| Strawberry is one of the most valuable fruits. Most cultivated strawberry varieties are octoploidies with high degree of heterzygosity in heredity while nearly all wild species are in low ploidy levels, which make it difficult to transfer merit characters from wild strawberries to cultivated strawberries by conventional breeding methods. Protoplast manipulations, which can expand the range of genetic recombination, transfer distant characters, and create new germplasm, have showed great potential in strawberry breeding.The research dealt with the induction of calli from anther of different genotypes, establishment and maintenance of suspension cell lines, protoplast isolation, culture, regeneration and fusion. The genetic variation of the materials was also investigated.1. The calli were induced from anther of 9 strawberry genotypes. The results showed that the efficiency of calli induction was affected by low temperature treatment before inoculation, genotypes of initial materials and plant growth regulators combination and ratio, et al. The pre-treated anther was inoculated on the medium of MS + 0.5~1.0mg/L 2,4-D + 1.0~2.0mg/L BA + 1000mg/L KNO3, high quality anther-derived calli were induced. Many types of calli were formed after the initial calli proliferation on MS medium with high 2,4-D level. Among them, yellowish and friable calli were transferred to the medium with high NO3-/NH4+ rate, high osmotic pressure and low 2,4-D level, and about 4-5 months after, fresh-yellow, friable and granular embryogenic callus was obtained. It was of great benefit to embryogenic calli that supplemented with CH 400mg/L + Gln 300 mg/L + ME 500mg/ L, modified medium according to calli growth states and selected by sub-cultured.2. Growth of suspension cell lines was affected by the concentration of PGR, the initial cell density and days after sub-cultured, et al. Low level of 2,4-D (0.2~1.0mg/L), 0.5g (FW) suspension cell/40ml MS liquid medium and 6d~8d after sub-cultured were essential to maintain the suspension cell lines of strawberry. By alternating solid and liquid culture pattern regularly and selecting calli of yellow, compact and small pellet, the embryogenic cell lines were improved. We were successful in establishing stable suspension cell lines of cultivar 'Mingxu', 'Hoko', 'Joho', 'Allstar' and 'Xinjiang No. 3'.3. Protoplasts were isolated from leaves of in vitro-grown plantlets, embryogenic calli and suspension cells. The results showed: (1) The optimal enzyme solution for isolation of protoplasts from suspension cells was: CPW13%Mannitol+0.5%PVP+0.1 %MES+0.5~1.0% Cellulase+0.5 %Macerozyme+0.05 %Pectolyase Y-23; whereas the enzyme solution for mesophyll and embryogenic calli was: CPW13%Mannitol+0.5%PVP+0.1 %MES+1.0% Cellulase+1.0 % Macerozyme+0.1% Pectolyase Y-23. (2) High yield and viability protoplasts were obtained when incubated on constant-temperature shaker with 35~40rpm at 27 +1C for 12 hours. (3) Genotype, explant source and their physiological state, speed and time of centrifugation during purification also affected protoplasts yield and viability.4. Appropriate medium for strawberry protoplast culture was found to be KM8p supplemented with 0.5mol/L Glucose + CH400mg/L + Gln 300mg/L + ME 500mg/L + 2,4-D 1.0mg/L + BA 1.0mg/L. The best result was achieved when protoplasts were cultured in liquid medium at a density of 1.0×105/ml. We also found that decreasing osmotic pressure was favorable for the growth and development of protoplast-derived cell colonies. Protoplasts from suspension cells divided for the first time in 3~4 days, formed multi-cell colonies in 20 days, and grew into visible mini-calli in 40-50 days. Protoplasts from mesophyll and calli only divided one to three times, and no multi-cell colony was obtained.5. The calli derived from protoplasts were transferred to the solid differentiation medium. The results showed that plant regeneration frequency was higher by increasing TDZ concentration gradually on MS basic medium than other treatments. The plantlets were regenerated from...
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