In vitro Multiple Shoot Induction in Soybean

Abstract

Genetic transformation is a crucial tool for cultivar improvement; however, soybean is regarded as highly recalcitrant and the success of transformation is affected by the explant source and the rate of in vitro shoot regeneration. Multiple shoot induction on soybean (Glycine max L.) cultivar LS 677 was carried out using single and double coty-node explants from soybean seedlings derived from seeds germinated on Murashige and Skoog (MS) culture media containing different concentrations of N 6 -benzyladenine (BA) and BA in combination with kinetin (KIN). Seedlings obtained from cytokinin treatments (2-4 mg L -1 BA) were stronger with thick hypocotyls, reduced epicotyls and primary roots with few or no lateral root formation. These seedlings showed a better potential as a source for excision of explants than the thin, elongated control seedlings grown on MS medium without cytokinins. The highest number of multiple shoots, from both explant types, was obtained on MS media supplemented with 1.57 and 2.00 mg L -1 BA. The induced shoots were efficiently elongated and rooted on MS medium without plant growth regulators (PGRs). Rooted plantlets were acclimatised under controlled conditions with 70% success and produced viable seeds. The results of this study indicate that the soybean double coty-node explants derived from cytokinin treated seedlings could be a better explant source for in vitro shoot multiplication than the single coty-node explants.

The Influence of Seed Viability on the Germination and In Vitro Multiple Shoot Regeneration of Soybean (Glycine max L.)

“…The jar was immediately closed tightly, and the seeds were surface-sterilised with the liberated chlorine gas for 16 h. Sterilised seeds were inoculated on Murashige and Skoog (MS) basal culture medium for germination and seedling development. The MS culture medium was prepared as described by Pierik [16], modified with 2.00 mg L −1 6-benzylaminopurine (6-BA) as reported by Mangena et al [4]. Other seeds were inoculated on MS basal culture medium without 6-BA, as a control.…”

“…Such protocols have been applied in the attempt to improve soybean growth under biotic and abiotic stress. Multiple shoot induction using immature embryos [1], cotyledonary node explants with or without axillary buds [2], shoot organogenesis on mature and immature cotyledons [3] and the use of double cotyledonary node explants prepared from 10-day-old seedlings [4] have been established for in vitro regeneration. However, due to the myriad of constraining factors involving both tissue culture conditions and gene manipulation, these advances still require optimisation.…”

Challenges of In Vitro and In Vivo Agrobacterium-Mediated Genetic Transformation in Soybean

“…The use of chlorine gas proved effective for surface sterilisation of soybean seeds in our studies [39]. Other sterilising methods include chemical sterilisation of the plant material using 70% alcohol for a few seconds, and 1% sodium hypochlorite (NaClO) containing few drops of Tween 20 for 10-30 min.…”

“…They are prepared by not splitting evenly the cotyledons and still contain meristematic tissues as for the single cotyledonary nodes. Soybean cotyledonary nodes obtained from matured 10-day-old seedlings developed on Murashige and Skoog [44] culture medium supplemented with 2.0 mg/l 6-benzylaminopurine (BA) showed high shoot multiplication [39]. Shoot regeneration can be improved by the development of explant source in soybean transformation from BA pretreated seeds.…”

“…Shoot regeneration can be improved by the development of explant source in soybean transformation from BA pretreated seeds. However, the advantage of using double coty-node or single coty-node explants is the efficient proliferation of higher shoot numbers [39].…”