| Kiwifruit is a kind of nutritious fruits, its unique flavor, and it’s known as the ’king of fruits’. Hongyang kiwifruit s fruit pulp is yellow-green, and the transverse cross is like radial blood red stripes, moreover, its sugar content is higher than other varieties of Kiwifruit and its juice is delicious, as a result, the ploidy is complex,and femal and male plants flowering is not the case,which makes interspecific hybridization difficult, and breeding cycle longer, so it has a strong uncertainty. The conventional breeding cycle longer, so it has a strong uncertainty. The conventional breeding methods is likely to cause degradation of varieties, while, the propagation by tissue culture can preserve excellent heredity and achive mass propagation.In recent years, the major domestic syudies focuses on Actinidia chinensis and foreign major in tasty kiwifruit, but their reproductive efficiency is low generally, at the same time, we did research on Hongyang kiwi too little. Therefore, there is an urgent require the use of new technology and means to change itstraditional breeding status on Hongyang kiwi. Meanwhile, production and cultivation of kiwi are vulnerable to pests and diseases, which including Hongyang kiwi. In many diseases, the canker is the mostly prominent, and the using of pesticides in the traditional production will bring a lot of side effects. However, plant genetic engineering technology provides a new way for the kiwi breeding for disease resistance. We built the genetic transformation system by filrering the level of various factors which affect the transformation frequence. By GUS staining and PCR analysis, we can preliminary evidence that exogenous resistance genes LJAMP2has been successfully integrated into the genome Hongyang kiwifruit. The main findings are as follows: 1. We used the stem of Hongyang kiwifruit as explants to select better media for inducing and proliferating callus and adventitious buds in order to establish efficient regeneration system. Results showed that the stem segment explants on MS+1.0mg·L-12,4-D+0.5mg·L-16-BA in four weeks culture period, the rate of explant dedifferentiation was100%. On MS+2.0mg·L-12,4-D+0.4mg·L-16-BA medium,1to4generation averation average callus proliferation multiples of up to3.38. The rate of adventitious bud induction up to83.33%after transferred on MS+5.0mg·L-16-BA+0.1mg·L-1NAA in two months culture period. And the suitable medium for adventitious buds proliferated was on MS+3.0mg·L-16-BA+1.0mg·L-1NAA. The rooting rate of adventitious buds was100%after culturing on1/2MS medium which treated with0.01%IBA for40minutes. The root system of plantlets developed better cultured in pearlite matrix which contained1/2MS solution for20days.85transplanted to the filed soil nuturient bowl.81plantlets (95.29%) were survived after transplanting to matrix soil for15days.2. Hongyang kiwifruit leaves as explant to explore the different effects of growth hormone on leaf regeneration. The results showed that the maximum adventitious buds regeneration rate achieved100%on the medium of MS+3.0mg-L-16-BA+1.0mg·L-1NAA, the maximum adventitious buds proliferation frequency achieved100%on the medium of MS+5.0mg·L-16-BA+0.2mg·L-1NAA+0.1mg·L-1GA3, and during1to6subcultures the average breeding coefficient on behalf of adventitious buds highest8.7. The regenerating buds grow fast and robust on MS+0.4mg·L-16-BA+0.2mg·L-1NAA. We also demonstrate that the rooting rate of the shoot achieves100%by stimulating seven days in1/2MS+0.8mg·L-1IBA. Then transferring into the medium of1/2MS.95plantlets (96.94%) were survived among98plantlets after transplanting to matrix soil after15days.3. Eeffets of different antbiioties on in vitro regeneration and ogrnaogenesis of Hongyang kiwi from leaf were examined. We concluded that kanamycin inhibited the differentiation of adventitious buds. Callus and shoot formation from leaf explants can proceed nomal differentiation when there were no kanamycin. When the concentration of kanamycin accomplished15mg·L-1, there were small amount of callus formed. With the growth of kanamycin concentration, the differentiation rate decreased sharply. The callus and adventitious bud was completely inhibited when kanamycin concentration exceed20mg·L-1. No root appeared on rooting medium acceed30mg·L-1kanamycin. So20mg·L-1kanamycin was selected in the transgenic plants. These results also showed that carbenicillin was superior to ceftoime in the genetic transformation. The ceftoime was unuseful to inhibit the growth of Agrobacterium tumefaciens even though the high level of it. When carbenicillin concentration was400mg·L-1, shoot differentiation rate reached the highest levels,100%. Besides, the eligible acetosyringone (AS) could increase the rate of transformation. It was suggested that for400mg·L-1carbenicillin was suitabie for elimination of Agrobacterium tumefaciens.4. On the base of establishing effective regeneration system, the optional parameters of the genetic transformation for Hongyang kiwifruit was examined. The study showed that pre-incubation time, bacterium concentration, infection time, co-culture and the concentration of acetosyringone have some influence on the conversionefficiency. At last, we sum up that the transient expression frequency was highest when leaf explants were preculture for3days, and then disseminated for10min with Agrobaeterium tumefaciens (OD600=0.5), and finally co-culture for2days. Besides, when the acetosyringone (AS) concentration touched100μmol·L-1the transformation efficiency could be improved.5. An nsLTPs-like antimicrobial protein genes LJAMP2from the seeds of motherwort was introduced into the leaf of Hongyang kiwifruit via Agrobacterium-mediated transformation. Twenty resistant plants were positive among forty plants by GUS histochemical staining. Besides PCR analysis also confirmated it. The results showed that LJAMP2gene had been integrated into the genome of Hongyang kiwifruit. |
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