| Strawberry is one of the most important commercial fruit crops in the world. In this research, strawberry cultivar'Benihope'is used as experiment material. During strawberry stolon tip disinfection and proliferation, we have studied the effects of a series of factors including surface disinfectants, processing time, explant growth and development period, initial medium, hormones, pH and sugar concentration. Then on the basis of shoot proliferation system, we have studied the effects of hormone concentration and combinations, dark culture time, leaf age and the way of placement on regeneration rate of strawberry leaves, and also studied the effects of hormone concentration and combinations on regeneration rate of strawberry petioles and stems. We have established an efficient and quick Agrobacterium-mediated trans genic system of strawberry, with which the AtCYP2 gene was successfully transformed into the strawberry genome by PCR detection. The major results can be summarized as below:1. Strawberry cultivar'Benihope'rapid propagation system(1)Studies on the surface disinfection of strawberry stolon tip have found: unexpanded stolon tips as explants,0.1% HgCl2+2% Tween-100 solution as disinfectant, MS or MS+0.5 mg/L 6-BA as the initial medium, being treated for 8 minutes, explant contamination rate and browning rate are at a relatively low level. And explants survival rate is at a higher level.(2)MS medium adding 0.5 mg/L 6-BA and MS medium adding 0.3 mg/L 6-BA are used in turn, proliferation of strawberry seedling plants are the most. In addition, adjusting hormone combinations should be based on the purpose of studies. On MS medium addiing 0.5 mg/L 6-BA and 0.3 mg/L GA, although the proliferation rate is not high, the plants are growing strong. On MS medium with 1.0 mg/L 6-BA and 0.3 mg/LIBA, strawberry shoots tend to be serious vitreous, but we can get stolons.(3)On 1/2MS medium containing 0.01 mg/L NAA and 20 g/L glucose (pH 6.0±0.2), the-rate of strawberry rooting is highest, and the plants have the highest average number of roots.(4) Keeping the total nitrogen stable, we used NO3 and (NH4)2SO4 respectively instead ofNO3- and NH4+ of NH4NO3 during strawberry propagation. Comparatively, there is little effect of nitrogen on the rooting.(5) Compared with common strawberry fruit, the contents of soluble sugar and free amino acid of virus-free strawberry fruit are higher.2. Leaves, petioles and stems regeneration of'Benihope'(1)MS containing 2.0 mg/L TDZ and 0.1 mg/L NAA are found to be the most efficient medium during the regeneration of'Benihope'leaves. Induction rate of callus was 91.4%, differentiation rate was 66.4%. Taking the second and third leaves as explants can promote the regeneration.(2) The best induction medium of petioles is MS containing 2.0 mg/L 6-BA and 0.5 mg/L IAA. The regeneration rate is 77.8%.(3) Stem regeneration medium is MS+0.5 mg/L 6-BA+0.05 mg/L NAA. On this medium, callus induction and differentiation rate was 71.9% and 68.7% respectively.3 Transformation of AtCYP2 gene to 'Benihope'(1) The sensitivity tests of HmB of strawberry have found that when the medium contains 20 mg/L HmB, only a small part of the buds are grow. When HmB up to 30 mg/L, the buds can not develop normally, all buds are dead.(2) Suitable pre-incubation and infection time can increase the induction rate of resistance buds. The most efficient time is (2-4) days and (8-10) minutes respectively.(3) Through HmB resistance screening and PCR detection, it has confirmed that AtCYP2 gene has integrated into the genome of strawberry. |
