The Establishment Of Fragaria Ananassa Duch Regeneration System And Obtaining Disease Resistant Germplasm By Introducing Chit42 Gene

Strawberry is one of the most important commercial fruit crops in the world.Because of the genetic limitations associated with high heterozygosity andpolyploidy, it is laborious, costly, time-consuming and low efficient to improvestrawberry quality and yield by the conventional methods of breeding. A majorchallenge remains in commercial strawberry cultivars for the lack of good varietieswith resistance to coldness, insects and disease. Recent advances and applications ingenetic engineering technology provide a new path for strawberry breeding by wayof introducing foreign genes into plant genome for desired traits. An efficientregeneration system is thought to be crucial to the success of plant geneticengineering, since the efficiency of Agrobacterium-mediated transformation isconsidered to be dependent on two primary factors, one being the regenerationability of the infected tissue, and the other, the infection efficiency of Agrobacterium.In this research, we have studied the effects of a series of factors (genotypes, basicmedia, plant growth regulators, explants, inoculation methods, leaf ages and darkperiod) in the inducing adventitious shoot of strawberry((Fragaria ananassa Duchcv. Toyonoka and F. ananassa Duch cv. Zoji)). Then on the basic of optionalregeneration system obtained above, we established an efficient and quickAgrobacterium-mediated transgenic system of strawberry, with which the chit42gene from Trichoderma harzianum was successfully transformed into the strawberrygenome. The major results can be summarized as below:1. The strawberry regeneration system has been established successfully.Genotype was the main factor influencing regeneration system of strawberry invitro. Regeneration frequency varied dramatically with genotype of strawberry,'Toyonoka' and 'Zoji' obtained rate of Shoot regeneration of 60% and 40%,respectively.Plant growth regulators play important roles in adventitious shoot inducingprotocol. It was necessary for the regeneration to supplement cytokinin and auxin inthe regeneration medium. The combination of MS+TDZ2.0mg/L+2, 4-D0.1mg/Land MS+TDZ 3.0mg/L+ NAA 0.5mg/L were found to be the most efficient of'Toyonoka' and 'Zoji', respectively. In addition, it was considerate to add TDZ intomedium to enhance the frequency of regeneration.It was suitable to induce shoot with 7～10 days dark period. And the rate of shoot regeneration was significantly enhanced by the MS basic medium.The regeneration capacity of leaf was higher far than petiole. Leaf age can affectalso the regeneration frequency and about 30 days was the best. There was notnotable difference for the frequency of regeneration whether leaf discs were placedabaxial side up or down.2. An efficient strawberry transformation system has been established.On medium containing 50 mg/L kanamycin, the shoot regeneration capacity ofstrawberry was inhibited. The growth of Agrobactium was inhibited effectively with300mg/l cefotaxine supplemented in the medium, and the regeneration frequencybegan to decrease on this medium. As a result, 50 mg/L ofkanamycin and 300 mg/Lof cefotaxine were established as the concentration effective for selection of shootsand inhibition of Agrobactium growth.Some factors that affect the transformation of strawberry were studied, such aspre-culture time, EHA105 concentration, infecting time, AS concentration,co-culture time et al. We have preliminarily determined an effective system forgenetic transformation. The protocol was follows: OD₆₀₀ 0.4, 10～20 minutes forinfection, 3days for co-culture, AS80～120μM as co-culture medium, Step-selectionor selection after delay for 14 days.3. Molecular analysis and disease-resistance testThe regenerated kanamycin resistant plants were analyzed by PCR andPCR-Southern reaction with the untransformed plants as negative control, plasmidDNA as positive control. Part of plants showed specific positive band same as theplasmid control, while untransformed plants have not any band. The result indicatedthat the chit42 gene has been integrated into the strawberry genome.Disease-resistance test of the plants demonstrated that the grey-mildew tolerance ofthe transgenic plants, which varied with different transgenic plants, was strongerthan that of the untransformed plants.