Establishment Of High Efficient Regeneration System And In Vitro Selection For Salt-Tolerant Callus In Diploid Potato

At present, progress in potato breeding is slow and this may be due to its tetrasomic inheritance and high heterogeneity. However, cross-breeding is still the most widely used breeding method. As a result of the narrow genetic base and inbreeding phenomenon in the common potato (Solanum tuberosum L.), it is difficult to develop varieties with breakthrough traits. Therefore, it is of importance to establish high efficient regeneration system and in vitro selection of salt-tolerant callus in diploid potato for improving potato varieties.In this research, in vitro plantlets of eight long-day adapted diploid potato hybrid clones of Solanum phureja and S. stenotomum were used as plant materials, and leaves and stems were taken as explants, respectively, to study callus induction, adventitious bud differentiation and selection of salt-tolerant callus using MS+3%sucrose+0.8%agar as basic medium. High efficient regeneration system of leaves and stems in diploid potato was established, influence of NaCl on callus induction and growth was studied and the relative growth of salt-tolerant callus in different screening methods was compared. The main results were as following:1. In analysis of variance, highly significant difference was found in diploid clones, treatments and diploid clone x treatment interaction for leaf and stem callus induction, suggesting that there is much great effect of clone and treatment and interaction on leaf and stem callus induction, and that the optimal medium is genotype dependent for callus induction on leaf and stem.2. Based on the induction rate and growth status, the optimal induction medium for leaf segments’ callus of472-1and566-1was MS+6-BA2.0mg/L+2,4-D1.0mg/L+3%sucrose+0.8%agar; the optimal medium for leaf callus of267-1and270-2was MS+6-BA1.0mg/L+2,4-D2.0mg/L+3%sucrose+0.8%agar; the optimal medium for leaf callus of J188-1was MS+6-BA2.0mg/L+2,4-D2.0mg/L+3%sucrose+0.8%ager; the optimal medium for leaf callus of354-1was MS+6-BA2.5mg/L+NAA0.1mg/L+3%sucrose+0.8%agar. The optimal induction medium for stem segments’ callus of472-1and566-1was MS+6-BA2.0mg/L+2,4-D1.0mg/L+3%sucrose+0.8%agar, the optimal medium for stem callus of270-2was MS+6-BA1.5mg/L+NAA0.2mg/L+3%sucrose+0.8%agar; the optimal medium for stem callus of354-1was MS+6-BA2.5mg/L+NAA0.1mg/L+3%sucrose+0.8%age. The results also showed that NAA may cause rooting in the process of callus induction, but2,4-D can ease the rooting situation.3. Results in orthogonal experiments indicated that order of the effect of three hormones on adventitious bud differentiation of472-1leaf callus was GA3>6-BA>IAA; the order of the effect of three hormones on adventitious bud differentiation of566-1leaf callus was6-BA>GA3=IAA; the order of the effect of three hormones on adventitious bud differentiation of472-1stem callus was6-BA3> IAA> GA; the order of the effect of three hormones on adventitious bud differentiation of566-1stem callus was6-BA>GA3>IAA. Thus the concentration of6-BA and GA3was the key factor to improve induction rate on callus adventitious bud differentiation.4. Based on the days needed for sprouting from callus and induction rate and growth status, the optimal medium for adventitious bud differentiation of472-1leaf callus was MS+6-BA1.0mg/L+IAA0.1mg/L+GA32.5mg/L; for adventitious bud differentiation of472-1stem callus was MS+6-BA1.0mg/L+GA31.0mg/L; and for adventitious bud differentiation of566-1stem callus was MS+6-BA5.0mg/L+IAA0.1mgL/L+GA35.0mg/L. The results also indicated that it was easy for callus on stem segments to differentiate into adventitious buds as compared with callus formed on leaf discs.5. NaC1had remarkable inhibitory action in the process of callus induction and the growth. There were large differences in salt-tolerance among callus of different genotypes and explants. When callus was induced directly through high level NaCl from explants, leaf and stem of472-1were tolerant to160mmol/L and200mmol/L, and leaf and stem of566-1were tolerant to160mmol/L and120mmol/L, respectively. When callus was stressed on media containing NaCl, leaf and stem of472-1were tolerant to240mmol/L, and leaf and stem of566-1were tolerant to320mmol/L and160mmol/L, respectively. Salt-tolerant callus induced directly with high level of NaCl from leaf and stem has lower salt-tolerance as compared with salt-tolerance of callus, which was the initial material for salt-tolerant callus induction. When callus was selected against NaCl, salt-tolerance of callus selected with different methods was different. Salt-tolerant callus selected with progressive method could endure higher level of NaCl.