| Apple is one of the most important fruit in China, but apple industry is hindered by low yield and poor quality. The resolution is to bread excellent cultivars and rootstocks. Protoplasts culture and fusion, which can expanded the genetic recombination and transfer distant traits, have great potential for breeding. To date only regeneration of plants from apple protoplast has been reported in limited genotypes. Therefore, it is significant to further study protoplast culture and fusion which could create new germplasm and be useful to genetic improvements of cultivars and rootstocks in apple.The research dealt with the induction of calli and establishment of suspension culture, protoplast isolation, fusion and regeneration from different explants and genotypes.1. Induction of calli from young ovule, mature ovule and leaves there was significant difference in the induction of calli when different combinations and ratios of hormone were tried. Friable callus from leaves had the capacity of morphogensis, and could be selected by subcultured.2. Growth of suspension culture was affected by sucrose, nitrogen nutrition, it is successful to establish stable and friable suspension culture. Suspension cultures of calli from leaves could experient morphogenesis.3. Viability of mesophyll protoplasts reduced when Pectinase was increased while enzyme concentration (both Pectinase and Cellulase) was below 1%. The highest yield of protoplast was obtained when 0.5% or 0.75% Pectinase were used. Yieldand viability of mesophyll protoplasts were increased as Cellulase concentration was increased. BSA(Bovine Serum Albumin) also had some effect on constant isolation of protoplasts. The optimal enzyme solution used for isolation of protoplasts from leaves and cotyledons was: CPW 12 + MES 0.1% + PVP1% + Cellulase 1% + Pectinase Y-23 0.05%; whereas enzyme for suspension culture was CPW 12 + MES 0.1% + PVP1% + Cellulase 0.5-1% + Pectinase 0.3-0.5%. Purification could be obtained by means of centrifugation and floating collection.4. Appropriate culture medium for protoplasts was modified MT supplemented with Vc, 2,4-D, glutamine, CH, ME and glucose. Glucose had a better effect on osmoregulation than sucrose. Low-density culture could reduce browning. Under the above culture conditions, protoplasts from suspension culture divided for the first time in five to six days, formed multi-cell clusters and grew into visible mini-calli. No multi-cell clusters were got from protoplasts of leaves and cotyledons were concerned. Mini-calli was transferred to solidified medium for proliferation.5. The calli derived from protoplasts changed to compact type and could be induced to form green bud spots which grew into green and hard pieces and subsequently adventitious shoots when transferred to the differentiation medium,The shoots rooted in rooting medium, and plantlets have regenerated from three genotypes including Mains hiipehemis, Mae, Gala6. Chromosomes of calli from protoplasts and suspension cultures were observed. Ploidy stability had great effect on morphogenesis of the calli as may be the key factor involving the regeneration capacity of different genotypes.7. Protoplasts both from suspension and mesophyll were electro-fused. Fusion of protoplasts from suspension cultures was carried out as well, the optimum parameters were alternating current(AC) 100V/cm, duration 60 s; direct current(DC) intensity 1250 V/cm, 5 times, duration 5 us. The fused protoplasts were adjusted to 0.5 105/ml. The first division was observed in eight to ten days and mini-calli in forty to sixty days. Individual clones,which could form friable and hard calli by changing medium,were acquired under the conditions of low density and controlled culture.8. POX izozyme was utilized to identify the protoplasts-derived calli, differences inizozyme which resulted from fusion was observed. Specific band of parents were included in the regenerated clones, additive band pattern demonstrated hybrid clone exist in the regenerated clones...
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