High Frequency Shoot Regeneration From In Vitro Leaves Of Cherry Dwarf Rootstock, Gisela (Prunus Cerasus X P.Canescens) And Obtainment Of Transgenic Plants With MB39 Gene

Cherry is an important tree for commercial fruit in the world. With the development of culture in our country, disease, such as bacterial canker, crown gall, brown rot, PNRV (prunus necrotic ringspot virus), PDV(prune dwarf virus) and so on.were become more inportant, especially, crown gall, caused by Agrobacterium tumefaciens, is one of the most important sweet and sour cherry pathogens, which causes leaf defoliation, yield reduction and even death. It is present in all the major cherry growing areas of the world. Therefore, to breed new varities of cherry that resist A.tumefaciens infection is very crucial in cherry production. In this study, we obtained several transgenic plants. A gene containing the osmotin promoter and cecrotin MB39 coding region was introduced into cherry dwarf rootstock, Gisela6, using Agrobacterium tumefaciens mediated transformation and using leaf disc as the explant.The leaf explant of GiselaS, Gisela6 (Prunus cerasus X P. Canescens) , mahaleb (P. mahaleb), china cherry c28 (P. pseudocerasus) and sweet cherry No.3 (P. avium) were culture and several factors including basal medium, hormone concentration and composition, concentratio of sugar, age of explant, and so on , have been examined for their effects on the frequency of shoot regeneration, basal medium WPM was consistently better than MS, B5 and N6; IBA was more effective than NAA for inducing adventitious shoots to form on excised leaves from in vitro; Up to 70% of leaves from micropropagated cultures of GiselaS and Gisela6 could be induced to form adventitious shoots, when cultured on WPM medium containing 5-7mg/l BA and 0.3-0.5mg/l IBA; the cultivars of Prunus Pseudocerasus, Prunus mahaleb and Prunus avium could also be made to regenerate shoots from leaf explant, but the frequency was very low, varied from a few to 30% depending on the cultivars, this indicated that shoot regeneration capacity of the different genotype was greatly different. The highest frequency was obtained from GiselaS and Gisela6 cultured on the optimal medium(WPM+BA7mg/l-HBA0.3mg/l+AgN03 4.5mg/!+CH300ppm, and WPM+ BA5mg/l -HBAO.Smg/l + AgNOS 4.5mg/l+CH300ppm respectively), in the dark for 15 days, counted for 76.8% and 100% respectively.On the basis of regeneration system from leaf explant of Gisela6, factors effecting on frequency of gene transfer were examined and an efficient gene transfer system for cherry was established. Young explanding leaf explants were first precultured on inducing medium for 1 day, then cocultivated on inducing medium with Agrobacterium tumefaciens EHA105 harboring a binary vector containing chimeric genes of npt II and MB39gene driven by osmotin promoter, and As 20 u m. After 3 days coculturation, these explants were transfered to selective medium with Kanamycin 50mg/l, Carb 250mg/l and Cef 250mg/l (WPM+BA5mg/l+IBA0.5mg/l+AgNO3 4.5mg/l+CH300ppm+Kan 50mg/l+Carb 250mg/l +Cef 250mg/l) to select transgenic shoots.PCR analysis of these kan resistant shoots confirmed that they contained cecropin MB39 gene and the foreign cecropin MB39 gene was introduced into the cherry genome.