High Frequency Shoot Regeneration And Gene Transformation From In Vitro Leaves Of Pear(Pyrus Communis L.)

Pear (Pyrus communis L.) is a major crop in the world. With thedevelopment of culture, diseases, such as Podosphaera leucotricha, Erwiniaamylovora, etc., cause leaf defoliation, yield reduction and even death, whichare present in all the major pear growing areas of the world. Therefore, tobreed new varities of pear that resist fungi and lacteria disease is very crucialin pear production. In this study, we established high frequency shootregeneration system from in vitro leaves of pear. And with Agrobacterium-mediated transformation we conducted "Fertility"pear transformation usingradish seed ?-thionin Rs-afp1 gene and uidA (gus) gene drived by greenspecific spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco)activase promoter (RCAP) and putative transgenic pear plants with nptⅡresistance were produced. Several factors, including basal medium, hormone concentration andcomposition, cotykinin, anxin, sucrose concentration, pH value, cotyledonaryexplant age and cultured ways, etc., were investigated for their effects on pearadventitious shoot regeneration. Basal medium NN69 was consistently betterthan MS, LE and C; The concentration and composition of hormone iscrucial to regeneration of pear; NAA was more effective than IBA for inducingadventitious shoots to form on excised leaves from in vitro; The genotype ofexplant greatly influenced regeneration, e.g. the regeneration frequency of 4山东农业大学硕士学位论文Jinhua and Fertility is up to 90%, higher than that of other cultivars; Theaddition of CH to the medium could promote regeneration; The highestfrequency was obtained from Jinhua and Fertility cultured on the optimalmedium (NN69 + BA5mg/L + NAA0.3mg/L + AgNO34.5mg/L+ CH300ppm+Sugar3%) in the dark for 20 days, counted for 100% and 95.3%, respectively.While the adventitious shoots elongate, they were transplanted to MS medium,to which BA1mg/L and IBA0.2mg/L were added. After shoots reached toabout 0.5cm, they were transferred to the medium containing IBA0.3mg/L toinduce roots. 20 days later, white and long roots emerged, and then they weretransplanted to the sands with the survive rate up to 85%. On the basis of regeneration system from in vitro leaves of pear, factorsinfluencing the frequency of gene transfer were examined and an efficientgene transformation system for pear was established. Young expanding leafexplants were first precultured on inducing medium for 1 day, thencocultivated on inducing medium with Agrobacterium tumefaciens EHA105harboring a binary vector containing chimeric genes of gus and Rs-afp1 genes,and AS 20μM. After 3 days coculturation, these explants were transfered topre-selective medium (NN69+BA5mg/L+NAA0.3mg/L+AgNO34.5mg/L+CH300ppm+Carb250mg/L+Cef250mg/L). 3 days later, they were transfered toselective medium (Kan25mg/L added to pre-selective medium). In the end, weobtained 15 putative transgenic lines. PCR and southern blot analysis of plantsconfirmed the integration of thionin and gus genes into pear genome. gusassay confirmed the function of the tissue-specific expression of RCAPpromoter. Now transgenic pears were transplanted in the field to identify theagronomy traits.