The Influence Of Priming Two Cucumber Cultivar Seeds

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J. Duhok Univ. Vol.13, No.1, (Agri. And Vet. Sciences) Pp 112-121, 2010 111 MICROPROPAGATION OF CROTONE (Codiaeum variegatum) MOSLEH M.S. DUHOKY AND LAYLA S. M. AL-MIZORY Dept. of Horticulture, College of Agriculture, University of Duhok, Kurdistan Region-Iraq (Received: June 9, 2010; Accepted for publication: February 14, 2011) ABSTRACT The present study was conducted to investigate the effects of different concentration and combinations of plant growth regulators on callus induction and plant regeneration of croton (Codiaeum variegatum). The leaf segments were used as explant and cultured on MS medium supplemented with different concentrations of NAA or 2,4-D combined with BA for callus induction. The best callus was formed on whole area of explant cultured on MS medium with different levels of BA combined with different concentrations of 2,4-D. MS medium supplemented with 2mgl -1 BA+ 0.2 mgl -1 2,4-D was used for percentage of callus induction (100%) and the highest average callus fresh weight (5.39 mg) was formed under dark condition. The highest percentage of callus induction (80%) and the highest average callus fresh weight (3.91 mg) was obtained under light condition compared with control treatment which did not formed callus. In multiplication stage the the maximum number of shoots, number of leaves / shoot and highest length of shoots (4.58 shoots/culture, 3.13 leaves / shoot and 2.33 cm) was obtained in MS medium supplemented with 2.5mgl-1 BA respectively, compared with all treatments including the treatment control. For shoot multiplication, the maximum (4.53) shoot/ culture were formed on MS medium having 3mgl -1 BA+ 4.5 mgl -1 Kin. At rooting stage, a large number of roots, root length and root percentage (2.67 roots /explant, 3.56 cm and 70%) respectively, was observed when 0.5 mgl -1 IBA was used in ¼ MS medium. KEYWORDS:- Somatic embryogenesiss, Codiaeum variegatum, Auxin and Callus induction C INTRODUCTION odiaeum variegatum, commonly known as Croton and sometimes called Joseph's Coat (Deshmukh and Borle, 1975; Kupchan et al., 1976), belongs to the family Euphorbiaceae, is one of the most popular ornamental plants because of vivid foliage colors and varied leaf shapes. Codiaeum variegatum is native to Indonesia, Malaysia, Philippines, India, Thailand and Sri Lanka. It is an evergreen shrub, up to 6 m in height but usually maintained at 60-90 cm and grows well in areas having humid climate. More than 200 varieties of croton exist on the globe, available in different leaf sizes, shapes and color patterns. Young leaves are usually green, bronze, yellow, or red, later changes to gold, cream, white, scarlet, pink, maroon, purple, black or brown (Fig 1). Croton can be propagated by various methods such as cuttings, grafting, seeds and air layering. From shoot tip cuttings, one mother/stock plant can yield only 20 plants per year (Nasib et al., 2008). Due to its slow rate of conventional multiplication, the plant is very high in demand. Micropropagation is a relatively new technology and applications of innovative methods have served to overcome barriers to progress in the multiplication of elite species and further improvements are anticipated. In vitro growth and development is considerably influenced by several factors like genotype, the age and size of mother plant and explant, the season, growth conditions, media composition, and various other physiological factors. As a mean of securing pathogen free plants, culture of shoot apical meristem is ideal. Other advantages that assure by this method include large number of plant production in shorter time period, irrespective of the season (Mulabagal and Tsay, 2004). Croton was chosen for micropropagation due to its rare success in conventional breeding and very little data is available for its in vitro production (Shibata et al., 1996 and Orlikowska et al., 1995; 2000). The objective of the present study is to develop an alternative micropropagation of croton (Codiaeum variegatum) through indirect somatic embryogenesis, using leaf as explant.
J. Duhok Univ. Vol.13, No.1, (Agri. And Vet. Sciences) Pp 112-121, 2010 Fig. (1): Croton shrub is one of the ornamental plants(Codiaeum variegatum). MATERIALS AND METHODS Pot croton (Codiaeum variegatum) was grown in the greenhouse of Horticulture Department, Agriculture College, University of Duhok at 15 \ 9\ 2008. Leaves with petioles were collected, and the petioles were removed after leaves washed under tap water for 30 minutes to remove soil and other superficial contaminations. The leaves were cut into 1 cm 2 in size under sterilized condition (Fig 2). The explants were surface sterilized with 70% ethanol for 3 minutes in a laminar air flow cabinet followed by immersion in a 4 % sodium hypochlorite solution (5% active chlorine) for 10 minutes and rinsed three times with sterile distilled water, and then cultured on MS medium (Murashige and Skoog , 1962). The medium was supplemented with 30 gl -1 sucrose and solidified with 7 gl -1 agar. The pH of the medium was adjusted to 5.7± 0.1 befor autoclaving. First experiment: The following growth regulators were added to the medium . BA (Benzyl adenine) at (0, 1, 2) mgl -1 . NAA (Naphthalene acetic acid) at (0, 0.2, 0.4) mgl -1 . 2,4-D (2,4-Dichlorophenoxy acetic acid) at (0, 0.2, 0.4) mgl -1 . Each treatment was replicated 10 times with 1 explant. The explants were cultured in growth room at 25°C in the dark to encourage callus formation or in the light condition for callus growth. When the calli were one month old, they were transferred to a growth chamber and maintained under a 16 hour photoperiod and 8 hour darkness under artificial light at an intensity of 1000 lux. Shoot regeneration was encouraged by 50 mgl -1 casein hydrolysis and 1 mgl -1 BA added to the medium. After 6-8 weeks, the following data were recorded: percentage of callus induction, average callus fresh weight (mg) and embryo induction percentage. All data were statistically carried out using Complete Randomized Design (CRD) using 10 replications. Significant differences among mean values were separated using Duncan Multiple Range Tests at P≤0.05 (Duncan, 1955). Second experiment: After somatic embryo formation from the callus. The embryogenic callus was weekly subcultured on MS medium for accelerating the induction of embryoids, with the removal of dead and dark-brown cells from the callus. After 6 weeks of subculture, greenish- yellow, globular pro- embryoids were observed under microscopic examination and these were selected and subcultured on the same medium for maturation according to Robinson et. al.(2009) and Buchheim et. al.(1989). Matured somatic embryos (heart- and torpedo shaped) were transferred to somatic embryo regeneration medium containing MS salts; After the complete regeneration of embryoids to plantlets the latter were transferred to the third experiment. After 4- 6 weeks ,the following data were recorded: No.of somatic embryo induced from callus, No. of shoot development, No. of leaves per culture and average of shoots length (cm). Third experiment: The explants were allowed to grow and then transferred to MS medium with different concentrations of cytokinin (0, 2.5, 3.0, 3.5, 4, 4.5 and 5) mgl -1 BA combine with (0, 2.5, 3.0, 3.5, 4, 4.5 and 5) mgl -1 Kin. After 4-6 weeks ,the following data were recorded: No. of shoots per culture, No. of leaves per culture and average of shoots length (cm). Fourth experiment: Shoots were transferred to (¼ and ½ strength) MS medium with different concentrations (0, 0.5 and 1) mgl -1 of NAA, IAA and IBA alone for in vitro rooting. After 4 weeks ,the following data were recorded: percentage of root per culture, No. of roots per culture and the average of roots length (cm). For acclimatization, rooted plants of croton with well developed roots and leaves were taken out of the culture jars washed with water to remove the agar around the roots. The plantlets were transferred to plastic pots containing soil mixture and peat moss (1 :1 by volume). The percentage survived plant were recorded after 4 weeks from transplanting. 111
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The Influence Of Priming Two Cucumber Cultivar Seeds

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