| Strawberry?Fragaria×ananassa Duch.?is one of the most important soft fruits.Along with the application of transgenic technology,strawberry gene functional verification has became an important part of strawberry molecular biology research.In the past,strawberry transformation used F.vescaas as the material,but it is different to phenotypic expression and economical value of F.vescaas with F.E×ananassa.Thus,it is more meaningful to use the cultivated strawberry as the material to verify the gene function.In this study,the clones with high regeneration ability and more sensitive to Agrobacterium were selected from hybrid seeds of different combinations of octaploid cv.strawberry.At the same time,the in vitro regeneration system and efficient Agrobacterium-mediated transformation system using leaves as explants get optimized.The main results are as follows:1.The regeneration ability of the six different combinations of hybrid progeny was compared.The combination of 'Allstar' x 'Shimei7' and 'Yongli' had higher regeneration ability.The proportion of single plant inducing adventitious buds in the hybrid progeny of'Allstar' x 'Shimei7,was 57.14%,the average regeneration efficiency was 51.15%.The proportion of single plant inducing adventitious buds in self-pollinated progeny of 'Yongli'was 46.67%,the average regeneration efficiency was 36.67%.2.A-19,D-10,and D-34,which were highly regenerated,were selected from different combinations of octaploid strawberries,establishing clones in vitro.In the several regeneration experiments,the regeneration efficiency of A-19 and D-10 was more than 90%,and the average number of adventitious buds was between 9.2 with 10.7.The regeneration efficiency of D-34 was more than 85%,the average number of adventitious buds was between 7.6 with 8.9,which were higher than the variety 'Allstar',which belongs to the genotype with high regeneration ability.3.Using the leaves of A-19,D-10,D-34,'Allstar,as explants,compared the sensitivity to Agrobacterium tumefaciens of clones A-19,D-10,D-34,'Allstar' by GUS histochemical analysis.Results display,they were all sensitive to Agrobacterium,and the sensitivity of A-19,D-10,D-34 were not significantly difference but significantly higher than 'Allstar'.4.Using the leaves of A-19,D-10,D-34,'Allstar' as explants to develope Agrobacterium-mediated stable genetic transformation.The transformation efficiency of A-19 and D-10 was 4.3%and 3.3%,higher than that of cultivars 'Allstar'?1.7%?.The explants of D-34 were gradually faded and no resistant buds were produced after infection.The results showed that A-19 and D-10 could be used for stable genetic transformation.5.The in vitro regeneration system and genetic transformation system of octaploid strawberry were optimized.The meidium with 0.2 mg-L-1 IBA + 2.0 mg·L-1 TDZ/6-BA can accelerate the regeneration of leaves from A-19.The meida with 2.0 mg·L-1TDZ + 0.1-0.2 mg·L-1 IBA can accelerate the regeneration of leaves of D-10.Adventitious buds from the strawberry explants were reduced in the regenerative medium supplemented with antibiotics.200 mg·L-1 Timentin,200 mg·L-1 Cefotaxime,200 mg·L-1 Timentin + 200 mg.L-1 Cefotaxime had no significant difference in the toxicity for explants.Except that,the pre-culture of the leaves can induce resistant buds before infecting Agrobacterium tumefaciens.The pre-culture for 6 days is the most beneficial in the genetic transformation of cultivated strawberries.6.A-19 has a high sensitivity to gibberellin.The treated plants had obvious elongation in height of plants and bigger in area of leaves in the media supplemented with different concentrations of GA3 and in the experiment of spraying GA3 on leaves,respectively.The plants of A-19 grew best in the media supplemented with 0.5 mg·L-1 GA3. |
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