , B.A. RAVI 2 AND S. GANGAPRASAD 

1 

Agricultural Research Station, UAS, Raichur, Hagari 583 138, Bellary District, Karnataka 

2 

Department of Bio-technology, UAS, GKVK, Bangalore 560 065, Karnataka 

e-mail: lnygenes@gmail.com 

ABSTRACT 

Field screening of 121 moth bean accessions for YMV resistance 

revealed sixty-four resistant genotypes, one moderately 

resistant and remaining all susceptible in summer whereas, 

only 57 entries showed resistance in Kharif 2008. MH 43, 

resistant line identified from germplasm screening was crossed 

with GMO 25, YMV susceptible line. The F 1 

and F 2 

progenies 

were phenotyped for YMV-reaction under natural conditions. 

A monogenic dominant control of YMV-resistance has been 

revealed from the F 2 

segregation ratio of 3:1 (resistant: 

susceptible). 

Key words 

Moth bean, YMV, germplasm, inheritance 

Moth bean [Vigna accontifolia (Jacq.) Marechal] is a 

native minor pulse crop found in hot and dry habitats of 

northern-western parts of India. In severe soil moisture deficit 

situations encountered with exceeding evaporative demands, 

moth bean has been rated as the most adaptive, economic 

and useful annual legume Moth bean is grown in India over 

an area of 13.53 lakh hectares with an annual production of 

2.91 lakh tonnes and productivity of 215 kg per hectare (Anon., 

2008). In Karnataka production of moth bean is least amongst 

other growing states in India and is mainly grown in northern 

districts. In India, the YMV disease was first noticed during 

early seventies in Uttar Pradesh and Rajasthan (Tyagi and 

Mathur, 1978 and Nene, 1972). YMV is transmitted through 

the vector white fly, Bemisia tabaci Genn., is one of the most 

devastating biotic stresses which can cause up to 100% 

damage to a large number of leguminous crops. Although 

chemical control of white flies may limit the local spread of 

YMV genetic resistance is the only economically viable 

method of disease control. Despite the severity of the damage 

caused by YMV, the information on inheritance pattern of 

resistance gene controlling this disease is limited. Hence in 

the present study an attempt was made to investigate the 

mode of genetic control of resistance to YMV under field 

conditions. 


Screening for YMV disease resistance 

One hundred twenty one accessions maintained at 

AICRP on Arid Legumes (Table 1) were screened against 

yellow mosaic virus under field conditions during summer- 

2008. Susceptible genotype was planted after every five test 

entries as disease spreader rows. The per cent leaf chlorosis 

as an indication of disease severity on each genotype was 

assessed regularly at 15 days intervals. The disease severity 

was recorded on five trifoliate leaves on each plant. Per cent 

incidence of YMV disease was recorded by counting the 

number of infected plants out of the total number of plants. 

The genotypes were grouped into different categories using 

disease scoring scale suggested by Muniyappa, et al., 1987 

(Table 2). 

Development of F 1 

s and evaluation of F 2 

population 

The parent MH 43 differed from GMO 25 with respect to 

high level of intrinsic resistance to yellow mosaic disease. F 1 

seeds obtained from the above crosses along with their 

parents were sown during September 2009 under Greenhouse 

conditions for rapid advancement to produce F 2 

generation. 

Morphological traits such as plant type, flower color, pod 

color, seed colour and seed size of respective male parents 

were used as markers to differentiate and confirm F 1 

plants 

from selfed ones. The 125 F 2 

seeds from the GMO 25 X MH 43 

cross along with their parents were sown in the field for YMV 

resistance screening during summer 2010 (disease expression 

is better in summer) to understand the inheritance of YMV 

resistance. Plants were scored for the incidence of YMV at 15 

days interval upto 75 days under natural conditions. The 

plants showing yellow in mosaic symptoms were considered 

‘susceptible’ failing which were considered ‘resistant’. The 

goodness of fit to Mendelian segregation ratio has been tested 

in the F 2 

segregating population by Chi- square test. 


Disease reactions of the genotypes evaluated are listed 

in Table 3. Among the genotypes evaluated, 65 entries showed 

less than 5% of disease incidence and hence were grouped 

under resistant category. Only one genotype (Local 2) had 

8.5% disease and hence it was grouped under moderately 

resistant category. 29 genotypes showed 15 to 30 % disease 

incidence and were grouped as moderately susceptible. Six 

genotype viz., GMO 01-04, GMO 01-09, RMB 100, RMB 25, 

GMO 14, and GMO 3 showed more than 30 up to 50% disease 

incidence and were grouped as susceptible. Remaining 20 

entries showed more than 75% disease incidence and were 

grouped under highly susceptible category to the mosaic virus 

disease.

YOGEESH et al., Inheritance of Resistance to Yellow Mosaic Virus in Moth Bean 185 

Inheritance of YMV disease 

Genetics of resistance to Yellow Mosaic Virus from F 2 

populations of GMO 25 X MH 43 cross has been worked out 

(Table 4). The F 2 

plants were grouped into two classes viz., 

resistant with no apparent symptoms and susceptible with 

severe yellow mosaic symptoms. The goodness of fit to the 

Mendelian segregation ratio for resistance and susceptibility 

in the F 2 

plants has been worked out based on the Chi-square 

test (Table 4). It was observed that F 2 

plants segregated into 

3:1 (Resistant: Susceptible) ratio with chi-square test for the 

cross non-significant. In this cross 82 plants were found to be 

resistant and 43 plants were susceptible. Similar findings were 

made by Anon, 2003 in mungbean and Dahiya, et al., 1977; 

Kaushal and Singh, 1998; Reddy and Singh, 1993; Gupta, et 

al., 2005 in blackgram. However the present results are not in 

agreement with the reports of Thakur, et al., 1977 who observed 

single recessive allele controlling Mungbean Yellow Mosaic 

Virus tolerance, Singh, 1980 and Solanki, et al., 1982 have 

claimed that Mungbean Yellow Mosaic Virus resistance was 

due to duplicate recessive genes. Verma and Singh, 1986; 

Reddy and Singh, 1993 reported that MYMV resistance was 

due to one dominant and one recessive gene. 

Monogenic dominant control of YMV-resistance has been 

revealed from the F 2 

segregation ratio of 3:1 (resistant: 

susceptible). This knowledge of the simple inheritance pattern 

of resistance and independent assortment of genes governing 

resistance to yellow mosaic disease could be used effectively 

in development of cultivars resistant to disease or 

Table 1. 

List of 121 accession of moth bean used in the study 

Sl 

no. Entries Source Sl 

no. Entries Source Sl 

no. 

Entries 

Source 

1 Bijapur 1 North Karnataka 42 Muddebihal 13 North Karnataka 83 Shikaripura 2 Central Karnataka 

2 Bijapur 2 North Karnataka 43 Honnali 1 Central Karnataka 84 Shikaripura 3 Central Karnataka 

3 Bijapur 3 North Karnataka 44 Honnali 2 Central Karnataka 85 Shikaripura 4 Central Karnataka 

4 Bijapur 4 North Karnataka 45 Honnali 3 Central Karnataka 86 Gulbarga 1 North Karnataka 

5 Bijapur 5 North Karnataka 46 Honnali 4 Central Karnataka 87 Gulbarga-Halisagar 2 North Karnataka 

6 Bijapur 6 North Karnataka 47 Honnali 5 Central Karnataka 88 Gulbarga-Kannoli 3 North Karnataka 

7 Bijapur 7 North Karnataka 48 Honnali 6 Central Karnataka 89 Gulbarga- Hosagar 4 North Karnataka 

8 Bijapur 8 North Karnataka 49 Honnali 7 Central Karnataka 90 Gulbarga- Vanadurga 5 North Karnataka 

9 Bijapur 9 North Karnataka 50 Honnali 8 Central Karnataka 91 Gulbarga-Shahapur 6 North Karnataka 

10 Bijapur 10 North Karnataka 51 Honnali 9 Central Karnataka 92 Gulbarga 7 North Karnataka 

11 Bijapur 11 North Karnataka 52 Honnali 10 Central Karnataka 93 Gulbarga-sindhi 8 North Karnataka 

12 Bijapur 12 North Karnataka 53 Honnali 11 Central Karnataka 94 Gulbarga-Golasar 9 North Karnataka 

13 Bijapur 13 North Karnataka 54 Soudatti 1 Central Karnataka 95 Gulbarga- Moratagi 10 North Karnataka 

14 Bijapur 14 North Karnataka 55 Soudatti 2 Central Karnataka 96 Gulbarga 11 North Karnataka 

15 Bijapur 15 North Karnataka 56 Soudatti 3 Central Karnataka 97 GMO 3 CAZRI, Rajasthan 



18 Bijapur 18 North Karnataka 59 Soudatti 6 Central Karnataka 100 GMO 01-04 CAZRI, Rajasthan 

19 Bijapur 19 North Karnataka 60 Soudatti 7 Central Karnataka 101 GMO 01-09 CAZRI, Rajasthan 

20 Bijapur 20 North Karnataka 61 Basavanabagewadi 1 Central Karnataka 102 RMO 40 CAZRI, Rajasthan 



23 Bijapur 23 North Karnataka 64 Basavandabagewadi 4 Central Karnataka 105 RMO 225 CAZRI, Rajasthan 


25 Bijapur 25 North Karnataka 66 Bhemarayanagudi 2 Central Karnataka 107 RMO 423 CAZRI, Rajasthan 

26 Bijapur 26 North Karnataka 67 Bagalkot 1 Central Karnataka 108 RMO 435 CAZRI, Rajasthan 

27 Bijapur 27 North Karnataka 68 Bagalkot 2 Central Karnataka 109 RMB 25 CAZRI, Rajasthan 

28 Bijapur 28 North Karnataka 69 Bagalkot 3 Central Karnataka 110 RMB 100 CAZRI, Rajasthan 

29 Bijapur 29 North Karnataka 70 Bagalkot 4 Central Karnataka 111 CZM 1 CAZRI, Rajasthan 

30 Muddebihal 1 North Karnataka 71 Bagalkot 5 Central Karnataka 112 CZM 2 CAZRI, Rajasthan 

31 Muddebihal 2 North Karnataka 72 Bagalkot 6 Central Karnataka 113 CZM 3 CAZRI, Rajasthan 

32 Muddebihal 3 North Karnataka 73 Maharastra 1 Maharastra 114 MH 43 CAZRI, Rajasthan 

33 Muddebihal 4 North Karnataka 74 Maharastra 2 Maharastra 115 MH 61 CAZRI, Rajasthan 

34 Muddebihal 5 North Karnataka 75 Maharastra 3 Maharastra 116 P1 CAZRI, Rajasthan 



37 Muddebihal 8 North Karnataka 78 Davanagere 1 Central Karnataka 119 P4 CAZRI, Rajasthan 



40 Muddebihal 11 North Karnataka 81 Local 2 North Karnataka 

41 Muddebihal 12 North Karnataka 82 Shikaripura 1 Central Karnataka


Table 2. 

Disease scoring scale for moth bean yellow mosaic virus Disease 

Rating Per cent foliage affected 

Reaction 

1 No visible symptoms on leaves 

Immune 

2 Small necrotic yellow specks with restricted spread covering 0.1-5% leaf area 

Resistant 

3 Mottling of leaves, covering 5.10-10% leaf area 

Moderately Resistant 

4 Yellow mottling of leaves, covering 10.10-15% leaf area 

5 Yellow mottling and discolouration of leaves, covering 15.10-30% leaf area Moderately Susceptible 

6 Yellow discolouration of 30.10-50% of leaves 

7 

Pronounced yellow mottling and discolouration of leaves, pods and reduction in leaf size and stunting of plants covering Susceptible 

50.10-75% of foliage 

8 

Sever yellow discolouration of leaves, stunting of plants, reduction in pod size, no pod formation, covering 75.1-85% of 

foliage 

Highly Susceptible 

9 Sever yellowing of entire leaves, stunting of plants and no pod formation covering 85-100% of foliage 

Table 3. 

Grouping of Moth bean genotypes/varieties into different degree of resistance against Moth bean yellow mosaic virus 

under field condition 

Rating percentage leaf area 

covered 

1 No visible symptoms 

on leaves 

2 Small necrotic yellow 

specks with restrict6ed 

spread covering 0.1- 

5% leaf area 

Reaction 

Resistant 

3 Mottling of leaves, 

covering 5.1-10% leaf Moderately 

area 

resistant 

4 Yellow mottling of 

leaves, covering 10.1 - 

15% leaf area 

5 Yellow mottling and 

discoloration of leaves 

covering 15.1-30% leaf 

area 

Moderately 

susceptible 

6 Yellow discoloration 

of 30-50% of leaves Susceptible 

7 Pronounced yellow 

mottling and 

discoloration of leaves, 

pods and reduction in 

leaf size and stunting 

of plants covering 


8 Sever yellow 

discoloration of leaves, Highly 

stunting of plants, Susceptible 

reduction in pod size, 

no pod formation, 

covering 75.1-85% of 

leaves. 

9 Sever yellowing of 

entire leaves, stunting 

of plants and no pod 

formation covering 


Genotypes 

(Summer season) 

Genotypes 

(Rainy season) 

RMO 257,RMO 40,RMO 435,RMO 225,RMO 0- MH 1, MH 43,MH 61, 1,H 2,H 3,H 4,H 5,H 6,H 

09,RMO 140,MH 1,MH 43, MH 61,RMO 42,RMO 423, 7,H 8,H 9,H 10,H 11,SOU 1,SOU 2,SOU 3,SOU 

H 1,H 2,H 3,H 4,H 5,H 6,H 7,H 8,H 9,H 10,H 11,SOU 4,SOU 5,SOU 6,SOU 7,BB 1,BB 2,BB 3,BB 

1,SOU 2,SOU 3,SOU 4,SOU 5,SOU 6,SOU 7,BB 1,BB 4,BB 5,BB 6,BK 1,BK 2,BK 3,BK 4, BK 5,BK 

2,BB 3,BB 4,BB 5,BB 6,BK 1,BK 2,BK 3,BK 4,BK 6,S 1,S 2,S 3,S 4,S 5,SP 1,SP 2,SP 3,SP 4,D 1,D 

5,BK 6,S 1,S 2,S 3,S 4,S 5,SP 1,SP 2,SP 3,SP 4,D 1,D 2, D 3,D 4,SINDI 8 ,GOLASAR,MORTAGI,P 

2,D 3,D 4,SINDI8,GOLASAR,MORTAGI,P 1,P 2,P 3,P 1,P 2,P 3,P 4,P 5, P 6, CZM 1, CZM 3,CZM 2 

4,P 5,P 6, CZM 1,CZM 3,CZM 2 

LOCAL 2 LOCAL 2, RMO 423, BIJAPUR 28, 

BIJAPUR 29 

BP 1,BP 2,BP 3,BP 4,BP 5,BP 6,BP 7,BP 8,BP 9,BP 

10,BP 11,BP 12,BP 13,BP 14,BP 15,BP 16,BP 17,BP 

18,BP 19,BP 20,BP 21,BP 22,BP 23,BP 24,BP 25,BP 

26,BP 27,BP 28,BP 29 

GMO 01-04, GMO 01-09,RMB 100, RMB 25,GMO 

14,GMO 3 

GMO 25, GB 1,GB 2,GB 3,GB 4,GB 5,GB 6,GB 7, MB 

1,MB 2,MB 3,MB 4,MB 5,MB 6,MB 7,MB 8,MB 9,MB 

10,MB 11,MB 12,MB 13 

BIJAPUR 1,BIJAPUR 2,BIJAPUR 3,BIJAPUR 

4, BIJAPUR 5,BIJAPUR 6,BIJAPUR 7, 

BIJAPUR 8, BIJAPUR 9, BIJAPUR 10, 


BIJAPUR 14, BIJAPUR 15,BIJAPUR 16, 


BIJAPUR 20, BIJAPUR 21,BIJAPUR 22, 


BIJAPUR 26, BIJAPUR 27 

GMO 01-09, RMB 100, RMB 25,GMO 14, 

GMO 3 

GMO 01-04, GMO 25, GB 1, GB 2, GB 3, GB 4, 

GB 5, GB 6, GB 7, MB 1, MB 2, MB 3, MB 4, 

MB 5, MB 6, MB 7, MB 8, MB 9, MB 10, MB 

11, MB 12, MB 13, RMO 225, RMO 257, RMO 

40, RMO 435, RMO 140

Table 4. 

YOGEESH et al., Inheritance of Resistance to Yellow Mosaic Virus in Moth Bean 187 

Reaction of the F 2 

segregating generation of the cross GMO 25 × MH 43 to Moth bean Yellow mosaic virus 

Sl. Crosse of F 2 generation 

Total 

Moth bean yellow mosaic virus disease 

Ratio χ 2 value χ 2 value 

No. 

plants Observed frequencies Expected frequencies R:S (Cal.) (Tab.) 

Resistant 

plants 

Susceptible 

plants 

Resistant 

plants 

Susceptible 

plants 

1. GMO 25 (S) x MH 43 (R) 125 82 43 93.75 31.25 3:1 5.88 6.64 

incorporating resistance in existing promising cultivars in moth 

bean. 


Anonymous, 2003. In: Final workshop and planning meeting on 

mungbean. Asian Vegetable Research and Development Center. pp. 

75-76. 

Anonymous, 2008. Directorate of economics and statistics, New Delhi. 

NHDF, Rajasthan. 

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to mungbean yellow mosaic virus in blackgram (Vigna mungo L.). 

Tropical Grain Legume Bull., 9:28-32. 

Gupta, S. Kumar, S. Singh, R.A. and Chandra, S. 2005. Identification of 

a single dominant gene for resistance to mungbean yellow mosaic 

virus in blackgram [Vigna mungo (L.) Hepper]. SABRAO J. Breed. 

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Vigna Taxon 28: 99-202. 

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B.L. 1987. Isolation and characterization of a gemini virus causing 

yellow mosaic disease of horsegram (Macrotyloma uniflorum) in 

India. Phytopathol. Z., 119: 81-87. 

Nene, Y. L. 1972. A study of viral disease of pulse crops in Uttar 

Pradesh. Res Bull. No.4., G. B. Pant. Univ. Agri. Tech., Pantnagar, 

pp.144. 

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Theor. Appl.Genet., 72: 737-738. 



Efficacy of IPM Modules against Tomato Leaf Miner, Liriomyza trifolii (Burgess) 

WAGH S.S. AND PATIL P.D. 

Department of Agricultural Entomology, Dr. B.S. Konkan Krishi Vidyapeeth, Dapoli 415 712 (Maharashtra) 

e-mail: shrirangwagh@gmail.com 

ABSTRACT 

The experiment was conducted to find out relative efficacy of 

different integrated pest management modules comprised of 

alternate spray of chemical pesticides, biopesticides and 

botanicals against tomato leaf miner. Though the results have 

indicated superiority of one of the insecticidal module in 

reduction of larval population of leaf miner, Liriomyza trifolii 

Burgess, the IPM module composed of alternate spray of Lamda 

cyhalothrin 5EC (0.005%), B. bassiana 1.25 kg ha -1 , Abamectin1.9 

EC (0.0009%) and Azadirachtin 1500 ppm-2 ml l -1 was at par 

with insecticidal module. Also the other insecticidal module, 

module and module recorded nearly same larval population 

per leaf and were comparable with module. 

Key words 

Tomato, Liriomyza trifolii and IPM modules. 

Tomato (Lycopersicon esculentum Mill.) is the world’s 

largest vegetable crop, occupies an outstanding place among 

the important vegetables. It is widely grown in the world 

because of its wide adaptability to different agro-climatic 

conditions and high yield potential. In India, productivity of 

tomato is very low as compare to its production potential of 

the developed countries. There are many reasons for low 

production potential, among them pest infestation is major 

one. 

Serpentine leaf miner (Liriomyza trifolii Burgess), a 

native of Southern United States and Central America was 

accidently introduced into India during 1991-92 along with 

plant material and has spread widely. It is polyphagous pest 

affecting more than 91 host plants including pulses, fiber and 

vegetables (Galande, 2001). The infestation is generally severe 

on cucurbits and tomato and observed throughout the year. 

Presently, chemical pesticides are used by farmers for 

the protection of tomato against leaf miner and other pests. 

The over dependence and indiscriminate use of chemical 

pesticides has resulted in several problems like development 

of resistance to pesticides, outbreak of secondary pest, 

reduction of biodiversity and natural enemies. Indiscriminate 

use of pesticides has resulted in failure of control of the tomato 

fruit borer (Lal and Lal, 1996). These drawbacks of chemical 

pesticides emphasized the need to identify alternate ecofriendly 

methods to manage the pests of tomato. 


Field experiment was conducted at ASPEE Agricultural 

Research and Development foundation Farm, Village Nare. 

Tal. Wada, Dist. Thane during Rabi 2008-2009. The seedlings 

of tomato variety NS 815 (M/s Namdhari Seeds Private Limited, 

Bidadi, Bangalore) were raised under shed net condition. 

Transplanting was done in plot (Gross- 4.8m x 3.0m, Net- 4.8m 

x 2.25m) in R.B.D. (Randomized Block Design) having three 

replications and eleven treatments. There were eleven 

predefined IPM modules including control (Table 1). Four 

sprays were given, each at interval of 15 days starting from 15 

days after transplanting. The quantity of spray solution 

required to treat all plants under each treatment was determined 

prior to the application of each spray. The spraying was done 

by using manually operated Knap-Sack sprayer. Seed treatment 

and seedling root dip treatment were given at the time of 

sowing and transplanting, respectively. 

Goneem has prepared in laboratory having constituentscow 

urine 80%, Neemazal-T/S 10%, leaf extract of Ocimum 

basilicum (Tulas) 6%, seed powder of Terminalia chebula 

(Harda) 2% and extract of Allium sativum (lasun) 2%. 

The pre-count was recorded 1 day prior to treatment 

and post treatment observations were recorded 3, 7, and 14 

days after each spray. For recording the observations, five 

plants per plot were selected randomly and labeled. 

Observations were recorded on five leaves on each randomly 

selected plants representing lower, middle and upper portion 

of the plant and number of larvae per leaf were worked out. 


Efficacy of first spray of IPM modules against tomato 

leaf miner, L. trifolii 

Data on mean larval population per leaf after first spray 

(Table 2) indicated that the modules M 5 

and M 6 

comprising of 

treatment with 0.005 per cent lamda cyhalothrin recorded 

significantly lowest larval population (0.46 and 0.48, 

respectively) followed by (M 7 

) 0.004 per cent acetamiprid (0.55) 

and (M 9 

) 0.01 per cent fipronil (0.64) which were at par with 

each other. The module M 3 

and M 4 

consisting of treatment 

with 0.0045 per cent imidacloprid 17.8 SL were observed as 

next best modules in order of merit for management of leaf 

miner infesting tomato. Module M 2 

and module M 10 

comprising 

of seedling root dip treatment with 0.04 per cent imidacloprid 

and Neemazal (1%), respectively also observed significantly 

effective over control. However, module M 1 

consisting of seed 

treatment with imidacloprid 70 WS @ 10 g kg -1 and module M 8 

with foliar spray of HaNPV @ 0.5 l ha -1 failed to show 

significant reduction over control.

WAGH S.S. AND PATIL P.D., Efficacy of IPM Modules Against Tomato Leaf Miner, Liriomyza trifoli (Burgess) 189 

The present findings regarding efficacy of lamda 

cyhalothrin 5 EC confirm the results of Naitam, et al., 1999. 

They reported the effectiveness of lamda cyhalothrin against 

leaf miner infesting tomato. Effectiveness of acetamiprid 50 g 

a.i. ha -1 and imidacloprid 17.8 SL 20 g a.i. ha -1 was reported by 

Choudary and Rosaiah, 2001. 

Efficacy of second spray of IPM modules against tomato 


The mean larval population per leaf after second spray 

(Table 2) indicated that the module M 6 

and M 7 

composed of 

treatment with 0.0009 per cent abamectin 1.9 EC recorded 

significantly lowest mean larval population (0.63 and 0.64, 

respectively) followed by (M 9 

) 0.004 per cent acetamiprid 20 

SP (0.77) which were at par with each other. The module M 8 

composed of treatment with goneem was observed next best 

in order of merit. The treatments with B. bassiana @ 1.25 kg 

ha -1 , HaNPV @ 0.5 l ha -1 and V. lecanii @ 2.5 kg ha -1 were 

found least effective against leaf miner infesting tomato. 

The present findings with the abamectin confirm the 

results of Eswarareddy, et al., 2004 and Kumar and Kotikal, 

2006. They reported effectiveness of abamectin against leaf 

miner infesting tomato. Similarly, effectiveness of acetamiprid 

@ 50 g a.i. ha -1 was reported by Choudary and Rosaiah, 2001. 

Efficacy of third spray of IPM modules against tomato leaf 

miner, L. trifolii 

After third spray mean larval population per leaf (Table 

2) revealed that the module M 5 

composed of treatment with 

0.0009 per cent abamectin 1.9 EC recorded significantly lowest 

population of larvae (0.45) followed by (M 9 

) 0.15 per cent 

carbaryl 50 WP (0.52) and (M 4 

and M 3 

) 0.015 per cent 

diflubenzuron 25 WP (0.56 and 0.58) which were at par with 

each other. The treatment with goneem @ 5 ml l -1 was found 

next best treatment in reducing larval population of leaf miner. 

The treatment with B. thuringiensis @ 1 kg ha -1 and HaNPV 

@ 0.5 l ha -1 were found least effective against leaf miner 

infesting tomato. 

From the observations 3, 7 and 14 days after third spray 

it is found that abamectin was highly effective against tomato 

leaf miner. The findings obtained during present investigation 

confirm the results of Eswarareddy, et al., 2004 and Kumar 

and Kotikal, 2006. 

Efficacy of fourth spray of IPM modules against tomato 


The mean larval population per leaf after fourth spray 

(Table 2) indicated that the module M 9 

comprised of treatment 

Table 1. 

Details of the IPM modules tested for management of tomato leaf miner, Liriomyza trifoli (Burgess) 

Module I spray/application II Spray III Spray IV Spray 

M 1 

Imidacloprid 

70 WS 10 g kg -1 

(Seed treatment) 

HaNPV 

0.5 l ha -1 Goneem 

5 ml l -1 Azadirachtin 

1500 ppm 

2 ml l -1 

M 2 

M 3 

M 4 

M 5 

M 6 

M 7 

M 8 

M 9 

Imidacloprid 

17.8 SL 0.04% 

(seedling root dip) 

Imidacloprid 

17.8 SL 

0.0045% 

Imidacloprid 

17.8 SL 

0.0045% 

Lamda cyhalothrin 

5EC 

0.005% 

Lamda cyhalothrin 

5EC 

0.005% 

Acetamiprid 

20 SP 

0.004% 

HaNPV 

Goneem 

Azadirachtin 

0.5 l ha -1 5 ml l -1 1500 ppm 

2 ml l -1 

HaNPV 

Diflubenzuron 

0.5 l ha -1 25 WP 

0.015% 

B. thuringiensis 

1kg ha -1 

Diflubenzuron 

25 WP 

0.015% 

B. bassiana 

Abamectin 

1.25 kg ha -1 1.9 EC 

0.0009% 

Abamectin 

1.9 EC 

0.0009% 

Abamectin 

1.9 EC 

0.0009% 

Goneem 

5 ml l -1 

Goneem 

5 ml l -1 

Azadirachtin 

1500 ppm 

2 ml l -1 

HaNPV 

Azadirachtin 

0.5 l ha -1 1500 ppm 

2 ml l -1 


1kg ha -1 

HaNPV 

Goneem 


0.5 l ha -1 5 ml l -1 1kg ha -1 

Fipronil 5 SC 

0.01% 

M 10 Neemazal 1% 

(seedling root dip) 

M 11 

Control (water spray) 

Acetamiprid 

0.004% 

20 SP 

Carbaryl 

50 WP 

0.15% 

Goneem 

5 ml l -1 

Azadirachtin 

1500 ppm 

2 ml l -1 

Endosulfan 

35 EC 

0.05% 

V. lecanii 

HaNPV 

Azadirachtin 

2.5 kg ha -1 0.5 l ha -1 1500 ppm 

2 ml l -1


Table 2. 

Relative efficacy of IPM modules against tomato leaf miner, L. trifolii 

Sr. 

No. 

Module 

Mean larval population/leaf 

Number of sprays 

Pooled 

Mean 

I II III IV 

1. M 1 1.16 (1.47)* 1.80 (1.67) 1.09 (1.45) 0.89 (1.37) 1.24 (1.49) 

2. M 2 0.97 (1.40) 1.76 (1.66) 1.02 (1.42) 0.84 (1.36) 1.15 (1.47) 

3. M 3 0.68 (1.30) 1.62 (1.62) 0.58 (1.26) 0.91 (1.38) 0.95 (1.40) 

4. M 4 0.67 (1.29) 1.33 (1.53) 0.56 (1.25) 0.84 (1.36) 0.84 (1.36) 

5. M 5 0.46 (1.21) 1.50 (1.58) 0.45 (1.21) 0.72 (1.31) 0.79 (1.34) 

6. M 6 0.48 (1.22) 0.63 (1.28) 1.33 (1.53) 0.79 (1.34) 0.81 (1.35) 

7. M 7 0.55 (1.24) 0.64 (1.28) 1.17 (1.47) 1.10 (1.45) 0.85 (1.36) 

8. M 8 1.18 (1.48) 1.24 (1.50) 1.31 (1.52) 0.83 (1.35) 1.14 (1.46) 

9. M 9 0.64 (1.28) 0.77 (1.33) 0.52 (1.23) 0.44 (1.20) 0.59 (1.26) 

10. M 10 0.92 (1.39) 1.63 (1.62) 1.81 (1.68) 0.90 (1.38) 1.32 (1.52) 

11. M 11 1.27 (1.51) 1.90 (1.70) 1.99 (1.73) 1.94 (1.71) 1.78 (1.67) 

S.E. ± 

C.D. at 5% 

0.03 

0.07 

*Figures in parentheses are n 1 transformations 

0.03 

0.07 

0.04 

0.12 

0.05 

0.13 

0.03 

0.07 

with 0.05 per cent endosulfan 35 EC was offered longer 

protection to the tomato crop from infestation of leaf miner 

even upto 14 days of treatment. The modules consisted of 

treatments with azadirachtin 1500 ppm @ 2 ml l -1 and goneem 

5 ml l -1 were also found effective in management of leaf miner 

infesting tomato. The mean larval population within treatments 

varied between 0.44 to 0.91. The maximum mean larval 

population of 1.94 was recorded in untreated control. 

The present findings about effectiveness of endosulfan 

against tomato leaf miner confirm the results of Kumar and 

Kotikal, 2006. Effectiveness of neem products against tomato 

leaf miner has been reported by many workers, azadirachtin 

Sushil, et al., 2006, NSKE and neemazal Choudhary and 

Rosaiah, 2001. 

Relative efficacy of different IPM modules against tomato 


The studies on overall efficacy of treatments (Table 2) 

indicated that among the various options used the treatment 

with chemical insecticides were significantly superior over 

other treatments including control at each spray. The 

cumulative effect of all the sprays also indicated that the module 

M 9 

composed of chemical insecticides used in all four sprays 

was significantly superior over other modules and recorded 

lowest number of leaf miner larvae (0.59per leaf). However, the 

module M 5 

(0.005 per cent Lamda cyhalothrin followed by B. 

bassiana @ 1.25 kg ha -1 followed by 0.0009 per cent abamectin 

followed by azadirachtin 1500 ppm @ 2 ml l -1 in four sprays, 

respectively), module M 6 

(0.005 per cent Lamda cyhalothrin 

followed by 0.0009 per cent abamectin followed by HaNPV @ 

0.5 l ha -1 followed by azadirachtin 1500 ppm @ 2 ml l -1 in four 

sprays, respectively), module M 4 

(0.0045 per cent Imidacloprid 

followed by B. thuringiensis @ 1kg ha -1 followed by 0.015 per 

cent Diflubenzuron followed by goneem @ 5 ml l -1 in four 

sprays, respectively) and module M 7 

(0.004 per cent 

Acetamiprid followed by 0.0009 per cent abamectin followed 

by B. thuringiensis @ 1kg ha -1 followed by goneem @ 5 ml l - 

1 

in four sprays, respectively) composed of alternate spray of 

chemical and biopesticides have also recorded nearly same 

larval population (0.79, 0.81, 0.84 and 0.85per leaf) and 

comparable with module M 9 

. 

Above results revealed that integrated approach 

consisting of alternate use of chemical pesticides, 

biopesticides and botanicals for management of leaf miner 

infesting tomato can be effectively adopted with minimum 

damage to the environment. 


Choudary, D.P.R., Rosaiah, B. 2001. Evaluation of different Insecticides 

against serpentine leaf miner Liriomyza trifolii (Burgess) 

(Agromyzidae: Diptera) on tomato. Pestology, 25(2): 37-39. 

Eswarareddy, S.G., Krishnakumar, N.K., Shivakumar, B. and 

Krishnamoorthy, P.N. 2004. Efficacy of abamectin (Vertimec 

1.9EC) against serpentine leafminer Liriomyza trifolii (Burgess) on 

tomato. Pestology, 28(8): 17-19. 

Galande, S.M. 2001. Studies on bio-ecology and management of 

serpentine leaf miner Liriomyza trifolii (Burgess) on tomato and 

cucumber. Ph.D. (Agri.) Thesis submitted to M.P.K.V. Rahuri (India). 

Kumar, P. and Kotikal, Y.K. 2006. Bioefficacy of Vertimec 1.9EC 

against leaf miner in tomato. Pestology, 30(6): 41-43. 

Lal, O.P. and Lal, S.K. 1996. Failure of control measures against 

Helicoverpa armigera (Hub.) infesting tomato in heavy pesticide 

application areas in Delhi and satellite towns in western Uttar 

Pradesh and Haryana (India). J. Ent. Res., 20(4): 355-364. 

Naitam, N.R. and Dorak, S.V. and Wase, V.S. 1999. Bioefficacy of 

lamdacyhalothrin for the control of pests of tomato. Pest 

management in Horticultural Ecosystem, 3(2): 151-153. 

Sushil, S.N., Mohan, M., Hooda, K.S., Bhatt, J.C. and Gupta, H.S. 2006. 

Efficacy of safer management tools against major insect pests of 

tomato and garden pea in northwest Himalayas. J. Biol. Control, 

20(2): 113-118. 



Association of ABO Blood Groups with Lipid Profile and the Level of Oxidative 

Stress in Hypertensive Patients 

SHALINI KAPOOR 1 , RAJESH KUMAR 2 AND NEERAJ ARORA 3 

1 

Department of Biochemistry, Rama Dental College, Hospital and Research Centre, Kanpur 

2 

Institute of Life Sciences Chhatrapati Shahu Ji Maharaj University, Kanpur 

3 

L.P.S. The Institute of Cardiology, Kanpur 

3 

e-mail: kapoorshalini37@yahoo.in 

ABSTRACT 

A case control study was undertaken to find out a possible 

relationship of ABO blood groups with serum triglyerides 

(TGs), total cholesterol (TC), low density lipoprotein cholesterol 

(LDL-C), high density lipoprotein cholesterol (HDL-C), very 

low density lipoprotein cholesterol (VLDL-C) and the level of 

oxidative stress in 60 patients with essential hypertension (SBP 

> 140, DBP > 90) of age group between (45 - 65 years) which 

were compared with 60 normal healthy controls. The results 

showed significantly elevated levels serum TGs, TC, LDL-C 

and VLDL-C and lipid peroxide (LPO) content in patients with 

antigen A and B (B+AB) along with decreased levels of HDL-C. 

A significant positive correlation between serum TGs and TC 

with LPO was observed in group A (r=0.62, P


used for the estimation of lipid profile using standard 

diagnostic kits. LDL-C and VLDL-C was calculated by applying 

Friedwald’s formula. Thiobarbituric Acid Test (TBA) was used 

for the estimation of LPO in plasma (Ohkawa, et al., 1979). 

Platelet count was determined with the help of Neubauer’s 

chamber. 

Statistical analysis was done by applying unpaired 

students t-test Karl Pearsons coefficient of correlation was 

used to findout the correlation between two parameters. 


Anthropometric and blood biochemical parameters of 

normal healthy and hypertensive patients of group A, B, AB 

and O are compared in Table 1.There was significant increase 

in BMI in group A (P

KAPOOR, et al., Association of ABO Blood Groups with Lipid Profile and the Level of Oxidative Stress 193 

also reported increased plasma lipid peroxides in hypertensive 

patients (Armas Padilla, et al., 2007, Vaziri, et al., 1998). An 

imbalance between reactive oxygen species and antioxidant 

reserve referred to as oxidative stress producing an excess of 

ROS/RNS mainly superoxide anions and this could result in 

altered structure and function of protein, lipids and DNA 

associated with hypertension (Halliwell, et al., 1989). An excess 

of oxidative stress is also known to play an aberrant role in 

the initiation and progression of microvascular complication 

and this may be involved with their pathogenic role in arterial 

damage related to essential hypertension. Furthermore 

evidence exists which appears to indicate that those who carry 

a B antigen (blood group B and AB) have some difficulty in 

regulating the activity of nitric oxide. It has been reported hat 

anti-B antibody helps NO in working and this might be possible 

because of some unknown gene associated with the ABO 

locus on chromosome ‘9Q34’ and would be influenced by 

ABO genetics. 

Excess of ROS and cluster of proteins in different blood 

groups may also interrupt the biosynthesis and availability of 

NO in hypertensive subjects by interfering with the cofactors 

BH 4 

of endothelial NOS or destabilizing the enzyme co-factor 

Zn-thiolate (Zn(Cys) 4 

). As a result the enzyme becomes 

uncoupled and produce superoxide anion rather than NO 

(Vasquez–vivar, et al., 1999, Stores, et al.,1998). Oxidative 

stress can modify the scaffolding property of structural protein 

and decreased activity of uncoupling protein could be 

involved in the inactivation of scavenging enzymes and 

occurrence of cardiovascular diseases in these patient and 

this could be mediated by tyrosine kinases, phosphatases, 

protein kinase, protein and transcriptional factors (Mattiason 

and Sullian, 2006, Touz, et al.,2003) which play an important 

role in normal function but can also lead to remodeling of the 

vascular wall increasing high vascular reactivity and 

hypertension in particular blood group. 

In earlier study it is reported (Kapoor, et al., 2010) 

suppressed activity of Superoxide dismutase, Catalase and 

reduced glutathione in hypertensive patients associated with 

greater than normal lipid peroxidation predominantly in Group 

A, B & AB could be due to involvement of glycoprotiens 

present on the terminal of the basic fucose structure of red 

blood cell antigen. 

It has been reported that some genetic predispositions 

promote the development of coronary heart disease and found 

a novel association at the ABO locus which was attributable 

to the glycosyl tranferase deficient enzymes that encodes the 

group O phenotype which seems to protect against heart 

diseases (Carapeggiani, et al., 2010). 

The data presented in Table 2 indicates significantly 

increased (P


al., 1994). Lithell, et al., 1981 suggested that in patients with 

essential hypertension the occurrence of dyslipidemia may 

impair the vasodilatory function which results in the loss of 

small blood vessel density and surface area (rarefaction) and 

this patho-physiological alteration suppress the activity of 

the enzyme lipoprotein lipase which are expressed on the 

endothelium at the vascular lumen surface and responsible 

for the catabolism of triglycerides. The other reason for the 

high level of serum lipid (TG and TC) in group A individuals 

could be the influence of red-cell antigen on the enzyme 

alkaline phosphatase which is manufactured in the small 

intestine and has the primary function of splitting dietary fats 

and cholesterol esters (Bayer, et al., 1980). It has been found 

that the red cells of blood group A and AB binds almost all 

intestinal alkaline phosphatase and this effect in group B or O 

was seen to a much lesser degree and this is in an accordance 

with the fact that intestinal alkaline phosphatase has shown 

to be found more frequently in individuals of blood group B 

and O than in group A and AB. Thus it seems likely that lower 

levels of this enzyme in blood group A and AB individuals 

may inactivate intestinal alkaline phosphatase or A antigen 

itself inactivate this enzyme or ABO gene might influence the 

rate at which the intestinal alkaline phosphatase enter the 

blood or its metabolism in the intestine may provide a lead to 

their role in the elevation and deposition of lipids in blood 

and generation of cardiovascular disease in them (Nakata and 

Tozava, 1995 and Domar, et al., 1991). Furthermore we have 

observed significant increase in serum TC, LDL-C and VLDL- 

C in all the hypertensives predominately in blood group A 

patients when compared with normal healthy. However the 

level of TC in HDL was found decreased. Our results are similar 

to that of previous observations where individuals with blood 

group A were linked with having higher serum TC 

concentration and were at higher risk of heart diseases 

(Whincup, et al., 1990). 

The studies carried out by other authors in hypertensive 

subjects also found increased level of TC and LDL-C in 

hypertensive male subjects with blood group A. A number of 

studies by several workers have found a significant association 

of elevated cholesterol and hypertension in group A 

individuals and suggested that blood group phenotype 

represent an important biophysio-pathological action in regard 

to cardiac consequences in hypertensive males only 

(Robinson, et al., 2004). 

As it is earlier reported that the concentration of LDL-C 

and VLDL-C were predominately elevated in group A patient 

and there increased concentration may cause uncoupling of 

eNOS, activation of NADPH oxidase and consequently 

increased production of superoxide anions in the vessel wall 

which leads to increased oxidative stress. Increased levels of 

superoxide anions combines with NO and forms peroxinitrite, 

contributes to endothelial dysfunction (Prior and Squadreto; 

1995) and increased risk of cardiovascular diseases in them 

and this is true as we have also observed increased oxidative 

stress in group A and B patients. There was significant positive 

correlation found between serum TGs and TC with lipid 

peroxides in group A (r=+0.62; P < 0.01, r = +0.52, P < 0.05), B 

(r = +0.28, P < 0.10, r = + 0.46, P < 0.05) and AB (r = + 0.0.18, r = 

+0.28, P < 0.10) whereas in group O, TG was found to be 

negatively correlated and TC was positively correlated with 

lipid peroxide (r = -0.12; r = -0.10, P < 0.10) 

Elevated levels of harmful cholesterol in LDL and VLDL 

may lead to the development of atherosclerosis and free 

radicals are known to be involved with the process of 

atherogenesis forming lipid rich atherosclerotic plaque leading 

to hardening of coronary arteries, obstruction of the carotid 

artery, peripheral artery disease that all may contribute to MI 

or sudden death. Since all these disorders have been shown 

higher rates of occurrence in blood group A individuals, might 

be due to hyperlipoprotenemia causing oxidative degradation, 

formation of peroxides and free radicals causing injuries and 

erosions in the wall of vessels and atherosclerosis. LDL-C 

may be modified by oxidative processes and may initiate the 

peroxidation with long chain PUFA giving rise to lipid peroxy 

radicals (LOO·) which is a self propagating reaction and can 

attack adjacent fatty acid until complete fragmentation of fatty 

acids occur and there is accumulation of highly reactive 

products like lipid peroxides and lysophosphatides in the LDL 

particle (Assadpoor Piranfar, et al., 2009 and Maharajan, et 

al., 2008). 

Hypertension associated with lipid abnormalities like 

elevated levels of serum total cholesterol & triglycerides and 

increased lipid peroxidation may synergestically affect 

coronary endothelial function in group A, B and AB patients. 

The patients with blood group O containing no specific 

antigen are more protected against oxidative stress. 


The author is grateful to Dr. S.L. Shyam, Dr. Kripa 

Shankar and Dr. Ramesh Chander for guidance and 

encouragement and also thankful to the Director Rama Dental 

& Medical College, Kanpur for encouragement. 


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phosphatase and the ABO blood group system – a new aspect. 

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between the blood groups, BMI and Pre-hypertension among medical 

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Effect of Feeding Mixed Leaves of Different Mulberry Varieties on Economic Traits 

of Silk Worm Bombyx mori L. 

G.B. DURANDE, R.S. GADE, R.N. JAGTAP AND D.S. TAYADE 

Department of Agricultural Entomology, College of Agriculture, Latur 413 512, (Marathwada Agricultural 

University, Parbhani) Maharashtra 

ABSTRACT 

All the economic traits of silkworm viz., larval weight (39.10 g), 

larval duration (22.83 days), pupal duration (10.22 days), cocoon 

weight (1.80 g), cocoon yield (17.35 kg), filament length (872 

m), filament weight (0.26g) and moth emergence percentage 

(95.33) were found to be superior in case of those larvae which 

were reared on 100 % leaves of V 1, S 1635 and mixed feed of 

50% leaves of V 1 and 50% leaves of S 1635. Hence the varieties 

V 1 and S 1635 are used for increasing silk yield and improving 

all the economic characters of mulberry silkworm. While All 

the economic traits of silkworm viz., larval weight (34.83 g), 

larval duration (28.46 days), pupal duration (11.35 days), cocoon 

weight (1.32 g), cocoon yield (13.60 kg), filament length (688 

m), filament weight (0.22 g) and moth emergence percentage 

(84.66) were found to be inferior in case of those larvae which 

were reared on 100% leaves of M 5. Thus the variety M 5 was 

unsuitable for increasing silk yield and improving the economic 

characters of mulberry silkworm. 

Key words 

Larval duration, pupal duration, moth emergence, 

Bombyx mori L., varietal effect 

Sericulture is a cottage industry par excellence. It is one 

of the most labour intensive sectors of the Indian economy 

combining both agriculture and industry, which provides for 

means of livelihood to a large section of the population i.e. 

mulberry cultivator, co-operative rearer, silkworm seed 

producer, farmer-cum-rearer, reeler, twister, weaver, hand 

spinners of silk waste, traders, etc. It is the only one cash crop 

in agriculture sectors that gives returns within 30 days. 

One hectare of land under mulberry in terms of 

productivity yields Rs. 80,000 worth of silk, which is quite 

attractive compared to the other commercial crops. In addition, 

it creates employment to 12-13 persons annually in mulberry 

cultivation, silkworm related activity (Meenakshisundaram, 

1983). 

Nutritional quality of leaves play vital role in the robust 

growth of silkworm larvae and improving the commercial 

characters of cocoon as well as in the reproductive 

performance (Yokayama, 1962; Krishnaswami, et al., 1970; 

Takano and Arai, 1978, Li and Sano, 1987). The major leaf 

components such as nitrogen and water content decide largely 

the growth of an insect and hamper the optimal growth (Hough 

and Pimental, 1978). The term quality leaf refers to the presence 

of constituents in required quantity in the leaf for proper 

growth and development of silkworm resulting in production 

of good quality cocoons. 


The experiment was conducted in a Randomised Block 

Design (RBD) with seven treatments and three replications at 

Department of Entomology, College of Agriculture, Latur. 

Disease free laying of silkworm hybrid race PM X CSR 2 

were 

used to feed on the leaves of three different mulberry varieties 

viz., V 1, S 1635, M 5 and their mixed leaves, ten disease free 

laying’s (dfl’s) of race PM X CSR 2 

were reared in the laboratory 

by adopting the technology suggested by Krishnaswami, 

1978. During early larval stage mass rearing was done. The 

larvae after emerging out of second moult were distributed 

into three replications. Each replications consituted of 100 

worms. The equal quantity of food on the basis of weight as 

per treatment was given to the larvae for feeding. The chopped 

mulberry leaves were fed to the larvae at 6, 10, 16 and 21 hours 

in a day. The rearing trays reshuffled daily within a treatment 

and were cleaned daily. 

The total larval period (days) was measured by 

recording the period from hatching to the of spinning; larval 

weight by taking the weight of randomly selected 10 matured 

larvae before the onset of spinning and pupal period (days) 

was measured by recording the period from spinning to the 

emergence of moth. 


Data presented in Table 1 revealed that the larval weight 

of mulberry silkworm varied from 34.83 to 39.10 g on leaves of 

mulberry variety VI. Patil, 2004 and Gawade, 2006 reported the 

larval weight of mulberry silkworm to the tune of 34.80 and 

40.78 g respectively when grown on the leaves of mulberry 

variety, V 1. The present findings in respect of larval weight 

are more or less in conformity with the findings of above 

mentioned workers. 

Larval duration was observed in the range of 22.83 to 

28.46 days. The variety S 1635 (22.83 days) recorded 

significantly lowest larval duration over rest of the treatments 

excepting V 1 (23.36 days) and mixed leaves of S 1635( 50%) + 

V 1 (50%) (23.66 days). Significantly, the highest larval duration 

was recorded by larvae fed on leaves of M 5 (28.46 days). 

Patil, 2004 and Gawade, 2006 reported the larval duration of

DURANDE et al., Effect of feeding mixed leaves of different mulberry varieties on economic traits of silk worm 197 

mulberry silkworm to the tune of 22.36 and 22.23 days 

respectively when grown on the leaves of mulberry variety V 

1. The present findings in respect of larval weight are more or 

less in conformity with the findings of above mentioned 

workers. 

Pupal duration was observed in the range of 10.22 to 

11.35 days. The variety, S 1635 (10.22 days) recorded 

significantly lowest pupal duration over rest of the treatments, 

excepting V 1 (10.24 days) and mixed leaves of S 1635+V 1 

(50%+50%) (10.32 days). However, they were at par with each 

other. Longest pupal duration was recorded by larvae fed on 

leaves of M 5 (11.35 days). Gaaboub, et al., 1977 reported 

shortened and lengthened pupal duration of Pure Mysore 

and bivoltine hybrid NB 7 

xD 18 

silkworm to the extent of 10.50 

and 12.58 days when their larval stages were fed on the leaves 

of mulberry. The corresponding values on S 54 and Kanva 

2+S 54 reported by Telang, 1990 were 11.50 and 12.08 days. 

Individual cocoon weight of silkworm B. mori varied 

from 1.32 to 1.80 g. Significantly the cocoon weight was 

recorded by the larvae fed on leaves of V 1 (1.80 g) and S-1635 

(1.80 g). Significantly the lowest cocoon weight was recorded 

by larvae fed on leaves of M 5 (1.32 g). Anonymous, 2001 

recorded single cocoon weight to the tune of 1.065 g when 

larva were fed on leaves of variety, V 1. Patil, 2004 and Gawade, 

2006 recorded cocoon weight of mulberry silkworm to the 

tune of 2.22g and 2.12g respectively when grown on the 

leaves of variety V 1. The present findings are more or less in 

agreement with the findings of Anonymous, 2001; Patil, 2004 

and Gawade, 2006. 

Cocoon yield per 10,000 larvae brushed varied from 13.60 

to 17.35 kg. Significantly the highest cocoon yield was obtained 

by feeding the leaves of variety V 1 (17.35kg) followed by S 

1635 (17.33 kg) and mixed leaves of S 1635+V 1 (50%+50%) 

(17.13 kg). However, they were at par with each other . Whereas, 

significantly the lowest cocoon yield was obtained from larvae 

fed on leaves of M 5 (13.60 kg). Anonymous, 2001 also 

recorded cocoon yield of silkworm to the tune of 18.13 kg 

when its larval stage were fed on leaves of variety V 1. The 

present findings corroborate with the findings of Anonymous, 

2001. 

Cocoon filament length of mulberry silkworm ranged 

from 688 to 872 m. Significantly the longest filament was 

recorded by those cocoons whose larval stage were fed on 

leaves of variety V 1 (872 m) followed by S 1635 (867 m). 

Whereas, the shortest cocoon filament length recorded in the 

case of those larvae which were fed on leaves of M 5 (688 m). 

Gawade, 2006 recorded the cocoon filament length of mulberry 

silkworm to the extent of 866.67 m when its larval stage was 

reared on the leaves of mulberry variety V 1. The present 

findings are in conformity his findings. 

Cocoon filament weight of silkworm ranged from 0.22 to 

0.26 g. Significantly the highest filament weight to the tune of 

0.26 g was recorded by those cocoons whose larval stage 

were fed on leaves of varieties viz., V 1, S 1635 , mixed leaves 

of S 1635+V 1 (50%+50%) (0.26 g) and mixed leaves of M 5+V 

1+S 1635 in equal proportion. Whereas, the lowest cocoon 

filament weight was recorded by cocoons whose larval stage 

were fed on leaves of M 5 (0.22 g). Singh and Raghuvanshi, 

2005 recorded the highest silk filament weight to the extent of 

0.26 g when the larvae of bi-voltine silkworm NB 4 

D 2 

x KA were 

fed on mulberry leaves treated with 2 per cent urea. 

The emergence of silkmoth varied from 84.66 to 95.33 

per cent. Significantly the highest moth emergence was 

observed due to larvae fed on leaves of variety V 1 (95.33 per 

cent) followed by S 1635 (95.00 per cent). Significantly the 

lowest moth emergence was observed in case of those larvae 

which were reared on leaves of M 5 (84.66 per cent). Tayade, 

1983 reported 80 per cent emergence of moths of different races 

of mulberry silkworm when grown on leaves of mulberry 

variety, during September to October. 

Table 1. 

Effect of feeding mixed leaves of different mulberry varieties on different economic traits of silkworm 

Bombyx mori L. 

Sr. 

No. 

Treatment Weight of 

full grown 

larvae (g) 

Larval 

duration 

(days) 

Pupal 

duration 

(days) 

Single 

cocoon 

weight (g) 

Yield per 

10,000 larvae 

brushed (kg) 

Filament 

length 

(m) 

Filament 

weight 

(g) 

Moth 

emergence 

(%) 

1 M 5 (100%) 34.83 28.46 11.35 1.32 13.60 688 0.22 84.66 (66.94) 

2 V 1 (100%) 39.10 23.36 10.24 1.80 17.35 872 0.26 95.33 (77.52) 

3 S 1635 (100%) 38.60 22.83 10.22 1.80 17.33 867 0.26 95.00 (77.08) 

4 

M 5+V 1 

36.00 26.26 10.50 1.48 14.66 728 0.23 89.67 (71.25) 

(50%+50%) 

5 

S 1635+V 1 

38.54 23.66 10.32 1.74 17.13 858 0.26 92.00 (73.57) 

(50%+50%) 

6 

M 5+S 1635 

35.50 27.10 10.88 1.42 14.03 705 0.23 89.00 (70.63) 

(50%+50%) 

7 

M 5+V 1+S 1635 36.33 25.92 10.38 1.74 15.26 857 0.26 91.00 (72.54) 

(in equal proportion) 

S.E.± 0.21 0.30 0.03 0.02 0.08 3.3 0.003 1.1 

C.D. at 5% 0.63 0.91 0.09 0.06 0.24 10.3 0.009 3.3 

*Figure in parenthesis indicate arcsine transformed values



Anonymous. 2001. Evaluation of mulberry varieties for rearing 

performance and economic traits of silkworm Bombyx mori L. 

Annual Report, Sericulture Research Unit, Marathwada Agricultural 

University, Parbhani. pp.8-10. 

Gaaboub, I.A., Rawash, I.A. and Mostafa, S.M. 1977. Studies of some 

factors affecting the output of silk in Bombyx mori L. (Lepidoptera: 

Bombycidae). 

Gawade, B.V. 2006. Evaluation of mulberry varieties for rearing 

performance and their different economic traits on silkworm 

(Bombyx mori). M.Sc. (Agri.) Thesis, Marathwada Agricultural 

University, Parbhani. 

Hough, J.A. and Pimental, D. 1978. Influence of host foliage on 

development, survival and fecundity of gypsy moth. Environ. 

Entomol., 7: 97-102. 

Krishnaswami, S., Asan, M. and Sridharan, T.D. 1970. Studies on the 

quality of mulberry leaves and silkworm cocoon crop production. 

Indian J. Seri., 9(1): 11-25. 

Krishnaswami, S. 1978. New technology of silkworm rearing. Bull. 

No. 3, CSR and TI, Mysore. 

Li, R. and Sano, X. 1987.The relationship between quality of mulberry 

Leaves and some economic characterrs during the later larval 

stage.Acta Sericologia Sinica (Canye Kexue), 10:196-201. 

Meenakshisundaram, S.S. 1983. Intensifying field oriented research: A 

must. Indian Silk, 21(2):3-8. 

Patil, S.N. 2004. Evaluation of mulberry varieties for rearing 

performance and their different economic traits on silkworm 

(Bombyx mori). M.Sc. (Agri.) Thesis, Marathwada Agricultural 

University, Parbhani. 

Singh, R.P. and Raghuvanshi, A.K. 2005. Studies on effects of urea 

sprayed mulberry leaves feeding on growth and development of 

Bombyx mori L. Multivoltine race Nistari and Bivoltine race NB 4 

D 2 

xKA. Indian J. Ent., 67(1): 48-52. 

Takano, K and Arai, N. 1978. Studies on food value on the basis of 

feeding and cocoon productivity in the silkworm Bombyx mori L 

The amount of food intake and cocoon productivity. J. Seric. Sci. 

Jpn., 47(2): 134-142. 

Tayade, D.S. 1983. Studies on the feasibility and profitability of 

mulberry silkworm Bombyx mori L. under marathwada conditions. 

Res. Bull. Marathwada Agricultural University, Parbhani. 

Telang, S.M. 1990. Effect of mixed mulberry leaves of different varieties 

on growth and development of silkworm.Bombyx mori L. M.Sc. 

(Agri.) Thesis, MAU, Parbhani. 

Yokayama, T. 1962. Synthesized Science of sericulture. Central Silk 

Board. Banglore, India, pp.273-274. 



Ecofriendly Approach for the Management of Shoot and Fruit Borer, Earias vittella 

(Fab.) on Okra in Allahabad (UP) 

K.P. TULANKAR 1 , A.D. GONDE 2, R.K. WARGANTIWAR, A. KUMAR AND P.S. BURANGE 3 

Department of Plant Protection, Sam Hingginbottom Institute of Agriculture, Technology and Sciences, 

Allahabad, U.P. 

3 

Department of Entomology, Punjab Agricultural University, Ludhiana 141 004 

e-mail: kishortulankar890@yahoo.com, atul.dgonde111@gmail.com 

ABSTRACT 

Lowest infestation of shoot and fruit borer was observed 

in treatment consisting of Neem oil 1% + quinalphos 0.03% 

which was significantly superior over other treatments for 

controlling shoot and fruit borer on okra however, it was on 

par with quinalphos @0.06% alone. Among different 

combinations, NSE 5% + quinalphos 0.03%, was found next 

superior treatment followed by Neem leaf extract 5% + 

quinalphos 0.03%, Neem oil 1% , NSE @ 5%. Neem leaf 

extract 5% recorded significantly lower shoot and fruit 

infestation over control. The data on incremental cost benefit 

ratio of different treatments revealed that the treatment 

Quinalphos @ 0.06% found to be the most economically 

viable treatment giving highest incremental cost benefit ratio 

(1:11.35) and was followed by NSE 5% + quinalphos 0.03% 

(1:8.58) > Neem seed extract 5% (1:7.61) > Neem leaf extract 

5% + quinalphos 0.03% (1:7.54) > Neem oil 1% + quinalphos 

0.03% (1:3.74) > Neem leaf extract 5 % ( 1:2.81) > Neem 

oil 1% (1:1.42). 

Key words 

Abelmoschus esculentus, Earias vittella, insecticides, 

plant products, managment. 

Okra (Abelmoschus esculentus L. Moench) is attacked 

by several insect pest like aphids Aphis gosypii, (Glov.), 

jassids, Amarasca divastans (Dist.); shoot and fruit borers, 

Earias vittella (Fab.) and Earias insulana (Biosd.), Thrips, 

Thrips tabaci (Tinn), whitefly, Bemisia tabaci (Ginn.), Blister 

beelte (Mylabris pustulata), stem fly (Melanagromyza hibisci) 

and mites, Tetranychus telarius (L.) etc. (Nag and Sharma, 

1993). 

Shoot and fruit borer, Earias vittella (Fab.) and Earias 

insulana (Biosd.) are the most serious pests causing 57.10 

per cent fruit infestation and 54.04 per cent net yield loss in 

okra (Dubey and Ganguly, 1998). Many plant products are 

known to have antifeedant and insecticidal properties Among 

these neem products are popularly used in pest management 

(Singh, 2003). Efforts were made to test the different neem 

products for their insecticidal and antifeedant properties which 

may avoid toxicity problems in human beings and domestic 

livestock leading to development of newer non toxic 

insecticides. 


The trial was conducted at the field of Department of 

Plant Protection, Sam Higginbottom Institute of Agriculture, 

Technology and Science. Allahabad. There were eight 

treatments including an untreated control and each treatment 

was replicated thrice in the randomized black design. Three 

sprays were given, each at interval of 15 days. The spraying 

was done by using manually operated Knap-Sack sprayer. 

Shoot infestation 

The shoot borer enters into tip of growing shoot and as 

a result the infested shoot droops down. For recording the 

observations on per cent shoot infestation, the total number 

of shoot per plant from each treatment and the number of 

shoots infested were recorded at 7 days interval. 

Fruit infestation 

Five plants were randomly selected from each plot and 

labeled. Only the post treatment observations were recorded 

at 7 days interval at each pickings. For this all the marketable 

size fruits of these plants which had attained 8 to 10 cm in 

length were picked up. 

In order to work out per cent fruit infestation on number 

as well as weight basis, fruits were picked from five selected 

plants, they were counted and weighed. Similarly from these 

total fruits, the infested fruits due to fruits borer were separated 

out and were counted and weighted. The per cent infested 

fruits on number as well as weight basis were worked out by 

using following formulae David and Kumarswami, 1991. 

Number of damaged fruits 

Per cent fruit infestation = —––—————————— x 100 

Total number of fruits 

Weight of damaged fruits 

Per cent fruit damage = ————————————— x 100 

Total weight of fruits 


1. Mean per cent shoot infestation after first and second 

spray 

The data presented on mean per cent shoot infestation


is presented in (Table 1). It was revealed that the minimum 

1.39 to 5.18% shoot infestation after 1 st and 2 nd spray, 

respectively was observed in treatment combination of Neem 

oil 1% + quinalphos 0.03% and it was found most effective 

against shoot borer. This was followed by quinalphos 0.06%> 

NSE 5% + quinalphos 0.03% > Neem leaf extract 5% + 

quinalphos 0.03% > neem oil 1% > NSE 5 % >NLE 5% in 

descending order of their effectiveness. All the treatment 

combinations were found significantly superior over control 

after both the sprays. 

The effectiveness of quinalphos has been reported by 

Prasad, et al., 2009. The effectiveness of neem oil 1% + 

quinalphos 0.03% against shoot and fruit borer was 

authenticated in the studies made by earlier researcher 

(Jumade, 1994; Singh, et al., 1998; Wargantiwar, et al., 2010). 

The findings were also in close conformity with Temurde, et 

al., 1992. 

2. Mean per cent fruit infestation after first, second and 

third spray 

Number basis 

Data on percent fruit infestation on number basis after 

first, second and third spraying Table 2 revealed that all the 

treatments were significantly superior over control. 

At 7 DAS, The minimum infestation of fruit 19.81% was 

found in the treatment of Neem oil 1% + quinalphos 0.03% 

(T 5 

) followed by quinalphos 0.06% (T 3 

) 20.96%, NSE 5% + 

quinalphos 0.03% (T 6 

) 22.04%, neem leaf extract 5% + 


) 23.76%. The least effective treatment 

was neem leaf extracts 5% (T 4 

) recording 29.86% infested fruits. 

Maximum (33.21%) infested fruits were observed in control. 

Fourteen days after spraying also, the minimum fruit 

infestation (19.80%) was found in the treatment consisting of 

Neem oil 1% + quinalphos 0.03% (T 5 

) followed by quinalphos 

@ 0.06% (T 3 

) (22.52%), NSE 5% + quinalphos 0.03% (T 6 

) 

(23.85%), neem leaf extract 5% + quinalphos 0.03% (T 7 

) 

(25.30%). The treatment neem leaf extracts 5% (T 4 

) was found 

least effective in all three sprays recording fruit infestation of 

28.23%. Maximum (33.21%) infested fruits were observed in 

control (water spray). 

Weight basis 

Data on average percentage fruit infestation on weight 

basis after first, second and third spray presented in Table 2 

revealed that all the treatments were significantly superior 

over control. 

At 7 DAS, The least infestation of fruit (19.90%) was 

found in the treatment of Neem oil 1% + quinalphos 0.03% 

Table 1. 

Mean shoot infestation (%) after first and second spray 

Treatment 

Mean per cent shoot infestation after first 

spraying 

Mean per cent shoot infestation after second 

spraying 

7 DAS 14 DAS 7 DAS 14 DAS 

Neem oil @ 1% 20.49 (4.52) 21.45 (4.63) 18.52 (4.30) 18.52 (4.30) 

Neem seed extract @ 5% 25.45 (5.04) 25.00 (5.00) 23.00 (4.79) 23.00 (4.79) 

Quinalphos 0.06% 5.79 (2.40) 5.55 (2.35) 5.55 (2.35) 5.55 (2.35) 

Neem leaf extract @ 5% 25.93 (5.09) 26.89 (5.18) 23.68 (4.86) 23.68 (4.86) 

Neem oil 1% + quinalphos 0.03% 1.39 (1.17) 4.00 (2.00) 5.18 (2.27) 5.18 (2.27) 

Neem seed extract 5% + quinalphos 

0.03% 

6.94 (2.63) 13.89 (3.72) 15.27 (3.90) 15.27 (3.90) 

Neem leaf extract 5% + quinalphos 0.03% 16.66 (4.08) 15.61 (3.95) 15.04 (3.87) 15.04 (3.87) 

Control (Water spray) 26.91 (5.18) 29.65 (5.44) 26.36 (5.13) 26.36 (5.13) 

`F’ test Sig. Sig. Sig. Sig. 

(SE)+ 0.22 0.84 0.16 0.16 

CD at 5% 0.47 1.81 0.35 0.35 

Table 2. 

Average percentage fruit infestation after first, second and third spraying 

Tr. No. Treatments 

Number basis 

Weight basis 

7 das 14 das 7 das 14 das 

T 1 Neem oil @ 1% 24.84 (4.98) 26.96 (5.19) 25.88 (5.08) 26.20 (5.11) 

T 2 Neem seed extract @ 5% 26.49 (5.14) 28.22 (5.31) 26.91 (5.18) 26.90 (5.18) 

T 3 Quinalphos @ 0.06% 20.96 (5.78) 22.52 (4.74) 21.78 (4.66) 21.22 (4.60) 

T 4 Neem leaf extract @ 5% 29.86 (5.46) 30.67 (5.53) 28.11 (5.30) 28.47 (5.33) 

T 5 Neem oil 1% + quinalphos 0.03% 19.81 (4.45) 19.80 (4.44) 19.90 (4.46) 20.66 (4.54) 

T 6 Neem seed extract 5% + quinalphos 0.03% 22.04 (4.69) 23.85 (4.88) 23.57 (4.85) 21.48 (4.63) 

T 7 Neem leaf extract 5% + quinalphos 0.03% 23.76 (4.87) 25.30 (5.02) 24.58 (4.95) 23.69 (4.86) 

T 8 Control (Water spray) 33.21 (5.76) 33.71 (5.80) 33.11 (5.75) 32.50 (5.70) 

`F’ test Sig. Sig. Sig. Sig. 

(SE)+ 0.10 0.12 0.12 0.14 

CD at 5% 0.22 0.26 0.26 0.30

Table 3. 

Treatments 

TULANKAR et al., Ecofriendly Approach for the Management of Shoot and Fruit Borer, Earias vittella (Fab.) on Okra 201 

Yield and incremental cost benefit ratio (ICAR) under different treatments 

Qty. of insecticide 

req. /ha for 3 

spray 

Approximate 

Cost of insecticide 

+Labour + sprayer 

charges 

(Rs) 

Avg. total 

Yield 

(q/ha) 

Increase yield 

over control 

(q/ha) 

Value of 

increased yield 

(Rs./ha) 

Approx. net 

profit 

(Rs./ha) 

Increamental cost 

benefit ratio 

Neem oil @ 1% 15 lit. 5475 57.00 16.62 13296 7821 1:1.42 

Neem seed extract @ 5% 75 kg 1350 55.33 14.53 11624 10274 1:7.61 

Quinalphos @ 0.06% 2.5 lit. 1675 66.66 25.86 20688 19013 1:11.35 

Neem leaf extract @ 5% 75 kg 1275 46.88 6.08 4864 3589 1:2.81 

Neem oil+quinalphos 15 lit + 1.25 lit. 5825 75.33 34.53 27624 21799 1:3.74 

Neem seed 

extract+quinalphos 

75 kg + 1.25 lit. 1700 61.16 20.36 16288 14588 1:8.58 

Neem leaf extract+quinalphos 75 kg + 1.25 lit. 1625 58.16 17.36 13888 12263 1:7.54 

Control (Water spray) - 40.80 - - - - 

(T 5 

) followed by quinalphos @ 0.06% (T 3 

) (21.78%), NSE 5% 

+ quinalphos 0.03% (T 6 

) (23.57%), neem leaf extract 5% + 


) 24.58%.The least effective treatment 

was Neem leaf extracts 5% (T 4 

) recording 28.11% infested 

fruit. Maximum (33.11%.) infested fruits were observed in 

control. 

Fourteen days after spray similar trend was observed. 

Minimum infestation of fruit 20.66% was found in the treatment 

consisting of Neem oil 1% + quinalphos 0.03% (T 5 

) followed 

by quinalphos @ 0.06%(T 3 

) (21.22%), NSE 5% + quinalphos 

0.03% (T 6 

) (21.48%), Neem leaf extract 5% + quinalphos 0.03% 

(T 7 

) (23.69%) in order of their efficacy. The treatment neem 

leaf extract 5% (T 4 

) was consistantly found least effective 

treatment in all three sprays recording fruit infestation of 28.47 

per cent. Maximum (32.50%.) nfested fruits were observed in 

control. 

The results of the present investigation are in closed 

conformity with those obtained by Jat and Pareek, 2001, who 

reported the effectiveness of neem oil 1% + quinalphos 0.03% 

against shoot and fruit borer infesting okra. 

3. Effect of different treatment on incremental cost 

benefit ratio (ICAR) 

The data in respect of incremental cost benefit ratio as 

influenced by various treatments are presented in Table 3. It 

is revealed that the amount of monetary returns in descending 

order were Neem oil 1% + quinalphos 0.03% (Rs. 27624/ha), 

Quinalphos 25 EC @ 0.06% (Rs. 20688/ha), Neem seed extract 

5% + quinalphos 0.03% (Rs. 16288/ha), Neem leaf extract 5% + 

quinalphos 0.03% (Rs. 13888/ha), Neem oil 1% (Rs.13296/ha), 

Neem seed extract @ 5% (11624/ha) and Neem leaf extract @ 

5% (Rs. 4864/ha) over the control. 

The highest incremental cost benefit ratio was obtained 

in the treatment of quinalphos @ 0.06% (1:11.35) followed by 

NSE 5% + quinalphos 0.03% (1:8.58), Neem seed extract 5% 

(1:7.61), Neem leaf extract 5% + quinalphos 0.03% (1:7.54), 

Neem oil 1% + quinalphos 0.03% (1:3.74), neem leaf extract @ 

5% (1:2.81) and Neem oil @ 1% (1:1.42), respectively. 


Authors are grateful to Dean and Director of Research 

for allotting field for research work at Central field of Sam 

Higginbottom Institute of Agriculture, Technology and 

Sciences,Allahabad. 

LITRATURE CITED 

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vittella (Fab.). Insect Environment., 4(1): 25-26. 

Jumde, Y.S. 1994. Management of shoot and fruit borer, Earias vittella 

(Fab.) on okra with botanicals, chitin inhibitor alone and in 

combination with insecticides. M.Sc. Thesis (unpub.), Dr. PDKV, 

Akola. 

Jat K.L. and Pareek, B.L. 2001. Field evaluation of ecofriendly against 

brinjal shoot and fruit borer, Lucinodes orbonalis (Guen). Indian 

journal of plant protection, 29(1&2):53-56. 

Nag, A. and Sharma, R.K. 1993. Insect pests of Bhendi (Abelmoschus 

esculentus L.) and their management. Pestology, 17(9): 8-10. 

Prasad, R., Sathi, S.K. and Prasad, D. 2009. Bioefficacy of conventional 

and neem insecticides against insect pest of okra, Uttar Pradesh 

Journal of Zoology, 29(1): 39-43. 

Singh, G., Bharadwaj, S.G. and Dhaliwal, G.S. 1998. Evaluation of some 

pesticides for the management of fruit borer Earias spp. on okra 

crop. Indian Journal of Ecology, 25(2): 187-189. 

Singh, P.K. 2003. Control of Brinjal shoot and fruit borer Lucenodes 

orbonalis with antifident and insecticidal properties. Indian journal 

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1992. Efficacy of neemark and its combinations with other groups 

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245-247. 



Diversity and Community Structure of Phytonematodes Associated with Guava in 

and Around Aligarh, Uttar Pradesh, India 

RIZWAN ALI ANSARI 1 AND TABREIZ AHMAD KHAN 2* 

Section of Plant Pathology and Nematology, Department of Botany, Aligarh Muslim University, Aligarh 

1 

e-mail: rizwans.ansari@gmail.com; 2 e-mail: tabreizkhan@hotmail.com 

ABSTRACT 

An extensive survey of plant parasitic nematodes from fourteen 

localities of Aligarh district was conducted during November, 

2011 to March, 2011. A total of 164 samples were collected and 

analyzed for plant parasitic nematodes and their importance 

through community analysis. Analysis of soil samples revealed 

the presence of twelve genera of plant parasitic nematodes viz; 

Meloidogyne, Hoplolaimus, Pratylenchus, Helicotylenchus, 

Tylenchus, Tyl enchorhynchus, Hemi criconem oides, 

Aphelenchoides, Longidorus, Trichodorus, Xiphinema and 

Rotylenchulus. Among plant parasitic nematodes, the highest 

absolute density and relative density were recorded in 

Meloidogyne spp. followed by Hoplolaimus spp., Moreover, in 

relation to absolute frequency and relative frequency the 

Hoplolaimus spp. ranked first. The prominence value of 

Meloidogyne spp. ranked first followed by Hoplolaimus spp. 

However, the saprozoic nematodes showed the highest absolute 

density, relative density, absolute frequency, relative frequency 

and prominence value as compared to the plant parasitic 

nematodes. The present investigations have clearly indicated 

that the association of plant parasitic nematodes with guava, 

especially the most frequently occurring ones like Meloidogyne 

spp., Hoplolaimus spp., Rotylenchulus sp., Helicotylenchus sp. 

and Hemicriconemoides sp., are highly pathogenic in nature. 

Key words 

Absolute frequency, Relative frequency, Absolute 

density, Relative density and survey 

The guava (Psidium guajava Linn.) is one of the most 

widely distributed fruit tree crops often referred as the apple 

of the tropics. In India, guava grows nearly throughout the 

country and is cultivated commercially in almost all states, 

the total estimated area being cultivated about 50,000 ha. The 

important guava growing states are Uttar Pradesh, Bihar, 

Assam, Maharashtra, West Bengal, Andhra Pradesh and 

Madras. About half of the total area under production is 

reported in Uttar Pradesh and Madras (Singh, et al., 1963). 

Plant parasitic nematodes have been recognized as one of the 

limiting factor in the normal production in several important 

crops including fruits. The present work was undertaken to 

get an idea about nematode diversity and community structure 

in guava orchards. 

fourteen localities of Aligarh district viz; Atrauli, Harduaganj, 

Mahrawal, Gabhana, Keshanpur, Khair, Iglas, Jatari, Chatari, 

Jawan, AMU campus, kasimpur, Sasni and Nanau was 

conducted during November, 2011 to March, 2011. The total 

numbers of plant selected for study were 164 from the above 

mentioned localities. The soil samples were collected from the 

rhizosphere of guava at a depth of 10-15 cm. with the help of 

an auger. Four soil samples were randomly collected around 

the rhizospheric soil of each guava tree. All the four samples 

taken from each plant were mixed together to form a composite 

sample of 250 cm 3 soil. Samples were stored at 5-10°C until 

processed for nematode extraction. Nematodes were extracted 

from soil by using the Cobb’s sieving and decanting method 

followed by Baremann funnel techniques. Killing and fixing 

of nematodes process were done in double strength TAF 

followed by mounting with glycerin for nematode identification 

(Southey, 1986). The nematodes were identified upto the 

generic level. Identified genera were also assigned to different 

trophic groups based on their feeding habit. For quantitative 

analysis, 5 ml nematode suspension was poured in counting 

dish and the nematodes were counted under the stereo 

microscope. At least three readings were taken to calculate 

the average number of nematodes in 100 ml of suspension. 

Community analysis was carried out by using the following 

standard formula; 

Number of samples containing a species 

 

 

Total number of samples collected 

 

 

Absolute frequency = 100 

Frequency of species 

Relative frequency = 

 

100 

Sum of frequency of allspecies 

 

 

Number of individual s of species in a sample 

 

 

Volume of sample 

 

 

Absolute density = 100 

Number of individual s of species in a sample 

Total of all individual in a sample 

 

 

Absolute density = 

100 


An extensive survey of plant parasitic nematodes from 

Prominence value = Absolute density 

Absolute frequency

ANSARI AND KHAN, Diversity and Community Structure of Phytonematodes Associated with Guava 203 


The result presented in Table I clearly revealed that in 

all, twelve genera of plant parasitic nematodes were identified 

and found associated with guava plants, grown in different 

localities of Aligarh district viz., of which, two nematodes 

species viz., Meloidogyne and Hoplolaimus were reported 

from all the fourteen localities , while genus Helicotylenchus 

was found to be present in all the localities except A.M.U. 

campus. However, occurrence of the rest of the nematodes, 

varied in different localities. Similarly, the population of 

different nematodes also varied in different localities surveyed. 

In addition, saprozoic nematodes were also found in all the 

localities. Among these phytonematodes, the average 

population of Meloidogyne spp. (1344.3) was highest followed 

by Hoplolaimus spp. (183.6), moreover, as far as the 

community analysis is concerned, it was revealed that 

Hoplolaimus spp. ranked first among the plant pathogenic 

nematodes as it showed cent per cent absolute frequency 

followed by Helicotylenchus sp. (90.8%) similarly, as far as 

the relative frequency is concerned, Hoplolaimus spp. (10.9) 

also ranked first followed by Helicotylenchus sp. (9.9). 

However, on the other hand, among the plant parasitic 

nematodes Meloidogyne spp. (7528.0) showed the highest 

absolute density followed by Hoplolaimus spp. (1028.0), 

similarly, the relative density of Meloidogyne spp. (36.3) also 

ranked first among the plant parasitic nematodes followed by 

Hoplolaimus spp. (5.0). In terms of prominence value, 

Meloidogyne spp. (66472.2) ranked first among the plant 

parasitic nematodes (Table 1). 

The results showed that guava is good host of plant 

parasitic nematodes particularly Meloidogyne spp., 

Hoplolaimus spp., Rotylenchulus sp. on the basis of 

prominence value as well as relative density, relative frequency, 

absolute frequency and absolute density and may be more 

pathogenic to guava as compared to others. Our results are 

also in agreement with those of Moura,1989, Zeidan, 1990, 

Carrillo Riversa, et al., 1990 and Ansari and Ahmad, 2000 who 

also reported the association of, Meloidogyne mayaguensis, 

M. incognita, M. javanica, Tylenchorhynchus brassicae, 

Hoplolaimus indicus, Helicotylenchus spp. and 

Table 1. 

Occurrence of plant parasitic nematodes associated with guava trees in and around Aligarh 

Population of phytonematodes / 250 cm 3 soil 

No. of 

Locality samples 

Population of saprozoic 

Aph.* Hel.* Hem.* Hop.* Lon.* Mel* Pra*. Rot.* Tri.* Tyl.* Tylen.* Xip.* 

nematodes 

Atrauli 21 

12 176 118 255 134 3234 

44 

159 1875 

- - - 

- 

(10) (21) (18) (21) (21) (19) 

(21) 

(15) 

(21) 

AMU 

135 78 108 125 95 

65 72 48 

1542 

10 - 

- 

- - 

Campus 

(16) (11) (10) (11) (6) 

(16) (16) (16) 

(16) 

Chatari 11 - 

77 67 68 77 1844 110 

34 13 124 1374 

- - 

(9) (11) (11) (5) (9) (11) 

(11) (11) (11) 

(11) 

Gabhana 14 

65 108 69 210 144 956 48 158 10 87 69 95 

795 

(12) (14) (14) (14) (14) (14) (14) (14) (14) (14) (14) (14) 

(14) 

Harduaganj 15 

35 85 167 277 95 2818 165 

105 84 

1385 

- - 

- 

(15) (12) (13) (15) (15) (13) (15) 

(13) (15) 

(15) 

Iglas 11 

7 44 

134 

855 28 222 

72 115 207 

618 

- 

- 

- 

(2) (11) 

(11) 

(8) (11) (11) 

(9) (11) (11) 

(11) 

Jatari 5 

28 56 121 35 

1577 

48 14 60 

1688 

- 

- 

- - 

(3) (5) (5) (5) 

(2) 

(5) (5) (5) 

(5) 

Jawan 16 - - 

57 108 

95 

174 

122 

1055 

- 

- 

- 

(10) (10) 

(6) 

(10) 

- (10) - 

(10) 

Kasimpur 7 - 

166 145 187 166 543 75 92 

215 1756 

- - - 

(7) (7) (7) (7) (5) (07) (7) 

(7) 

(7) 

Keshanpur 8 

44 94 

95 

165 

367 

45 35 

2018 

- 

- 

- 

- 

- 

(8) (8) 

(8) 

(4) 

(8) 

(8) (8) 

(8) 

Khair 12 - 

33 

187 18 256 

765 

17 

176 2443 

- 

- 

- 

- 

(14) 

(14) (14) (11) 

(14) (14) 

(14) 

(14) 

Mahrawal 10 - 

122 90 307 164 1215 

267 

174 1268 

- 

- - - 

(10) (10) (10) (10) (17) 

(10) 

(10) 

(10) 

Nanau 4 

26 114 84 134 26 1545 87 339 

644 

- - - - 

(4) (4) (4) (4) (4) (3) (4) (4) 

(4) 

Sasni 18 

77 204 133 318 85 2761 18 34 

18 64 

944 

- - 

(18) (18) (18) (18) (18) (12) (18) (14) 

(18) (18) 

(18) 

Total (164) (72) (149) (121) (164) (119) (128) (80) (113) (35) (111) (87) (100) (164) 

Average 

population 

21.0 101.0 80.6 183.6 73.8 1344.3 37.9 180.9 6.8 36.6 32.6 86.7 1514.71 

In parentheses number of samples containing a species is given; 

Aph.*= Aphelenchoides, Hel.* = Helicotylenchus, Hem.* = Hemicriconemoides, Hop.* = Hoplolaimus, Lon. *= Longidorus, Mel*.= Meloidogyne, 

Pra.*= Pratylenchus, Rot* = Rotylenchulus, Tri.*= Trichodorus, Tyl.*= Tylenchus, Tylen.*=Tylenchorhynchus, Xip.*= Xiphinema.


Table 2. 

Community analysis of plant parasitic nematodes on guava in and around Aligarh. 

Nematode Trophic group Average/ Range A.F. R.F. A.D. R.D. P.V. 

Aphelenchoides 

Myceliophagus 

21.0 

(7-77) 

43.9 4.8 117.6 0.6 778.5 

Helicotylenchus 

Ecto and semi endoparasite 

101.0 

(33-204) 

90.8 9.9 565.6 2.7 5390.2 

Hemicriconemoides 

Sedentry ectoparasite 

80.6 

(57-167) 

73.8 8.1 451.6 2.2 3874.7 

Hoplolaimus 

Migratory endo- and ectoparasite 

183.6 

(35-307) 

100.0 10.9 1028.0 5.0 10280.0 

Longidorus 

Migratory ectoparasite 

73.9 

(26-166) 

72.6 7.9 413.6 2.0 3672.9 

Meloidogyne 

Sedentry endoparasite 

1344.3 

(95-3234) 78.0 8.5 7528.0 36.3 66472.2 

Pratylenchus 

Migratory endoparasite 

37.9 

(18-165) 

48.7 5.3 212.4 1.0 1482.5 

Rotylenchulus 

Semi-endoparasite 

180.8 

(34-765) 

68.9 7.5 1012.4 4.9 8402.9 

Trichodorus 


6.8 

(10-72) 

54.7 6.0 38.4 0.2 283.8 

Tylenchorhynchus 


32.6 

(13-122) 

53.0 5.8 182.4 0.9 1327.9 

Tylenchus 

Ectoparasite 

36.6 

(17-105) 

67.7 7.4 204.8 1.0 1683.4 

Xiphinema 

Ectoparasite 

86.7 

(64-207) 

61.0 6.7 485.6 2.3 3787.7 

Saprozoic nematodes 

saprozoic 

1514.7 

(618-1875) 

100.0 10.9 8482.4 40.9 84824.0 

A.F. = Absolute Frequency, R.F. = Relative Frequency, A.D. = Absolute Density, R.D. =Relative Density, P.V. = Prominence Value. 

Tylenchorhynchus projectus with guava trees. It is confirmed 

from the results that the diversity and the population of the 

nematode in all the soil samples taken were not-uniform. 

Fluctuation in population and distribution of nematode genera 

might be due to soil types having different physico-chemical 

characteristics. The present investigation clearly indicated 

that the association of plant parasitic nematodes, especially 

the most frequently occurring ones like Meloidogyne spp., 

Hoplolaimus spp., Rotylenchulus sp., Helicotylenchus sp. 

and Hemicriconemoides sp. are highly pathogenic in nature. 

Therefore, their occurrence in high densities may pose a 

serious threat to guava trees, if the management practices are 

not governed to keep the population under check. Therefore, 

it needs immediate attention of the growers and researchers 

to manage the damage to guava trees infested with nematodes. 


Ansari, M.A. and Ahmad, W. 2000. Community analysis of plant parsitic 

and predatory nematodes in guava (Psidium guajava) orchard. 

Med. Fac. Landbouww.univ.Gent., 65(2b): 557-562. 

Carillo Rivera, J., Carillo Fonseca, C. and Dominguez Alvarez, J.L. 

1990. Nematodes attacking guava trees (Psidium guajava L.) and 

chemical control in the Juchipila Canyon, Zac. Mexico. Revista 

Chapingo, 15(67-68): 94-97. 

Moura, R.M. De and Moura, A.M. De. 1989. Root–knot on guava: a 

series disease In Pernambuco state, Brazil. Nematologia Brasileria, 

13: 13-19. 

Singh, S., Krishnamurthi, S. and Katyal, S.L. 1963. Fruit culture in 

India, Indian council of Agricultural Research, New Delhi. pp.456. 

Southey, J.F. 1986. Laboratory methods for work with plant and soil 

nematodes. Ministry of Agriculture, Fisheries and Food Reference 

Book No. 402, HMSO, London, UK. pp.202. 

Zeidan, A.B. and Geraert, E. 1990. Helicotylenchus from Sudan, with 

description of two new species (Nematoda: Tylenchida). 

Nematologia Mediterranea, 18(1): 33-45. 



Anti Fungicidal Activity of Secretion from Scent Glands of Heteropteran Bugs 

CH. SRINIVASULU 1 *, V. KARUNAKAR 1 , S.M. REDDY 2 AND C. JANAIAH 3 

1 

Department of Zoology Govt. Degree College Peddapalli, Karimnagar Dist. A.P. 505 172 

2 

Department of Botany, Kakatiya University, Warangal A.P. 

3 

Department of Zoology, Kakatiya University, Warangal A.P. 

*e-mail: chsri39@gmail.com 

ABSTRACT 

Effect of volatile scent components of heteropteran bugs on the 

growth of two fungi, Alternaria alternata and Aspergillus clavatus 

was investigated by Glass cup method .The scent components 

of n-dodecane showed differential toxicity towards A.alternata 

and A.clavatus. Undecane significantly reduced the growth of 

A. alternate where as its effect on A. clavatus was negligible. n- 

tridecane and n-tetradecane exhibited fungicidal activity against 

both the fungi under study. 1-Hexanol and Propan -1-ol showed 

lack of fungicidal activity against both the fungi. 1-Butanol 

exhibited a strong fungicidal activity against both the fungi 

under study. 2-Butanone a scent component had fungicidal 

activity but was more inhibitiry than to A. alternata and 

A. clavatus. 

Key words 

Heteropteran bugs, Scent components, Fungicidal 

activity, Alternaria alternate, Aspergillus clavatus. 

Effect of some natural scent compounds was observed 

on pathogenic fungi Alternaria alternata and Botrytis cinera 

by Hamitton Kempt, et al., 1992. Volatile compounds produced 

in the mandibular gland of leaf cutting ants of genera Atta and 

Acromyrmex had significant inhibitory effect on germination, 

growth and development of certain fungi (Knapp, et al., 1994). 

Certain volatile chemical compounds like hexanol, 

acetone, formaldehyde, acetic anhydride, pyridene, chloroform 

and phenol inhibited the growth and sporulation of Fusarium 

oxysporum completely (Kantilal,1980). n-hexanol, hexanal and 

trans-hept-2-enal, 5-hexenoic acid and trans-hex-2-enylacetate 

and n-hexyl acetate were the components of scent secretions 

from C. purpueus and T. javanica. They showed a strong 

fungicidal activity against plant pathogenic fungi Duchsheria 

specifera, F. oxysporum (Surender, et al., 1987). 

n-dodecane, n-tetradodacane, n-undecane, Benzyle 

alcohol, propan-1-ol, 1- butanol, 2-butanone, Benzyldehyde, 

the components of scent secretion from C. purpureus, T. 

javanica, H. dentatus, C. lectularius, amblybeltanitida, 

Gelastocoris culatus, Biprorulus bibax, Graphosoma 

linatum, Chlorochroa uhleri, C. sayi, C. ligata and N. viridula 

showed fungicidal activity against the both Curvularia lunata 

and Fusarium oxysporum (Vidyasagar, 1995). 


Secretions of scent glands of some heteropteran bugs 

Graphosoma lineatum, C. purpureus, T. javanica, Pallantia 

macunaim, H. denatus, C. lectularius, A. nitida, Gelastocoris 

oculatus, Biprorulus bibax, N. viridula, Chlorochroa uhleri, 

C. sayi, C. ligata were collected and assayed for their 

antifungal activity. The collection of scent secretion from 

abdominal scent glands of larvae and metathoracic scent 

glands of adults of Heteropteran bugs was described earlier 

(Janaiah, et al., 1979). The secretions were subjected to 

chemical analysis by Gas liquid chromatography and Mass 

spectrometry (GC-MS). The unknown individual components 

of secretions were compared with authentic sample (ICN, Lab, 

New York) some known compounds from different 

heteropteran bugs, reported earlier, were also evaluated for 

their antifungal activity. 

Antifungal activity of scent component was assayed as 

per the method described by Fries ,1961 Glasscup (1 ml) 

capacity were placed in a petridish and sterilized at 150 0 C for 

30mn. The sterilized medium was poured into these 

petridishes. After cooling at room temperature 1 ml of test 

compound was poured in a glass cup asceptically. The 

petridishes thus prepared were seeded with the test fungi 

(Alternaria alternata and Aspergillus clavatus) and incubated 

at 27+2 0 C for 7days, 1 ml sterilized water is placed for test 

compound and it served as control. At the end of incubation 

period and growth of colony was measured and percentage 

of growth inhibition was calculated, minimum of five replicates 

were employed. 


Effect of scent components of heteropteran bugs on 

the growth of two fungi (Alternaria alternata and Aspergillus 

clavatus) was investigated and the results are presented in 

Table 1. 

Hydrocarbons: n-dodecane,the hydrocarbon from the 

abdominal scent glands of Chrysocoris purpureus (Janaiah, 

1978), Graphosoma lineatum (Dilek and Kalender, 2008) and 

Halys dentatus (Srinivasulu, et al., 1996) was responsible total 

differential toxicity towards A.alternata and A. clavatus. 

Undecane, the scent component from the metathoracic scent 

glands of bronze orange bug, Musgraevia sulciventris, 

Biprorulus bibex (Macleoid, et al., 1975) and Nymph of the 

Pallantia macunaima (Carla, et al., 2011) exhibited moderate 

inhibition of growth on A. altenata, little or no inhibition of


growth on A. clavatus. n-tridecane a scent component from 

the abdominal scent glands of Tessaratoma javanica (Janaiah, 

et al., 1979) and H. dentatus (Srinivasulu, et al., 1996) and 

also from the metathoracic scent glands H. dentatus (Surender 

and Janaiah, 1990) C. ligata, C. sayi, C. uhleri (Hsiar - Young 

Ho, et al., 2001) Showed fungicidal activity against A. 

alternata and A. clavatus. While n-tetradecane reported in 

the scent secretion of heteropteran bugs Caura rufiventris 

(Prestwitch, 1976) and Chlorochroa uhleri (Hsiao-Young and 

Jocelyn, 2001) showed a fungicidal activity on both A. 

alternata and A. clavatus. 

Alcohols: Benzyl alcohol,the abdominal scent glands of 

Coreoid bugs Leptoglossus australis (Gough, et al., 1985) 

showed median toxicity on both the fungi under study 1- 

Hexanol, reported from the metathoracic scent glands of adult 

Coreoid bugs. Gelastocoris oculatus (Staddon, 1973) showed 

lacked fungicidal activity against both fungi. Propan-1-ol, the 

volatile alcoholic compound from the abdominal scent glands 

of larvae of Libiapis angolensis (Cmelik, 1969) and from the 

metathoracic scent glands of Chrysocoris stolli (Chowdhari 

and Dass, 1968) was also showed no fungicidal activity against 

both fungi understudy. 1-Butanol reported from the scent 

secretions of Coreoid bugs also showed strong toxicity of 

both the fungi understudy. 

Ketone: 2-Butanone (methyl ethyl ketone) found in the 

metathoracic scent secretion of C. stolli (Chowdari and Dass, 

1968) showed more sensitive of A. alternata than A. clavatus 

while it was being mild toxicity on A. clavatus. 

Aldehyde: Benzaldehyde, from the predatory insects Podisus 

maculiventris (Aldrich, et al., 1984) was mild inhibited of 

growth of both fungi. 

From the present investigations, it is clear that the scent 

components undecane, n-dodecane, n-tridecane and n- 

tetradecane of certain heteropteran bugs exhibit strong 

fungicidal activity against of A. alternata while n-tetradecane 

was high toxic to the growth of both fungi. Benzyl alcohol 

and 1-Butanol exhibited strong fungi toxicity on both the fungi 

2-Butanone and Benzaldehyde also strong fungicidal activity 

against both fungi under study, while 2-Butanone showed 

median toxicity on A. clavatus. 

n-dodecane, which showed differential toxicity towards 

A. alternata and A. clavatus was also reported to have varied 

effect. The fungicidal activity, Surender, et al., 1987 observed 

that D. specifera and F. oxysporum were insensitive, which C. 

lunata was totally inhibited by this compound n-undecane, 

was also effect two fungi differentially. A. clavatus was 

insensitivity while A. alternata was partially inhibited. Similarly 

Vidyasagar, 1995 reported that this compound has lack of 

fungicidal activity against fungi studied by him. 

n-tridecane and n-tetradecane also exhibited fungicidal 

activity against both the fungi under investigation it can be 

concluded the A. alternata was more sensitive to low 

molecular weight hydrocarbons and with the increase of 

molecular weight, the sensitivity decreased. Reverse was true 

with A. clavatus. Acetone, Formaldehyde, Acetic anhydride, 

Pyridine, Chloroform and Phenols exhibited total inhibition of 

growth on D. specifera and F. oxysporum (Kanthilal, 1980). 

Benzyle alcohol showed median toxicity on both the 

fungi under study 1-Hexanol and Propan-1-ol lacked fungicidal 

activity against present fungi. On the other hand, Surender et 

al., 1987 reported to effective fungicidal against D. specifera 

and F. oxysporum. Similarly Vidyasagar, 1995 reported the 

sensitive of C. lunata towards these compounds. It was 

effective enough to cause total spore germination inhibition 

of F. oxysporum at higher concentrations. (Vidyasagar, 1995). 

1-Butanol, exhibited strong fungicidal activity against fungi 

understudy. Vidyasagar, 1995 has also reported that it was 

responsible for total germination inhibition at higher 

concentration. 2-Butanone and Benzaldehyde had almost 

same in their fungicidal activity. A. alternata was more 

Table 1. 

Effect of certain scent components of insects on the growth of two fungi 

S.No. Name of the Compound 

I 

1. 

2. 

3. 

4. 

II 

5. 

6. 

7. 

8. 

III 

9. 

IV 

10. 

HYDROCARBONS 

n- dodecane 

Undecane 

n-tridecane 

n-tetradecane 

ALCOHOLS 

Benzyl alcohol 

1-Hexanol 

Propan-1-ol 

1. Butanol 

KETONE 

2- Butanone 

(Methyl Ethyl Ketone) 

ALDEHYDE 

Benzaldehyde 

Diameter of the colony 

(mm) 

20 

35 

40 

30 

30 

Nil 

Nil 

05 

15 

Alternaria alternata 

Percentage of 

inhibition 

70.6 

48.5 

41.2 

55.9 

55.9 

Nil 

Nil 

92.6 

Diameter of the colony 

(mm) 

24 

64.7 

40 

Control 68 90 

67.6 

90 

85 

70 

35 

30 

Nil 

Nil 

05 

55 

Aspergillus clavatus 

Percentage of 

inhibition 

Nil 

5.6 

22.2 

61.6 

66.6 

Nil 

Nil 

94.4 

38.9 

55.5

SRINIVASULU et al., Anti fungicidal activity of secretion from scent glands of Heteropteran Bugs 207 

sensitive than A. clavatus. The response of C. lunata and F. 

oxysporum was also similar to A. alternata. 


Thanks are due to Head, Department of Zoology, 

Kakatiya University, Warangal, for providing working facilities 

and to the ICN, Lab New York for supplied authentic samples. 


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metathoracic scent glands of Gelastocoris oculatus (Fabricius) 

(Heteroptera:Gelastocoridae). Entomol. 106: 253-255. 

Surender, P. Janaiah, C. 1990. Chemical constituents of metathoracic 

scent glands of the bark bug Halys dentatus (Febr.) Indian J. Comp. 

Anl. Physiol., 8(2): 60-64. 

Surender, P., Janaiah, C., Krishna Reddy, V. and Reddy, S.M. 1987 Anti 

fungal activity of secretion of scent glands from heteropteran bugs. 

Indian J. Exp. Biol., 25: 233-234. 

Vidyasagar, C.H. 1995. Identification and functions of scent secretion 

from the scent glands of pentatomid bugs. Ph.D. Thesis, Kakatiya 

University, Warangal. 



Effect of Different Concentrations of SA on the Morphological and Agronomical 

Characteristics of Wheat (Triticum aestivum L. em. Thell.) 

JYOTSNA SUBHA, S. MARKER, A. KRUPAKAR AND ATUL TRIPATHI 

Department of Genetics and Plant Breeding, Sam Higginbottom Institute of Agriculture, Technology & Sciences, 

Deemed to be University, Naini, Allahabad 211 007 (U.P.) 

ABSTRACT 

Mutation breeding is resorted to achieve rapid genetic changes 

in the targeted material for its genetic enhancement. Induced 

polygenic variability in respect of growth and yield contributing 

traits were studied in M 2 

generation of cultivar Kalyan sona. 

Results indicated that higher concentration of sodium azide 

reduces the germination percentage, root and shoot length. 

However, at low concentration it was at par with control. Yield 

attributing characters showed both positive and negative shift 

in mean value in different concentrations in comparison to 

control. The magnitude of polygenic traits for genotypic and 

phenotypic variability, heritability and genetic gain decreases 

with the increases in concentration of sodium azide. Some of 

the mutant lines (eight progeny for earliness, one for plant 

height, three for spike length and grain yield each, two for 

tillering and four for test weight) were found desirable. These 

lines were either comparable to or better than control for yield 

and its components. It can be concluded that sodium azide with 

0.02 per cent concentration appear to be most effective mutagenic 

treatment for induction of micro-mutation in yield component 

traits in wheat. Selection in M 2 

populations of these treatment 

would be effective in rectificatio n of simply inherited 

morphological deficiencies and bringing out lines with yield 

improvement. 

Key words 

Mutagenesis, sodium azide, agro-morphometric 

traits, crop improvement and wheat 

Presently wheat crop is witnessing a plateau in yield 

level and it has become imparative to break this, which is only 

possible by creating new variation. One way of creating 

variability in such an important self pollinated crop is 

attempting crosses between two genotypes complementing 

the characters of each other but due to autogamous nature of 

the crop, hybridization at appropriate time is a difficult process 

(Micke, et al., 1985). The another alternative left with breeders 

to create variability is mutation breeding. This method can be 

used as a potential source of creating variability. Mutations 

have played a great role in increasing world food security, 

since new food crop varieties embedded with various induced 

mutations have contributed to the significant increase of crop 

production. It offers the possibility of inducing desired 

attributes that either cannot be found in nature or have been 

lost during evaluation (Reddy, 1998). The present studies have 

provided evidence on the induction of genetic variability 

connected with yield and yield components in wheat crop. 

Chemical mutagenesis is regarded as an effective and 

important tool in improving the yield and quality characters 

of crop plants. Sodium azide has also proved its worth as 

chemical mutagens to induce genetic variation in the character 

of economic importance (Mendulkar, 2002). Thus, this chemical 

mutagen was used in present investigation to isolate and 

identify the genes used in designing wheat crop with improved 

yield, increase biotic resistance and reduced agronomic inputs. 


The genotype used for mutagenic treatment was Kalyan 

sona, a promising and leading wheat variety of India but 

susceptible to leaf rust. Three different concentrations of 

sodium azide (0.02 per cent, 0.04 per cent, and 0.06 per cent) 

were used in present investigation along with control. After 

pre-soaking ( 6hrs in distilled water) the seeds were blotted, 

dry and treated ( 6hrs at room temperature 26 0 C + 2 0 C with 

intermittent shaking) with freshly prepared chemical mutagen 

solution of sodium azide in above mentioned three 

concentrations. An equal number of seeds were soaked in 

distilled water, which served as control. After the treatment 

seeds were thoroughly washed in running tap water for two 

hours and then blotted dry. Out of 100 seeds, 30 seeds were 

sown in petridishes for all the three concentrations and in 

control for laboratory experiment, where observations on 

germination per cent, root and shoot length were recorded. 

Remaining 70 seeds of each concentrations were used for 

raising M 1 

generation. 

M 1 

Generation: The sowing of M 1 

generation was done on 

25 th November , 

2007 in four different plots keeping 25 x 5 cm 

spacing at Field Experimentation Centre of the Department of 

Genetics and Plant Breeding, Sam Higginbottom Institute of 

Agriculture, Technology and Sciences, Allahabad, U.P. 

Observations on germination, flowering, seedling survival and 

other characters were noted. The first spike of randomly 

selected plants was selfed by using paper bags in treated 

population. After the crop was harvested, 16 plants from each 

concentration were selected randomly for post harvest 

observations. Plants within the population of different 

concentrations were further designated as F 1 

P 1, 

F 1 

P 2 

——— 

—F 1 

P 16 

. Similarly, in all others concentrations 16 random plants 

were selected and designated as above. 

M 2 

Generation: The seeds of all randomly selected 16 plants 

were harvested from each concentration separately and were 

grown in augmented randomized compact family block design

SUBHA et al., Effect of different concentrations of SA on the morphological and agronomical characteristics of Wheat 209 

with three replications during next crop season i.e. winter 2008- 

09. Each entry was sown in two rows plot of 2 m length with a 

spacing of 30 × 5 cm. All the recommended agronomic packages 

and practices were followed to raised a healthy crop. The data 

recorded for eight polygenic traits (Table 1) were subjected to 

the analysis of variance according to the procedure outlined 

by Steel and Torrie, 1980. The estimation of variability was 

done on family and population basis using the standard 

statistical procedure (Panse and Sukhatme, 1967). The 

observed F-value was compared against the corresponding 

table value given by Fisher and Yates, 1963 for deciding the 

significance of between progenies and within progenies 

component of variances. PCV and GCV were calculated by 

the formula given by Burton, 1952, heritability in broad sense 

(h 2 ) by Burton and De Vane, 1953 and expected genetic gain 

were calculated by using the procedure given by Johnson, et 

al., 1955. 


Analysis of variance revealed that variance between 

families was significant for all traits studied, whereas variance 

within progenies were non-significant for most of the 

characters studied (Table 1). There was general decrease in 

germination percentage with increase mutagenic 

concentration . Similarly,the root and shoot length showed a 

similar pattern of growth i.e. with the increase of concentration 

of chemical mutagen there was adverse effect on the growth 

of root and shoot length of wheat seedlings in the laboratory 

conditions (Table 2).The reduction in seedling survival is 

attributed to cytogenetic damage and physiological 

disturbances (Sato and Gaul, 1967). Thus, the probable reason 

of this may be the hindrance caused by the sodium azide on 

different metabolic pathway of the cells. Similar findings have 

also been reported by Reddy, 1998 and Rachovska and Dimova, 

2000. 

Progeny and family mean values (Table 3) reveals that 

high and/or at par progeny mean value was observed for all 

the quantitative traits studied in 0.02 per cent concentration 

of sodium azide. In M 2 

generation variation is expected to be 

high for any character because of the segregation and a number 

of mutants for different quantitative characters can be identified 

in this generation. A progeny with a high or low mean 

(depending on the character under consideration) over the 

mean of control, with a high coefficient of variation is expected 

to yield desirable segregants in successive generations. At 

the same time families with a desirable shift in the mean were 

also considered desirable. The role of mutation breeding in 

increasing the genetic variability for quantitative traits in 

various crop plants have been proved beyond doubt (Khan 

and Goyal, 2009). 

Individual character has great role in mutation breeding. 

In the present investigation due emphasis was given to 

individual yield contributing traits. The mean values indicated 

that sodium azide did not caused any significant changes in 

mean for days to maturity. Semi dwarf plant height (85-90 cm) 

is desirable attribute in wheat as too tall plants are susceptible 

for lodging and height below the D 2 

type causes significant 

reduction in straw weight, ( an essential by-product of wheat). 

In the present investigation, most of the progenies in the 

treated concentration and in control were not in the desirable 

plant height group, which indicates that sodium azide did not 

cause any significant reduction in plant height. However, in 

higher concentration of sodium azide, progenies showed 

reduction in plant height, indicating their undesirability for 

selection. Sharma, et al., 1989 showed similar results in wheat, 

Table 1. 

S. 

No. 

Analysis of variance of different characters in M 2 

generations of wheat 

Source of variance 

d.f 

Days to 50% 

flowering 

Days to 

maturity 

Plant 

height 

*,** Significant at 5% and 1% level respectively WF = within family 

Mean sum of squares 

Spike Width of 

length flag leaf 

Number of 

tillers per 

plant 

Test 

weight 

1 Bet/Fam 63 4.67* 26.36* 74.10* 0.35 0.44* 72.12* 309.13* 0.55* 

2 Rep Fam 15 1.46 8.61* 1.92 0.51* 0.02 1.08 2.48 0.91 

3 WF 1 15 3.70* 8.96* 59.44* 0.22 0.02 3.25* 14.91* 4.70* 

4 WF 2 15 6.90* 13.81* 10.28 0.17 0.07** 9.66* 10.94* 9.10* 

5 WF 3 15 2.03 7.38* 70.47* 0.29* 0.06* 10.37* 23.15 4.82* 

6 WF 4 15 28.39* 33.28* 17.70 0.63* 0.009 2.02* 28.21 2.54* 

7 WF 5 3 1.41 2.10 15.70 0.13 0.01 0.56 1.92 1.25 

Table 2. 

Germination %, root length (cm) and shoot length (cm) at 7, 10 and 14 DAS in SA treated wheat variety Kalyan Sona 

Treatment 

Germination (%) Root length (cm) Shoot length (cm) 

Mean 

7 DAS 10 DAS 14 DAS 7 DAS 10 DAS 14DAS 7 DAS 10 DAS 14 DAS 

Control 100.00 100.00 100.00 7.1 7.5 7.8 7.2 7.4 7.6 95.55 

0.02% 98.00 98.00 97.00 6.5 6.7 6.9 5.0 5.3 5.7 97.00 

0.04% 96.00 92.00 93.00 6.2 6.4 6.6 5.0 6.5 6.8 92.33 

0.06% 90.00 89.00 82.00 5.6 5.7 5.8 6.5 7.0 7.1 85.66 

Grain 

yield/ 

plant


Table 3. 

Family means for different quantitative characters in M 2 

generation of wheat 

S. No. Family Days to 50% 

flowering 

Days to 

maturity 

Plant height 

(cm) 

Spike 

length 

(cm) 

Width of flag 

leaf 

(cm) 

Number of 

tillers per 

plant 

Test weight 

(g) 

Grain yield/ 

plant 

(g) 

1 F1 72.88 103.13 84.44 9.23 2.04 11.23 31.56 22.28 

2 F2 72.44 101.91 83.71 9.38 1.9 10.03 33.41 23.02 

3 F3 72.88 103.34 79.59 9.16 1.88 8.72 29.25 19.5 

4 F4 73.38 104.09 78.99 9.15 1.75 7.19 26.06 16.73 

Gm 72.89 103.12 84.18 9.23 1.89 9.29 30.07 20.38 

SE 0.211 0.256 0.701 0.066 0.022 0.133 0.245 0.198 

CD (5%) 0.596 0.725 1.982 0.186 0.061 0.376 0.694 0.56 

CD (1%) 0.792 0.964 2.637 0.247 0.082 0.5 0.923 0.744 

CV 1.634 1.406 4.708 4.026 6.495 7.129 4.613 5.967 

Spike length combining with more number of tillers per 

plant and test weight is desirable attributes of a genotype in 

order to obtain higher grain yield per plant. Based on overall 

mean performance for most of the yield contributing characters 

progenies viz., F 2 

P 6, 

F 2 

P 8, 

F 2 

P 10 

, F 2 

P 12 

and F 2 

P 13 

of 0.02 per cent 

sodium azide concentrations were identified to possess 

desirable combination of characters with high mean value. It 

is expected that true breeding genotypes with better 

performance would be expected in advanced generations and 

therefore these characters can be considered as a selection 

criteria for yield. These findings are in accordance with the 

findings of Choudhary and Das, 2001. 

An overall perusal of family mean revealed that with the 

increase in concentration of mutagen, there was a decrease in 

family mean of different progenies for different quantitative 

characters. It is evident that family means of control and smaller 

dose of sodium azide (0.02 per cent concentration) were almost 

at par for different quantitative traits whereas all the yield 

Table 4. 

Estimates of genetic parameters for different quantitative characters in different SA treated families of wheat 

S. No. Character Family CV GV PV GCV PCV h 2 GG 

1 2.64 1.04 2.43 1.40 2.14 42.73 1.88 

1 Days to 50% flowering 

2 7.36 12.61 14.00 4.90 5.17 90.06 9.58 

3 1.96 10.20 11.7 4.38 4.69 87.17 8.42 

4 3.58 2.54 3.93 2.17 2.70 64.59 3.59 

1 2.90 13.24 15.17 3.52 3.77 87.27 9.60 

2 Days to maturity 

2 5.66 14.23 16.97 3.70 4.04 83.90 6.99 

3 3.57 5.76 7.19 2.31 2.58 80.10 4.25 

4 2.63 2.30 4.62 1.47 2.08 49.87 2.14 

1 9.13 21.79 57.51 5.52 8.98 37.88 7.00 

3 Plant height 

2 5.03 17.41 29.19 4.98 6.45 59.64 7.92 

3 10.55 12.65 23.41 4.46 6.08 54.09 6.76 

4 3.60 4.33 5.32 2.34 2.59 81.44 4.35 

1 5.16 0.06 0.15 2.76 4.16 43.89 3.76 

4 Spike length 

2 8.52 0.25 0.35 5.28 6.34 69.84 9.07 

3 5.92 0.05 0.23 2.43 5.19 22.01 2.35 

4 4.55 0.04 0.21 2.18 5.00 19.04 1.96 

1 6.95 1.23 2.02 5.43 6.96 60.89 8.73 

5 Width of flag leaf 

2 5.02 1.00 2.01 5.26 7.46 49.75 4.02 

3 14.00 0.09 1.20 1.95 5.82 75.00 8.53 

4 15.18 0.06 1.04 1.39 4.88 57.89 6.92 

1 32.11 4.62 5.10 21.43 22.53 90.49 38.00 

6 Number of tillers/plant 

2 15.50 4.44 4.62 17.23 21.05 90.49 34.89 

3 16.07 1.02 2.03 8.99 12.70 50.15 13.12 

4 16.30 0.66 1.24 9.30 12.75 53.21 12.12 

1 12.23 15.73 18.25 12.50 13.50 86.19 24.00 

7 Test weight 

2 15.90 13.00 13.45 10.79 10.97 96.70 21.86 

3 18.33 9.03 12.68 11.44 13.57 71.15 19.88 

4 11.38 4.59 5.66 7.38 8.19 81.12 13.68 

1 12.11 1.38 3.03 6.56 9.72 85.61 19.13 

8 Seed yield/plant 

2 8.34 4.06 4.47 10.58 11.10 90.77 20.76 

3 11.60 1.02 3.50 5.34 9.88 69.18 15.94 

4 15.84 0.96 1.43 5.12 6.25 47.07 8.63 

Where CV= coefficient of variation, GV= Genotypic variance, PV= Phenotypic variance, GCV= Genotypic coefficient of variation, PCV= Phenotypic 

coefficient of variation, H 2 = Heritability, GG= Genetic gain

SUBHA et al., Effect of different concentrations of SA on the morphological and agronomical characteristics of Wheat 211 

attributing characters like spike length, number of tillers per 

plant and test weight showed marked decrease in family means 

consequently low yield at higher dose of sodium azide. 

Rachovska and Dimova, 2000 showed similar results for yield 

attributing characters. 

A close perusal of results for variability parameters 

further revealed that the genetic component of induced 

variability (GCV) varied with mutagenic treatment and 

characters studied (Table 4). Heritability estimates depicted 

that for test weight, number of tillers per plant, spike length, 

days to maturity and days to 50 per cent flowering it was high 

for most of the concentrations of sodium azide, thus indicating 

that these characters are govern by additive gene action and 

there is greater scope of selection. Similar trends of heritability 

for different quantitative traits in mutagenic populations have 

been also reported by Reddy, et al., 1994, Chaudhary and 

Das, 2001 and Mendulkar, 2002. 

Genetic advance as per cent of mean under selection in 

the M 2 

populations varied with treatments and characters 

studied. The study revealed that selection in the treated 

populations may lead to improvement up to 20.76 g in yield 

per plant, 21.86 g in test weight, 9.07 cm in spike length, 7.92 

cm in plant height and 9.60 days for maturity. Genetic advance 

as per cent of mean also increased in most of the treatments 

and it was relatively higher for different quantitative characters 

studied. Similar differential estimates of genetic advance in 

different mutagenic treatment populations for different traits 

have also been reported by Kalia, et al., 2001. 

In general, sodium azide was found effective in inducing 

mutations with respect to all the quantitative characters in 

Kalyan sona. The most effective concentration being 0.02 

per cent. Thus, in the present experiment, sodium azide with 

0.02 per cent concentration appear to be most effective 

mutagenic treatment for induction of micro-mutation in yield 

component traits and selection in M 2 

populations of these 

treatment was found effective in bringing out lines with better 

yield potential. 

From the above foregoing results and discussion, it is 

concluded that different doses of Sodium Azide (SA) in Kalyan 

sona wheat cultivar provide enough scope by developing a 

wide range of variation in desirable plant attributes to select 

the high yielding mutants. High genetic variability was induced 

through different doses of SA. It indicates that inducing 

genetic variability and improvement in quantitative traits 

would be possible through SA. Hence, it played a pivotal role 

in crop breeding through mutation and stability of genetic 

variability should be analyzed in succeeding generations and 

selection of desirable mutants could be performed for a 

successful breeding programme. 


Burton, G.W. and De Vane, E.H. 1953. Estimating heritability in tall 

fescue (Festuca arundinacea) from replicated clonal material. 

Agronomy Journal, 45:478-481. 

Burton, G.W. 1952. Quantitative inheritance of grasses. Proc 6th Int, 

Grassland Congress, 1:277-283. 

Chowdhary and Das, P.K. 2001. Induced variability in protein content 

and quality in hexaploid wheat. Journal of Interacademicia, 1(5): 

1-6. 

Fisher, R.A. and Yates, F. 1957. Statistical tables for biological, 

agriculture and medical research New York, Hafner, table 38, pp.134. 

Johnson, H.W., Robinson, H.F. and Comstock, R.E. 1955. Estimates of 

genetic and environmental variability in soybeans. Agronomy Journal 

47: 314-318. 

Kalia, C.S., Kharkwal, M.C. Singh, M.P. and Vari, A.K. 2001. Mutagenic 

affects of environmental industrial chemical agents in inducing 

cytogenetical changes in wheat. Indian Journal of Genetics and 

Plant Breeding, 61(3):203-208. 

Khan, S. and Goyal, S. 2009. Improvement of mungbean varieties 

through induced mutations. Africa. Journal of Plant Sciences, 3: 

174-180. 

Micke, A., Maluszynski, M. and Donini, B. 1985. Plant cultivars derived 

from mutation for the use of induced mutants in cross breeding. 

Mutation Breeding Review, 3: 90-92. 

Mendhulkar, V.D. 2002. Synergistic effect of Sodium Azide in 

combination with maleic hydrazide in Triticum aestivum Linn. 

Advances in Plant Sciences, 15(1): 213-219. 

Panse, V.G. and Sukhatme, P.V. 1967. Statistical Methods of agricultural 

Workers. 2 nd edition, I.C.A.R Publication, New Delhi, pp.381. 

Rachovska, G. and Dimova, D. 2000. Effect of Sodium Azide and gamma 

rays on M 2 

quantitative characteristics of the productivity and their 

connection with M 2 

mutation changes in winter common wheat. 

Rasteniev dni-Nauki, 37(7): 413-419. 

Reddy, V.R.K., Suganthi, C.P. and Edwin, R. 1994. Mutation breeding in 

some cereals III mutagenic parameter. Advances in Plant Sciences, 

7:323-329. 

Reddy, V.R.K. 1998. Mutagenic parameters in single and combined 

treatments of gamma rays, EMS and SA in triticale, barley and 

wheat. Advances in Plant Sciences, 5(2): 542-553. 

Sato, M. and Gaul, H. 1967. Effect of EMS on fertility in barley. 

Radiation Botany, 7: 7-10. 

Steel, R.D.G. and Torrie, J.H. 1980. Principles and Procedures of 

Statistics. 3rd edn., McGraw Hill Konga Kusha Ltd. Book Co. Inc., 

New York. 



Seroprevalence of Brucellosis in Cattle and Bufallo in Some Districts of South Bengal 

PRABIR KUMAR KARMAKAR, BIKASH KANTI BISWAS, SOURAV CHANDRA AND MRS. R. DAS 

Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, 37, K.B. 

Sarani, Kolkata 700 037 

ABSTRACT 

In the present study all the 263 sera samples collected from 

cattle and buffalo from adult female animals with the history 

of abnormal termination of pregnancy, sterility, repeat breeding 

or retention of placenta, were screened for Brucella antibodies 

using Rose Bengal Plate Test (RBPT) and a total of 119 (45.25%) 

sera samples found to be positive and rest were negative. Among 

130 cattle sera samples from organized farm 99 (76.15%) were 

positive and among 57 cattle sera samples from unorganized 

farms 4 (7.02%) sera samples were positive reactor. Out of 76 

buffalo serum samples collected from organized farm 16 

(21.05%) were positive reactor to Brucella antibody. 

Key words 

Brucella, antibody, antigen, abortion, premature birth, 

infertility, RBPT. 

Reproductive performance plays a fundamental role in 

profitability of dairy herds; indeed the primary causes of 

economic loss accrue from infertility or sub fertility leading to 

culling of dairy cattle. There are many etiology i.e. brucellosis, 

campylobacteriosis, trichomoniasis and leptospirosis cause 

reproductive tract infection. Among them brucellosis 

obviously inflicts heavy economic losses causing abortion, 

premature birth, decrease in milk yield and the temporary or 

permanent infertility in infected cattle Blood, et al., 1983. 

Schwabe, 1971 stated that brucellosis alone in cattle and 

buffaloes in India causes an annual economic loss to the tune 

of Rs.240 million. This disease not only responsible for 

considerable economic losses in animal industry but it has 

great public health significance. The disease can be transmitted 

horizontally as well as vertically. Infected animal even after 

cure may release the organism through secretions. These 

types of latent infections can be detected through 

seroprevalence survey. The present programme has been 

undertaken to study the seroprevalence of brucellosis among 

cattle and buffalo in organized and unorganized farms 

employing serological test. 


Rose Bengal antigen was procured from Division of 

Biological Products, I. V. R.I., Izatnagar, U. P. for this study. 

Test cattle serum samples were collected from organized 

(130 nos.) and unorganized (57 Nos.) farms of North 24 

parganas, Nadia and Purulia districts of West Bengal. buffalo 

serum sample (76Nos.) was collected only from organized 

farms. All the sera samples were collected from adult female 

animal with the history of abnormal termination of pregnancy, 

sterility, repeat breeding or retention of placenta. After 

inactivated at 56° C in a water bath for 30 minutes the serum 

samples were stored at -20° C. Rose Bengal Plate test (RBPT) 

was done as per method described by Alton, et al. 1975a. 


In the present study all the 263 sera samples collected 

from cattle and buffalo were screened for Brucella antibodies 

using RBPT and 119 (45.25%) sera samples found to be 

positive and rest were negative (Table 1). Among 130 cattle 

sera samples from organized farm 99 (76.15%) were positive 

while 4 (7.02%) sera samples give positive reaction among 57 

cattle samples collected from unorganized farms. Out of 76 

buffalo serum samples collected from organized farm 16 

(21.05%) were positive reactor to Brucella antigen. 

These results support the observation of Hadad and 

Jamaluddin, 1992 where 77.8% serum samples were positive 

to Brucella antibody, but the results reported by Nag, et al., 

1979 i.e. 24.5% positivity were much lower than the present 

observations. The seropositivity of the samples in the study 

from organized and unorganized farms are similar to the 

observation of Mehra, et al., 2000 where seropositivity of 

samples from organized farms were much higher than the 

samples from unorganized farms. 

Table 1. 

Seroprevalence of brucellosis in cattle and buffalo by RBPT 

Species Source No. of sera sample tested 

RBPT 

Positive 

Negative 

Cattle 

Organized farm 130 99 (76.15%) 31(23.85%) 

Un-organized farm 57 4 (7.02%) 53 (92.98%) 

Buffalo Organized farm 76 16 (21.05%) 60 (78.95%) 

Total 263 119 (45.25%) 144 (54.75%)

KARMAKAR et al., Seroprevalence of Brucellosis in Cattle and Bufallo in some Districts of South Bengal 213 


Alton, G.G., Jones, L.M. and Pietz, D.E. 1975a. Laboratory Technique 

In Brucellosis. 2 nd Edn. W.H.O. Monograph Series No. 55, Geneva. 

Blood, D.C., Rodostita, O.M., Henderson, J.A., Arundel, J.H. and Gay, 

C.C. 1983. Veterinary Medicine, ELBS, Bailliere, Tindal. 

Hadad, J.J. and Jamaluddin, N.M.A. 1992. The prevalence of brucellosis 

in cattle in Ninevah province, Iraq. Iraqi Journal of Veterinary 

Science, 5(2): 159-164. 

Mehra, K.N., Dhaneswar, N.S. and Chaturvedi, V.K. 2000. 

Seroprevalance of brucellosis in bovines in Madhya Prodesh, Indian 

Vet. J., 77(7): 571-573. 

Nag, N.C., Kanjilal, B.C. and Roy, J.P. 1979. Brucellosis in cows and 

buffalos in West Bengal. Indian J. Anim. Hlth., 16(1): 89-90. 

Schwabe, C.W. 1971. Report of the first WHO regional seminar on 

Veterinary Public Health, held at Mukteswar from 8-18 th , 1970, 

(WHO Project; SEARO, 0168) Wld. Hlth. Org. Regional Office for 

South East Asia, SEA/VPH/9, 11 January. 



Production of Cellulase and Bioethanol from Lignocellulosic Materials Using 

Aspergillus niger and Sacchromyces cerevisiae 

AKHILESH BIND 1 , BUDH PRAKASH KANOUJIA 1 , NABEEL AHMAD 3 , SAMA. MASIH 2 AND 

ABHISHEK SHARAN 1 

1 

Department of Biochemistry and Biochemical Engineering, Sam Higginbottom Institute of Agriculture, 

Technology and Sciences, Allahabad 211 007 

2 

Centre for Transgenic Studies, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad 

211 007 

3 

School of Biotechnology, IFTM University, Moradabad 244 001 

e-mail: horton037@yahoo.co.in 

ABSTRACT 

Agricultural crop residues viz. sugarcane bagasse, wheat straw, 

wheat bran, rice husk, banana peel and orange peel have great 

potential to produce bioethanol as they have immense quantity 

of carbon source. These agricultural crop residues were pretreated 

with NaOH for delignification and then subjected to 

solid state fermentation for the production of cellulase enzyme 

using Aspergillus niger. Cellulase production parameter such 

as pH, incubation period and temperature were optimized. 

Activity of cellulose was checked by FPase method which was 

found as 3.69 U/ml from wheat bran at pH 6 and at 28 0 C after 6 

days of incubation and the cellulase was produced under these 

optimum conditions. Subsequently enzymatic hydrolysis was 

done as cellulase play a catalytic role in the hydrolysis of 

cellulose to glucose monomer units, so that it could be further 

converted into ethanol. After enzymatic hydrolysis, glucose 

released was inoculated with Sacchromyces cerevisiae (MCCB 

0278) under optimum condition which converted glucose into 

ethanol. After this, fractional distillation was done for the 

purification of bio-ethanol produced and specific gravity was 

also checked. 

Key words 

Bioethanol, Aspergillus niger, cellulase enzyme, solid 

state fermentation, saccharomyces cerevisiae, specific 

gravity 

Aspergillus spp. is the best producer of cellulose in 

twenty nine fungal strains that were isolated from agriculture 

wastes, Abostate, et al., 2010. Aspergillus niger and 

Saccharomyces cerevisiae can be chosen for ethanol 

fermentation because of its high ethanol tolerance and high 

rate of fermentation activity (Yang and Zhang, 2005). Ethanol 

fermented from renewable sources for fuel or fuel additives 

are known as bio-ethanol. Additionally, the ethanol from 

biomass-based waste materials is considered as bio-ethanol. 

Currently, there is a growing interest for ecologically 

sustainable bio-fuels. Using ethanol as a fuel decreases fossil 

fuel consumption and increases energy supply security. It 

can be used neat or blended with gasoline. Additionally, it is 

considered biodegradable and sulphur free. Its carbon content 

has a vegetable origin and as a consequence, when it is 

released during the combustion process, it does not contribute 

to the increase of CO 2 

in the atmosphere, reducing global, 

warming (Liimatainen, et al., 2004). 

Ethanol production from cellulosic materials by direct 

bioconversion is highly encouraging and its commercial 

production is established in countries like Brazil, Canada and 

USA. The economics of ethanol production by fermentation 

is significantly influenced by the cost of raw materials,which 

accounts for more than half of the production cost. In recent 

years, efforts have been directed towards the utilization of 

cheap renewable agricultural resources (Manikandan, et al., 

2007). 


Collection of culture 

Pure culture of Aspergillus niger (MCCB0202) and 

Saccharomyces cerevisiae (MCCB0278) were procured from 

the research laboratory of Microbiology and Fermentation 

Technology, SHIATS, Allahabad. The cultures were 

maintained on PDA medium at 4 0 C. The slants were grown at 

28±2 0 C for 3 days. 

Preparation of substrates 

The agro residues were screened for bioethanol 

production and collected from local market of Naini, Allahabad. 

The substrates were powdered in grinder to get the small size. 

The substrates were hydrolysed at 10psi for 30min, with 2% 

NaOH at concentration range 1% of crushed substrates, and 

the alkali hydrolyzates were cooled at room temperature. The 

solution was neutralized with Acetic acid to remove the lignin 

surface. The treated substrates were subjected to heat 

treatment by autoclave. The substrates were autoclave for 1 

hr at 121 0 C.After the heat treatment, substrates were washed 

using distilled water and then neutralized by acetic acid and 

NaOH. The substrates were dried at 60 0 C in oven for 12 h.

BIND et al., Production of cellulase and bioethanol from lignocellulosic materials using Aspergillus niger 215 

Inoculum preparation 

The production media was inoculated with 1 % of revived 

yeast culture and incubated under constant condition at 28±2 0 

C for 4 days. Fungal culture was inoculated onto PDA medium 

in the slant, after 72 hr, the spores were harvested using 

sterilized water with 0.1% Tween 80. For inoculation, 3 ml of 

spore suspension was used. 

Optimization of process parameters for the production of 

cellulase 

Optimization parameters 

Different optimization parameters such as pH, 

temperature and incubation period was carried out for maximum 

cellulose production by varying physical conditions of 

culturing biomass in substrate to find out suitable optimised 

condition for enzyme production on combination of different 

agro-residues. 

For pH optimisation the inoculation of A.niger was 

carried out at different pH i.e. 3, 4, 5, 6, 7, 8 and 9 for maximal 

enzyme (cellulase) production. For temperature optimisation, 

the inoculation of A. nigerwas carried at different temperature 

i.e. 20, 22, 25, 28, 33, 37 and 40 for maximal enzyme production 

and for incubation period the inoculation of A. Niger was 

incubated for 48, 72, 96, 120, 144, 168 and 192 hrs for maximal 

enzyme production. 

Extraction of Cellulase 

After 5 days of incubation, the cellulose extraction was 

done from the fermented matter by citrate buffer. 50 ml of 0.1M 

citrate buffer, pH 4.8 was added to the SSF medium for cellulase 

extraction. The mixture was vigorously homogenized on a 

rotary shaker for 30 min at 200 rpm. The solid biomass residues 

were separated from the suspension by filtration through 

Whattman filter paper no. 1. The filtrate was used as crude 

enzyme activity. 

Estimation of Cellulase Activity 

The cellulase activity was measured as filter paper 

(FPase) assay. In a test tube, 1.5 ml of 0.05 M sodium citrate 

buffer having pH 4.8 was taken. To this 0.5 ml of enzyme was 

added. One strip of Whatman no. 1 filter paper (weighing 50 

mg) was put into test tube. The test tube along with blank was 

kept in a water bath at 50 0 C for 60 min. After 60 min the tubes 

were taken out and 3 ml of DNSA reagent was added to the 

tubes. The tubes kept on a vigorously boiling water bath for 

5 min. After cooling, 5 ml of water was added to equalize the 

reaction mixture to a volume of 10 ml. The contents were mixed 

well. Then the filter paper pulp was allowed to settle down. 

After this, the colour formed was read at 540 nm using UV 

spectrophotometer. The amount of glucose was known by 

referring to standard graph of glucose. One unit of enzyme 

activity is the one micromole of glucose released per min. 

Enzymatic Hydrolysis/Saccharification 

Enzymatic hydrolysis was carried out in reaction mixture 

containing 5 g of pre-treated substrates in 100 ml of citrate 

buffer (pH 4.8) with 5 U/ml of crude cellulase. The pH was 

adjusted to 4.5 in all the flasks. The reaction mixtures were 

incubated on rotary shaker at 50 0 C, 75 rpm for 24 hrs. After 24 

hrs, the samples were boiled for 2 min to denature enzyme and 

then centrifuged at 5000 rpm for 15 min. The supernatants 

were collected and used for fermentation. 

Bioethanol Fermentation 

The fermentation was carried out by solid state 

fermentation. The volume was again adjusted to 100 ml. The 

pH of the suspension adjusted to 4.5. The flasks containing 

the hydrolyzed samples were covered with cotton wool, 

wrapped in aluminium foil, autoclaved for 15 min at 121 0 C and 

allowed to cool room temp. The sterilized flasks were inoculated 

with 2 ml of 24 hrs old yeast culture i.e S. cerevisiae and under 

anaerobic condition. Harvesting was done by centrifugation 

at 5000 rpm for 20 min. 

Purification of Bioethanol by Fractional Distillation 

After 10 days of fermentation, the fermented substrates 

were filtered and dispensed into round bottom flask fixed to a 

distillation column enclosed in running tap water. A conical 

flask was fixed to the other end of the distillation column to 

collect the distillate. A heating mantle with the temperature 

adjusted to 78 C for 6-7 hrs was used to heat the roundbottomed 

flask containing the fermented broth. 

Specific Gravity of Bioethanol 

Specific gravity bottle was cleaned with a mixture of 

chromic acid and nitric acid followed by thorough washing 

with water. It was rinsed with dist. water and finally with 

alcohol, it was dried in oven. The empty bottle was weighed 

with stopper. With the help of pipette the ethanol was added 

to the bottle and its weight was measured along the stopper. 

The alcohol was removed from the bottle and dried in oven. 

The dried bottle was filled with water avoiding any air bubbles 

and weighed. 


Effect of Incubation 

The effect of different incubation period at constant 

temperature and pH on cellulase production is shown. Similar 

pattern of enzyme production and productivity profiles were 

exhibited for all the different incubation days used. The 

maximum activity was obtained at 144 hrs of incubation for all 

substrates (Table 1). This result was more or less similar with 

that obtained by Jeeu, 2000 and Narasimha, et al., 2006. They 

reported that optimum incubation period of cellulolytic 

enzymes during SSF of lignocellulosic residues was 3 to 8


days. Based on the results obtained, the incubation period of 

the medium was adjusted to 144 hrs.for A. niger in subsequent 

studies. 

Effect of pH 

As shown in figure 1 that initial pH of the medium has 

profound effect on cellulase production. The cellulase 

production by A. niger in varying pH of showed highest values 

of cellulose activity (3.69 U/ml from wheat bran) at pH 6. The 

initial pH of the medium has a great effect on the growth of the 

organism, permeability membrane, as well as on the 

biosynthesis and stability of the enzymes (Table 2, Fig. 2). 

These data suggest that the enzyme systems within the same 

species may vary, depending on the strain under study. Based 

on the results obtained, the initial pH of the medium was 

adjusted to pH 6 for A. niger in subsequent studies. 

The Effect of Incubation Temperature 

Incubation temperature plays an important role in the 

metabolic activities of a microorganism. In the present study 

the optimum temperature for maximum enzyme production 

(3.65 U/ml from wheat bran) was recorded at 28ºC under SSF 

(Table 3, Fig.3). The results of the present study confirm the 

findings of Ali, et al., 1991 reported maximum yield of cellulase 

from Aspergillus niger Z10 strain and A. terreus at 40ºC, 

respectively in SSF. In general the temperature maintained in 

SSF system is in the range of 25 to 35ºC and depends on the 

growth kinetics of the microorganism employed rather than 

on the enzyme produced. Asquieri and Park, 1992 found that 

the optimum temperature for production of CMCase from 

thermostable Aspergillus sp. was 37ºC, whereas the maximum 

cellulase production was observed at 40ºC for Aspergillus 

terreus QTC 828. 

Specific gravity of Ethanol 

Specific gravity bottle was cleaned thoroughly with a 

mixture of chromic acid and nitric acid followed by distilled 

water and finally with alcohol and dried in oven.Weight of 

empty specific gravity bottle with stopper was taken. Ethanol 

was pipette into the specific gravity bottle and its weight is 

again measured along with stopper. Alcohol was removed 

from the specific gravity bottle and dried in oven.The dried 

specific gravity bottle was filled with distilled water avoiding 

any air bubble and stopped in the same manner and weighted 

accurately. Calculation for specific gravity was done as 

follows: 

Fig. 1. Activity of cellulase at different incubation period Fig. 2. Cellulase activity at different pH 

Fig. 3. Activity of Cellulse at different incubation temperature Fig. 4. Specific activity of cellulose enzyme

BIND et al., Production of cellulase and bioethanol from lignocellulosic materials using Aspergillus niger 217 

Table 1. 

Cellulase activity at different incubation period 

Incubation 

FPase activity (U/ml) 

period (hrs) Banana 

peel 

Orange 

peel 

Rice 

husk 

Sugarcane 

bagasse 

Wheat 

bran 

Wheat 

straw 

48 1.11 1.21 1.78 1.32 1.89 1.55 

72 1.77 1.56 1.77 1.67 1.99 1.78 

96 1.80 2.01 2.13 1.77 2.23 1.83 

120 2.58 2.20 2.89 2.20 2.97 2.22 

144 3.06 3.03 3.34 2.91 3.43 3.16 

168 2.97 2.91 3.09 2.82 3.21 3.13 

192 2.66 2.87 2.97 2.67 3.02 2.99 

Table 2. 

Cellulase activity at different pH concentration 

pH 

FPase activity(U/ml) 

Banana 

peel 

Orange 

peel 

Rice 

husk 

Sugarcane 

bagasse 

Wheat 

bran 

Wheat 

straw 

3 2.10 1.43 1.95 1.34 2.32 1.87 

4 2.82 1.61 2.21 1.69 2.54 1.72 

5 2.26 2.86 2.97 2.60 3.16 2.91 

6 3.55 3.18 3.24 3.53 3.69 3.58 

7 2.61 2.85 2.99 2.19 3.11 2.11 

8 2.34 2.98 3.11 2.71 3.01 2.18 

9 2.09 2.11 1.09 1.12 0.97 0.98 

Specific gravity = Weight of alcohol * density of water 

Weight of water 

The specific gravity of bioethanol was found 0.77 (Table 

4, Fig. 4 ) for wheat bran which is closest to the absolute value 

of 0.74 

Bioethanol production by solid state fermentation using 

lignocellulosic materials has been considered as an excellent 

alternative for reusing these wastes with additional 

technological and economic advantages. Production of 

bioethanol was carried out from these materials using 

Aspergillus niger and Sacchromyces cerevesiae. This study 

conducted for the determination of cellulase activity and 

glucose concentration at varying pH, for different incubation 

period and at altering incubation temperature. The results 

indicates the highest production of cellulase (3.69 U/ml) and 

hence ethanol at pH6 for incubation period of 6days at 

incubation temperature 28 o C from wheat bran. The specific 

gravity of bioethanol was found 0.77 for wheat bran which is 

closest to the absolute value of 0.74. This result concludes 

that wheat bran is more efficient than any other substrate for 

cellulase and ethanol production.The other substrates too 

have great potential for the same. So it will be beneficial if 

further detailed studies on bioethanol production from 

agricultural wastes using A. niger and S. cerevisiae through 

SSF method be conducted. 

Table 3. 

Cellulase activity at differe nt incubation 

temperature 

Incubation 

FPase activity (U/ml) 

temperature Banana Orange Rice Sugarcane Wheat Wheat 

( 0 C) peel peel husk bagasse bran straw 

20 1.21 1.01 0.91 0.98 1.43 1.24 

22 2.66 2.51 2.43 2.48 2.13 2.23 

25 2.22 3.11 3.22 2.55 3.59 3.11 

28 3.17 3.22 3.34 3.01 3.69 3.21 

33 2.91 3.03 2.83 2.89 3.12 2.91 

37 2.41 2.93 2.27 2.11 2.29 1.99 

40 2.21 2.77 2.22 2.01 2.33 2.11 

Table 4. Specific gravity 

Substrates 

Specific gravity 

Banana peel 0.91 

Orange peel 0.81 

Rice husk 0.79 

Sugarcane bagasse 0.89 

Wheat bran 0.77 

Wheat straw 0.83 


Abostate, M.A.M., Hammad, A.I., Swelim, M. and Gannam, R.B. 2010. 

Enhanced Production of Cellulase(s) by Aspergillus spp. Isolated 

From Agriculture Wastes by Solid State fermentation American- 

Eurasian. J. Agric. and Environ. Sci. 8: 402-410. 

Ali, S., Sayed, A., Sarker, R.I. and Alam, R. 1991. Factors affecting 

cellulase production by Aspergillus terreus using water hyacinth. 

World J. Microbiol. Biotechnol. 71: 62-66. 

Asquieri, E.R. and Park, Y.K. 1992. Production of extracellular cellulases 

from the thermostable Aspergillus sp. Rev. Microbiol., 23: 183- 

188. 

Jeeu, L. 2000. Solid state fermentation of agricultural wastes for 

endogluconase production industry. Crops Production.11: 1-5. 

Liimatainen, H., Kuokkanen, T. and Kaariainen, J. 2004. Development 

of Bio-ethanol Production from Waste Potatoes. Oulu University 

Press. 123-129. 

Manikandan, K., Saravanan, V. and Viruthagiri, T. 2008. Kinetics studies 

on ethanol production from banana peel waste using mutant strain 

of Saccharomyces cerevisiae. Ind. J. of Biotechnol., 7: 83-88. 

Narasimha, G., Sridevi, A., Viswanath, B., Chandra, M.S. and Rajasekhar, 

R.B. 2006. Nutrient effects on production of cellulolytic enzymes 

by Aspergillus niger. Afric.J. of Biotech., 5: 472-476. 

Yang, X.G. and Zhang, W.G. 2005. A Shortcut to the Production of 

High Ethanol Concentration from Jerusalem Artichoke Tubers. 

Food Technol. Biotechnol., 43: 241-246. 



Lipid Peroxidation as Biomarker for Evaluating the Level of Toxicity in Nostoc 

muscorum under Multiple Stress 

KHAN UZMA AFTAB 1 , RAJESH CHATURVEDI AND IFFAT ZAREEN AHMAD 2 

Department of Biotechnology, Integral University, Dasauli, Kursi Road, Lucknow 

e-mail: uzma.aftab@rediffmail.com 1 , iffat77@rediffmail.com 2 

ABSTRACT 

Heavy metals are chemically or biologically non degradable. 

Malondialdehyde (MDA) is a cytotoxic product of lipid per 

oxidation and an indicator of free radical production. We have 

use lipid per oxidation as biomarker to check the level of 

toxicity of heterocystous cyanobacteria Nostoc muscorum under 

single (NaCl, 100mM) and multiple stress (NaCl and chloride 

of heavy metal, 10µM). Each metal with salt stress has been 

used as a multiple stress individually. Increase in biomass was 

observed in some of the test culture show great adaptation in 

the stress. Result could be summarized as Pb > Mg, Zn > Cd, 

Mn > Cont > Co. Multiple stressed culture however depicts 

different results than single stress. Order of toxicity shown as 

Pb > Mg > Mn > Cd > Zn > Cont > Co. Heterocystous 

cyanobacterial biofertilizer is an excellent replacement of 

chemical fertilizer. 

Key words 

MDA, lipid per oxidation, ROS, free radical, cellular 

damage 

The use of different chemical fertilizers resulted in 

different abiotic stresses, especially salinity and heavy metals, 

and thus reduced soil fertility and created stagnant crop 

productivity (Chaoui, et al., 1997). Salinity alters the 

physicochemical properties of soil, but is also harmful to 

microbial flora including cyanobacteria (Heath and Packer, 

1968). Photosynthetic microorganisms including 

cyanobacteria are highly sensitive to heavy metal ions (Lang, 

1968, LEs and Walker, 1984, Mittova, et al., 2000). The 

prokaryotic cyanobacteria serve as an excellent tool for 

studying the effect of heavy metals on photosynthetic activity 

owing to their close similarity with the chloroplast (Rama and 

Prasad, 1998). Microorganisms could be used to clean up metal 

contamination. Organisms respond to heavy metal stress using 

different defence systems, making complexes and the 

synthesis of binding proteins such as metallothioneins (MTs) 

or phytochelatins (PCs). 


The culture of Nostoc muscorum was obtained from the 

University of Allahabad, Allahabad, Uttar Pradesh, India. 

Cultures were grown and maintained in nitrate-free BG 11 

medium [8] sets of culture was grown in Batch culture under 

heavy metal stress, Nostoc muscorum under single (NaCl, 

100mM) and multiple stress (NaCl and chloride of heavy metal, 

10µM). Alkaline earth metal used in this study are Pb, Zn, Cd, 

Co, Mg and Mn individual metal with salt stress has been 

used as a multiple stress individually with no stress culture 

serve as control for single stress and salinity stressed culture 

as control for multiple stress. 

Cultures were maintained at 27°C±1°C under fluorescent 

illumination of 30-40 µEm -2 s -1 , exposed to a 12 h light / 12 h 

dark photoperiod and swirled manually for five minutes, thrice 

daily. 

Enzyme extraction 

The level of lipid peroxidation (LPX) was measured in 

terms of malondialdehyde (MDA), a product of LPX estimated 

by thiobarbituric acid (TBA) reaction [9]. Fresh algal sample 

(0.5 g) was homogenized in 5 mL of 10% (W/V) trichloro acetic 

acid (TCA), and the homogenate was centrifuged at 7000 ×g 

for 10 min. One ml of the supernatant was mixed with 2 mL of 

0.5% TBA solution (in 10% TCA). Then the mixture was heated 

at 95°C for 45 min and then cooled under room temperature. 

The supernatant was read at 532 nm. 


Data pertaining to cellular damage and free radical 

accumulation in terms of malondialdehyde under single and 

multiple stress are compiled in Fig. 1 and Fig. 2. MDA content 

was estimated after 10 th day the microorganism was subjected 

to stress. Some of the test culture show great adaptation in 

Malondialdehyde% 

Fig. 1. 

120 

100 

80 

60 

40 

20 

0 

Conc Mg Mn Co Cd Zn Pb 

Heavy metal stress (10µM) 

Day 10 

Day 12 

Day 14 

Effect of single stress [heavy metal chloride (10µM)] 

on the growth of Nostoc muscorum measured on the 

10 th , 12 th and 14 th days. Each set (from left to right) 

represents stress exerted by metals in terms of MDA 

due to lipid per oxidation. 1st, 3rd, 5th, 7th, 9th and 

11th days. Each set (from left to right) represents 0.0, 

0.05, 0.10, 0.15, and 0.20mg Cu/L, respectively.

AFTAB et al., Lipid Peroxidation as Biomarker for Evaluating the Level of Toxicity in Nostoc muscorum 219 

Malondialdehyde% 

Fig. 2. 

120 

100 

80 

60 

40 

20 

0 

Conc Mg Mn Co Cd Zn Pb 

Heavy metal(10µM) and NaCl (100mM) stress 

Day 10 

Day 12 

Day 14 

Effect of multiple stress [heavy metal chloride (10µM) 

and NaCl (100mM)] on the growth o f Nostoc 

muscorum measured on the 10 th , 12 th and 14 th days. 

Each set (from left to right) represents stress exerted 

by metals in terms of MDA d ue to lipid per 

oxidation. 

the stress and show increased cell division than control 

(culture serving with no stress). Lead has shown greater 

production of MDA which is considered as a measure of LPO 

status, Zinc and magnesium show similar level of toxicity 

similarly cadmium and manganese show same ROS production. 

Test culture in cobalt stress shows excellent adaptation less 

production of ROS. Result could be summarized as Pb > Mg, 

Zn > Cd, Mn >Cont > Co. Multiple stressed culture however 

depicts different results than single stress. No two metals 

showed similar level of toxicity. Order of toxicity shown as Pb 

> Mg > Mn > Cd > Zn > Cont > Co Culture in single stress 

shows lesser formation of free radicals than multiple stress 

except magnesium and zinc .Cadmium, lead, magnesium, zinc, 

show enhancement in lipid per oxidation than control. Level 

of toxicity was shown was recorded for all the three alternate 

days. 

The present study provide evidence of oxidative damage 

to the test microorganism under multiple and single stress.The 

interaction between heavy metal and NaCl appeared to be 

antagonistic as the depression of growth and physiological 

activities by a mixture of the two was lesser than that caused 

by either of these. These observations form the report on the 

response of a metal resistant strain of cyanobacterium to 

salinity.The level of MDA in the tissue is considered a measure 

of lipid peroxidation status. ROSs are known to damage cellular 

membranes. They also can damage DNA, proteins, lipids and 

chlorophyll. Culture in multiple stress show reduction in 

growth could be due to inhibition of normal cell division by 

metal is primarily attributed to their binding to sulphahydryl 

group which is necessary for cell division. Thus, the increased 

level of MDA suggests that metal ions stimulate free radical 

generating capacity of the microorganism. Increased MDA 

level has been reported in several higher plants also. 

Salinity-induced lipid peroxidation in N. muscorum (Fig. 

1) finds support from the results reported in A. doliolum and 

Scytonema javanicum, respectively, exposed to salt stress. 

Among the different ROS, H 2 

O 2 

is of great significance owing 

to its enormous stability and role as a secondary messenger 

to regulate the expressions of defense genes. The result of 

salinity promoting H 2 

O 2 

production in N. muscorum (Fig. 2) 

finds support from observations in hyper-accumulation in saltexposed 

wheat. The foremost reason of ROS production in 

cyanobacteria is altered energy transfer from excited Chl to 

molecular oxygen during photosynthesis. 


Chaoui, et al. 1997. Cadmium and zinc induction of lipid peroxidation 

and effects on antioxidant enzyme activities in bean (Phaseolus 

vulgaris L.). Plant Sciences, 127:139-147. 

Lang, N.J. 1968. The fine structure of blue-green algae. Annu Rev 

Microbiol, 22: 15-46. 

LEs, A. and Walker. 1984. Toxicity and binding of copper, zinc and 

cadmium by the blue-green alga, Chroococcus paris. Water Air Soil 

Pollution, 23: 129-139. 

Mittova V., et al. 2000. Activitiesof SOD and the ascorbate-glutathione 

cycle enzymes in subcellular compartments in leaves and roots of 

the cultivated tomato and its wild salt-tolerant relative Lycopersicon 

pennellii. Physiol. Plant. 110: 45. 

Rama and Prasad 1998. Copper toxicity in Ceratophyllumdemeresum 

L. (Coontail), a free floating macrophyte: Response of antioxidant 

enzymes and antioxidants. Plant Sciences, 138: 157. 



Isolation and Identification of Algae from Dhemaji District of Assam, India 

JYOTI PRASAD LAHAN, RITUPARNA KALITA, SUDIPTA SANKAR BORA, ARCHANA DEKA, 

ROBIN CH. BORO AND MADHUMITA BAROOAH* 

Department of Agricultural Biotechnology Assam Agricultural University Jorhat 13, Assam 

*e-mail: m17barooah@yahoo.co.in 

ABSTRACT 

The occurrence of diverse crops and microbes in Assam indicate 

the potential existence of algal diversity in this region. However, 

reports on the occurrence and diversity of the algae resources 

in this region are very scarce. Although algae have received 

significant interest due to their pivotal role in agriculture, 

environment and industry, relatively little is known about the 

existence and extent of algal diversity in Assam. Keeping this 

in mind, a study was undertaken to document the diversity of 

algae occurring in Dhemaji district of Assam. A total no. fifty 

nine species belonging to Chlorophyceae, Cyanophyceae, 

Euglenophyceae, Bacillariophyceae and Xanthophyceae 

families were documented. Chlorella species was most 

frequently encountered during the study. 

Key words 

Chlorophyceae, Cyanophyceae, Euglenophyceae, 

Bacillariophyceae, Xanthophyceae, Chlorella 

Assam with its unique bio-geographical features is a 

store house of biological resources and genetic diversity. 

Assam being at the centre of the North-Eastern region has 

been reported as a part of biological hot spot (Bhagabati, et 

al., 2006). The occurrence of diverse crops and microbes 

indicates to the potential existence of algal diversity in this 

region. 

The shift in climate change and rapid industrialization 

has increased the vulnerability of algae and accelerated the 

depletion of species. The distribution of algae in North-East 

India is scanty, have hitherto remained untapped. So it is 

important to evaluate their diversity and make effort to 

conserve. Despite of its importance, a little has been known 

about the existence and extent of algal diversity in Assam. 

Rout and Dey, 1999 contributed to the algal flora of different 

habitats in the district. Deb, 2005 investigated on the epilithic 

algae of a temporary stream at Dargakona, Cachar district of 

Assam. Devi, 2006 worked on the distribution and diversity 

of algal communities growing in pond ecosystem. Biodiversity 

of epilithic cyanobacteria from one of the unexplored habitats 

of freshwater streams of Kakoijana reserve forest of Assam 

has been estimated to 29 species representing 18 genera of 12 

families and 4 orders (Saha, et al., 2007). Till now, there is no 

work on detailed investigation of algal diversity in Dhemaji 

district of Assam has been reported. Therefore, the present 

investigation was made to explore and document the algal 

diversity in Dhemaji District of Assam. 


The present study site Dhemaji is at an altitude of 104 m, 

North-27°-28°, East- 94°-96°. It is a seasonal wetland where 

people are engaged in agriculture during dry period while the 

wetland acts as a fishery during wet period. The algal samples 

were collected at regular intervals (monthly) for a period of 

one year (2010-2011). The algae were identified by relevant 

monographs and recent available literature. Collected samples 

were cultured in laboratory by pour plate method (Toledo and 

Palenik, 1997) using different media i.e., BG-11, Chu-10 etc 

and incubated at 28 ± 2°C under 2-3 Klux light intensity with 

10:14 hrs photoperiods. Purification was done by repeated 

sub-culturing. 

Table 1. Details of Sample collection 

Sub-Division Area No. of samples GPS data pH 

Dhemaji 

Batgharia 32 

N= 27 0 23 / -24 / , E=94 0 31 / -32 / , Alt=97-103 m 

5.5-7.5 

Bordoloni 26 

N= 27 0 24 / -25 / , E=94 0 25 / -26 / , Alt=101-104 m 

5.0-7.5 

Chamarajan 29 

N= 27 0 28 / -29 / , E=94 0 28 / -29 / , Alt=101-103 m 

6.3-7.5 

Gogamukh 18 

N= 27 0 26 / -27 / , E=94 0 13 / -14 / , Alt=91-95 m 

5.5-7.3 

Maridhal 37 

N= 27 0 43 / -44 / , E=94 0 11 / -12 / , Alt=99-102 m 

5.0-7.8 

Machkhowa 33 

N= 27 0 23 / -24 / , E=94 0 31 / -32 / , Alt=105-107 m 

6.0-7.1 

Panchali 23 

N= 28 0 36 / -37 / , E=93 0 71 / -72 / , Alt=106-107 m 

5.7-7.4 

Jonai 

Akajan 15 

N= 27 0 33 / -34 / , E=94 0 38 / -39 / , Alt=104-106 m 

5.4-7.3 

Jonai 31 

N= 27 0 37 / -38 / , E=94 0 38 / -40 / , Alt=101-103 m 

6.1-7.7 

Silapather 19 

N= 27 0 34 / -35 / , E=94 0 39 / -40 / , Alt=105-107 m 

5.5-7.5 

Sisi Baogaon 21 

N= 27 0 32 / -33 / , E=94 0 36 / -37 / , Alt=101-103 m 

6.1-7.5

LAHAN et al., Isolation and Identification of Algae from Dhemaji District of Assam, India 221 

Table 2. 

Distribution of total algal groups in the study area 

Class Genus No. of species 

Chlorophyceae Chlorella spp. 5 

Scenedesmus spp. 2 

Spirogyra spp. 3 

Neochloris spp. 1 

Chlamydomonas spp. 2 

Cosmarium spp. 2 

Microspora spp. 1 

Pleodorian spp. 1 

Chlorococcum spp. 1 

Tetracystis spp. 2 

Cladophora spp. 2 

Haematococcus spp. 1 

Ankistrodesmus spp. 2 

Cyanophyceae Oscillatoria spp. 3 

Nostoc spp. 2 

Anabaena spp. 2 

Lyngbya spp. 3 

Asterocapsa spp. 1 

Microcystis spp. 2 

Calothrix spp. 1 

Phormidium spp. 3 

Spirulina spp. 2 

Nodularia spp. 1 

Xanthophyceae Stigeoclonium spp. 2 

Ulothrix spp. 1 

Zygnema spp. 2 

Vaucheria spp. 2 

Bacillariophyceae Diatom spp. 4 

Navicula spp. 1 

Euglenophyceae Euglena spp. 2 

Fig. 1. 

Geographical location of the s tudy area (Dhemaji 

District, Assam) 


In the present investigation a total no. 59 species 

belonging to Chlorophyceae, Cyanophyceae, 

Euglenophyceae, Bacillariophyceae and Xanthophyceae 

families were documented. All the sites were dominated by 

green algae (Chlorophyceae), among which Chlorella was 

most abundant followed by spirogyra. In case of 

cyanophyceae, Oscillatoria and Lyngbya were often 

encountered. Algae diversity was found to be highest in the 

months of June-July and was lowest in the month of December. 

Four species of diatoms were found in the study. (Table 1 and 

Table 2). 

Algae are promising sustainable oil source for biodiesel 

production. They are increasingly predicted to play a crucial 

role in clean environmentally sustainable future. From the 

present study, it was found that Dhemaji District of Assam is 

very rich in algal diversity where chlorophycean algae are 

very common. 

The feasibility of using algal lipids as a fuel precursor 

on a large scale requires not only productivity but also an 

environment conducive for growth of microalgal culture. The 

study also concluded that urbanization has led to pollution of 

surface water bodies resulting in decline/extinction of some 

species. 

Fig. 2. 

Pie chart showing comparative algal diversity in the 

study area. 


Bhagabati, A.K., Kalita, M.C. and Baruah, S. 2006. Biodiversity of 

Assam: Status Strategy & Action Plan for Conservation, (eds.), 

Eastern Book House, New Delhi. 

Deb, P. 2005. Investigation on the epilithic algae of a temporary 

stream at Dargakona, Cachar district, Assam. M.Sc. dissertation, 

Dept of Ecology, Assam University, Silchar, Assam. 

Devi, D. 2006. Distribution and diversity of algal comm\U1ity growing 

in Barambaba Temple Pond,Cachar District (Southern Assam) N.E. 

India,M.Sc. dissertation, Dept. of Ecology, AssamUniversity, Silchar, 

Assam. 

Rout, J. and Dey, A. 1999. A study of algal flora from rice field 

ofIrongmara (Barak Valley, Assam). Phykos., 38(1 and 2): 19-25. 

Saha, S.K., Das, R., Bora, K.N. and Uma, K. 2007. Biodiversity of 

epilithic cyanobacteria from freshwater streams of Kakoijana 

reserve forest, Assam, India, Indian Journal of Microbiology, 47(3): 

219-232. 

Toledo, G. and Palenik, B. 1997. Synechococcus diversity in the 

California Current as seen by RNA polymerase (rpoC1) gene 

sequences of isolated strains. Appl. Environ. Microbiol., 63: 4298- 

4303. 



A Study on Impact of Watershed Development Programme in Uttar Pradesh 

PUSHPENDRA SAROJ * BASVAPRABHU JIRLI ** AND VINOD KUMAR*** 

*KVK, Lower Subansiri, Arunachal Pradesh 

**Department of Extension Education, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, 

U.P. 

***Agricultural Economics & A.B.M., Sam Higginbottom Institute of Agriculture, Technology and Sciences, 

Deemed to be University, Allahabad 

ABSTRACT 

Natural resources viz., soil and water need to be conserved, 

developed and utilized efficiently due to ever depleting ground 

water which is the only feasible solution for conserving fresh 

water. The water tables are not getting recharged due to more 

run off (approximately 46% of the annual precipitation) 1 

during the monsoon season which lasts only for three months 

(July–September). Study was conducted in district Unnao 

during 2005-08. Out of sixteen blocks of the district, seven 

blocks were selected for the study and tow villages from each 

block were short listed for selecting the beneficiaries of 

watershed development programme. Thus a total of 210 

respondents from 14 villages constituted the sample for research 

work, beside farmers, seventy officers working in watershed 

development projects were selected for delineating constraints 

in implementation of watershed development projects. A pretested 

standardized interview schedule was used to collect data 

from the respondents using personal interview method, and 

cost of cultivation was calculated to measure net income of 

respondents. The highest increment in net income was recorded 

(340.00 per cent) in Maize crop. The beneficiaries who were 

having 4-20 plants have gone to 92 as compared to 48 during 

pre-project period, positive increment were recorded in majority 

(153.33 per cent) of the respondents having above two cows and 

highest increment (37.50 per cent) recorded in the number of 

respondents having one buffalo. The highest increment (33.33 

per cent) in productivity of milch animals (cows and goats both) 

was recorded. 

Key words 

Impact, production, watershed development 

India shares near about 19% of the world’s population, 

15% of the live stock population, while it has only 1% of the 

forest area, 0.5% of the pasture land and 2% land of the total 

geographical area of the world. Naturally, there leading 

pressure on the productive lands and forest. Therefore, the 

lands especially the farmlands in India are in the constant 

process of various degrees of degradation and are going very 

fast turning into wastelands. In accordance with the National 

Remote Sensing Agencies (NRSA) findings 75.05 million 

hectares wastelands in the country. It has been estimated that 

out of these around 58 million hectares are treatable and can 

be brought back to productive levels through watershed 

technology. 

In our country out of the total geographical area 329 

million ha, 143 million ha is under cultivation, 108 million ha 

area is rainfed (75%). Rainfed agriculture contributes about 

44% of the total food grain production in the country and 

supports 40% of the population. Bulk of pulses, oil seeds, 

millets, coarse grains and commercial crops like cotton and 

ground nut etc., are accounted by the rainfed agriculture. Thus, 

dryland holds great prospect of contributing substantially to 

country’s food production and unless the production from 

these areas increases, the real breakthrough in agriculture 

may not be possible. 


Study was conducted in Unnao district during 2005- 

08.Out of sixteen blocks of the district, seven blocks were 

selected for the study and two villages from each block were 

short listed for selecting the beneficiaries of watershed 

development programme. Thus a total of 14 villages were 

selected for the study. A group of 15 respondents from each 

selected village were selected randomly. Thus, a total of 210 

respondents from 14 villages constituted the sample for 

research work. Besides, seventy officers from the identified 

watershed development projects were also selected for this 

study. A pre-tested standardized interview schedule was used 

to collect data from the respondents by personal interview 

method and cost of cultivation was calculated to measure net 

income of the respondents. 


The impact analysis of watershed project on yield of 

various crops during post project period against pre project 

period is presented in following tables. 

The findings revealed that highest increment in net 

income were recorded (340.00%) in Maize crop followed by 

103.17% in Mung, 86.66% in Sesamum, 75.97% in Gram, 73.33% 

in Bajra, 73.06% in Mustard, 47.36% in Linseed and minimum 

22.62% increment in net income were recorded in wheat crop. 

Therefore, the findings reveal that there has been significant 

and positive incerement in the yield and net income of all the 

major crops of watershed. However, the yield level obtained 

are still quite low than the state average which may be 

attributed to the poor lands and also as the area has been 

recently undertaken in the watershed development

Table 1. 

Season crops 

Rabi 

SAROJ et al., A Study on Impact of Watershed Development Programme in Uttar Pradesh 223 

Changes in production, cost of production, gross income and net income per hectare 

Cost of 

cul. 

Rs/ha 

Prod. 

qt/ha 

Pre-project 

Gross 

income 

Rs/ha 

Net income 

Rs/ha 

Cost of cul. 

Rs/ha 

Prod. 

qt/ha 

Gross 

income 

Rs/ha 

Post- project 

Net income 

Rs/ha 

Percentage increase 

(+)or decrease(-)In 

net income 

Bajra 7000 20 11500 4500 7800 26 15600 7800 73.33(+) 

Sesamum 6500 8 8000 1500 7200 10 10000 2800 86.66(+) 

Mungneam 4715 6 7080 2365 6225 8.50 11030 4805 103.17(+) 

Maize 8000 15 10500 2500 9000 25 20000 11000 340.00(+) 

Wheat 16000 42 49900 25900 18000 50 49500 31500 21.62(+) 

Mustard 13200 19 25700 12500 15800 25 37500 21700 73.06(+) 

Gram 12600 20 28000 15400 14500 26 41600 27100 75.97(+) 

Linseed 2200 3 6000 3800 2400 4 8000 5600 47.36(+) 

programme. Similar trends of results were reported by Kumar, 

1990, Chandregowda and Jayararnaiah, 1990, Patel, et al., 1995, 

Singh and Prasad, 1998 and Verma, et al., 2004. 

Table 2. Change in number of trees during project period 

N= 210 

S. Number 

Number of households 

No. of trees Pre-project Post-project Percentage 

frequency % frequency % increase (+) or 

decrease 

(-) 

1. 0-10 130 (61.90) 75 (35.71) (-)42.30 

2. 11-20 48 (22.85) 92 (43.80) (+) 91.66 

3. Above 

20 

32 (15.23) 43 (20.47) (+)34.37 

The impact analysis of watershed project on trees during 

post project period against pre project period is presented in 

following Table 2. 

The findings clearly reveal that there has been 

tremendous increments were recorded in the plantation during 

post project period against pre-project period. Though number 

of watershed beneficiaries were having up to 10 plants, 

gradually decreased after the commencement of the project. 

The members of beneficiaries who were having 4-20 plants 

have gone to 92 as compared to 48 during pre-project period. 

The members of watershed beneficiaries who were having 

over 20 plants have also registered an increment of about 

34.37%. Therefore it is quite clear that the watershed 

development programme has created impact in terms of 

increasing plantations through various campaigners and 

Table 3. 

S. 

No. 

Number of 

cows 

Status of cows during pre-project and post-project 

period 

N= 210 


Pre-project Post-project 

frequency % % % 

% 

increase 

(+)or 

decrease(-) 

1. No cow 115 54.76 84 40.00 (-)26.95 

2. One cow 45 21.42 50 23.80 (+)11.11 

3. Two cows 35 16.67 38 18.09 (+)8.57 

4. Above two 

cows 

15 7.14 38 18.09 (+)153.33 

awareness programme. The obtained results are in consonance 

with results of earlier researchers Dhar, 1994 and Singh and 

Prasad, 1998. 

The watershed development programme and the gradual 

change in the mind set of the people resulted into the changes 

in the member of livestock reared by them, which is given in 

the following Table 3. 

The data presented in the tables 3 indicated that positive 

increment were recorded in majority (153.33%) of the 

respondents having above two cows, followed by 11.11% 

having one cow and 8.57% having two cows during post 

project period in comparison to pre-project period. Whether, 

number of the respondents decreased 26.95% which do not 

have cow during post project period in comparison to preproject 

period. This finding is in accordance with the result 

of Arya, et al., 1994 and Purohit, 1995. 

Table reveals that highest increment (37.50%) recorded 

in the number of respondents having one buffalo, followed 

by 27.02% and 12.90% having two buffalos and above two 

buffalos respectively during post project period in comparison 

to pre-project period. Whether, number of the respondents 

having no buffalo was decreased 23.63% during post project 

period in comparison to pre-project period. This finding is in 

contradiction with the result of Arya, et al., 1994 and Purohit, 

1995. 

The data presented in the Table 5 reveal that numbers of 

watershed beneficiaries having two goats were increased 

Table 4. 

S. Number of 

No. buffaloes 

Status of buffalos during pre-project and postproject 

period 

N= 210 



frequency % frequency % 

% 

increase 

(+)or 

decrease(-) 

1. No buffalo 110 52.38 84 40.00 (-)23.63 

2. One buffalo 32 15.23 44 20.95 (+)37.50 

3. Two 

37 17.61 47 22.38 (+)27.02 

buffaloes 

4. Above two 

buffaloes 

31 14.76 35 16.67 (+)12.90


Table 5. 

S. 

No. 

Number of 

goats 

Status of goats during pre-project and post-project 

period 

N= 210 



frequency % frequency % 

% 

increase 

(+)or 

decrease(-) 

1. No goat 65 30.95 35 16.67 (-)46.15 

2. One goat 39 18.57 47 22.38 (+)20.51 

3. Two goats 56 26.66 69 32.85 (+)23.21 

4. Above two 

goats 

50 23.80 59 28.09 (+)18.00 

23.21% followed by 20.51% and 18.00% having one goat and 

two goats respectively during post project period in 

comparison to pre-project period. Whether, numbers of 

watershed beneficiaries having no goats were decreased 

46.15% during post project period in comparison to pre-project 

period. 

The impacts of watershed development programme on 

livestock production (in liters) are given in Table 6. 

The results presented in the table indicated that highest 

increment (33.33%) was recorded in the productivity of cows 

and goats both and in case of buffalo’s productivity increased 

(17.64%). It might be due to more availability of green fodder 

and grasses grown in pasture land. And also due to increased 

ground water level and water resources during post project 

period in comparison to pre-project period. Similar trends of 

results were reported by Mahnot, et al., 1992, Purohit, 1995, 

Dhar, 1994, Arya, et al., 1994 and Patil, 1999. 

These results are comparatively better as compared to 

number of PRA based studies conducted in other parts of 

different states (SREPUPDASP). This is because of the fact 

that the watershed area selected in this study falls on uneven 

area where lighter weight cows are preferred more than to 

buffaloes. The findings also conclude that there has been 

tremendous increase in population of goat and cattle rearing 

after the introduction of the projects. The study also indicates 

that still the households rear livestock as supplementary 

enterprise, whereas agriculture still remains number one 

enterprise. Thus, the study suggested bringing in change in 

the farming system by motivating and making the farmers 

aware about the commercial livestock rearing. 

The results of the study revealed that the watershed 

development programme is effective due to increased income 

of watershed beneficiaries and also increased number of trees, 

number of milch animals and production and productivity 

Table 6. 

Change in productivity of milch animals 

(including live stock) in watershed area. 


N= 210 

S. Animals 

Productivity( liter/animal ) 

No. 

Pre-project Post-project Percentage 

increase (+)or 

decrease(-) 

1. Cow 6.00 8.00 (+) 33.33 

2. Buffalo 8.50 10.0 (+) 17.64 

3. Goat 0.75 1.00 (+) 33.33 

Arya, S.L., Agnihotri, Y. and Sharma, J.S. 1994. Watershed Management: 

Changes in animal populatioon structure, income and cattle 

migration, Shiwaliks, India. Ambio., 23(7): 446-450. 

Chandregowda, M.J. and Jayaramaiah, K.M. 1990. Impact of watershed 

development programme on socio-economic status, land 

productivity and income of small and marginal farmers. Indian 

Journal of Extention Education, 25(3&4): 44-47. 

Dhar, S.K. 1994. Rehabilitation of degraded tropical forest watersheds 

with people’s participation. Amblo, 23(3): 216-221. 

Kumar, P.N. 1990. Soil Conservation - Impact on Crop Production. A 

case study, Yojana, 34(20): 22-25. 

Mahnot, S.C., Singh, P.K. and Sharma, Y. 1992. Socio-economic 

evaluation of Watershed Management Project- A Case Study. Journal 

of Rural Development, 11(2): 219-227. 

Patel, N.R., Pandya, D.N. and Patel, B.J. 1995. Techno-economic 

change among beneficiaries farmers in watershed area. Maharashtra 

Journal of Extension Education, 14: 26. 

Patil, J.B. 1999. Evaluation of land treatment for in-situ moisture 

conservation in maize and sesamum crop on medium deep soil. M. 

Sc.(Agri), Thesis, Dr. Panjabrao Deshmulch Krishi Vidhtapeeth, 

Akola. 

Puroit, S.D. 1995. Evaluation of engineering structure under Soil 

Conservation Scheme- A Case Study of Chambal RVP and Sahibi 

FPR in Rajashtan, pp.87. 

Raghunandan, H.C. 2004 A study on knowledge and adoption level of 

soil and water conservation practices by farmers in northern 

Karnataka. M.Sc. (Agri) Thesis, University of Agricultural Sciences 

Dharwad. 

Singh, K.P., Prasad, R. 1998. Experience on some watershed based 

operational research project of Uttar Pradesh for watershed 

development. National Seminar on Wasteland Development : 

Challenges and Opportunities at Kanpur, 15. 

Verma, A.R., Rajput, A.M. and Srivastava 2004. Economic evaluation 

of national watershed development programme for rainfed 

agriculture in Indore district of Madhya Pradesh. Ind. J. Agric. 

Econ., 59(3): 368-369. 



Combining Ability Analysis for Yield and its Components in Indian Mustard (Brassica 

juncea L. Czern and Coss) 

KANHAIYA LAL 1 , RAM KRISHNA 2 , RANJEET 2 , HASMAT ALI1 AND RAMA KANT 2 

1 

Indian Institute of Pulses Research, Kanpur 208 024 

2 

Department of Genetics and Plant Breeding, C.S.A. University of Agriculture & Technology, Kanpur 208 002 

e-mail: kanhaiya_vis04@yahoo.co.in 

ABSTRACT 

The combining ability analysis of 10 parents and their 45 F 1 

s 

generated through diallel system of mating revealed that 

significant differences existed for general and specific 

combining ability for all the characters. Gca and sca variances 

were highly significant for all the characters indicating 

importance of both additive and non-additive gene effects in 

controlling the expression of various characters. Vardan, Krishna 

and Pusa Bahar were found to be good general combiners for 

seed yield and some of its component traits. Twenty crosses 

exhibited good combining ability for seed yield. Yield was found 

to be controlled predominantly by non-additive gene action. 

The crosses with high sca effects did not always had parents 

with good gca effects. Such a relationship between gca and sca 

indicates the importance of epistasis and the crosses are expected 

to produce desirable transgressive segregants if the additive 

ge netic system of good ge nera l co mbiner a nd the 

complementary epistatic effect of F 1 

act in the same direction 

to maximize the desirable yield attribute 

Key words 

Brassica juncea, combining ability, yield components. 

High yielding varieties contribute significantly towards 

increasing both production and productivity. A suitable 

breeding methodology and identification of superior parents 

are the important pre-requisites for the development of high 

yielding genotypes. Sound understanding of gene effects 

involved in the expression of various yield attributes is of 

prime importance in formulating any breeding methodology 

.Combining ability analysis provides a guideline for the 

assessment of relative breeding potential of parental material 

which can be utilized in pursuing a systematic breeding 

programme. (Asthana and Pandey, 1977). This possibility was 

explored in the present investigation and the combining ability 

of the desirable lines was studied. 


Eight cultivars viz., B 85, Urvashi, Krishna, Pusa Bahar, 

Pusa Jaikisan , Pusa Basant, Vardan and Sanjokta and two 

experimental strains viz., RK 9902, RK9901 representing a wide 

range of diversity for various characters were selected for the 

study.These parents had been maintained by self pollination 

for several generations and therefore may be considered as 

homozygous inbred lines. A complete set of 55 entries 

comprising 45 F 1 

s and their 10 parents was grown in a 

randomized block design with three replications at Oilseeds 

Research Farm, Kalyanpur of CSAUA&T, Kanpur. The parental 

and F 1 

populations were sown in single row plots. The inter 

spacings were kept 45 cm and 20 cm, respectively. All the 

recommended agronomic practices were adopted for raising a 

good crop. Data for 10 characters were recorded on five 

randomly selected plants from each row. Diallel mating 

technique was employed for the program. 


The variances due to gca and sca were found highly 

significant. The magnitude of gca variances was high than 

sca variences except in harves index. The estimates of ä 2 g/ ä 2 s 

ratio indicated that values varied in F 1 

generation from 0.02 to 

1.58 suggesting thereby that all the characters except number 

of secondary branches and days to maturity showed 

preponderance of non-additive genetic variance whereas 

additive variance played a major role in respect of number of 

secondary branches and days to maturity 

The estimates of gca effects for 10 parents are given in 

Table 2. For the developmental traits like days to flower and 

days to maturity, the negative and significant values of gca 

were considered desirable, keeping in view the fact that early 

flowering and early maturity are more sought after traits. On 

the other hand, component traits namely, number of primary 

branches, number of siliquae per plant, number of seeds per 

siliqua, 1000-seed weight and seed yield per plant including 

biological yield per plant and harvest index-developmental 

traits, the highest positive and significant values of gca were 

considered appropriate for selecting good general combiners. 

The estimates of sca effect for 45 crosses are presented 

in Table 3. The norms for the selection of F 1 

hybrids with 

desirable and significant sca effects were the same as listed 

under gca effects. The combining ability estimates obtained 

under certain assumptions (Griffing,1956, Kempthome and 

Curnow, 1961) can be translated into the variance due to 

additive and dominance gene effects. Such information 

provides nature and magnitude of gene action involved in the 

expression of a particular character. It helps in identification 

of promising parents with gca and selection of superior cross 

combinations having high sca effects. The combining ability


Table 1. 

Source of 

variation 

ANOVA for combining ability for 10 characters in 10 parent diallel cross of Indian mustard [Brassica juncea (L.) 

Czern and Coss] 

d. f. Days of 

flower 

Number of 

primary 

branches 

*Significant at 5% level of significance 

Number of 

secondary 

branches 

Days to 

maturity 

Number of 

siliquae per 

plant 

**Significant at 1% level of significance 

Number of 

seeds per 

siliqua 

Biological 

yield per 

plant 

Harvest 

index 

1000 seed 

weight 

Seed yield 

per 

plant 

GCA 9 25.25** 3.13** 56.21** 71.65** 83412.36** 5.40** 1986.99** 26.33** 3.47080** 193.99** 

SCA 44 5.79** 11.85** 27.93** 5.69** 36246.29** 3.88** 589.91** 45.32** 051720** 124.85** 

Error 108 0.04 0.02 0.06 0.03 8.77 0.03 0.18 0.67 0.00002 0.08 

Table 2. 

Parent 

Estimate of gca effects of 10 parent for 10 characters in 10 x 10 diallel cross of Indian mustard [Brassica juncea L.) 

Czern and Coss] 

Days to 

flower 

Number of 

primary 

branches 


Number of 

secondary 

branches 

Days to 

maturity 

Number of 

siliquae per 

plant 


Number of 

seeds per 

siliqua 

Biological 

yield per 

plant 

Harvest 

index 

1000- seed 

weight 

Seed yield per 

plant 

RK9902 1.23** -0.74** -3.63** 1.60** -107.32** 1.25** -6.57** 0.60** 0.17** -1.46** 

RK9901 1.56** -0.31** 0.02 3.65** 19.13** -0.79** 2.31** 0.73** 0.21** 1.19** 

B85 -2.38** -0.59** -1.71** -3.71** -38.05** -1.05** -25.43** -2.43** -0.93** -8.64** 

Urvashi 0.34** -0.34** -1.13** 0.96** -41.55** 0.80** 2.97** -2.65** -0.09** -1.93** 

Krishna 0.97** 0.20** 1.07** 0.77** 40.79** -0.26** 4.61** 0.38** 0.13** 1.98** 

Pusa Bahar 0.52** 0.39** 1.49** 0.22** 47.90** -0.09 20.03** -0.29** 0.23** 4.36** 

Pusa Jaikisan 0.25** 0.02 -0.54** 1.00** -105.94** 0.06 -11.85** 0.64** 0.66** -3.37** 

Pusa Basant 1.14** -0.04 -1.40** 1.60** -43.96** -0.19** 4.42** 0.50** 0.74** 2.91** 

Vard an -1.75** 0.63** 3.97** -3.51** 160.14** -0.03 13.65** 0.29** -0.59** 4.54** 

Sanjokta -1.89** 0.80** 1.85** -2.58** 68.86** 0.27** -4.14** 2.23** -0.51** 0,42** 

SE ( gî) 0.05 0.04 0.07 0.05 0.81 0.05 0.12 0.07 0.0013 0.08 

SE(gî-g?) 0.08 0.05 0.10 0.07 1.21 0.07 0.17 0.11 0.0019 0.12 

analysis (Table 4) exhibited significance of gca and sca for all 

the traits under study. The estimates of variances due to sca 

( ä 2 s) were greater than gca (ä 2 g ) variances for all the traits 

except number of secondary branches and days to maturity 

emphasizing thereby the greater role of non-additive genetic 

effects for seed yield and its related attributes. 

The general combining ability is primarily a function of 

additive gene action and additive x additive interaction. This 

information on seed yield and yield component characters is 

important and would greatly help in the proper classification 

of parental lines. 

Considering significant and desirable gca effects in F 1 

generation, parent RK 9902 was good general combiner for 

number of seeds per siliqua, harvest index and 1000-seed 

weight. While parent RK 9901 was judged to be good for 

number of siliqua per plant, biological yield per plant, harvest 

index, 1000-seed weight and seed yield per plant Parent. B 85 

was observed for days to flower and days to maturity, parent 

Urvashi emerged as good general combiner for number of 

seeds per siliqua and biological yield per plant, whereas parent 

Krishna was found so for number of primary branches, 

number of secondary branches number of siliquae per plant 

biological yield per plant, harvest index, 1000 seed weight and 

seed yield per plant. Parent Pusa Bahar showed significant 

and desirable gca effects for number of primary branches, 

number of secondary branches, number of siliquae per plant, 

biological yield per plant ; 

1000-seed weight and seed yield per 

plant. Pusa Jaikisan was identified as good general combiner 

for harvest index and 1000-seed weight. Parent Pusa Basant 

exhibited good general combining ability for biological yield 

per plant, harvest index ,1000- seed weight and seed yield per 

plant. Vardan was identified as good general combiner for 

days to flower, number of primary branches, number of 

secondary branches, days to maturity, number of siliquae per 

plant, biological yield per plant, harvest index and seed yield 

per plant. Parent Sanjokta revealed high general combining 

ability effects for early flowering, number of primary branches, 

number of secondary branches, days to maturity, number of 

siliquae per plant, number of seeds per siliqua, harvest index 

and seed yield per plant. 

Based on per se performance and general combining 

ability best parents for general combining ability were identified 

(Table 5a). Similarly best crosses were identified on the basis 

of per se performance and specific combining ability (Table 

5b). 

For seed yield per plant, 10 best F 1 

s were selected in 

descending order on the basis of their relative sca effects 

(Table 5c). The genotypes identified as good general 

combiners on the basis of gca may serve as worth while 

parents for the improvement of various characters through 

hybridization programme. The ranking of genotypes on the 

basis of seed yield per plant showed an association with the 

ranking of gca effects. Thus in the present investigation, the 

high yielder parents are having high general combining ability

Table 3. 

F1 

LAL et al., Combining ability analysis for yield and its components in Indian Mustard 227 

Estimate of sca effects for 10 characters in 10 x 10 diellel cross of Indian mustard [Brassica juncea (L.) Czern and 

Coss] 

Days to 

flower 

Number of 

primary 

branches 

Number of 

secondary 

branches 

Days to 

maturity 

Number of 

siliquae per 

plant 

Number of 

seeds per 

siliqua 

Biological 

yield per 

plant 

Harvest 

index 

*Significant at 5% level of significance **Significant at 1% level of significance 

Table 4. Estimate of components of variances ratio (ä 2 g/ä 2 s) and average degree of dominance (ä 2 s/ä 2 g) 0.5 

1000- 

seed 

weight 

Seed yield 

per plant 

RK 9902 xRK 9901 1.54** 0.67** 2.06** -0.53** 148.42** -0.30* 22.81** 7.40** 0.89** 15.75** 

RK 9902x385 -0.53** 0.95** 2.27** -2.30** 128.20** 1,81** 33,88** 3.55** 0.38** 11.98** 

RK 9902 x Urvashi -3.85** 0.50** 3.49** 1.10** 244.83** -0.04 32,75** 11.12** 0.62** 23.13** 

RK 9902 x Krishna 0.46* 0.16 -1.32** 1.21** -98.38** -0.08 2.13** -1.79** 0.83** -1.71** 

RK 9902 x Pusa Bahar -1.49** -1.03** -7.67** -5.24** -166.89** -1.15** -40.85** -9.48** -1.01** -17.89** 

RK9902 x PusaJaikisan 3.24** 0.34** -0.97** 3.86** -88.78** -1.50** -21.43** -6.98** -0.25** -10.70** 

RK 9902 x Pusa Basant 2.22** 1.20** 5.49** 1.19** 99.30** 0.01 -27.76** -4.07** -050** 1.82** 

RK 9902 x Vardan 1.24** -1.27** -4.15** -1.50** -229.40** 2.46** 2.27** -11.65** -0.56** -12.27** 

RK 9902 x Sanjokta 5.18** 0.22* 4,44** 3.57** -23.44** 0.29 2.12** 4.39** 0.66** 5.11** 

RK9901 xB85 0.15 20.5** 9.89** 2.58** 37.01** 0.80** 14.87** -0.59* -0.11** 2.13** 

RK 9901 x Urvashi 1.43** -2.53** -2.90** 0.98** -66.09** -0,45** -3.13** 1.61** 0.51** 0.61* 

RK 9901 x Krishna -2.19** -0.28* -0.64** -3.84** -87.56** 2.88** -4.57** -4.80** -1.33** -6.63** 

RK 9901 x Pusa Bahar 1.58** 0,33** -2.79** 0.71** 12.46** 0.64** -0.93* 2.36** -0.11** 3.66** 

RK9901 x Pusa Jaikisan 1.86** 0.84** -20.2** 0.74** -99.83** 1.49** 10.69** -4.31** 0.21** -0.88** 

RK9901 x Pusa Basant 0.83** 1.03** 1.64** -0.06 -43.48** 0.74** -6.58** 1.58** 0.26** -0.88** 

RK 9901 x Vardan 2.72** 0.30* 0.94** 2.51** 93.82** -4.28** -15.01** -3,26** -0.14** -8.12** 

RK 9901 x Sanjokta 1.06** -0.81** -4.54** 4.32** -231.16** 1.62** -6.22** -3.24** 0.76** -4.60** 

BS5 x Urvashi 2.63** 2.35** 6.38** -0.66** -54.71** -0.88 2.14** 2.44** 0.67** 2.10** 

B85 x Krishna 2.07** -1.00** 1.57** -0.67** 316.55** -3.75** -6.63** 1.99** -0.45** -3.87** 

B85 x Pusa Bahar 0.38* -1.19** -5.65** 0.08 -95.56** 2.15** -22.79** 4.88** 0.22** -1.91** 

B85 x Pusa Jaikisan 0.46* 0.12 0.32** -0.83** 209.35** -1.20** 24.30** 4.89** 0.16** 10.35** 

B85 x Pusa Basant -0.24 1.18 2.11** -2.44** 75.50** -1.09** 10.22** 0.56* 008** 1.00** 

B85 x Vardan 2.66** -0.96** -4.59** 1.74** 2.00 -0.24 2.60** -2.88** 0.05** -3.56** 

B85 x Sanjokta 0.93** 0.33** 0.93** 0.88** -54.71** 1.92** -6.75** -.14** -0.37** -3.57** 

Urvashi x Krishna -0.78** 0.56** -7.01** -1.21** -263.69** -2.20** -25.77** -4.63** 1.69** -11.39** 

Urvashi x Pusa Bahar -0.34 0.30* -1.36** -.39* -32.26** 2.90** -14.46** -2.56** -1.34** -6.30** 

Urvashi x Pusa Jaikisan -2.00** 0.74** 0.60** -0.56** -78.69** 0.15 0.09 -4.88** 0.42** -3.17** 

Urvashi x Pusa Basant 3.04** 1.00** -2.01** 1.23** 76.13** -1.67** 9.42** -2.82** -0.32** -2.25** 

Urvashi x Vardan 1.07** -1.67** -3.51** -2.06** -147.64** -0.89** -15.08** 3.05** 1.05** -1.88** 

Urvashi x Sanjokta 0.34 0.16 -1.52** 3.79** -129.21** 1.87** 16.98** -5.12** 0.14** -1.22** 

Krishna x Pusa Bahar 1.24** -0.98** 2.36** 2.79** 12.12** 1.56** 3.23** 8.39** 0.67** 12.93** 

Krishna x Pusa Jaikisan 238** 0.20 3.46** 0,82** -9,36** 0.21 19.65** -1.95** 0.37** 3.59** 

Krishna x Pusa Basant 0.42* 1.74** -7.08** -0.92** -202.61** -1,88** -24.69** -8,88** -0.01* -16.56** 

Krishna x Vardan 1.38** 0.58** -0.91** -2.67** -278.38** 2.10** -37.98** -2.53** -0.61** -14.25** 

Krishna x Sanjokta -0.48** 2.41** 6.61** -6,60** 458.84** -3.27** 36-34** 1.73** -0.17** 13,87** 

Pusa Bahar x Pusa Jaikisan -0.11 1.21** 2.44** 0.37* -79.07** 1.24** 3.36** -1.70** 0.49** 0.48 

Pusa Bahar x Pusa Basant -3.07** 1.27** 5.24** -0.50** 105.95** 2.29** -3.51** 19.34** 0.85** 21.86** 

Pusa Bahar x Vardan -0.44* 0.40** 2.07** -2.19** -75.29** 0.80** -22.14** 0.14 -0.56** -5.76** 

Pusa Bahar x Sanjokta 3,69** 2.42** 1.72** -1.25** -97.26** -0.97** -9.95** -1.15** 0.54** -2.57** 

Pusa Jaikisan x Pusa Basant 0,33 -1.16** -3.43** 1.19** 111.32** 1.94** 20.04** 12.60** 0.75** 18.46** 

Pusa Jaikisan x Vardan -1.97** -0.23 -4.63** -3.23** 66.09** -2.02** -2.92** -2.25** -0.67** -3.24** 

Pusa Jaikisan x Sanjokta 0.03 0.80** 12,15** -3.02** 219.84** -0.52** 19.07** -1.68** -1.13** 4.42** 

Pusa Basant x Vardan -0.07 2.76** 12,16** -1.50** 191.44** 2.03** 22.94** 11.03** 0.14** 20.88** 

Pusa Basant x Sanjokta -0.73** 0.06 2.18** -2.70** 78.59** 0.73** 28.06** -2.72** -053** 4.54** 

Vardan x Sanjokta 0.03 -1.95** -2.02** 1.68** 445.76** -2.49** 31.50** 8.31** 0.13** 13.84** 

SE (î?) 0.18 0.12 0.22 0.16 2.73 0.15 0.39 0.25 0.0043 0.27 

SE (Sî? -Sîk) 0.26 0.18 0.33 0.24 4.01 0.23 0.57 0.36 0.0063 0.39 

Source of 

variation 

d.f. 

Days of 

flower 


Number of 

primary 

branches 

Number of 

secondary 

branches 

Days to 

maturity 

Number of 

siliquae per plant 


Number of 

seeds per 

siliqua 

Biological yield 

per plant 

Harvest 

index 

1000 seed 

weight 

Seed yield per 

plant 

δ 2 g (Parents) 9 2.10 0.03 44.15 5.97 6950.30 0.45 165.57 2.19 0.28923 16.16 

δ 2 s(F 1s) 44 5.75 1.83 27.87 5.66 36237.52 3.85 589.53 45.25 0.51718 124.77 

δ 2 e 108 0.04 0.02 0.06 0.03 8.77 0.03 0.18 0.07 0.00002 0.08 

δ 2 g/δ 2 s 0.37 0.02 1.58 1.05 0.19 0.12 0.28 0.05 0.55924 0.13 

(δ 2 s/δ 2 g) 0.5 1.65 7.81 0.79 0.97 22.8 2.92 1.89 4.55 1.33721 772


Table 5a. Ranking of desirable parents based on per se performance and gca effects for 10 characters in Indian mustard 

[Brassica juncea (L.) Czern and Coss] 

Character 

Best parents on the basis of desirable 

gca effects 

Days to flower B85, Sanjokta Vardan, Pusa Jaikisan and 

Urvashi 

Number of primary Sanjokta, Vardan, Pusa Bahar, Krishna and 

branches 

Pusa Jaikisan 

Number of secondary Vardan, Sanjokta Pusa Bahar, Krishna RK 

branches 

9901 

Days to maturity B85, Vardan, Sanjokta, Pusa Bahar and 

Krishna 

Number of siliquae Vardan, Sanjokta, Pusa Bahar, Krishna and 

per plant 

RK 9901 

Number of seed per RK 9902, Urvashi, Sanjokla, Pusa Jaikisan 

siliqua 

and Vardan 

Biological yield per Pusa Bahar, Vardan, Krishna, Pusa Bashant 

plant 

and Urvashi. 

Harvest index Sanjokta, RK9901, 

Pusa Jaikisan, RK 9902 and Pusa Basant 

1000- seed weight Pusa Basant, Pusa Jaikisan, Pusa Bahar 

RK 9901and RK 9902 

Seed yield per plant Vardan, Pusa Bahar, Pusa Basant, Krishna 

and RK 9901 

Best parents on the basis of per se 

performance 

B85, Sanjokta, Vardan, Pusa Jaikisan, and RK 

9902 

Vardan, Krishna, Pusa Bahar and Pusa 

Jaikisan 

Vardan, Pusa Bahar, Krishna, Urvashi and 

RK9901 

B85, Sanjokta, Vardan Pusa Bahar and Pusa 

Jaikisan 

Pusa Bahar, Vardan, Krishna, RK 9901 and 

Urvashi 

Urvashi, RK 9902, Krishna, Vardan and 

Sanjokta 

Pusa Bahar, Vardan, Krishna, Uravashi and 

RK9901. 

Krishna, RK 9902, Sanjokta, Pusa Jaikisan 

and RK 9901 

Pusa Jaikisan, Pusa Basant Pusa Bahar, RK 

9901 and RK 9902 

Vardan, Krishana, Pusa Bahar, RK 9901 and 

Urvashi 

Best common parents both on the basis of per se 

performance and gca effects 

B85, Sanjokta, Vardan and Pusa Jaikisan 

Vardan, Sanjokta, Krishna, Push Bahar and Push Jaikisan 

Vardan, Pusa Bahar, Krishna and RK 9901 

B85, Sanjokta, Vardan and Pusa Bahar 


RK 9902, Uarvashi, Sanjokta and Vardan 

Pusa Bahar, Vardan, Krishna and Uravshi. 

Sanjokta, RK 9901, Pusa Jaikisan and RK9902 

Pusa Basant, Pusa Jaikisan, Pusa Bahar, RK 9901 and 

RK 9902 


Table 5b. Ranking of desirable crosses based on per se performacne and sca effects in F1 generation for 10 traits in Indian 

mustard [Brassica juncea (L.) Czern and Coss] 

Character 

Days to flower 

Number of primary branches 

Number of secondary 

branches 

Days to maturity 

Number of siliquae per plant 

Best cross combination on the basis 

of per se performance 

Vardan x Sanjokta 

Pusa Jaikisan x Vardan B85 x 

Sanjokta 

RK 9902 x Urvashi Pusa Bahar x 

Vardan B85 x Pusa Jaikisan 

RK 9902 x B 85 

Pusa Bahar x Sanjokta Krishna x 

Sanjokta 

Pusa Basant x Vardan Pusa Jaikisan 

x Sanjokta Pusa Bahar xPusaBasant 

Pusa Bahar PusaJaikisan B 85 x 

Urvashi 

Pusa Basant x Vardan Pusa Jaikisan 

x Sanjokta Krishna x Sanjokta 

RK 9901 x B 85 

Pusa Bahar x Vardan Pusa Bahar 

xPusaBasant 

PusaBaharxPusaJaikisan B 85 x 

Urvashi 

Pusa Jaikisan x Vardan Pusa Bahar 

x Vardan 

B 85 x Vardan 

B 85 x Sanjokta Krishna x Vardan 

Urvashi x Sanjokta 

Pusa Jaikisan x Sanjokta 

Vardan x Sanjokta Krishna x 

Sanjokta 

B 85 x Krishna 

Pusa Basant x Vardan RK 990 Ix 

Vardan 

Pusa Jaikisan x Sanjokta Pusa Bahar 

x Vardan 

Best cross combination on the basis of desirable 

sca effects 

RK 9902 x Urvashi 

Pusa Bahar x Pusha Basant RK9902 x Krishna 

Urvashi x Pusha Jaikisan 

Pusha Jaikisan x Vardan 

RK 9902 x Pusa Bahar 

Urvashi x Krishna 

Pusa Basant x Vardan Pusa Bahar x Sanjokta 

Krishna x Sanjokta 

B 85 x Urvashi 

RK 9901 x B 85 

Pusa Bahar x Pusa Basant 

Pusa Basant x Pusa Jaikisan 

Pusa Basant x Vardan 

Pusa Bahar x Sanjokta 



RK 9901 x B 85 



RK 9902 x Pusa Basant 


RK 9902 x Pusa Bahar 

RK9901 x Krishna 


Pusa Jaikisan x Vardan 


Pusa Basant x Sanjokta 

Krishna x Vardan 



B85 x Krishna 



B 85 x Pusa Jaikisan 


Best common crosses both on the basis of per se 

performance and sea effects 

RK 9902 x Urvashi Pusa Jaikisan x Vardan 

Pusa Bahar x Sanjokta 





Pusa Bahar x Pusa Jaikisan 

Pusa Basant x Vardan Pusa Jaikisan x Sanjokta 

RK 9901 x B 85 




Pusa Jaikisan x Vardan 





B 85 x Krishna 



effects and hence they can be utilized in the breeding 

programmes. For effective utilization of additive genetic 

variance, population breeding techniques with some 

modifications could be effective in improving self pollinated 

species. Under such situation careful selection could be of 

weighted importance.

LAL et al., Combining ability analysis for yield and its components in Indian Mustard 229 

Table 5b. Contd.... 

Character 

Number of seeds per 

siliqua 

Biological yield per 

plant 

Harvest index 

1000-seed weight 

Seed yield per plant 

Best cross combination on the 

basis of per se performance 

RK9901xVardan 

Urvashi x Pusa Bahar Urvashi x 

Sanjokta 

RK 9902 x B 85 


Pusa Bahar xPusaBasant 

RK9901xKrishna 

Pusa Basant x Vardan Krishna x 

Sanjokta 


Pusa Basant x Sanjokta Krishna x 

Pusa Bahar RK 9902 x Pusa 

Basant RK 9901 x Pusa Bahar 

Pusa Bahar x PusaBasant 

PusaJaikisanxPusaBasant Pusa 

Basant x Vardan Vardan x 

Sanjokta 

RK 9902 x Urvashi RK9902 

xRK9901 Krishan x Pusa Bahar 

PusaJaikisanxPusaBasant 

Pusa Basant x PusaBahar 

Urvashi x Krishan 

Pusa BaharxPusaJaikisan 

RK9902 xRK9901 

RK9901xPusaBasant 

Krishna x Pusa Jaikisan 

Pusa Bahar x PusaBasant 

Pusa Basant x Vardan RK 9902 x 

Urvashi Krishna x Pusa Bahar 

Vardan x Sanjokta Pusa Jaikisan x 

Pusa Basant Krishna x Sanjokta 

Best cross combination on the basis of 

desirable sea effects 

Urvashi x Pusa Bahar 

RK 9901 x Krishna 

RK 9902 x Vardan 


B 85 x Pusa Bahar 




RK 9902 x B 85 


Pusa Basant x Sanjokta 

B 85 x Pusa Jaikisan 


RK 9902 xRK 9901 


Pusa Jaikisan x Pusa Basant RK 9902 x 

Urvashi 


Krishna x Pusa Bahar 


RK9902 xRK 9901 


Urvashi x Vardan 

RK9902 xRK9901 

Pusa Basant x Pusa Bahar 


RK9901 x Sanjokta 

Pusa Jaikisan x Pusa Basant 



Pusa Basant x Vardan Pusa Jaikisan x Pusa 

Basant 

RK9902 xRK9901 


Vardan x Sanokta 

Best common crosses both on the basis of 

per se performance and sea effects 

Urvashi x Pusa Bahar 


RK 9902 x Vardan 






Pusa Jaikisan x Pusa Basant RK 9902 x 

Urvashi 


Krishna x Pusa Bahar 


RK9902 xRK9901 


RK9902 xRK9901 


Pusa Jaikisan x PusaBasant 








Table 5c. Promising specific combiners for yield per plant in F1 generation and their per se performance gca effects of the 

parents involved and desirable sca effects for other characters in Indian mustard [Brassica juncea (L.) Czern and 

Coss.] 

Cross 

Per se 

performance 

sca 

effects 

gca effects 

Desirable sca effects in other characters 

RK9902 xUrvashi 49.33 231.13** -146** -153** Xi (-3.85**), X 2 (0.52**), X 3 (3.49**), X 5 (244.83**) X 7(32.75**), X g (1 1.12**) and X 9 

(0.62**) 

Pusa Bahar x Pusa Basant 58.73 21.86** 4.36** 2.91** Xi (-3.07**), X 2 (1.27**), X 3 (5.24**), X* (-0.50**) X 5(105.95**), Xe (2.29**), X 8 

(0.62**) and X 9 (0.85**) 

Pusa Bahar x Pusa Vardan 57.93 20.88** 2.91** 4.54** X] (-0.07), X 2 (2.76**), X 3 (12.16**), X 4 (-1.50**) X 5(191.44**),X 6 (2.03**) 

X 7(22.94**) ?X g(l 1.83**) and X 9 (0.14**) 

Pusa Jaikisan x Pusa Basant 47.60 18.46** -3.37** 2.91** X 5 (1 11.32**), X 6 (1.94**), X 7 (20.04**), Xg (12.60**) and X 9 (0.75**) 

RK 9902 xRK 9901 45.07 15.75** -1.46** 1.19** X 2 (0.67**), X 3 (2.06**), X, (-0.53**), X 5 (148.42**) X 7(22.81**), X 8 (7.40**) and X 9 

(0.89**) 

Krishna x Sanjokta 45.87 13.87** 1.98** 0.42** Xj (0.48**), X 2 (2.41**), X 3 (6.61**), X4 (-1.60**) X 5(458.84**),X 7 (36.34**) andXg 

(1.73**) 

Vardan x Sanjokta 48.40 13.84** 4.54** 0.42** X 5 (445.7^X7(1 1.50**), X» (8.31**) and X 9 (0.13**) 

Krishna x Pusa Bahari 48.87 12.93** 1.98** 4.36** X 3 (2.36**), X 5 (12.13**), X 6 (1.56**) and X 7 (19.65**) 

RK 9902xB85 31.47 12.93** -1.46** -8.64** X! (0.53**), X 2 (0.95**), X 3 (2.27**), X4 (2.30**) X 5(128.20**), Xe (1.81**) X 7 

(33.8**), X 8 (3.55**) and X 9 (0.38**) 

B85 x Pusa Jaikisan 27.93 11.98 -8.64** .337** X 2 (0.12), X 3 (3.32**), X A (-0.83**), X 5 (209.35**) X 7(24.20**), Xg (4.89**) and X 9 

(0.16**) 

*Significant at 5% level of significance **Significant at 1% level of significance


The crosses showing positive sca effects for seed yield 

were also having positive sca effects for almost all the yield 

components and developmental traits studied. The parents 

identified as good or poor general combiners for seed yield 

were also same for some of the component characters. This 

suggested that combining ability for seed yield to some extent 

was related to combining ability of yield contributing 

characters. The traits influencing combining ability effects 

for seed yield may be given due consideration in selection of 

parents for hybridization programme. 

ACKNOWLDGEMENT 

The first author gratefully acknowledges Uttar Pradesh 

Krishi Utpadan Mandi Parisad, Lucknow for providing 

financial assistance in the form of scholarship during the 

period of study. 


Asthana, A.N. and Pandey, V.K. 1977. Combining ability and correlation 

in a diallel cross of Indian mustard. Experimental Agriculture, 18:71- 

79. 

Griffing, B. 1956 a. Concept of general and specific combining ability 

in relation to diallel crossing system. Australian Journal of Biological 

Sciences, 9: 463-493. 

Kempthorne, O. and Curnow, R.N. 1961. The partial diallel corss. 

Biometrics, 17: 229-250. 



A Key to the Indian Species of Schizoprymnus Foerster (Hymenoptera: Braconidae: 

Brachistinae) with Description of A New Species 

MOHAMMAD SHAMIM 

Section of Entomology, Department of Zoology, Aligarh Muslim University, Aligarh 202 002 

e-mail: shamimento@gmail.com 

ABSTRACT 

A new species of braconid genus Schizoprymnus Foerster viz., 

Schizoprymnus sharifi Shamim sp. nov. is described and 

illustrated from India. The new species unambiguously similar 

with Schizoprymnus tortilis Papp in having antennal segments 

20, head in dorsal view 2× as wide as long, face 2.2× as wide as 

long, apical rim of carapace semi circularly excised with a pair 

of denticles, length of hind femur 3.7× its width and length of 

pterostigma 2.7 × its width. However, it differs in having: length 

of eye in dorsal view 1.87 × temples; length of malar space 2× 

basal width of mandible; hind tibia one third longer than hind 

tarsus. 

Key words Hymenop tera , Bracon idae, Brachistin ae, 

Schizoprymnus, new species, India. 

Brachistinae is a large subfamily of moderately small to 

large parasitoid wasps with 413 species in eleven genera 

worldwide (Yu, et al., 2009). It includes the genera Calyptites 

Scudder 1878; Calyptoides Cockerell, 1921; Chelostes van 

Achterberg, 1990; Cuniculobracon van Achterberg and Falco, 

2001; Dicyrtaspis van Achterberg, 1980; Eubazus Nees, 1812; 

Foersteria Szepligeti, 1896; Nealiolus Mason, 1974; 

Polydegmon Foerster, 1862; Schizoprymnus Foerster, 1862 and 

Triaspis Haliday, 1835. Members of the subfamily live as 

parasitoids on the families Curculionidae and Apionidae 

(Coleoptera), which also include very important agricultural 

pests (Belokobylskij, et al., 2004). 

The genus Schizoprymnus Foerster recognized by the 

presence of anterior three metasomal tergites immovably fused 

to form a metasomal carapace and absence of two transeverse 

sutures on the carapace. However, some species of 

Schizoprymnus have the carapace with first suture almost 

entirely and the second one at least laterally developed (Papp, 

1984, 1991, 1993; Belokobylskij, 1994, 1998). The members of 

this genus are endoparasitoids of larval Coleoptera (Shaw 

and Huddleston, 1991). 

The genus is cosmopolitan in distribution containing 

122 species worldwide (Yu, et al., 2009) and 35 species from 

Indo-Australian region, of which only two species i.e. 

Schizoprymnus indicus Ahmad and Schizoprymnus transiens 

Ahmad are reported from India. In the present work, a new 

species i.e. Schizoprymnus sharifi Shamim sp. nov. is 

described and illustrated with 10 photographs. A key to the 

Indian species also proposed. 


The specimens were collected by using a sweeping net. 

Photographs were taken with the help of a digital camera 

attached to a Stereozoom binocular (Nikon-SMZ1500). 

Measurements of slide-mounted parts (antenna, hind leg, 

forewing and hind wing) and card mounted specimens were 

taken with the help of an ocular micrometer (linear side of 100 

divisions) placed in the eye piece of the Stereozoom 

microscope. The divisions of the ocular micrometer were 

converted to millimeters. 

The terminology and venation follows van Achterberg, 

1993 and surface sculpture follows Eady, 1968. Abbreviations 

used in the text are: POL: Posterior ocellar line (distance 

between the posterior ocelli); OOL: Ocello-ocular line (distance 

between posterior ocellus and eye); OD: Ocellar diameter; F: 

Flagellomere. 


Body length: 5.2mm, Forewing, 4.7mm, Antenna: 3.6mm 

Head: Width of head in dorsal view 2.48× its length, 1.37× its 

height; antennal segments 20; F 1 

shorter than F 2 

(11:14); 

lengths of F 1 

, F 2 

, F 3 

, F 4 

__ 

F 5 

, F 6 

, F 7 

__ 

F 10 

, F 11 

__ 

F 17 

and F 18 

2.2×, 

2.8×, 2.4×, 2×, 1.33×, 1.2×, 1× and 2× their widths respectively; 

F 1 

without sensillum, F 2 

__ 

F 4 

with two sensillae; F 5 

__ 

F 7 

with 

three sensillae; length of posterior side of stemmaticum 1.8× 

its lateral side; occipital carina complete; OOL: POL: OD= 16: 

11: 4; length of eye in dorsal view 1.66× its width and 1.87× 

temple; temple smooth, sparsely setose, temple rounded 

behind eyes; vertex 2.75× its width, smooth, sparsely setose; 

frons 1.6× as wide as long, punctate, sparsely setose; antennal 

sockets depressed, mid longitudinal carina between antennal 

sockets, near antennal sockets densely punctate, setose; face 

2.2× as wide as long, impressed medially, densely punctate, 

setose; intertentorial line 1.66× tentorio-ocular line, tentorial 

pit deep and its area depressed; clypeus 2.2× its width, slightly 

convex, punctate; length of malar space 2× basal width of 

mandible. 

Mesosoma: Length of mesosoma 1.7× its width and its height; 

pronotal side dorsally smooth, anteriorly, remaining reticulate, 

sparsely setose; mesopleuron medially smooth, dorsally 

reticulate, setose; precoxal sulcus wide, large, reticulate, 

setose; notauli anteriorly narrow, crenulate, posteriorly broad, 

reticulate with small indistinctly mid longitudinal carina; middle


lobe of mesoscutum punctate, sparsely setose, lateral lobes 

smooth, sparsely setose; scutellar sulcus, deep, smooth with 

one longitudinal strong carina and four weak lateral carinae; 

scutellum smooth, sparsely setose, strongly convex; side of 

scutellum reticulate; medio-posterior depression deep with 

one longitudinal carina and two weak lateral carinae; 

metanotun crenulate; propodeum basally reticulate rugose, 

apically rugose, setose. 

Wings: Forewing 2.5× as long as wide, 0.9× as long as body 

length, shorter than body; marginal cell large; pterostigma 

2.8× its width; length of vein 1 __ R1 0.77× length of pterostigma; 

r issuing proximally from its middle of pterostigma, its length 

0.42× width of pterostigma; vein SR1+3 __ SR curved; m __ cu 

antefurcal; cu __ a postfurcal; r: 2 __ SR: SR1+3 __ SR= 6: 15: 42; 

1 __ CU1: 2 __ CU1: 3 __ CU1= 6: 30: 10; hindwing 4× as long as 

wide; 1 __ M: 1 __ r-m: 2 __ SC+R = 9: 10: 10. 

Legs: Hind coxa sparsely punctate, setose; lengths of hind 

femur, tibia and basitarsus 3.75×, 11.33× and 7.33× their widths 

respectively; hind tibia one third longer than hind tarsus; 

hind tibial spurs 0.36× and 0.27× as long as hind basitarsus. 

Metasoma: Length of metasoma 1.42× its width and 2.5× its 

height; reticulate, densely setose; suture between first and 

second tergites distinct only laterally; apical rim of carapace 

semi circularly excised with a pair of denticles; carapace longer 

than mesosoma; ovipositor sheaths slender, setose, shorter 

than carapace, its length 0.42× forewing, 0.87× hind basitarsus; 

hypopygium setose; ovipositor depressed apically, pointed. 

Colour: Black except mandibles basally, maxillary palp, labial 

palp yellowish brown; mandibles apically, antenna, ovipositor 

sheaths brown; pterostigma, wing veins, femur, tibia and 

tarsus brownish yellow; forewing veins 1 __ M and M+CU1 

pale yellow; ovipositor yellow; ocelli transparent yellow; eyes 

grayish. 

Variation: Antennal segments 21; face strongly convex, 2.2× 

as wide as long, mesopleuron entirely reticulate; forewing 

vein SR __ 1+ 3 __ SR strongly curved; length of body: 5.5mm, 

forewing, 5mm, antenna: 4mm; ocelli transparent; ovipositor 

sheaths 0.46× forewing, 0.9× hind basitarsus and lengths of 

hind femur, tibia and basitarsus 3.8×, 11.5× and 8× their widths 

respectively. 

Host: Unknown 

Type material: Holotype, female, INDIA: Uttar Pradesh, 

Etawah, 26° 47' N 79° 02' E, 14 15. vii. 2008, Coll. M. Sharif. 

(ZDAMU). Deposited in Insect collection, Department of 

Zoology, A.M.U. Aligarh (ZDAMU). 

Paratypes, 2 females, same as holotypes. INDIA: Uttar 

Pradesh, Aligarh, 20. i. 2012, 27° 30' N 79° 40' E Coll. Mohammad 

Shamim. (ZDAMU). 2 females, same as holotypes. INDIA: 

Uttar Pradesh, Aligarh, 08. iv. 2012, 27° 30' N 79° 40' E Coll. 

Mohammad Azam. (ZDAMU). Deposited in Insect collection, 

Department of Zoology, A.M.U. Aligarh (ZDAMU). 

Etymology: The species is named in memory of my late father, 

Mohammad Sharif, who collected these braconid parasitoids. 

Remarks: The new species Schizoprymnus sharifi Shamim 

sp. nov. closely resembles with Schizoprymnus indicus 

Ahmad 2010. However, it differs in having (1) Antennal 

segments 20 (antennal segments 23 in indicus) (2) vein r of 

forewing issuing proximally from its middle; (vein r of forewing 

issuing slightly distally from its middle in indicus) (3) hind 

tibial spurs 0.36 and 0.27× as long as hind basitarsus (hind 

tibial spurs about 0.3× as long as basitarsus in indicus) (4) 

carapace 1.4× as long as wide, reticulate, densely setose 

(carapace 1.8× as long as wide, reticulate rugose in indicus) 

(5) middle lobe of mesoscutum punctate, sparsely setose 

(mesoscutum smooth in indicus) (6) propodeum basally 

reticulate rugose, apically rugose (propodeum reticulaterugose 

in indicus). 

The new species Schizoprymnus sharifi Shamim sp. nov. 

also closely resembles with Schizoprymnus tortilis Papp, 1984. 

However, it differs in having (1) length of malar space 2× basal 

width of mandible, length of malar space either as long as or 

somewhat longer than basal width of mandible in S. tortilis 

(2) length of eye in dorsal view 1.87× temple (eyes longer than 

temple in S. tortilis (3) hind tibia one third longer than hind 

tarsus (hind tibia one sixth longer than hind tarsus in S. tortilis 

(4) ovipositor sheaths shorter than carapace (ovipositor 

sheath either as long as or somewhat longer than carapace in 

S. tortilis. 

Key to Indian species of Schizoprymnus Foerster based 

on Females 

1. Face finely punctuate; eye in dorsal view about as long 

as temple; malar space 1.2× basal width of mandible; 

forewing 0.8× as long as body length; pterostigma 2.3× 

as long as wide; length of vein 1 __ R1 as long as 

pterostigma————————————————— 

—————————————————————— 

— —— —— —- — —— —— — —— —— —— S . 

transiens Ahmad 

— Face punctate; eye in dorsal view 1.87 __ 2× as long as 

temple; malar space 2 __ 2.5× basal width of mandible; 

forewing 0.9 __ 1.15× as long as body length; pterostigma 

2.6 __ 2.8× as long as wide; length of vein 1-R1 0.3 __ 0.77× 

as long as pterostigma————————————— 

—————————————————————— 

————————————————————-2 

2. Antennal segments 23; vein r of forewing issuing slightly 

distally from its middle; hind tibial spurs about 0.3× as 

long as basitarsus; carapace 1.8× as long as wide, 

reticulate rugose; mesoscutum smooth; propodeum 

reticulate rugose——————————————— 

—————————————————————— 

———————————————-S. indicus Ahmad

MOHAMMAD SHAMIM, A Key to the Indian Species of Schizoprymnus Foerster 233 

Figs. 1-7.S. sharifi Shamim sp. nov. Head in dorsal view 2. Head in ventral view 3. Carapace 4. Ovipositor and sheaths 5. Hind leg 6. 

Fore wing 7. Antenna 

— Antennal segments 20; vein r of forewing issuing 

proximally from its middle; hind tibial spurs about 0.36× 

and 0.27× as long as basitarsus; carapace 1.4× as long 

as wide, reticulate, densely setose; middle lobe of 

mesoscutum punctate; sparsely setose; propodeum 

basally reticulate rugose, apically rugose————— 

————-S. sharifi sp. nov. 

Schizoprymnus sharifi sp. nov. (Figures 1 __ 7) 


I thank Dr. M. Hayat for his review and suggestions on 

this manuscript. I also thank the Chairman, Department of 

Zoology, for the laboratory facilities. This research was 

supported by the Department of Science & Technology, New 

Delhi (Grant no. SR/FT/LS-065/2008). 


Ahmad, Z. and Ahmed, Z. 2010. Record of the genus Schizoprymnus 

Foerster (Hymenoptera: Braconidae) from India, with descriptions 

of two new species. J. Bombay Nat. Hist. Soc.107 (1): 45 __ 47. 

Belokobylskij, S. A., 1994. To the knowledge of the braconid fauna of 

the Russian Far East (Hymenoptera, Braconidae): new species of 

the subfamily Brachistinae. Russ. Entomol. Journal. 3: 81–108. 

Belokobylskij, S. A. 1998. Subfam. Brachistinae (Calyptinae). In: Lehr 

PA (ed.) Opredelitel’ nasekomykh Dal’nego Vostoka Rossii. 

Setchatokryloobraznye, skorpionnitzy, ereponchatokrylye [Keys to 

Insects of the Russian Far East. Neuropteroidea, Mecoptera, 

Hymenoptera], Vol. 4, pp. 440-489. 

Belokobylskij, S. A., Güçlü, C. and Özbek H., 2004, A new species of 

the genus Schizoprymnus Foerster (Hymenoptera, Braconidae, 

Brachistinae) from Turkey. Zoosyst. Rossica, 12: 245-248. 

Papp, J. 1984. First survey of the Triaspidini species of the Indo- 

Australian Region (Hymenoptera: Braconidae, Calyptinae). I. The 

genus Triaspis Haliday. Acta Zool. Hung. 30: 137–158. 

Papp, J. 1991. First survey of the Triaspidini species ofthe Indo- 

Australian Region (Hymenoptera, Braconidae, Calyptinae). II. The 

genus Schizoprymnus Foerster, 1. Acta Zool. Hung. 37: 75–99. 

Papp J. 1993 First survey of the Triaspidini species of the Indo-Australian 

Region (Hymenoptera, Braconidae, Calyptinae). III. The genus 

Schizoprymnus Foerster, 2. Acta Zool. Hung., 39: 129–173. 

Van Achterberg, C 1980. Three new Palearctic genera of Braconidae 

(Hymenoptera). Entomol. Berichte. Amsterdam. 40: 72–80. 

Yu, D. S., van Achterberg, C., Horstmann, K. 2009. Biological and 

taxonomic information of world Ichneumonoidea, 2004. Electronic 

compact disk. Taxapad, Vanouver, Canada. http:www.taxapad.com. 



Effect of Organic and Inorganic Sources of Nitrogen on N, P, K and S Content of 

Rice Grain at Harvest and Straw at Different Stages of Rice (Oryza sativa) Crop 

Growth 

DEBIPRASAD DASH 1 AND HRUSIKESH PATRO 2 

1 

Krishi Vigyan Kendra(OUAT), Balasore, Orissa; 2 AICRP on Groundnut, OUAT, Bhubaneswar 751 003 

3 

email: pranati_hkp@hotmail.com 

ABSTRACT 

The soil of experimental site was sandy clay loam in texture 

with normal pH, low in nitrogen and phosphorus and medium 

in organic carbon and potassium contents. The experiment 

was laid out in Randomised Block Design with nine treatments 

replicated thrice. The treatments were applied to rice crop 

during kharif season. Application of N through chemical 

fertilizer (T 9 

) increased the N, P, K and S contents significantly 

at different stages including grain and straw at harvest by the 

crop over no fertilizer/manure (T 1 

). Among various organic N 

sources, N application through combination of D.S, P.M and 

C.W @ 40 kg N ha -1 each (T 8 

) resulted in higher N, P, K and S 

contents in plants over all other organic N sources at all the 

stages of straw and grain. Application of N through chemical 


) brought about significant improvement in grain 

and straw yields of rice crop and established superiority over 

rest of the treatments. Among organic N sources, supplication 

of N through combination of D.S + P.M + C.W @ 40 kg N ha - 

1 

each (T 8 

) increased the grain and straw yield significantly as 

against the application of rest of the organic N sources and the 

control (T 1 

) except the straw yield due to incorporation of P.M 

alone (T 3 

) which remained at par. 

Key words 

Organic sources, N, P, K, S content, grain yield, rice. 

Rice is a heavy nitrogen feeder, however, fertilizer N 

efficiency in rice is very low under tropical conditions where 

it rarely exceeds 50% and usually ranges between 15 to 35 per 

cent (De Dutta, 1984). Most of the N taken up by rice plant is 

supplied through soils own natural resources. In wetland rice 

cultivation, the significance of native soil N is apparent from 

the fact that 60-80% of N absorbed by the crop is derived from 

native pool (Broadbent, 1979). A major portion of N in wetland 

soils occur in organic pool, though this is usually very low. 

Conclusive evidences indicate that in production of irrigated 

rice, improvement in organic carbon content of soil and initial 

soil nitrogen content and efficiency of applied nutrient are 

more important. Several hypotheses were proposed: (i) high 

N uptake crops take up ammonium, amino acids or relatively 

high molecules of organic N preferentially; (ii) rice has stronger 

activity in competing with soil microorganisms than the other 

crops; (iii) rice secretes organic substances that support 

multiplication of microfauna, resulting in rapid decomposition 

of organic matter; and (iv) rice has superior Km (Michaelis 

constant), Vmax (maximum uptake velocity) and Cmin (minimum 

concentration of nutrient) for N uptake (Yamagata, et al., 1996). 

Keeping these facts in consideration, the present 

investigation entitled, “Effect of organic and inorganic sources 

of nitrogen on N, P, K and S content of rice grain at harvest 

and straw at different stages of rice (Oryza sativa) crop 

growth” was undertaken at the Agricultural Research Farm, 

Department of Soil Science and Agricultural Chemistry, 

Institute of Agricultural Sciences, Banaras Hindu University, 

Varanasi, with an objective to study the nutrient acquisition 

efficiency of rice under organics in comparison to synthetic N 

source. 


The present investigation was at the Agricultural 

Research Farm, Department of Soil Science and Agricultural 

Chemistry, Institute of Agricultural Sciences, Banaras Hindu 

University, Varanasi. 

The soil of experimental site was sandy clay loam in 

texture with normal pH, low in nitrogen and phosphorus and 

medium in organic carbon and potassium contents. The 

experiment was laid out in Randomised Block Design with 

nine treatments replicated thrice. The treatments were applied 

to rice crop during kharif season. 

The treatments are T 1 

- Control (without chemical 

fertilizers and organics), T 2 

-120 kg N through digested sludge 

(D.S, 6936.4 kg), T 3 

-120 kg N through press mud (P.M, 11428.6 

kg), T 4 

-120 kg N through woolen carpet wastes (C.W, 960 kg), 

T 5 

-60 kg N through D.S (3468.2 kg) + 60 kg N through P.M 

(5714.29 kg), T 6 

-60 kg N through D.S (3468.2 kg) + 60 kg N 

through C.W (480 kg), T 7 

-60 kg N through P.M (5714.29 kg) + 

60 kg N through C.W (480 kg), T 8 

-40 kg N through D.S (2312.1 

kg) + 40 kg N through P.M (3809.5 kg) + 40 kg N through C.W 

(320 kg), T 9 

- Recommended doses of fertilizers [120 : 60 : 60 :: 

N (209.8 kg Urea) : P (130.4 kg D.A.P) : K (100 kg M.O.P)]. 

Nitrogen was applied at the rate of 120 kg ha -1 through 

different organic sources along with two additional treatments 

which were recommended doses of NPK through chemical 

fertilizer (120 kg N : 60 kg P 2 

O 5 

: 60 kg K 2 

O) and without N 

(control). The organic sources of N were digested sludge, 

press mud and carpet wastes and inorganic N source was 

urea. Phosphorus and potassium were applied through 

diammonium phosphate and muriate of potash, respectively.

DASH, et al., Effect of Organic and Inorganic Sources of Nitrogen on N, P, K and S Content of Rice Grain at Harvest 235 

The half of recommended dose of nitrogen with full doses of 

phosphorus and potassium were applied as basal at the time 

of transplanting and rest 50 % N was top dressed in two equal 

splits (coinciding maximum tillering and panicle initiation 

stage) at the interval of one month after transplanting of rice 

seedlings. The total amount of organic manures/wastes viz. 

digested sludge, press mud and woolen carpet wastes were 

applied 14 days before transplanting of the rice var. Sarju-52 

at a spacing of 20 cm x 10 cm. 

Composite plant samples of rice were collected randomly 

at 40 and 65 days after transplanting (DAT) and at the time of 

harvesting. The plant samples (straw and grain) were , then, 

dried in oven at 65 0 C for 48 hours, powdered with the help of 

willey mill and were digested and analyzed for N, P, K, S as per 

procedure described in the following paragraphs and in Table 

1. 

The digestion of dried plant samples (straw and grain) 

for total N estimation were done using conc. H 2 

SO 4 

and 

digestion mixture (K 2 

SO 4 

: CuSO 4 

. 5 H 2 

O : metallic Selenium 

powder :: 50 : 10 : 1) as described by Yoshida, et al., 1976. The 

digested samples were then steam distilled with 40 % NaOH 

in KEL PLUS distillation apparatus. 

For the estimation of P, K, S the straw and grain samples 

were digested in diacid mixture of conc. HNO 3 

and HClO 4 

in 

the ratio of 10 : 4 as described by Jackson, 1967. 

RESULTS AND DICUSSION 

N content in plant at different stages of growth and grain 

at harvest: 

Data recorded on N content in rice plant at successive 

growth stages (40 DAT, 60 DAT and straw at harvest) 

pertaining to different treatments are presented in Table 1. 

Perusal of data revealed significant variations due to different 

treatments during both the years of experimentations. Initially 

higher N contents in plant were observed but thereafter it 

decreased with the advancement in age. It might be due to 

dilution effect (Josheph and Prasad, 1992) i.e. higher rate of 

dry matter production than rate of nutrient uptake. It is 

perceptible from the data that various treatments induced 

significant variations in the N content of plants at different 

growth stages. The various N sources brought about 

significant improvement in the N content of the plant over 

control (T 1 

) and application of N through chemical fertilizer 

(T 9 

) increased the N content at different stages significantly 

including in grain at harvest except straw at harvest where the 

treatment T 9 

remained at par with the application of three 

organics @ 40 kg N ha -1 (T 8 

), over rest of the treatments. The 

amount of mobilizable N or soil solution concentration usually 

decides the absorption of N from soil and the plant N content 

is directly related to the level of N supplied at each stage. The 

absorption of N was high at active tillering and panicle initiation 

stages. Thus, split application of inorganic sources 

compensated the N requirement, thereby, N concentration in 

the plant increased at these stages. 

Among various organic N sources, N application 

through combination of D.S, P.M and C.W @ 40 kg N ha -1 

each (T 8 

) resulted in significantly higher N content over all 

other organic N sources along with control (T 1 

) at all the stages 

of straw and in grain. This might be attributed to fast 

mineralization rate of all the three organics in combination in 

soil because of their narrow C:N ratio and also due to the 

synergistic effect. The enhancement in N content in dry matter 

and grain was observed due to application of D.S, C.W or P.M 

(Biswas and Dravid, 1998; Ram, et al., 2000 and Singh, et al., 

2001). 

Though there was a decreasing N content in plant straw 

at successive stages, significant increased N content was 

recorded due to application of C.W alone (T 4 

) at 40 DAT over 

the application of organic N sources except P. M alone (T 3 

) 

and combination of three organics at 40 kg N ha -1 each (T 8 

). 

But the N content due to application of C.W alone (T 4 

) got 

reduced over the N content due to application of other organic 

N sources except D.S + C.W @ 60 kg N ha -1 each (T 6 

), D.S + 

P.M @ 60 kg N ha -1 each (T 5 

) and D.S alone (T 2 

) at 65 DAT and 

further reduced in straw at harvest except the N content due 

to application of D.S alone (T 2 

). It might be attributed to the 

quicker mineralization rate of C.W as compared to D.S and 

P.M due to which the availability of N was more before 40 

DAT increasing the N content at 40 DAT and further decreased 

in the subsequent stages. 

Table 1. 

Effect of organics and inorganics on N content (%) of rice straw at different stages and grain at harvest. 

Treatments Ist year 2nd year Pooled Ist year 2nd year Pooled Straw at harvest Pooled Grain at harvest Pooled 

40 DAT 40 DAT 

65 DAT 65 DAT 

Ist year 2nd year 


T 1 1.59 1.74 1.67 1.19 1.31 1.25 0.31 0.33 0.32 1.01 1.17 1.02 

T 2 1.98 2.14 2.06 1.53 1.62 1.58 0.36 0.37 0.39 1.09 1.20 1.15 

T 3 2.27 2.45 2.36 1.63 1.83 1.73 0.44 0.45 0.48 1.15 1.25 1.24 

T 4 2.31 2.48 2.40 1.46 1.84 1.65 0.34 0.39 0.40 1.08 1.20 1.14 

T 5 2.20 2.28 2.24 1.55 1.71 1.63 0.37 0.42 0.43 1.12 1.23 1.20 

T 6 2.21 2.24 2.22 1.57 1.70 1.64 0.38 0.40 0.43 1.13 1.22 1.20 

T 7 2.24 2.39 2.32 1.60 1.84 1.72 0.41 0.44 0.46 1.14 1.24 1.22 

T 8 2.37 2.52 2.45 1.66 1.87 1.76 0.46 0.47 0.51 1.16 1.26 1.25 

T 9 2.46 2.60 2.53 1.76 1.90 1.83 0.48 0.49 0.53 1.22 1.28 1.30 

C.D. (P=0.05) 0.12 0.07 0.07 0.10 0.06 0.06 0.04 0.04 0.03 0.05 0.04 0.03


Almost similar trend was observed in the N content of 

straw and grain at harvest with the only exception that N 

content due to application of D.S alone (T 2 

) increased slightly 

over the N content due to supplication of C.W alone (T 4 

). 

P content of rice straw at different stages and grain at 

harvest: 

The data based on P content of rice straw at different 

stages and grain at harvest (Table 2) revealed that there was 

significant variation due to different treatments during both 

the years of experimentation during 2001 and 2002. P content 

(%) of rice straw at different stages was less than the P content 

of grain at harvest. P content of rice straw was recorded 

maximum at 40 DAT; but decreased as the crop proceeded 

towards maturity. It might be due to dilution effect and fixation 

of applied P in soil. It is evident from the pooled data that 

incorporation of N through chemical fertilizer (T 9 

) and D.S + 

P.M + C.W @ 40 kg N ha -1 each (T 8 

) did not produce any 

significant variation in P content of rice straw at different 

growth stages while differing significantly in grain at harvest. 

However, incorporation of N through organics increased 

P content over control (T 1 

) and application of N through 

chemical fertilizer (T 9 

) followed by the treatment receiving all 

the organics @ 40 kg N ha -1 each (T 8 

) showed superiority 

over all the N sources along with control (T 1 

). This may be 

ascribed to the increased solubility of P with application of 

organics. Increased P content might also be attributed to the 

increase in soil available P due to appreciable amount of P 

present in organics (particularly press mud) and also due to 

solubilization of insoluble forms of phosphate by organic acids 

produced during the decomposition process (Subramaniyan 

and Wahab, 1997). The observation of increased yield as well 

as P content as a result of NPK fertilization (T 9 

) is self 

explanatory and substantiates the findings of Dixit and Gupta, 

2000. 

The presence of N in soil favoured the utilization of P 

and K efficiently by plants. The improvement in utilization of 

P is brought about through the effect of N on root proliferation, 

extent of distribution of P in soil (Grune and Krant, 1958 and 

Subramaniyan and Wahab, 1997). Phosphorous is directly 

related to the vegetative and reproductive phases of the crop 

and attributes complex phenomenon of P utilization in plant 

metabolism. Since this element enters into the oxidative 

disintegration process of carbohydrate to yield hexose 

phosphate which further transformed due to development of 

meristematic tissues, cell division, leaf-area development and 

grain filling. It has also helped in the efficient absorption and 

utilization of the other required plant nutrients which ultimately 

increased the grain and straw yields, confirming the findings 

of Singh and Rai, 2002. 

K content of rice straw at different stages and grain at 

harvest: 

The data pertaining to the K content (%) in plant at 

different growth stages are presented in Table 3. The K 

content was higher at 40 DAT but it decreased in the later 

stages (65 DAT) and further increased in straw at harvest. 

Analysis of data revealed significant variations in K content 

Table 2. 

Effect of organics and inorganics on P content (%) of rice straw at different stages and grain at harvest. 


40 DAT 40 DAT 

65 DAT 65 DAT 



T 1 0.187 0.211 0.199 0.157 0.151 0.154 0.055 0.059 0.057 0.242 0.241 0.242 

T 2 0.235 0.239 0.237 0.172 0.179 0.176 0.079 0.083 0.081 0.274 0.284 0.279 

T 3 0.256 0.280 0.268 0.197 0.201 0.199 0.103 0.114 0.109 0.309 0.326 0.318 

T 4 0.231 0.226 0.229 0.168 0.175 0.172 0.071 0.072 0.071 0.270 0.278 0.274 

T 5 0.240 0.258 0.249 0.181 0.187 0.184 0.096 0.101 0.099 0.284 0.307 0.296 

T 6 0.242 0.251 0.247 0.184 0.182 0.183 0.096 0.095 0.096 0.289 0.299 0.294 

T 7 0.253 0.269 0.261 0.192 0.195 0.194 0.102 0.109 0.106 0.301 0.312 0.307 

T 8 0.261 0.284 0.273 0.203 0.210 0.207 0.108 0.119 0.114 0.317 0.331 0.324 

T 9 0.267 0.289 0.278 0.213 0.219 0.216 0.116 0.127 0.122 0.334 0.342 0.338 

C.D. (P=0.05) 0.013 0.007 0.007 0.019 0.013 0.011 0.016 0.011 0.010 0.020 0.008 0.011 

Table 3. 

Effect of organics and inorganics on K content (%) of rice straw at different stages and grain at harvest. 


40 DAT 40 DAT 

65 DAT 65 DAT 



T 1 1.61 1.58 1.59 1.03 1.17 1.10 1.28 1.41 1.35 0.194 0.205 0.200 

T 2 1.82 1.86 1.84 1.35 1.48 1.42 1.46 1.63 1.55 0.214 0.229 0.222 

T 3 1.98 2.22 2.10 1.46 1.61 1.54 1.63 1.81 1.72 0.238 0.243 0.240 

T 4 1.80 1.78 1.79 1.33 1.44 1.38 1.43 1.59 1.51 0.210 0.223 0.217 

T 5 1.91 2.11 2.01 1.41 1.52 1.47 1.53 1.78 1.66 0.220 0.235 0.228 

T 6 1.91 2.10 2.01 1.42 1.51 1.46 1.51 1.74 1.62 0.221 0.235 0.228 

T 7 1.97 2.12 2.05 1.44 1.58 1.51 1.60 1.78 1.69 0.233 0.240 0.237 

T 8 2.06 2.23 2.15 1.51 1.63 1.57 1.65 1.89 1.73 0.245 0.250 0.248 

T 9 2.11 2.34 2.22 1.59 1.70 1.65 1.74 1.98 1.86 0.260 0.262 0.261 

C.D. (P=0.05) 0.11 0.11 0.08 0.09 0.08 0.06 0.13 0.08 0.07 0.009 0.011 0.007


of plant at different growth stages and in grain at harvest due 

to different treatments in both the years of field trial. It is 

perceptible from the pooled data that application of N through 

chemical fertilizer (T 9 

) being at par with the application of 

combination of three organics @ 40 kg N ha -1 each (T 8 

) at 40 

DAT increased significantly the K content over rest of the N 

sources and the control (T 1 

). Increase in K contents in grain 

due to NPK fertilization has also been reported by Dixit and 

Gupta, 2000. The observation in increased yield as well as K 

content as a result of NPK fertilization (T 9 

) is self explanatory 

and substantiates the findings of Dixit and Gupta, 2000. 

Application of three organics @ 40 kg N ha -1 each (T 8 

) also 

induced significant increment of K content over other 

treatments except application of P.M alone (T 3 

) at 40 DAT, 

P.M alone (T 3 

) and P.M + C.W @ 60 kg N ha -1 each (T 7 

) at 65 

DAT, P.M alone (T 3 

), P.M + C.W @ kg N ha -1 each (T 7 

) and D.S 

+ P.M @ 60 kg N ha -1 each (T 5 

) in straw at harvest which 

remained at par among themselves. 

Among organic N sources, all the sources alone or in 

combination enhanced the K content in plant and grain at 

harvest at all the stages over without N (T 1 

). This might be 

due to presence of K in organics (P.M/sludge). This result is 

in agreement with those of Subramaniyan and Wahab, 1997. 

Further application of organics might be helpful in making 

mineralizable K available from native sources to plants that 

increased K content in rice straw at different stages and in 

grain at harvest. 

Presence of N in soil favoured the utilization of K 

efficiently by plants. Enhanced utilization of K is brought 

about through the effect of N on root proliferation, extent of 

distribution of K in soil (Grune and Krant, 1958 and 

Subramaniyan and Wahab, 1997. 

S content (ppm) of rice straw at different stages and 

grain at harvest: 

Data on S content in plant recorded at successive growth 

stages and in grain at harvest as affected by different 

treatments are summarized in Table 4. The perusal of the data 

further show that various treatments significantly influenced 

the S content in the plant at different growth stages and in 

grain at harvest, during both the years of experimentation. 

Highest S content was observed at 40 days after transplanting 

and thereafter it decreased at subsequent stages and in grain 

at harvest which may be due to dilution effect. 

It is obvious from the pooled data that application of N 

through chemical fertilizer (T 9 

) and combination of D.S, P.M 

and C.W @ 40 kg N ha -1 each (T 8 

) were at par with each other 

significantly and increased S content in plants as against other 

N sources along with control (T 1 

) at all the stages. Rice takes 

up sulphur, which is oxidized as SO 4 

2- 

on the root surface 

(Engler and Patrick, 1975). Results corroborated increased S 

contents due to higher fertility levels. Similar findings are 

observed by Rathore and Singh, 1978. 

The S supplying capacity of different organic wastes 

depended on the amount present in them and on the differential 

rate of mineralization. This might be the reason due to which 

the S content of rice straw at all the stages varied, the treatment 

receiving all the organics @ 40 kg N ha -1 each (T 8 

) was found 

to have more S in plants. However, all the organic N sources 

produced significant improvement in S content in plant over 

the control (T 1 

). The availability of easily degradable material 

in the organic wastes supported greater biological activity 

leading to higher mineralization of S (Singh, et al., 2001). N 

application through chemical fertilizer (T 9 

) established 

superiority over rest of the treatments on S content in plant 

and grain. The S contents in plants were recorded in the 

decreasing order as T 9 

> T 8 

> T 3 

> T 7 

> T 5 

> T 6 

> T 4 

> T 2 

> T 1 

. 

Application of green leaf manure along with S fertilizer 

increased S content of rice crop (Poongothai, et al., 1999). 

Application of FYM increased S uptake by wheat when applied 

in conjunction with fertilizer. The increased S concentration 

was observed in the leaves of cane treated with SPMC 

(Yaduvanshi, 1998). Increase in root proliferation as a result 

of better plant growth through applied S has been observed 

in maize (Pasricha, et al., 1977). Increasing levels of S increased 

straw and grain S content (Sarkunan, et al., 1998). 

Grain Yield 

It is quite obvious from the pooled data that application 

of N through chemical fertilizer (T 9 

) brought about significant 

improvement in grain yield and established superiority over 

Table 4. 

Effect of organics and inorganics on S content (%) of rice straw at different stages and grain at harvest. 


40 DAT 40 DAT 

65 DAT 65 DAT 



T 1 0.207 0.232 0.220 0.166 0.183 0.175 0.136 0.141 0.139 0.101 0.111 0.106 

T 2 0.240 0.266 0.253 0.185 0.218 0.202 0.155 0.170 0.163 0.116 0.125 0.121 

T 3 0.293 0.301 0.297 0.234 0.261 0.248 0.189 0.202 0.196 0.139 0.148 0.144 

T 4 0.243 0.253 0.248 0.191 0.229 0.210 0.160 0.173 0.167 0.118 0.130 0.124 

T 5 0.261 0.277 0.270 0.209 0.237 0.223 0.169 0.183 0.176 0.123 0.138 0.131 

T 6 0.254 0.271 0.263 0.212 0.230 0.221 0.171 0.177 0.174 0.128 0.132 0.130 

T 7 0.286 0.294 0.290 0.228 0.254 0.241 0.183 0.195 0.189 0.132 0.144 0.138 

T 8 0.305 0.317 0.311 0.241 0.269 0.255 0.195 0.213 0.204 0.145 0.155 0.150 

T 9 0.311 0.322 0.317 0.246 0.276 0.261 0.197 0.216 0.207 0.148 0.159 0.154 

C.D. (P=0.05) 0.010 0.011 0.007 0.008 0.010 0.006 0.009 0.007 0.006 0.007 0.009 0.005


Table 5. 

Effect of organics and inorganics on grain and straw yield of rice. 

Treatments 

Ist year 2nd year Pooled Ist year 2nd year Pooled 

Grain Yield 

(kg ha -1 ) 

Grain Yield 

(kg ha -1 ) 

Straw yield 

(kg ha -1 ) 

Straw yield 

(kg ha -1 ) 

T 1 1783.3 1966.7 1875.0 3316.7 3516.7 3416.7 

T 2 3266.7 3833.3 3550.0 4883.3 5633.3 5258.3 

T 3 3833.3 4433.3 4133.3 5333.3 6166.7 5750.0 

T 4 3116.7 3716.7 3416.7 4733.3 5550.0 5141.7 

T 5 3516.7 4150.0 3833.3 5016.7 5933.3 5475.0 

T 6 3566.7 4083.3 3825.0 5100.0 5916.7 5508.3 

T 7 3783.3 4316.7 4050.0 5333.3 5983.3 5658.3 

T 8 4233.3 4716.7 4475.0 5716.7 6300.0 6008.3 

T 9 4683.3 5283.3 4983.3 6183.3 6850.0 6516.7 

C.D. (P=0.05) 198.0 190.0 131.9 363.3 530.0 308.8 

the application of organic N source and the control (T 1 

). 

Among organic N sources, supplying N through combination 

of D.S, P.M and C.W @ 40 kg N ha -1 each (T 8 

) significantly 

increased the grain yield as against the application of rest of 

the organic N sources. The addition of N through chemical 

fertilizers (T 9 

) and different combination of organic N sources 

(T 8, 

T 7, 

T 6, 

T 5 

) produced significantly more grain yield over the 

incorporation of organic N sources alone (T 2 

, T ) with the 

4 

exception of the addition of P.M alone (T 3 

) and the control 

(T 1 

). However, all the treatments increased the grain yield 

significantly as against no fertilizer/manure (T 1 

). The trend 

observed in increasing order was: T 1 

> T 4 

> T 2 

> T 6 

> T 5 

> T 7 

> 

T 8 

> T 9. 

It is, by and large, true that dwarf indica rice varieties 

have high rate of responsiveness towards fertilizer application 

and more particularly for N because of their conducive genetic 

make up. The findings of the present investigation revealed 

profound effect of N on yield and yield attributes of rice. It 

was noticed that the grain yield due to application of N 

through chemical fertilizer and various organics was 

associated with the number of grains per panicle, effective 

tillers m -2 and test weight of 1000 grains. Correlation studies 

have shown that grain yield is highly correlated with yield 

attributes (Hernandez, 1956). 

The present investigations revealed significant increase 

in the yield attributes under N application through chemical 


) followed by N through D.S + P.M + C.W (T 8 

) due 

to increased absorption of nutrients and their assimilation. 

Supply of N in balanced quantity enabled the rice plants to 

assimilate sufficient photosynthetic products and, thus, 

increased the dry matter accumulation. With increased dry 

matter and photosynthetic products, coupled with efficient 

translocation, plant produced more panicles with more number 

of fertile grains with increased test weight and ultimately higher 

grain yield. 

Straw Yield 

Application of N through chemical fertilizer (T 9 

) 

enhanced the straw yield significantly as against organic N 

sources and no N (T 1 

), which might be attributed due to quicker 

conversion of urea making N available to rice plants easily as 

compared to organics which release most of N after 

mineralization. 

Straw yield of a crop is closely related to the vegetative 

growth viz., plant height, tiller numbers, leaf numbers and 

final stand of a crop (Singh and Verma, 1971). The beneficial 

effect of any treatment on one or more of these characters 

without a corresponding decrease in one or more of them will 

result in increased straw yield. In the present investigation, 

the N application through any means enhanced the growth 

attributes that ultimately led to higher straw yield. 

Among organic N sources, incorporation of D.S, P.M 

and C.W @ 40 kg N ha -1 each (T 8 

) being at par with 

incorporation of P.M alone (T 3 

) significantly improved the 

straw yield over rest of the treatments treated with organics 

along with control (T 1 

). The availability of mineralizable N and 

other nutrients might be more in case of the treatment T 8 

followed by the treatment T 3 

than the treatments with organics 

due to differential rate of organic N mineralization in soil 

(Mukherjee, et al., 1995). 

Incorporation of organics such as D.S, P.M and C.W 

increased the straw yield of rice (Ram, et al., 2000; and Tiwari, 

2002). 

Application of N through chemical fertilizer (T 9 

) increased 

the N, P, K and S contents significantly at different stages 

including grain and straw at harvest by the crop over no 

fertilizer/manure (T 1 

). Among various organic N sources, N 

application through combination of D.S, P.M and C.W @ 40 

kg N ha -1 each (T 8 

) resulted in higher N, P, K and S contents in 

plants over all other organic N sources at all the stages of 

straw and grain. 


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Philippines. 



Incidence of Pod Borer, Helicoverpa armigera Hub. and Their Management Through 

Newer Insecticides in Chickpea. 

JEEWESH KUMAR, D. C. SINGH AND A. P. SINGH 

Department of Entomology, Narendra Deva University of Agriculture Technology, Kumarganj, Faizabad 

U.P. 224 229 

email: singhjeewesh@gmail.com 

ABSTRACT 

The present investigations were carried out at Students 

Instructional Farm of Narendra Deva University of Agriculture 

Technology, Kumarganj, Faizabad and farmers fields during 

Rabi, 2007-08. Activity of H. armigera started with flowering 

and continues to harvesting stage of the crop. The peak 

population were noticed 3 larva/ 10 plants on Sidhauna village 

and 2 larva/ 10 plants at Pandepurwa, Pithla and Jorium in 10 th 

standard weeks at a minimum temperature of 13.8 0 C, maximum 

temperature of 30.1 0 C, relative humidity 68.0% and no rainfall 

recorded during crop season. The larval population was positive 

correlation with minimum temperature, maximum temperature 

and relative humidity in village of Sidhauna. In case of 

Padepurwa and Pithla larval population was positively 

correlated with minimum temperature, relative humidity and 

negatively correlated with minimum temperature. In Jorium 

larval population was positive correlation with relative humidity 

and negative correlation with minimum and maximum 

temperature. Studied for management of H. armigera on 

chickpea carried out by using five insecticides, in which 

Spinosad 45 SC @ 90 g a.i./ ha was most effective treatment 

followed by Indoxacarb 14.5 EC @ 50 g a.i/ ha and Novaluron 10 

EC @ 100 g a.i/ ha. Endosulfan 35 EC @ 700 g a.i/ ha was least 

effective on basis of larval reduction and seed yield. The 

maximum cost: benefit ratio was obtained from Indoxacarb 

14.5 EC @ 50 g a.i/ ha. 

Key words 

Chickpea,H. armigera, population dynamics, 

Management 

One of the most practical means of increasing chickpea 

(Cicerarietimum L.) production is to minimize losses caused 

by major insect-pests, important among them is gram pod 

borer (Helicoverpa armigeraHubner. Lepidoptera :Noctuidae), 

the black aphid (Aphis craccivora Koch. Homoptera 

:Aphidideae) and the semi-looper (Autographa nigrisigna 

Walker. Lepidoptera :Noctuidae). Gram pod borer, H. armigera 

(Hub) is known to be the key pest due to high reproduction 

rates, a fast generation on turn over, wide genetic diversity 

occurs location and an ability to withstand, metabolize and 

avoid toxic chemicals. The young larvae feed on the tender 

portion of the leaves on shoots by making scratches. Second 

and subsequent developed up larvae consume whole leaf, 

leaf buds, flower buds and flower. Under severe pest 

infestation, whole crop may get defoliated. With the 

availability of pods, the 3 rd instar larvae make a hole in the 

pods and move inside to feed on the grains, where as fully 

developed larvae feed after making a hole in the pods and 

thrusting its head, there in while keeping hind parts of the 

body outside. The yield loss in chickpea due to H. armigera 

was reported as 10-60% in the normal weather condition 

(Vaishmpayan and Veda, 1980). The single larva of H. armigera 

may destroy 30-40 pods in its lifetime (Atwal and Dhaliwal, 

2005). 


The experimental site lies between 26.47 0 N latitude, 

82.12 0 E longitude and 113 m above from the sea level in the 

sub-tropical belt of the country and soil is alkaline to normal. 

For recording the incidence of H. armigera during crop season, 

the population of larvae was observed on 10 randomly selected 

plants at a location at weekly intervals. There were four sites 

for observation of this insect at farmer’s field and correlated 

between larval population and abiotic factors i.e. minimum 

temperature, maximum temperature and relative humidity were 

worked out. To find out the effective insecticides against pod 

borer an experiment was laid out in a Randomized Block Design 

with five treatments including control in four replication at 

the Student Instructional farm of Narendra Deva University 

of Agriculture and Technology Kumarganj, Faizabad. Larval 

populations of gram pod borer were recorded at weekly 

intervals on 10 randomly selected plants/ plot starting with 50 

% flowering till harvest. The spraying of insecticides were 

applied when infestation was reach at ETL (1.5 larvae/ row 

length). In addition, the seed yield was recorded after harvest 

of the crop. The cost effectiveness was determined by working 

out cost: benefit ratio. 


The data recorded on larval population of H. armigera 

during Rabi 2007-08 have been presented in Table-1 It is 

evident from data that pest activity started with flowering and 

continued till harvesting stage of crop. The presence of larvae 

of H. armigera could be noticed first time in 7 th standard week 

in villages Sidhauna and Jorium and in 8 th standard weeks on 

Padepurwa and Pithla village. The populationsat above 

periods of observation were 1 larva/ 10 plants were at all four 

villages. 

The maximum population were recorded 3 larva/ 10 

plants on Sidhauna village and 2 larva/ 10 plants at 

Pandepurwa, Pithla and Jorium in 10 th standard weeks at a

KUMAR, et al., Incidence of Pod Borer, Helicoverpa armigera Hub. and Their Management Through 241 

minimum temperature of 13.8 0 C, maximum temperature of 30.1 0 

C, relative humidity 68.0 per cent and 0.00 rainfall. Thereafter, 

decline in number of larval population was recorded in 

subsequent weeks and reached to its minimum i.e. 1 larva/ 10 

plants in 12 th SW at Sidhauna and Jorium. In case of 

Pandepurwa and Pithla population was reached to its 1 larva/ 

10 plants in 13 th SW when crop was ready to harvest. Similarly 

Shan and Shahzad, 2005 reported that pest population was 

low during 49 th to 6 th standard weeks but increased from 7 th 

standard weeks onward and declined again 14 th standard 

weeks and Singh, et al., 2005 found that population started to 

increase again from mid-February (1.0-1.8 larvae/ m 2 ) until the 

second week of April (8.0-10.8 larvae/ m 2 ), then declined 

abruptly. Vishwa Dhar, et al., 2003 reported that a sudden rise 

(75 0 C) in the minimum temperature around 7-8 standard weeks 

(Second fortnight of February) and rainfall during 1-9 standard 

weeks (January to February) were associated with a 

considerable adult population during 5-7 standard weeks, 

which triggered a major rise in the pest population around 10- 

14 standard weeks (Second fortnight of March). Gupta, et al., 

2004 observed that infestation by H. armigera occurred in 

March and April, with maximum moth catches of 70 per cent 

during the last fortnight of March and first fortnight of April. 

Singh and Ali, 2006 reported that the larval activity of H. 

armigera continued throughout the crop season with two 

peaks, first from 45 to 49 standard weeks and second from 5- 

13 standard weeks. The highest larval population was observed 

from 45 to 12 th standard weeks respectively. 

Correlation between larval population and weather 

parameters viz., minimum temperature, maximum temperature, 

relative humidity and rainfall have been given in Fig. 1. It is 

evident from data that larval population showed positive 

correlation with minimum temperature (+ 0.384), maximum 

temperature (+ 0.206) and relative humidity (+ 0.523), at 

Sidhauna. In case of Pandepurva and Pithala, larval population 

had positive associationship with minimum temperature (+ 

0.500), relative humidity (+ 0.156) and negative correlation 

with maximum temperature (- 0.031). Larval population of Jorium 

was positively correlated with relative humidity (+ 0.833) and 

negatively correlated with minimum temperature (-0.019) and 

maximum temperature (- 0.026). Singh, 2003 observed that 

temperature and rainfall had significant positive correlation 

with larval population. The maximum and minimum humidity 

had significant negative correlation and sunshine hours had 

no significant effect on larval population. It is reported that a 

positive correlation existed between the eggs, larval instar 

and overall density of H. armigera and the average maximum 

and minimum temperature. However, a negative correlation 

existed between the eggs, larval instar and overall density of 

H. armigera and the average morning per cent relative 

humidity. The eggs, larval instar and overall density of this 

pest had no relationship with evening per cent relative 

humidity. Singh, et al., 2005 reported that maximum temperature 

and minimum temperature had positive correlation with the 

larval population. Rainfall had a positive correlation with larval 

population may during 1998-99. The maximum relative humidity 

was negatively correlated with the larval population of H. 

armigera. Singh, et al., 2005 also found positive correlation 

with minimum temperature, maximum temperature and rainfall, 

while negative correlation with relative humidity. 

Effect of treatments on the larval population of H. 

armigera represented in Table 2, indicated the larval 

population was homogenously distributed throughout 

experimental plots before the application of treatments. All 

treatments were significantly superior over the control when 

observations were made 7 at days after treatments. Spinosad 

45 SC @ 90 g a.i./ ha showed maximum larval population 

reduction 75.00% followed by Indoxacarb 14.5 EC @ 50 g a.i./ 

ha in which reduction of larval population was 71.43% then 

Novaluron 10 EC @ 100 g a.i./ha with 62.50% was noticed. 

Endosulfan 35 EC @ 700 g a.i./ ha was least effective with 

Fig. 1. 

Correlation between larval population of H. armigera 

and abiotic factors during crop season. 

Table 1. 

Helicoverpa armigera intensity in chickpea crop in villages of Faizabad district 

Standard week 

Larval population/10 plants 

Abiotic factors 

Villages 

Mean 

Temperature ( 0 C) 

Sidhauna Padepurva Pithla Jorium 

R.H. (%) Rainfall (mm) 

Min. Max. 

6 0 0 0 0 0.00 9.9 23.1 67.3 0.00 

7 1 0 0 1 0.50 4.7 23.7 55.8 0.00 

8 1 1 1 1 1.00 8.7 26.4 63.1 0.00 

9 2 1 1 2 1.50 10.5 27.9 68.0 0.00 

10 3 2 2 2 2.25 13.8 30.1 68.0 0.00 

11 2 2 2 1 1.75 19.5 31.8 51.0 0.00 

12 1 1 1 1 1.00 16.5 34.9 51.5 0.00 

13 0 1 1 0 0.50 15.5 35.5 42.7 0.00


Table 2. Effectiveness of treatments against larval population of H. armigera in chickpea during Rabi 2007-08 

Treatments 

Reduction in larval population 

(%) days after spraying 

7 days 14 days Mean 

Yield 

(q/ha) 

Saved seed yield 

due to treatment 

(%) 

Common name 

Trade 

name 

Dose 

(g a.i./ha) 

Novaluron 10 EC Rimon 100 62.50 33.33 47.91 18.75 22.47 

Indoxacarb 14.5 EC Steward 50 71.43 50.00 60.71 19.31 26.13 

Spinosad 45 SC Tracer 90 75.00 50.00 62.50 21.25 38.80 

Endosulfan 35 EC Thiodan 700 50.00 33.33 41.67 17.50 14.30 

Control (water spray) - 14.28 16.67 15.47 15.31 - 

SEm± 2.57 0.75 2.11 0.16 - 

C.D. 7.79 2.30 6.86 0.62 - 

Table 3. Cost benefit ratio of treatments used for the management of H. armigera in chickpea during Rabi 2007-08. 

Treatments 

Dose 

(g a.i./ha) 

Cost of 

Treatments 

(Rs/ha) 

Yield 

(q/ha) 

Saved yield due to 

treatment 

(q/ha) 

Benefit due to 

treatment 

(Rs/ha) 

Cost : 

benefit ratio 

Common name 

Trade 

name 

Novaluron 10 EC Rimon 100 3930.00 18.75 3.44 10320 1:2.62 

Indoxacarb 14.5 EC Steward 50 1692.70 19.31 4.00 12000 1:7.09 

Spinosad 45 SC Tracer 90 2808.67 21.25 5.94 17820 1:6.34 

Endosulfan 35 EC Thiodan 700 1330.00 17.50 2.19 6750 1:5.97 

Control (water spray) - 430.00 15.31 - - - 

50.00% reduced in larval population. Data recorded 14 days 

after spray revealed that all treatments were effective and 

significantly better than control. Maximum reduction in larval 

population (50 %) was obtained by both treatments (Spinosad 

45 SC @ 90 g a.i./ ha and Indoxacarb 154.5 EC @ 100 g a.i./ 

ha),Novaluron 10 EC @ 100 g a.i./ha and Endosulfan 35 E.C. 

@ 700 g a.i./ ha showed 33% reduction. Pooled data from 7 

and 14 days after spraying also spinosad performed better in 

comparison to other treatments with 62.50% larval reduction 

and saved yield due to treatment 38.80% followed by 

Indoxacarb 60.71% larval reduction and 26.12% saved yield. 

Spinosad 45 SC when tested by Gowda, et al., 2003 at different 

doses also recorded significantly lower pod damage and higher 

grain yield as compared to Endosulfan 35 EC @ 700 g a.i./ ha. 

Indoxacarb 14.5 EC treated plots gave highest cost: benefit 

ratio 1:7.09 followed by Spinosad 45 SC treated plots (1:6.34). 

But Rahman, et al., 2006 observed that Steward 14.5 EC 

(Indoxacarb) was the most effective with highest larval 

mortality 97.38% also Babaria, et al., 2010 reported that 

indoxacarb 0.0075% caused highest 89-96% mortality of the 

H. armigera followed by spinosad 0.009% with 86-95%. 

Sharma, et al., 2011 indicate Emamectin benzoate 5 SG in 

combination with Acetamiprid 20 SP or Dimethoate 30 EC gave 

lowest pod damage (13.30 and 11.95%). Singh and Ali, 2005 

observed that maximum larval mortality was recorded in 

Endosulfan (85%) followed by Bt formulation (80%) and 

HaNPV at 450 LE/ ha (75%). NSKE was least effective 

treatment. 

Table 2 revealed that produced of all treatments 

significantly higher yield than control (water spray). Spinosad 

45 SC (90 g a.i./ ha) produced maximum yield of 21.25 q/ ha 

and benefit due to treatment Rs. 17820 followed by Indoxacarb 

14.5 EC (50 g a.i./ ha) with yield 19.31/ ha and benefit Rs. 

12000 and Novaluron 10 EC (100 g a.i./ ha) yield recorded was 

18.75 q/ ha and benefit Rs. 10320. Endosulfan 35 EC (700 g 

a.i./ ha) gave lowest yield of 15.31 q/ ha and benefit Rs. 6750 

as compared to other insecticidal treatments. Maximum cost 

benefit ratio was recorded by Indoxacarb 1: 7.09 followed by 

Spinosad 1: 6.34. The effectiveness of Spinosad was highly 

effective in comparison to other treatment but due to more 

costly insecticides it have not gave maximum cost benefit 

ratio. Sharma, et al., 2011 recorded Emamectin benzoate 5 SG 

in combination with Acetamiprid 20 SP or Dimethoate 30 EC 

gave higher grain yield of 1399 and 1392 kg/ha. Effectiveness 

of emamectin benzoate, which is based on green chemistry, 

will help in achieving less yield losses through reduction in 

H. armigera incidence. Also Babaria, et al., 2010 highest grain 

yield of pigeonpea 1486 kg /ha and net return Rs. 19824 / ha 

were recorded with indoxacarb 0.0075 per cent treatment which 

was followed by spinosad 0.009 per cent (1451 kg /ha and net 

return Rs. 18844). 

LITERATUTRE CITED 

Atwal, A.S. and Dhaliwal, G.S. 2005. Agricultural Pests of South Asia 

and their Management, Kalyani Publisher, New Delhi. 

Babaria, P. M., Kabaria, B. B., Patel, V. N. and Joshi, M. D. 2010. 

Chemicalcontrol of gram pod borer, Helicoverpa armigera Hubn. 

infesting pigeonpea. Legume Research. 33 (3):224-1-226. 

Gupta, S., Kanaujia, S. and Kanaujia, K.R. 2004. Population dynamics 

of Helicoverpa armigera and C. chlorideaeon chickpea and 

sunflower using sex pheromone. Pl. Protec. Bull., 56 (1/2): 14-16. 

Shahzad, M.K. and Shan, Z.A. 2003. Screening of the pest insecticide 

in including the chickpea pod damage inflicted by gram pod borer, 

Helicoverpa armigera (Lapidoptera: Noctuida) in Faislabad. Pakistan 

J. Biological Sci., 6(3): 1156-1158. 

Sharma, O. P., Bhosle, B. B., Kamble, K. R., Bhede, B. V. and Seeras, N. 

R. 2011. Management of pigeonpea pod borers with special

KUMAR, et al., Incidence of Pod Borer, Helicoverpa armigera Hub. and Their Management Through 243 

reference to podfly (Melanagromyza obtusa). Indian Journalof 

Agriculture Science, 81(6):313-316. 

Singh, R. and Ali, S. 2005. Efficacy of bio-pesticides in management of 

Helicoverpa armigera (Hub.) in chickpea. Ann. Pl. Protec. Sci., 

13(1): 94-96. 

Singh, R. and Ali, S. 2006. Seasonal incidence of Helicoverpa armigera 

and Campoletischlorideae on chickpea. Ann. Pl. Protec. Sci., 14(1): 

234-235. 

Singh, V., Singh, R.K. and Prakash, V. 2005. Seasonal occurrence of 

larval population of Helicoverpa armigera on chickpea in northwest 

Rajasthan. Indian J. Pulse Res., 18(1): 92-93. 

Vaishampayan, S.M. and Veda, O.P. 1980. Population dynamics of 

gram pod borer (Helicoverpa armigera) in chickpea at Pantnagar 

(U.P.), Indian J. Pl. Prot., 15: 39-41. 

VishwaDhar, Trivedi, T.P., Yadav, C.P. and Das, D.K., Dhandapani, A., 

Singh, S.K., Choudhary, R.G., Devraj and Kumar, M. 2003. 

Protection of Helicoverpa armigera attack on pigeonpea in central 

Uttar Pradesh. IIPR, Newletter, 14(2): 3. 



Efficacy of Some Triazole and Strobilurin Fungicides against Rust Disease of Field 

Peas 

R S BAL AND A KUMAR 

PAU, Regional Research Station, Gurdaspur, Punjab 143 521 

e-mail: rsbalgsp@gmail.com 

ABSTRACT 

Rust of pea caused by Uromyces pisi is a major constraint to pea 

production. An experiment was conducted at Regional Research 

Station, Gurdaspur during Rabi, 2010-11 to control the pea rust 

disease with foliar sprays of the strobilurin and triazole 

fungicides in different combinations. All the fungicides caused 

reduction in disease severity. The highest disease reduction of 

81.8% was obtained with Score @0.1% followed by 66.5% with 

Score @0.05%. The minimum disease severity of 15.05% was 

observed with Score @0.1% as compared to 82.7% in the control 

plot. There was a significant effect of the fungicidal sprays on 

the grain weight. The highest 1000 grain weight of 174.25 g 

was obtained with Score @0.1% as compared to 122.75g in the 

control plot, thus giving an increase of 41.95% in grain weight 

over the control plot. 

Key words 

Field pea, Rust, Fungicides, Efficacy 

Field pea (Pisum sativum L.) is an important leguminous 

pulse crop. During the year 2009-10, it was grown on 1.6 

thousand hectares in Punjab with a production of 1.8 thousand 

tones (Anonymous, 2011). The field pea being a good source 

of vitamins, proteins and carbohydrates, is mainly consumed 

as pulse. The crop is attacked by many fungal and viral 

diseases. Among these, rust caused by Uromyces pisi (Pers) 

de Barry is one of the destructive diseases of peas which can 

cause high yield reduction within a short period of time. The 

disease can cause yield losses in the range of 20-100% (Amin 

and Ramachander, 1989). 

Different management strategies like biological, cultural, 

chemical and growing resistant varieties can be used to control 

the disease (Marshi, et al., 1982). Many scientists have triedto 

control this disease using various fungicides like Sedozole 

and Bavistin (Alam, et al., 2007), Triforine (Amin and 

Ramachander, 1989), Mancozeb (Singh, et al., 1992), 

Chlorothalonil (Dobson and Giltrap, 1991) and Mancozeb 

and Bayleton (Khan, et al., 2009). The pea crop suffers badly 

by the rust disease year by year. So far, no sources of resistance 

to Uromyces pisi have been reported in pea. Therefore, this 

study was planned to control the pea rust disease with some 

new triazole and strobilurin fungicides. 


The experiment was conducted at Regional Research 

Station, Gurdaspur during Rabi, 2010-11. The seed of field 

pea variety PG 3 was sown on 23 rd November, 2010 in a plot 

size of 6.0 m 2 in a randomized block design with four 

replications. The recommended agronomic practices were 

adopted for all treatments. The efficacy of four fungicides 

viz., Score (difenoconazole), Tilt (propiconazole), Amistar 

(azoxystrobin) and Indofil M45 was tested in different 

combinations. Score, Tilt and Amistar were used at 

concentrations of 0.1% and 0.05% while Indofil M45 was 

used at 0.2% concentration. The first spray was given at the 

appearance of the disease. Indofil M45 was sprayed at an 

interval of 10 days while the other fungicides were sprayed at 

20 day intervals. Data were recorded on disease severity and 

1000 grain weight. The data on disease severity were recorded 

on the basis of symptoms on leaves. The scoring was done 

according to the rating scale of Mayee and Datar, 1986. 

Rating Scale used for Scoring Pea Rust 

The per cent disease index (PDI) was calculated by using 

the formula (Wheeler, 1969) as follows: 

Table 1. 

PDI = 

Per cent disease index 

Disease Disease severity description 

rating 

0 No symptoms on leaf. 

1 Rust pustules small, scattered covering 1% or less of leaf area. 

3 Rust pustules more in number covering 1-10% of leaf area. 

5 Typical rust pustules covering 11-25% of leaf area. 

7 Typical rust pustules covering 26-50% of leaf area. Leaf 

shedding. 

9 Typical rust pustules covering 51% or more of leaf area. 

Defoliation severe. 

Total sum of individual ratings 

× 100 

Number of leaves examined × Maximum rating 

Data were analyzed following the statistical procedure 

of Gomez and Gomez, 1983. 


Effect of the fungicides on the severity of pea rust and 

1000 grain weight 

The effect of different fungicides on the severity of pea 

rust is presented in Table 2. The severity of the rust disease 

was reduced significantly in all the treatments than the control 

plots. The average percent disease index varied from 15.0% to 

82.73%. The minimum PDI of 15.0% was recorded with two 

sprays of Score @0.1%, followed by 27.73% with two sprays 

of Score @0.05%. However, when a single spray of Score 

@0.1% was given in combination with two sprays of Indofil

BAL AND KUMAR, Efficacy of some triazole and strobilurin fungicides against rust disease of field peas 245 

Table 2. Effect of fungicides on PDI of pea rust and 1000 grain weight. 

Treatment 

Concentration 

(%) 

% Disease 

index 

(PDI) 

% Reduction 

over control 

1000 Grain 

weight 

(g) 

% Increase in 

grain weight over 

control 

T1= Score+Score 0.1 15.00 81.87 174.25 41.95 

T2= Amistar+ Amistar 0.1 54.27 34.40 148.25 20.77 

T3= Tilt+ Tilt 0.1 66.47 19.65 143.25 16.70 

T4= Score+Score 0.05 27.73 66.48 161.25 31.36 

T5= Amistar+ Amistar 0.05 69.40 16.11 143.25 16.70 

T6= Tilt+ Tilt 0.05 65.80 20.46 134.0 9.16 

T7= Indofil M45 + Score + Indofil M45 0.2+0.1+0.2 31.63 61.77 160.0 30.35 

T8= Indofil M45+ Amistar+ IndofilM45 0.2+0.1+0.2 50.23 39.28 153.25 24.84 

T9= Indofil M45+ Tilt+ IndofilM45 0.2+0.1+0.2 57.13 30.94 147.0 19.75 

T10= Indofil M45+ Indofil M45+ Indofil M45 0.2 58.13 29.73 146.25 19.14 

T11=control - 82.73 - 122.75 - 

CD(5%) 7.52 6.86 

M 45 @0.2%, the PDI of rust disease was 31.63%. The highest 

disease index of 82.73% was observed in case of the control 

plot. The highest reduction (81.87%) in PDI was obtained 

with two sprays of Score @0.1%, followed by 66.48% disease 

reduction with Score @0.05%. The minimum reduction 

(16.11%) in the PDI of pea rust was noted with two sprays of 

Amistar @0.05%. The 1000 grain weight was also significantly 

increased in all the treatments over the control plot. The 

highest thousand grain weight of 174.25g was achieved with 

two sprays of Score @0.1% as compared to 122.75 g in the 

control plot. The maximum (41.95%) and minimum (9.16%) 

increase in grain weight over the control plots was noted with 

Score@0.1% and Tilt @0.1%, respectively. 

Relationship between PDI and grain weight 

The correlation coefficient (r) of -0.9595 showed a 

significant negative correlation between the PDI of rust disease 

and 1000 grain weight. It further showed that there was a 

progressive decrease in grain weight with the increase in the 

severity of rust disease. The contribution of the regression 

(R 2 = 0.9207) was 92% (Fig.1). 

From the above results, it is clear that Score@0.1% was 

the most effective fungicide in reducing the severity of pea 

rust. Hence, it can be concluded that the rust disease of field 

1000 Grain weight (g) 

200 

150 

100 

50 

Fig. 1. 

y = -0.6569x + 183.05 

r = -0.9595 

0 

0 20 40 60 80 100 

PDI of pea rust (%) 

Relationship between per cent disease index of pea rust 

and grain weight 

peas caused by Uromyces pisi can be effectively controlled 

by spraying Score@0.1% two times at 20 day intervals starting 

at the initiation of the disease symptoms. 


Alam, M.M., Sadat, M.A., Hoque, M.Z. and Rashid, M.H. 2007. 

Management of Powdery Mildew and Rust Diseases of Garden Pea 

Using Fungicides. Int. J. Sustain. Crop Prod., 2(3): 56-60. 

Amin, K.S. and Ramachander, P.R. 1989. Effect of Saprol on epidemic 

development of rust and powdery mildew on pea and their influence 

on yield. J. Pulses Res. 2(2): 140-146. 

Anonymous, 2011. Package of practices for crops of Punjab. PAU, 

Ludhiana. pp.36. 

Dobson, S.C. and Giltrap, N.J. 1991. Timing of sprays for control of 

rust and chocolate spot in spring and winter beans. Aspects of App. 

Biol., 27(6): 111-116. 

Gomez, K. A. and Gomez, A. A. 1983. Statistical procedures for 

Agricultural Research. 2 nd Ed. Intl. Res. Inst. Manila, Philippines. 

pp.139-207. 

Khan, A.I., Khan, H., Ali, A., Raziq, F., Hussain, S., Ahmad, M. and 

Attauddin. 2009. Evaluation of various fungicides and cultivars for 

the control of pea rust under natural conditions. Sarhad J. Agric., 

25(2): 261-268. 

Marshi, R.P., Gupta, R.B.L. and Matur, A.K. 1982. Response of Pea 

varieties to powdery mildew and rust in Rajastan. Indian Phytopath., 

35(2):232-235. 

Mayee, C.D. and Datar, V.V. 1986. Phytopathometry. Technical 

Bulletin-1 (Special Bulletin 3), Marathwada Agric. Univ. Parbhani. 

pp.218. 

Singh, S.K., Rahman, S.J., Gupta, B.R. and Kalha, C.S. 1992. An 

integrated approach to the management of the major diseases and 

insect pests of peas in India. Tropical Pest Mgt., 38(3): 265-267. 

Wheeler, B.E.J., 1969. An Introduction to Plant Diseases, John Wiley 

and Sons Limited, London. 


Trends in Biosciences 5 (3): 246, 2012 

SHORT COMMUNICATION 

Pseudomonas aeruginosa: Of Course I am An Arsenic Eater 

POONAM GUSAIN*, RAJESH KUMAR AND VIR SINGH 

Department of Microbiology, College of Basic Sciences and Humanities, GB Pant University of Agriculture 

and Technology, Pantnagar 263 145 

email: poonamgsn@gmail.com 

Present study was aimed at metal chelator siderophores 

produced by fluorescent Pseudomonas cultures. 

Pseudomonas strain RK3 was recovered from Pantnagar, 

screened for arsenic tolerance traits. Isolate RK3 showed 99% 

homology with Pseudomonas aeruginosa using partial 16SrRNA 

sequencing. FT-IR study revealed that characteristic 

hydroxamate ligands were synthesized by isolate RK3 under 

arsenic stress. This finding, sidero-metal interaction warrants 

future investigation that may help reduce heavy metals in 

contaminated soils. 

Transmittance% 

90 

60 

30 

2363.43 

2325.21 

2075.50 

980.70 

1090.30 

Table 1. Characterstic hydroxamate ligand determined in 

strain RK3 based on FT-IR 

Bonding Wavenumber cm -1 

Prim.Aliphatic.group 1090.80 

C=O Streching 1638.45 

C-OH Streching 1642.83 

C=N Streching 2325.21 

OH Group 3560 

As-O stretching 980.70 

0 

1642.83 

5000 4000 3000 2000 1000 

Wavenumber (cm -1 ) 

Fig.1. Infra red spectra of hydroxamate ligand 

430.90 


Nair, A., Juwarkar, A.A., Singh, S.K. 2007. Production and 

characterization of siderophores and its application in arsenic 

removal from contaminated soil. Water Air and Soil Pollution, 

180: 199–212. 

Paul, A., Geguere., Leu, I.D. 1952. Infrared spectrum, molecular structure 

and thermodynamic functions of hydroxalamine. Canadian Journal 

of Chemistry, 30: 648-962. 


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