| Tissue culture is widely used in the product of fruit trees. On the one hand, it provides the ideal receptor materials for genetic transformation. On the other hand, it can short the period of the breed of fruit trees, and breed better cultivars in a shorter time. As one of the most important stone fruit trees, plum (Prunus salicina Lindl) has many characters in traditional breeding, such as long juvenility, high heterozygosity, high rate of pistil abortive, low breeding rate, long generation, and hard work, likes other major fruit trees. At the same time, plum is easy infected by virus. Those facts impose restriction on the production and the development of plum. So, it seems important and urge to establish the in vitro regeneration system, improve the efficiency of the breeding, and improve the cultivars by tissue culture and the transgenic technology.In this study, we selected hypocotyl and epicotyl as the explants of ’Nubiana’, petiole and leaf as the explants of ’Tardicots’, and established the in vitro regeneration system, independently, in addition, the smallRNA of petiole and leaf explants calli of ’Tardicots’ were sequenced by Solexa, and the petiole explant was used as target gene acceptor in transformation pilot test. The main results are as follows:1. The result from the screening test showed:the optimum composition of the initial medium for hypocotyl of ’Nubiana’ was MS+6-BA 0.5 mg/L+IBA 0.05 mg/L+ sucrose 30.0 g/L+agrose 6.0 g/L, pH:6.0; the optimum composition of the medium for the epicotyl of the seedlings of ’Nubiana’ was MS+6-BA 2.0 mg/L+IBA 0.1 mg L+ sucrose 30.0 g/L+agrose 6.0 g/L, pH:6.0; the optimum composition of the medium for the petiole of ’Tardicots’ was M WPM+TDZ 2.0 mg/L+2,4-D 0.2 mg/L+sucrose 30.0 g/L+agrose 7.0 g/L, pH:6.0; the optimum composition of the medium for the leaves of ’Tardicots’was WPM+TDZ 2.0 mg/L+2,4-D 0.2 mg/L+sucrose 30.0 g/L+agrose 7.0 g/L, pH:6.0; The sucrose was the best carbon source for hypocotyl and epicotyl explants of ’Nubiana’ plum, meanwhile, it was the best carbon source for petiole and leaf explants of ’Tardicots’ plum; The test of the successive propagation and the proliferation showed that:the optimum medium for the successive propagation of ’Nubiana’ was MS+6-BA 0.1 mg/L+IBA 0.1 mg/L+Ad 2.0 mg/L+sucrose 30 g/L+agrose 6.0 g/L, pH:6.0; the optimum medium for the conservation of the seedling of’Nubiana’ was MS+6-BA 0.2 mg/L+IBA0.1～0.2 mg/L+Ad 2.0 mg/L+sucrose 30.0 g/L+agrose 6.0 g/L, pH:6.0; the optimum medium for the successive propagation of’Tardicots’was MS+6-BA 0.1 mg/L+IBA 0.1 mg/L+Ad 2.0 mg/L+sucrose 30.0 g/L+agrose 6.0 g/L, pH:6.0; The optimum medium for the rooting culture of ’Nubiana’ was 1/2WPM+IBA 0.6 mg/L+ sucrose 30 g/L+agrose 6.0 g/L, pH:6.0; the optimum medium for the rooting culture of ’Tardicots’was MS+IBA 1.0 mg/L+sucrose 30 g/L+agrose 7.0 g/L, pH:6.0.2. Antioxidant was used to decrease the influence of the browning for the explants. hypocotyl of ’Nubiana’ has the best efficiency of antioxidation when 5.0 g/L PVP was added in the medium. The epicotyl of ’Nubiana’ has the best efficiency of antioxidation when 3.0 g/L CA was added in the medium.3. The influence of the dark culture period for the regeneration of ’Tardicots’ was studied. For the petiole and leaf of ’Tardicots’, the best duration of the dark culture was 14d。4. There was no variance between the regeneration plants and the maternal plants of ’Tardicots’in their genetic background through randomly amplified polymorphic DNA analysis (RAPD).5. There was no significant variance in the process of the differentiation of the callus for the petiole and leaf of ’Tardicots’, through paraffin section. Solexa was used in sequencing the miRNA of the callus which was in the adventitious bud critical state of the differentiation from the explant of the petiole and leaf of ’Tardicots’. Their miRNA sequences were compared and analyzed. There were 5,612,766 Unique sequences and 2.33% of which was miRNA in the callus of the petiole of ’Tardicots’. For the callus of the leaf of ’Tardicots’ there were 4,942,180 Unique sequences and 2.42% of which was miRNA. By homologous alignment with miRNA of Arabidopsis Thaliana,10 micRNA were selected and verified by RT-PCR. To find the significant differentiation of the callus between the petiole and leaf of ’Tardicots’, targets genes of the 10 micRNA were predicted.6. Resistance seedlings of ’Tardicots’ were obtained in gene transformation experiment by using petiole as acceptor, the regeneration rate of resistance seedling was 1.67%. |
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