Induction Of Adventitious Buds In Phaseolus Vulgaris Recalcitrant To Regeneration

Plant regeneration technology was the basis of genetic transformation, it was aslo widely applied to agricultural production, plant protection and so on. But different kinds of plants had different capacity of regeneration. Some plants were difficult to be regenerated, the Phaseolus vulgaris L. was one of them. A protocol to induce adventitious buds of Phaseolus vulgaris L. in vitro was developed. The main results were as follows:First of all, the Phaseolus vulgaris hypocotyls were used as explants, the effects of auxin (2,4-D, NAA, IBA) and cytokinin (6-BA, TDZ, KT) on callus induction and differentiation were investigated. The callus can not be induced by Murashige and Skoog medium (MS). The calli with maximum fresh weight (1.0322±0.0386 g) and dry weight (0.0868±0.0016 g) were induced when hypocotyl explants were cultured on MS supplemented with 1.5 ?mol·L-1NAA and 1 ?mol L-1TDZ. But all hypocotyl calli appeared to brown,these calli were incompact, the callus failed to induced adventitious bud. And then we attempted to use the medium with activated carbon, silver nitrate,and ascorbic acid to reduce callus browning. The results showed that these methods failed to reduce callus browning.Because the hypocotyl calli appeared to brown, and these calli was unable to be induced adventitious bud, so we used root segments as explants. The effects of auxin(2,4-D, NAA, IBA) and cytokinin (6-BA, TDZ, KT) on callus induction and differentiation were investigated. The callus can not be induced by MS. The best medium for root callus induction was MS+1 ?mol·L-12,4-D+10 ?mol·L-1TDZ, these calli had the maximum fresh weight (0.2971±0.0189 g) and dry weight (0.0317±0.0017 g), all calli were pale green, these calli were compact, but there was no adventitious bud.In order to obtain adventitious bud, the calli induced by MS+1 ?mol·L-12,4-D+10?mol·L-1TDZ were used as experiment materials, the effects of auxin (NAA, IBA) and cytokinin (6-BA, TDZ, KT) on callus induction and differentiation were studied. The results showed that the calli which were cultured on MS supplemented with IBA and TDZ had the maximum fresh weight and dry weight, but these calli appeared to brown and had incompact structure. Then, the effects of one kind of auxin (NAA, IBA) (1?mol·L-1) and two kinds of cytokinins (6-BA, TDZ, KT) on callus subculture and differentiation were studied. The results proved that too much kind of cytokinins were bad for callus subculture, the callus appeared to brown.Based on the above research and comprehensive analysis of relevant literature, the Phaseolus vulgaris epicotyl slices were used as explants,B5 liquid culture medium was used as basal medium. The effects of different concentrations of 6-BA or TDZ on callus induction and differentiation were explored. The results showed that the best medium for adventitious bud induction was B5+15 ?mol L-1TDZ,the induction rate of adventitious bud was 25%.This protocol for adventitious bud induction of Phaseolus vulgaris L. could provide reference to other plants regeneration.