Sesame Shoot Regeneration-Using Different Combinations of Growth Regulators  

Bangaremma S. Wadeyar , Lokesha R. , Gayatree G. S. , Sharanamma Sharanamma
Plant Tissue Culture and Molecular Laboratory Department of Genetics and Plant Breeding, College of Agriculture, University of Agricultural Sciences, Raichur, India
Author    Correspondence author
Molecular Plant Breeding, 2013, Vol. 4, No. 33   doi: 10.5376/mpb.2013.04.0033
Received: 26 Aug., 2013    Accepted: 11 Oct., 2013    Published: 15 Oct., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Bangaremma et al., Sesame Shoot Regeneration-Using Different Combinations of Growth Regulators, Molecular Plant Breeding, Vol.4, No.33 267-269 (doi: 10.5376/mpb.2013.04.0033)

Sesame (Sesamum indicum L.) an important and oldest oil seed crop known to man still, the regeneration studies remained as a bottleneck. Here an attempt was to develop shoot via tissue culture approaching using hypocotyls callus, for this five genotypes viz GLW (Gulbarga local white), Western-II (W-II), DS-I, E-8 and Tumkur local (TL). Healthy and moderately compact callus was obtained from hypocotyls segments placed in the plane MS media were used as explants. These obtained callus were placed on the media containing TDZ of concentration 25 µM and IAA (2 µM &3 µM). The maximum number of shoot regeneration was in variety DS-I with 83.3% & 75% response achieved on MS media containing TDZ 25 µM and IAA 3 µM, 2 µM respectively, followed by 58% shoot response in variety E8 and TL respectively on MS media containing TDZ 25 µM and IAA 3 µM, 2 µM respectively and the mean shoot regeneration percent was 52.35 and number of shoots pre callus were 1.59. The shoot regeneration will increase the chances of genetic improvement of sesame through biotechnological ways such as genetic transformation and in vitro cell line selections. 
Sesamum indicum; Hypocotyl’s callus; High frequency shoots regeneration; TDZ

Sesame (Sesamum indicum L.) is the oldest oil seed crop known to man (Brar and Ahuja 1979) and believed to have originated from South-Western Africa, nicked as “Queen of oil crops", owing to excellent oil stability due to the presence of natural antioxidants such as sesamolin, sesamin, sesamol and alfa-tocopherol (Brar and Ahuja, 1979). Genetic improvement of sesame through conventional breeding methods is not rapid unlike other crops (Sardana, 1998). Recent advances in tissue culture techniques offer an immense promise for sesame improvement yet it has remained a trance. The successful application of these techniques depends on the callusing ability and shoots regeneration, the former has been achieved by several workers while the later, undeniably, is considered to be a bottleneck (Rao et al., 1997; Taskin and Turgurt, 1997; Kim Young Hee, 2001; Kariyllappa, 2003; Kariyallappa et al., 2003; Shashidhara, 2005). Intriguingly shoot regeneration is a vital step in tissue culture.

Globally, the best-achieved in vitro shoot regeneration in Sesame is placed up to 46 per cent (Japan) and up to 66% in India (Shashidhara, 2005). However, Seo et al (2006) reported high regeneration ability of deembryonated cotyledon explants via adventitious shoot formation. It is sober that in India, the shoot regeneration has been very low (<2%). Hence, it was felt necessary to enhance shoot regeneration of indigenous sesame genotypes which are land races/varieties but popular at farmer’s level. This could become a crucial breakthrough to further advance the genetic improvement of sesame through biotechnological ways like transgenic development or in vitro cell lines selection that are resistant to biotic and abiotic stresses. Hence, an attempt was made to achieve high frequency regeneration of shoots via callus in sesame. The results have been discussed in the light of sesame improvement through biotechnological ways particularly in vitro cell line selections.
Results and Discussion
Shoot regeneration is a vital step in tissue culture attempts in sesame. Indeed, it is found that the shoot induction via callus in sesame crop is a bottleneck (Taskin and Turgut, 1997; Kim 2001; Kariyallappa, 2003). Explants, genotypes and growth regulators are critical factors for shoot organogenesis and somatic embryogenesis (Venkatachalam et al., 1999). A variety of explants were used for organogenesis and regeneration but hypocotyls were found better followed by cotyledon. There are several reports of regeneration obtained from hypocotyls explants either from root or shoot or both (Lee et al., 1988; Shi and Cai, 1989; Batra et al., 1991; Rao and Vaidyanath, 1997; Kim 2001). The highest percentage of shoot regeneration was observed from hypocotyls derived callus on MS with TDZ 25 µM and 3 µM of IAA with 83.3% in variety DS-1 followed by 58% in variety E8 and 50% in variety TL with 53.2% of shoot regeneration and 1.5 mean number of shoots per callus. The shoot regeneration was also observed on MS with TDZ 25 µM and 2 µM of IAA with 75% in variety DS-1 followed by 58% in variety TL and 50% in variety E8 with 51.5% of mean shoot regeneration and 1.6 mean number of shoots per callus (Table 1; Figure 1). TDZ (Thidiazuran), a cytokinin-a synthetic phenylurea, is considered to be one of the most active cytokinins for shoot induction in plant tissue culture (Huetteman and Preece, 1993; Murthy et al., 1998) and known to induce shoot regeneration from different explants of many recalcitrant species (Thomas and Puthur, 2004). Several reports suggested that TDZ results in shoot regeneration better than other cytokinins (Thomas, 2003). TDZ-induced morphogenesis probably depends on the level of endogenous growth regula- tors and TDZ modulates endogenous auxin level. Multiple high frequency shoot induction in several crops by TDZ is known (Kumar et al., 2003). However, the report of its usage in Sesame crop is scares except (Shashidhara, 2005).

Table 1 Shoot regeneration on MS with 25 μM TDZ with variable concentrations of IAA

Figure 1 Shoot regeneration of sesame genotypes on MS with TDZ and Variable IAA

Materials and Methods
Five genotypes/varieties of Sesamum indicum were used for study viz. Tumkur Local (TL) and Gulbarga local white (GLW): the two land races; Western-II (W-II) (released variety from Rajasthan which is moderately tolerant to Alternaria blight and Phyllody), E-8 (white seeded, nationally released variety and most widely grown but susceptible to Alternaria blight and phyllody) and Dharwad Selection-1 (DS-1) (a white seeded variety released from UAS Dharwad, susceptible to Alternaria, phyllody and powdery mildew but resistant to Cerospora leaf spot. (Prakash, 2001). The surface sterilized seeds with 0.05% HgCl2 were directly inoculated on to MS supplemented with the growth regulators (NAA 0.5 mg/L, BAP 1.5 mg/L, Kn 1.5 mg/L) and incubated in the dark and transferred to continuous illuminating light at 1,500 lux in culture room temperature maintained at 27 as per the direct seeding method (Shashidhara, 2005; Lokesha et al., 2007). Callus induction was achieved within 15~17 days from different explants. The callus of hypocotyls region was separated and used for shoot induction studies. Thirtydayold callus were subcultured on MS supplemented with 0.5 mg/L NAA, 1.5 mg/L Kn and 1.5 mg/L BAP. Every fortnight, the callus was subcultured. Light green, healthy and moderately compact callus, subcultured for two cycles, was taken as small pieces of 1.5 cm3 and were inoculated on to MS supplemented with TDZ and variable concentrations of IAA (Ahn et al., 2006) Four callus pieces were inoculated per flask or bottles and were replicated three times for each concentration. All the flasks and bottles were incubated to 55~65 days for shoot regeneration.
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