Establishment of Tissue Culture Regeneration System for Medicago ruthenica L. cv. ‘Zhilixing’

Abstract

Background: Medicago ruthenica L. ‘Zhilixing’ is a new variety with superior forage and seed yield compared to the wild type. The cold, drought and salt tolerance of Zhlixing are better than those of alfalfa, suggesting that this variety can serve as a high-quality genetic resource for improving the stress resistance of alfalfa. However, because of the lack of tissue culture regeneration system, it is difficult to perform genetic transformation studies on stress resistance genes. This study aimed to establish an efficient tissue culture regeneration system for Zhilixing variety. Methods: Three types of explants were selected and tested on four types of basal media supplemented with different combinations of auxin and cytokinin for callus induction and differentiation, based on orthogonal tests to select the combinations of auxin and cytokinin suitable for callus induction and differentiation. Two-factor combination method was used to formulate a suitable rooting medium. Result: The hypocotyledonary axis was found to be an excellent explant for callus induction on MS medium. The optimum callus induction medium contained thidiazuron (TDZ, 0.5 mg/L), 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/L) and naphthaleneacetic acid (NAA, 0.5 mg/L) where the callus induction rate was 93.33%. The differentiation medium was supplemented with TDZ (0.75 mg/L), 2,4-D (0.25 mg/L) and 6-benzyladenine (6-BA, 1.5 mg/L) where the differentiation rate was 63.33%. Thidiazuron played the key role in both processes of callus induction and differentiation. Half-strength MS containing 0.1 mg/L of NAA was the most efficient rooting medium.