Somatic embryogenesis and plantlet regeneration in Sorghum ...

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Somatic embryogenesis and plantlet regeneration in Sorghum ...

Journal of Cell and Molecular Biology 5: 99-107, 2006.

Haliç University, Printed in Turkey.

Somatic embryogenesis and plantlet regeneration in Sorghum bicolor (L.)

Moench, from leaf segments

Sudhakara Rao Pola* and N. Sarada Mani

Department of Botany, Andhra University, Visakhapatnam, India, 530 003

(*author for correspondence)

Received 25 June 2006; Accepted 12 July 2006

Abstract

Efficient plant regeneration system from leaf disc segments of S o rghum (Sorghum bicolor (L.) Moench) was

developed. The factors affecting the somatic embryo formation and regeneration capacity of leaf segments of six

genotypes; IS 3566, SPV 475, CSV 13, CSV 15, CAV 112 and IS 348, were investigated. The highest number of

somatic embryos was obtained on MS medium supplemented with 2 mgl -1 2,4,5-T + 1 mgl -1 Zeatin in dark

conditions. Highest frequency of embryogenic callus and somatic embryo formation were observed in IS 3566. The

highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented

with 2.5 mgl -1 TDZ (14 plantlets per segment). Rooting of shoots was noticed on the NAAmedium. A mean of 30 ±

1.05 root intact plantlets was recovered per explant. The rooted plantlets were well accomplished with a survival

frequency of 96%. Moreover, there were no phenotypic differences observed between the in vitro regenerated and i n

v i v o plantlets.

Key Words: S o rghum bicolor, leaf, 2,4,5-T, somatic embryogenesis, TDZ, plantlet regeneration

Sorghum bicolor L. Moench yaprak segmentlerinden somatik embriyogenez ve bitki

rejenerasyonu

Özet

Sorghum (Sorghum bicolor (L.) Moench) yaprak disklerinden h›zl› bitki rejenerasyon sistemi gelifltirildi. Somatik

embriyo oluflumunu ve yaprak parçalar›n›n rejenerasyon kapasitesini etkileyen faktörler alt› genotipte; IS 3566, SPV

475, CSV 13, CSV 15, CAV 112 ve IS 348, araflt›r›ld›. En fazla somatik embriyo karanl›k koflulda 2 mgl -1 2,4,5-T +

1 mgl -1 Zeatin ilave edilmifl MS besiyerinde elde edildi. En fazla embriyonik kallus ve somatik embriyo oluflum

s›kl›¤› IS 3566’da gözlendi. En fazla bitki rejenerasyonu embriyonik kalluslar›n 2.5 mgl -1 TDZ ilave edilmifl

rejenerasyon besiyerine aktar›lmas›ndan sonra elde edildi (her bir parçada 14 bitki). Sürgün köklenmesinin NAA

besiyerinde oldu¤u dikkat çekmifltir. Her bir eksplantta köklenen bitki ortalamas› 30 ± 1.05 dir. Köklenen bitkilerde

sa¤kal›m s›kl›¤› %96 olarak belirlendi. Ayr›ca, in vivo bitkiler ve in vitro rejenere olanlar aras›nda fenotipik

farkl›l›klar gözlenmedi.

Anahtar Sözcükler: S o rghum bicolor, yaprak, 2,4,5-T, somotik embriyogenez, TDZ, bitki rejenerasyonu

99


100 Sudhakara Rao Pola and N. Sarada Mani

Introduction

S o rghum [S o rghum bicolor (L.) Moench] a tropical

plant belonging to the family of Poaceae, is one of the

most significant crops in Asia, Africa and Latin

America. S o rg h u m is the fifth most important cereal

crop after wheat, rice, maize, and barley in terms of

production (Dicko et al., 2006, FAO 2005). In India,

S o rg h u m is the fourth important cereal crop after

wheat, rice, and maize and is the third important cereal

for consumption after rice and wheat (Kishore et al.,

2006). Genetic improvement of S o rg h u m crop plants

can be achieved by transferring useful alleles at

existing loci through conventional breeding or by

adding new loci across diverse sources through

genetic transformation (Mifflin 2002). As for most

plant species, improvement of S o rg h u m v i a

biotechnology depends on improved tissue culture

responses, especially in plant regeneration, and a

successful scheme to introduce useful transgene.

S o rg h u m is considered to be one of the most

recalcitrant species for in vitro response (Manjula e t

a l., 2000, Hagio et al., 2002, Harshavardhan et al.,

2002, Jeoung et al., 2002, Chandrakanth et al., 2002,

Visarada et al., 2003, Gao et al., 2005 and Kishore

et al. 2006). Moreover, for the initial materials to

produce callus and plants, the most important factor is

that the material used can be obtained in any season, at

any time, and be easy and efficient for regeneration.

Some materials used to produce callus have been

successful, e.g. mature seeds, immature seeds or

embryos (Fromm et al., 1990; Vasil et al., 1992;

Akashi et al., 1992). However, all of these materials

are reproductive organs, so they are limited to

collection in certain seasons. In this regard, the

objectives of this study were to establish a simple and

efficient plant regeneration system from leaf tissue of

"S o rghum bicolor, which can be used as source

material for in vitro culture in any season and at any

time.

Previously several authors have studied the

somatic embryogenesis in leaf explants. Wernicke and

Brettell (1982) and Cai et al., (1987) reported

morphogenesis and callus initiation from cultured leaf

tissues, Bhaskaran and Smith (1989) observed the

control of morphogenesis in S o rg h u m by 2,4-D and

cytokinins in the leaf segment derived cultures.

Elkonin et al., (1993) cultured 15-60 mm size leaf

segments on 0.5 mgl -1 2,4-D and 1.0 mgl -1 BAP. A high

frequency of regeneration in callus cultures derived

from leaf tissues were reported by Elkonin et al.,

(1994). Plant regeneration from leaf sheath cultures of

some rabi S o rg h u m cultivars were reported by Patil

and Kuruvinashetti (1998). A simple and reproducible

protocol for callus induction and regeneration of

plantlets from leaf base cultures of S o rg h u m b i c o l o r

(296 B and RS 585) has been developed by Archana

and Paramjit (2003). Anju and Anandakumar (2005)

reported efficient regeneration from leaf base as an

source explant. In the majority of these cases, plant

regeneration takes place by means of somatic

embryogenesis. On the other hand, the rate of plant

regeneration per explant is not satisfactorily high to be

useful. Difficulties still exist in establishing and

propagating regenerable cultures for long periods, for

that reason it is essential to develop an efficient and

reproducible procedure for plant regeneration in vitro.

The aim of this study was to determine the most

favorable conditions for embryogenic callus initiation,

its development and improving its regeneration

e ff i c i e n c y, and categorize the S o rg h u m g e n o t y p e s

having enhanced in vitro response.

Materials and methods

G rowth conditions

Seeds of S o rghum (Sorghum bicolor (L.) Moench )

varieties viz., IS 3566, SPV 475, CSV 13, CSV 15,

C AV 112 and IS 348 were procured from the

I C R I S AT, Patancheru, India. Seeds were surface

sterilized with 3% sodium hypochlorite for 45 min,

rinsed three times with sterile distilled water, and

inoculated on MS (Murashige and Skoog, 1962)

medium supplemented with 0.8% bacteriological

grade agar. Seedlings were allowed to grow under a

daily schedule of 16 h light and 8h dark. Light was

provided by fluorescent lamps (Philips, TL40W/54) at

an irradiance of 5.27 Wm 2 and temperature maintained

at 25±2 0 C and 23±2 0 C during light and dark phase,

respectively (Pola 2005). Data represents the mean of

2 to 4 independent experiments, each consisting of 25

explants.

Callus induction

To determine the effects of hormones on callus

formation, approximately 25 explants were aseptically

collected from the leaf. The explants were placed on

MS medium for callus formation with upper surface in


Table 1. E ffect of different P.G.R. s on embryogenic callus induction from leaf segments*

contact with the callus induction medium, 25-30 ml of

solid agar medium containing 3%sucrose, 2,4-D (2,4-

Dichlorophenoxyacetic acid), 2,4,5-T ( 2 , 4 , 5 -

trichlorophenoxyacetic acid), IBA(Indole-3-butyric

acid), IAA (Indole-3-acetic acid ), NAA ( 1 -

naphthylacetic acid), Kinetin (6-furfurylaminopurine),

and Zeatin (4-hydroxy-3-methyl-trans-2-butenylamino

purine) either alone or in combination at various

concentrations from 0.2 to 3.0 mgl-1 , depend upon the

- 1

P.G.R (plant growth hormone) type, 200 mgl

Somatic embryogenesis in Sorghum bicolor 101

Concentration of P.G.R mgl -1 Explants with Embryogenic callus

2,4-D 2,4,5-T IBA IAA NAA Kinetin Zeatin E. callus frequency %

1.0 - -

1.5 6 24

2.0 8 32

2.5 8 32

3.0 6 24

1.0 10 40

1.5 16 64

2.0 18 72

2.5 14 56

3.0 12 48

1.0 - -

1.5 - -

2.0 8 32

2.5 8 32

3.0 6 24

1.0 - -

1.5 - -

2.0 6 24

2.5 8 32

3.0 6 24

1.0 8 32

1.5 10 40

2.0 10 40

2.5 8 32

3.0 8 32

0.2 - -

0.5 - -

1.0 6 24

1.5 6 24

2.0 - -

0.2 6 24

0.5 10 40

1.0 14 56

1.5 10 40

2.0 8 32

*25 explants per treatment, each treatment repeated thrice

asparagine and proline were used in the culture

medium (Mani and Sudhakar 2003). Inoculated sterile

Petri dishes were incubated for one week at 25±2 0 C in

continuous darkness, until the beginning of the

embryogenesis, tissue remained on callus initiation

medium, when embryogenic tissue found, and the calli

were transferred to a callus maintenance and/or

maturation medium. Subsequently the somatic

embryos produced were transferred to regeneration

medium.


102 Sudhakara Rao Pola and N. Sarada Mani

Table 2. Comparative effect of 2,4,5-T and Zeatin and their combination in callus induction frequency from leaf explants*

Genotype P.G.R con. mgl -1 Explants with calli E.C Frequency %

IS 3566 2,4,5-T 2 mgl -1 18 72

F requency of embryogenic calli

Frequency of embryogenic calli (%) was calculated as

the number of segments bearing regions of

embryogenic calli.

R e g e n e r a t i o n

For regeneration of plantlets, the MS medium was

supplemented with 0.5 -3 mgl - 1 of BAP ( 6 -

benzylaminopurine), Kinetin, zeatin, GA 3 (Gibberellic

acid) and TDZ (Thidiazuron) either alone or in

combination. Cultures incubated in continuous cool

white fluorescent light at 25 µ mol m -2 S -1 (photon flux

density = 33.3 μ mol m -2 s -1 , 16 h) at 25±2 0 C (Pola

2005). All tissue culture conditions were performed

under aseptic conditions. All cultures were transferred

to fresh medium every twenty-one days except where

changes were found necessary due to repeated

1.0 mgl-1 zeatin 14 56

2,4,5-T 2 mgl -1 +1.0 mgl -1 zeatin 20 80

SPV 475 2,4,5-T 2 mgl -1 18 72

1.0 mgl-1 zeatin 14 56

2,4,5-T 2 mgl -1 +1.0 mgl -1 zeatin 20 80

CSV 13 2,4-T 2 mgl -1 16 64

1.0 mgl-1 zeatin 12 48

2,4,5-T 2 mgl -1 +1.0 mgl -1 zeatin 18 72

CSV 15 2,4,5-T 2 mgl -1 14 56

1.0 mgl -1 zeatin 12 48

2,4,5-T 2 mgl -1 +1.0 mgl -1 zeatin 16 64

CSV 112 2,4,5-T 2 mgl -1 8 32

1.0 mgl-1 zeatin 8 32

2,4,5-T 2 mgl -1 +1.0 mgl -1 zeatin 10 40

IS 348 2,4,5-T 2 mgl -1 8 32

1.0 mgl -1 zeatin 8 32

2,4,5-T 2 mgl -1 + 0.5 mgl -1 zeatin 12 48

*25 explants per treatment, each treatment repeated thrice

formation of phenolics or medium contamination.

Shoots and leaves formed from different globular

somatic embryos regenerated into young plantlets on

cytokinin medium. The shoots were removed from the

cultures and rooted on hormone-free or MS+NAA

medium. These plantlets were finally transformed to

the soil through a gradual acclimatization process.

The rooted plants were potted in washed sand and

covered with sealed plastic vinyl bags to keep full

humidity at 25 °C in light condition (photon flux

density = 33.3 μ mol m -2 s -1 , 16 h). As the plants grew

vigorous, the bags were poked with chopsticks to

allow air enter inside the bags until to the plants selfsupported.


Table 3. E ffect of P.G.R s on regeneration from leaf segments*

Results

Callus initiation

Callus initiation was observed from the cut ends of the

leaf discs on 12 th day after inoculation. In the present

study, the structure of callus was dependent on the

variety and plant growth hormone used and it ranged

from hard, nodular, and yellowish to soft, watery,

loose, friable, creamy white to translucent. Yellowish

and crumby calli originated on the medium with 2,4-D

and white, compact and intensively growing and the

formation of somatic embryos was observed on the

medium with 2,4,5-T.

The nature of callus after 18 days was compact,

hard, white and the formation of somatic embryos was

observed as well. We found out that the frequency of

callus induction varied from 16-80% in dependence on

Somatic embryogenesis in Sorghum bicolor 103

Concentration of P.G.R mgl -1 Explants responded

BAP Kinetin Zeatin TDZ GA3 No. of shoots No. of roots

1.0 - -

1.5 4.88 16.6

2.0 6.34 18.3

2.5 8.82 18.7

3.0 6.63 16.24

1.0 -

1.5 - -

2.0 - -

2.5 3.93 16.4

3.0 6.4 17.82

1.0 8.26 16.8

1.5 10.82 23.82

2.0 - -

2.5 - -

3.0 - -

1.0 4.86 16.4

1.5 6.84 17.8

2.0 9.84 11.4

2.5 14 35.6

3.0 12.42 12.8

1.0 - -

1.5 - -

2.0 - -

2.5 - -

3.0 - -

*25 explants per treatment, each treatment repeated thrice

the genotype and auxin used. In callus induction

medium, when single auxin used in the medium only

72% response was observed (Table 1), but

combination of 2 mgl -1 2,4,5-T with 1.0 mgl -1 zeatin,

the callus induction frequency increased upto 80%

(Table 2).

Six varieties were tested in this experiment, variety

IS 3566 and SPV 475 gave good response. Among the

different auxins used in this experiment only 2,4,5-T

and 2,4-D were able to produce high frequency of

somatic embryos. However, IBA, IAA and NAA

produced callus but the frequency was inadequate.

Despite the fact that, zeatin and kinetin are cytokinins,

they also induced callus initiation from the leaf tissue

in the present study.


104 Sudhakara Rao Pola and N. Sarada Mani

Figure 1. Average number of shoots in different varieties Figure 2. Average number of roots in different varieties

Plant re g e n e r a t i o n

Different cytokinins were investigated on regeneration

medium. They were BAP, kinetin, zeatin, TDZ and

GA 3 (1.0-3.0 mgl -1 ) (Table 3). Plant regeneration was

observed in all regeneration media. Numerous green

spots appeared over the surface of embryogenic calli

within 12-14 days followed by the regeneration of

shoots. The highest plant regeneration was obtained in

regeneration medium containing MS + 2.5 mgl -1 TDZ

(14 shoots per explant) (Fig.1)) and followed by

regeneration medium containing MS + 3 mgl -1 TDZ

(12.42 shoots per explant). Kinetin with regeneration

Table 4. A N O VA test on shoot induction between genotypes

Source of Variation SS d.f. MS F

Between genotypes 380 5 240 32.6*

error 2.26 294 1.813

total 382.26 299

*significant at p=0.001.

Table 5. A N O VA test on root induction between genotypes

Source of Variation SS d.f. MS F

Between genotypes 2731 5 630.25 140.2*

error 2.742 294 2.814

total 2733.742 299

*significant at p=0.001.

capacity of 3-6 shoots per explant performed the

lowest plant regeneration. Based on the results

obtained from this study, plant regeneration generally

follows the somatic embryogenesis pathway.

Six varieties were employed in our study somatic

embryo formation and plantlet regeneration was

observed in all genotypes. Genotype IS 3566 produced

average 14 shoots per explant used. On the other

hand, genotype SPV 475 produced 13 shoots per

explant used, where as CSV13, CSV15, CSV 112 and

IS 348 which was lower response than IS 3566 variety.

According to the table 4, showing the analysis of

variance, the variability of a no. of regenerants was

significantly influenced by genotypes. Interactions of

the genotypes were statistically significant at p=0.001

(f value is 32.6). Shoots after attaining 3cm height,

were transferred on to rooting medium containing 1

mgl -1 NAA. Root number was highest in IS 3566 (36

roots) and low in IS 348 (17) (Fig.2). The root number

was subjected to analysis of variance test and was

significantly influenced by genotypes. Interactions of

the genotypes were statistically significant at p=0.001

(f value is 140.2) (Table-5).

When the regenerated plantlets had well developed

root system. They were transferred to soil in growth

c h a m b e r. Plants regenerated from leaf segments

grown as normal plants. No albino plant was observed

in vitro originated plants. The regenerated plants

appeared normally and uniform in their growth in the

green house, plants did not reveal any variation.


Figure 3. Different stages of plantlet regeneration from leaf explants of S o rghum bicolor.

Legends of Figure 3.

A. Callus initiation from cut ends of leaf explants

B. Well developed embryogenic callus on 2 mgl mg- 1 2 , 4 , 5 - T m e d i u m

C. Shoot initiation on 2.5 mgl mg- 1 TDZ medium

D. Shoot initiation after 2 weeks of subculture

E. Shoot development on 2.5 mgl mg- 1 TDZ medium

F. Nine week old regenerated plants in Baby Jar

G. Twelve week old regenerated plants in Baby Jar

H. Well developed shoots and root initiation on 1 mgl mg- 1 N A A medium

I. Well developed plantlets with prominent shoot and root system

Somatic embryogenesis in Sorghum bicolor 105


106 Sudhakara Rao Pola and N. Sarada Mani

Discussion

Kresovich et al., (1987) have made detailed study on

application of cell and tissue culture techniques for the

genetic improvement of S o rg h u m. They revealed that,

reports of plant regeneration from S o rg h u m tissue

cultures are limited due to the phenol secretion in the

cultures. They also reported that, an efficient method

of plant regeneration is necessary for the new genesplicing

and genetic manipulation technology to be

applicable in a crop improvement program. Isenhour

et al., (1991) found that tissue culture derived

S o rg h u m plants exhibits resistance to leaf-feeding by

the fall Armyworm. They reported that, tissue culture

induced variations can be a viable means of generating

new sources of genetic diversity for use in crop

improvement. The findings of Miller et al., (1992) are

another indication that tissue culture induced

variations can be a viable means of generating new

sources of genetic diversity for their applications in

crop improvement. They observed acid soil stress

tolerance in tissue culture derived S o rg h u m lines.

However, the present study fulfills these needs in

some extent.

Leaf segments in cereals serve as an excellent

system to investigate competence of dedifferentiated

cells due to the presence of a basal meristem

(Wernicke and Brettell 1982, Haliloglu 2006). It is

well known that plant growth hormones acting

decisive role in in vitro regeneration (Mahalakshmi e t

a l., 2003, D’Onofrio and Morini 2005, Haliloglu

2006). Our consequences also be evidence for that the

presence of 2,4-D or 2,4,5-T in initiation medium was

important for callus induction and somatic embryo

formation from leaf segments of S o rg h u m. Use of

cytokinins in combination with auxins to induce

somatic embryogenesis in callus cultures has been

reported for cereals (Bhaskaran and Smith 1990,

Gaspar et al. 1996) and Recently, Haliloglu (2006)

reported somatic embryo formation from wheat leaf

base segments, using auxin and cytokinin

combination.

Bhaskaran and Smith (1989) observed the control

of morphogenesis in S o rg h u mby 2,4-D and cytokinins

in the leaf segment derived cultures. Patil and

Kuruvinashetti (1998) reported embryogenic callus

from young leaf sheath segments with MS + 2 mgl -1

2,4-D and 0.5 mgl -1 BAP for regeneration. Archana

and Paramjit (2003) also reported genotypic depended

somatic embryogenesis and regeneration using 2 mgl -1

2,4-D for callus initiation and 0.1 mgl -1 BAP for

regeneration in the in S o rg h u m b i c o l o r genotype 296

B. In this study we observed varietal differences in

both embryogenic callus induction as well as plantlet

regeneration, previously broad range of varietal

differences in callus formation and plant regeneration

was reported by Hagio (1994) he reported that

sustained varietal differences exits in callus formation

and plant regeneration in S o rg h u mas well as in other

major cereals.

Anju and Anandakumar (2005) reported efficient

regeneration from leaf base explants. They reported

that the optimum hormone combination for maximum

regeneration was 2 mgl mg -1 BAP and 0.1 mgl mg -1

kinetin at this combination they obtained 5.40

shoots for each explant.

It can be concluded that, successful S o rg h u m

regeneration efficiency is affected by genotypes,

medium composition including P.G.R s and conditions

of culture. The age of six-eight day old leaf segments,

used by us, proved to be the best for callus induction

and plant regeneration.

Acknowledgements

We are thankful to the UGC-New Delhi, India for

providing the financial support for the present study.

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