Study On The Establishment Of In Vitro Regeneration System From Pear (P. Pyrifolia Nakai) And Chit42 Gene Transformation

Pear (Pyrus communis L)is one of the most important fruits in China. The cultivation history is glorious and the distribution spreads the whole nation. It is a popular fruit in the market due to it's excellent quality, attractive color, and marvelous fragrance. Sand pear(Pyrus pyrifolia Nakai)cultivars also have the characteristics of fruit-setting earlier, and stronger ability to anti-heat In recent year the cultural area in south port of China, the yield and quality of fruit rise fast. But most cultivars of sand pear still have a lot of shortages such as sensitive to moist and diseases, special in high moist and temperature So it is very important for sand pear to improve an ability of anti-disease.With the development of biotechnology and plant gene transfer technique, it makes it is possible to breed a fruit new cultivar. In this paper sand pear tissue culture and gene transformation technique were studied by using huangjin and guiguan as materials Shoots were regenerated from the young leaves of sand pear huangjin, guiguan based on regeneration system the condition of Agrobacterium-mediated transformation were explored. The results showed:1. Adventitious shoots regeneration from in vitro leaves and stems of sand pear cultivars, Huangjin and Guiguan pear widely cultivared in south of China were studied Among the two cultivars Huangjin leaves show the relatively higher ability of regeneration ,the stem of Huangjin was weaker,the leaves of Guiguan was more weaker and the stem of Guiguan had no response to regeneration. 15 d Dark treatment was necessary for regeneration of Huangjin leaves in the experiments. The best medium for Shoots regeneration was MS +6-BA1.0mg / L + NAA2.0mg / L, the best medium for rooting was 1/2MS +IBA3.5mg / L.2 Scanning electron microscopy (SEM)observation on the and adventitious root of Huangjin revealed that The callus induction appeared at 10d, meristem-like structures were already visible at 15d , adventitious shoots were observed after 40d and well development shoots were observed after 80 d.SEM also showed that more meristems were observed than the shoots eventually formed from them, and formed adventitious shoot was short far than the adventitious shoot which occurred.3. Through experiments of the effect of Cef on the callus induction and shoots regeneration from leaves of Huangjin and the effect of Kan on callus induction,factors influencing the frequency of gene transformation were examined and an efficient gene transformation system which the concentration of Cef was 300mg/L,Kan was 75 mg/L for pear was established.4. Leaf explants were first precultured on inducing medium for2 days, then cocultivated on inducing medium with Agrobacterium tumefaciens EHA105 which OD₆₀₀ was 0.6,then 3days coculturation, In the end, we obtained 4 putative transgenic lines. PCR analysis of plants showed two of them were positive.