Optimized System For Plant Regeneration And Agrobacterium-mediated Transformation Of Balsam Pear(Momordica Charantia L)

Balsam pear(Momordica charantia L) is a climbing annual vine weak herbage plant belonging to family Cucurbitaceae. It is an important horticultural crop in subtropical and tropical regions now widely cultivated throughout the world. But its output and quality have been severely influenced due to plant diseases. So improvement of its disease-resistance has always been an important research field. Conventional breeding methods are often limited by germplasm resources scarcity and long breeding cycle, but plant gene engineering can overcome these problems to some extent.The target of this study is to establish in vitro regeneration system in balsam pear, and introduce tomato disease-resistance transcription factor Pti4gene into balsam pear by agrobacterium-mediated transformation, and carry out PCR detection in transgenic plant.The main results are as follows:1. The establishment of in vitro regeneration balsam pear system.(1) By comparing the ability of adventitious bud inducing between’Changbai’,’Youlv’and’Dabai’, it was found that the genetypes had bigger effect on shoot inducing. There was the highest inducing rate from’Youlv’(80.65%), the next was’Changbai’(78.79%) and there was the lowest inducing rate from’Dabai’(67.74%). However, the average buds per explant of’Changbai’ were significant more than other two genetypes,which reached to4.38. The optimal variety of balsam pear was’Changbai’.(2) With the increase of seedling age, differentiation rate of multiple buds and the average number of multiple buds both increase at first and then decrease,8day is the best seedling age of explants in balsam pear regeneration was significant more than6day and10day, the inducing rate was also highest, which reached to78.63%.(3)The optimal explants screened in this test was cotyledonary nodes with cotyledon and the inducing rate was71.67%both are higher than other explants; the explants of cotyledon cannot be induced multiple buds.(4) The effect of IBA inducing was better than NAA and IAA;the optimal working concentration of6-BA was2mg/L-3mg/L in cotyledonary nodes; The best differentitation medium are MS+2.5mg/L6-BA+0.1mg/LIBA. (5)7day is the best dark period of explant in balsam pear. The inducing rate was highest (80.91%), the average buds per explant was also maximum which reached to4.38. With the extension of dark periods, differentiation rate of adventitious bud and the average number of multiple buds both increase at first and then decrease.(6)This experiment can be found that AgNO3had the obvious inhibiting effect to induce multiple buds, when the concentration of AgNO3was5mg/L, the induction rate was0%.(7)The best rooting medium was1/2MS+0.4mg/LIBA, the rate of rooting induction was100%,2. The establishment of genetic transformation in balsam pear system.(1)There was the highest rate of resistant buds (10.0%),when the inducing time was10min. It was significant higher than those of5min and15min.(2) The best pre-culture time was3d, which had the highest rate of resistant buds (11.3%). It was significant higher than those of pre-culture time.(3)The best co-culture time was4d, which had the highest rate of resistant buds (15%). It was significant higher than those of co-culture time.(4) The best selection medium was:MS+6-BA(2.0mg/L)+Kan (100mg/L)+Cef (500mg/L).(5) The exogenous target gene Pti4was introduced into balsam pear by agrobacterium-mediated transformation and got52Kan-resistance plants finally. NPTⅡ primer was used to carry out PCR detection in these Kan-resistance plants. The results show that36Kan-resistance plants which have the expected gene fragment were obtained and the conversion rate was69.2%.