Effect of explant type and growth regulators on in vitro micropropagation of Begonia rex

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1 International Research Journal of Applied and Basic Sciences. Vol., 3 (4), , 2012 Available online at ISSN X 2012 Effect of explant type and growth regulators on in vitro micropropagation of Begonia rex Kabirnataj Sara 1, Ghasemi Yousef 1*, Nematzadeh Ghorbanali 1, Asgharzadeh Roghayeh 1, Shahin Kaleybar Behzad 1, Yazdani Mohammad 2 1- Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT). 2- PhD Student, Department of Agronomy, Tabriz Branch, Islamic Azad University, Tabriz, Iran. *Corresponding author: ghasemiyousef83@gmail.com ABSTRACT: This research was conducted for optimizing in vitro micropropagation of Begonia Rexas an important ornamental pot plant. Leaf disc and petioleex plants were cultured on Agar solidified MS media in factorial experiment based on complete randomized design consisting 3 levels of BA (0.2, 0.5 and 1 mg/l) in combination with NAA (0.05, 0.2), IBA (0.1, 0.5) and were maintained in a growth room at 25 ± 1ºC with 16 height daily (Approximately 2500 Lux). The highest adventitious shoot regeneration with an average number of 41.6 was obtained from leaf disc explants after 5 weeks culture on MS medium supplemented with 1 mg/l BA and 0.5 mg/l IBA. The lowest adventitious shoots were observed on petioles which were cultured in a medium containing 0.2 mg/l BA and 0.2 mg/l NAA. Medium fortified with 0.2 mg/l GA 3 was selected as the best medium for increasing the length of adventitious shoots. Root induction occurred appropriately in 1/2 MS medium without using any hormones and finally the micro propagated shoots were successfully transferred to soil and adapted with a 95 % surviving rate. Keywords: Begonia, organogenesis, cytokinin, auxin, explant. Introduction Commercial production of ornamental plants is growing these days and the relatedmarket has been developing fast for the two last decades(jain, 2002). Major pot plants such as Ficus, Chrysanthemum, African violet, rose and Spathiphyllum are produced in developing centuries (Anonymous, 2003). Begonia, a genus in the flowering plants family Begoniaceae is one of the ten largest angiosperm genera. Begoniacea family contains 5 Genera and 920 species, majority of that belong to begonia genera and the most famous species is Rex (Gardening Australia, 2008). Planting material of ornamental plants is so important for commercial and international production. High quality of this material is necessary and critical for growers (Chebetet al, 2003). Biotechnology can improve the ornamental propagation for low cost production. Tissue culture method is part of biotechnology that is used for massive propagation especially for horticulture crops and ornamental plants. So many factors such as growth regulator, plant and explants type, environmental condition (temperature, light) influence organogenesis and in vitro multiplication. It seems that among these factors, growth regulators have the most effect on plant in vitro micro propagation (Jain, 2002). Organogenesis and in 2002, Espino et al, 2002). Simmonds and werry (1978) succeed in propagation of Begoniahimallis in liquid culture. Mendi et al (2009) indicated that MS medium containing 2 mg/l BA and 0.5 mg/l NAA was the best medium for Begonia organogenesis. This paper carried out effect of growth regulators like BA as cytokinin source and IBA and NAA as auxin source in organogenesis of begonia leaf and petiole.

2 Materials and methods Plant material and explant sterilization Plant material has been washed under running tap water in 30 minutes for cleaning of dust and then sterilized with 70% ethanol in one minute and 1.5 % sodium Hypochloride including a few drops of tween -20. For 15 minute. After sterilization explants were rinsed in distilled water for three times. Leaf disc were cut in 1 cm 2 and petiole in 1 cm under laminar air flow. Shoot organogenesis For shoot organogenesis, explants were cut and then were cultured in MS basal medium containing BA(6- Benzylaminopurine) with three levels (0.2, o.5, 1 mg/l) in combination with IBA (Indole-butyric acid) with (0.1, 0.5 mg/l) and NAA (0.05, 0.2). After the primary shoot emerging on explants some characteristic like days to shoot formation, shoot number and shoot elongation were measured after 5 weeks. Shoot elongation Adventitious shoots derived from best medium were transferred in five mediums include MS basal medium as control, MS medium with 0.05 and 0.2 mg/l GA, MS with 0.05 and 0.2 mg/l combination with 0.2 mg/l BA. Explants were maintained for 4 weeks in elongation medium and the heights of plants were measured after this time. Root induction 1/2 MS medium contained different concentration of IBA and NAA were selected for root induction. IBA and NAA were used in 0.2, 0.5, 0.75, 1 mg/l separately and after 4 weeks plant was assessed as root numbers and length. Experiment design and Data analysis All investigations were performed as factorial experiments in a completely randomized design with four replications (glass jars) per treatment and 4 explants per replication. Data of each experiment were subjected to analysis of variance (ANOVA) by the General Linear Models procedure using SAS software. Means were compared using the Least Significant Difference (anova) method at P Results and discussion Factors that were studied in this investigation have a good effect on in vitro plant proliferation so that interaction amongex plant, auxin, and cytokine in were significant at P 0.05.According to table (1), explant type without considering growth regulator, illustrated that the numbers of plantlet from disc was more than petiole but length of adventitious shoots from petiole were higher. On the other hand, two types of hormones, Indole-3-butyric (IBA) acid and Naphthalene acetic acid (NAA) combination with cytokinin were investigated on regeneration. Results indicated that auxin type and its concentration had the major effect on regeneration. According to table (2), IBA was better than NAA on regeneration of adventitious shoots and with increasing the concentration of Indole-3-butyric acid from 0.1 to 0.5 mg/l number of shoots were raised but the height of plant in medium containing NAA was higher. Various plants and explants type indicated different responses to type and concentration of auxin in micro propagation. Mandiet al (2009) indicated that in begonia regeneration, between IAA and NAA,1 mg/l NAA with 2 mg/l BA were more effective on regeneration. In investigation of growth regulators effects on regeneration of Gerbera, Kumar and Kanwar (2006) reported that regeneration percentage and plantlet numbers in explants that were cultured in medium containing BA with IBA were more than those of in BA with NAA while in this study IBA was more proper than NAA. Plants reaction to Auxin was different among various plants and each plant needs special auxin and suitable concentration for proper regeneration. Presence of auxin with cytokine in regeneration medium increase regeneration efficiency (Mendi et al, 2009). They reported that at high concentration of Cytokinin, auxin presence had positive effect on regeneration but at lower concentration of of Cytokinin and auxin presence had negative effect on regeneration.

3 Figure 1- A: adventitious shoots regeneration from disc and petiole 5 weeks after culturing B: complete rooted plant after 4 weeks in MS media without hormone Table1: average comprehensive of explant affection on numbers and adventitious branches length of begonia 5 weeks after cultivation. Explant Shoot number Shoot length(cm) Disc 15.6 a 0.42 b Petiole 10 b 0.95 a Table2: average comprehensive of auxin affection on numbers and adventitious branches of begonia 5 weeks after cultivation Auxin(mg.l -1 ) Shoot number Shoot length(cm) IBA: b 0.4 b IBA: a 0.4 b NAA: c 0.27 b NAA: b 1.6 a Benzyl adenine effect on regeneration separately, adventitious shoots numbers were increased by adding benzyl adenine concentration up to 1mg/l while the most adventitious shoots length were observed at 0.2 mg/l (table3). With increasing cell division, cytokinin causes plant growth and development especially when the cytokinin with increasing cell dividing specially when combined with auxin (Pierik, 1997). Benzyl adenine in lower concentration had less effect on apical dominance and so length of shoot increased. Table3.Average comparison of benzyl adenine effect on number and length of regenerated shoots in begonia 5 weeks after culturing 6-Benzylaminopurine(mg.l -1 ) Shoot number Shoot length(cm) c 1.29 a b 0.32 b a 0.45 b According to the table (4), there are differences days to shoot regeneration among different treatments. Disc and petiole explants formed shoots earlier than other treatments so they regenerated 18 days after culturing on medium containing 1 mg/l BA and 0.05 mg/l NAA. On the other hand disc and petiole explants were regenerated 27 days after culturing in medium containing 0.2 BA mg/l and 0.05 mg/l NAA that it shows 11 days delay in regeneration compared with optimum medium. As it is obvious from data, BA increasing concentration up to 1 mg/l accelerate the regeneration time. Cytokinin effect on start time of regeneration was

4 more than explant type. Maximum number of adventitious shoot was recorded in MS medium containing 1 mg/l BA and 0.5 mg/l IBA with the average number of 41.6 in disc segment. In investigation of four different begonia genotype by Espino (2004) it was reported that BA was more effective than kinetin in regeneration treatments (BA with NAA, Kin with NAA). Minimum number of adventitious shoots was recorded with the average number of 6.3 from petiole in the regeneration media containing 0.2 mg/l BA with 0.05 mg/l NAA the longest adventitious shoot from petiole caused by 0.2 mg/l BA and 0.2 mg/l NAA and in discs caused by 1 mg/l NAA. Table 4.Average comparison of cytokinin, auxin and explant on number and length of regenerated begonia 5 weeks after regeneration Explant 6-Benzylaminopurine(mg.l -1 ) Auxin(mg.l -1 ) Days to shoot Shoot number Shoot length(cm) Disc 0.2 IBA: g 0.46 b Disc 0.2 IBA: g 0.26 b Disc 0.2 NAA: g 0.3 b Disc 0.2 NAA: g 0.26 b Disc 0.5 IBA: fg 0.33 b Disc 0.5 IBA: cde 0.53 b Disc 0.5 NAA: cde 0.33 b Disc 0.5 NAA: cde 0.4 b Disc 1 IBA: b 0.63 b Disc 1 IBA: a 0.76 a Disc 1 NAA: cd 0.33 b Disc 1 NAA: b 0.46 b petiole 0.2 IBA: fg 0.3 b Petiole 0.2 IBA: def 0.2 b Petiole 0.2 NAA: g 0.2 b Petiole 0.2 NAA: g 0.8 a Petiole 0.5 IBA: def 0.23 b Petiole 0.5 IBA: g 0.26 b Petiole 0.5 NAA: fg 0.23 b Petiole 0.5 NAA: def 0.26 b Petiole 1 IBA: ef 0.46 b Petiole 1 IBA: de 0.4 b Petiole 1 NAA: fg 0.23 b Petiole 1 NAA: cde 0.36 b Adventitious shoots elongation: plantlets obtained from the best regeneration culture medium were subcultured to 5 different medium with the purpose of elongation and changes were determined 4 weeks later. Among various MS medium, the one containing 0.2 mg/l GA3 had the greatest effect on elongation while 0.05 mg/l gibberellic acid with 0.2 mg/l BA show the minimal effect on it that were smaller compared with the control. It seems that BA hormone in elongation media was limiting factor in adventitious shoot elongation. Gibberellic acid was used to plantlet elongation in different plants. With the addition of 2mg/l GA to MS medium by Saharan (2010), the length of asparagus in vitro plants was increased.

5 Graph1.Effect of differentmedium on length of regenerated shoots 4 weeks later Adventitious shoot rooting: ½ MS medium containing different IBA and NAA acid concentration were used to root inducing. The highest root was obtained in control medium with the average of 4.1 roots in each plantlet and the lowest one was in presence of NAA 1 mg/l with the average of 1.5 roots in each plant. As can be seen, auxin presence is not essential for root inducing in begonia and rooting were occured in auxin free medium. Also GA usage in root elongation media facilitated the formation of adventitious root in auxin free medium. If the plant treats with GA in the long term, cause root inducing (Zryd, 1988), on the other side, increasing the concentrations of Indole-3- butyric acid and NAA will reduce the number of roots. 0.5 mg/l NAA and 0.5 mg/l IBA caused the highest root length and concentration higher than 0.5 mg/l had inhibitory effect on it. Table 5. Average comparison of auxin effect on number and length of begonia root after 4 weeks Hormonal treatment(mg.l -1 ) Root number Root length(cm) a 15.6 abc NAA: b 16.6 ab NAA: b 18.6 a NAA: cd 14 cd NAA:1 1.5 d e IBA: b 13 cde IBA: b 18 a IBA: cd 12.3 de IBA:1 2.1 c 13 cde Plantlet Compatibility: resulted seedlings were transmitted to Perlite and peatmo use ratio of 1to 4 with 70-80% humidity and light condition of 5000 lux and then were compatibilited for 3 weeks. Conclusion Discs had more potential in regeneration in contrast to petioles. In Some other ornamental flowers such as African violet, disc explants were regenerated with high efficiency than petiole. Probably hormonal condition and food reserves of petiole are the most effective factors in increasing the efficiency. Simultaneous use of benzyl adenine with I-butyric acidwas more suitable media in adventitious shoot inducing compared withthe use of benzyl adenine and Naphthalene acetic acid also benzyl adenine to auxin ratio is very important too, as the highest regeneration efficiency was seen when BA concentration was 2 fold as IBA. Applying different combination of auxin and cytokinin hormones and also determine the appropriate concentration and their ratio are essential to increase the regeneration efficiency.

6 In this investigation the highest regeneration efficiency was observed in MS medium containing 1mg/l BA with 0.5mg/l IBA and it was decreased in higher or lower concentrations. In spite of the influence of auxin on rooting, ½ MS medium recorded as the best medium and it was not necessary to use auxin. Root number was reduced with increasing concentration of auxin because roots are more sensitive than leaf and stem segments and its optimal hormone level for growth is less than other parts of plant. Finally compatibility of regenerated plants was done with efficiency of 95% in 70-80% humidity and 5000 lux light condition. Acknowledgments This research was supported by Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT). References Anonymous (2003) Omzettabel Kamerplanten.Vakbal voorde Bloemisterij. 21a: Appelgren M (1991) Regeneration of Begonia hiemalis in vitro. Acta Hortic. 289: Bouman H, De Klerk GJ (2001) Measurement of The extent of somaclonal variation in Begonia plants regenerated under vrious conditions. comparison of three assays, Theor. Appl. Genet. 102(1): Chebet DK, Okeno JA, Mathenge P (2003) Biotechnological approaches to improve horticultural crop production. Acta Hortic. 625: Espino FJ, Linacero R, Rueda J, Vazquez AM (2004) Shoot regeneration in four Begonia genotypes, Biol. Plant. 48(1): Jain SM (2002) Feeding the world with induced mutations and biotechnology. Paper presented at Proceeding International Nuclear Conference 2002 Global trends and Perspectives. Seminar 1: agriculture and bioscience. Bangi, Malaysia: Kishimoto S, Aida R, Shibata M (2002) Agrobacterium tumefaciens mediated transformation of Elatior Begonia (Begonia X Hiemalis Fotsch). Plant Sci. 162(5): Kumar S, Kanwar K S (2006) Regeneration ability of petiole, leaf and petal explants in gerbera cut flower cultures in vitro. Folia Horticulture. 18(2): Mendi Y Y, Curuk P, Kocaman E, Unek C, Eldogan S, Gencel G, Cetiner S (2009) Regeneration of begonia plantlets by direct organogenesis. African Journal of Biotechnology. 8(9): Pierik R L M (1997). In vitro culture of Higher Plants. First edn. Springer. Saharan V (2010) Effect of gibberellic acid combined with saponin on shoot elongation of Asparagus officinalis. Bioloia plantarum. 54 (4): Simmonds J, Werry T (1987) Liquid shake cultures for improved micropropagation of Begonia hiemalis. Hortic Sci. 22: Zrÿd D (1988) Culture de cellules, tissuset organes végétaux. Presses. Polytechniques Romandes Ed.Tech & Doc Diffusion. P: 308.