Establishment Of A High Efficient Regeneration System In Vitro And Transformation Mediated By Agrobacterium Rhizogenes In Begonia Semperflorens

Many cultivars of Begonia semperflorens have been widely applied in urba greening beacause of theirs stable characters, good combinging ability, orderly florescence and good effect of viewing and admiring. The Begonia semperflorens are mainly considered to be native to Brazil and other places of the South America,which optimum growth temperature is20℃and very sensitive to chillinginjury.With the rapid development of molecular biological techniques,it is feasible to improve the cold resistance of Begonia semperflorens by genetic engineering and pro vied a new pathway to increase the cold resistance of Begonia semperflorens. Agrobacterium tumefaciens,as the genetic transformation vector with the advantages of low cost and high transformation efficiency,is a widely used in introduction of external genes. Previous studies showed that the over-expression of Chimonanthus praecox Cpcor413pml gene can improve the cold resistance of the transgenic tobacco.There are seldom report about the genetic transformation system of Begonia semperflorens..This paper transfered Chimonanthus praecox Cpcor413pml gene into Begonia semperflorens plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens based on the establishment of the high efficient regeneration system of Begonia semperflorens leaves,in order to the obtain the new variety or material of Begonia semperflorens with high cold resistance. The main results in our rasearch are as follows:(1) Establishment of a high efficient regeneration system of Begonia semperflorensWe established the high efficient regeneration system of Begonia semperflorens by using young leaves as explant, MS as the basic medium supplemented with different concentration of6-BA and IB A to study the optimal condition of the adventitious buds derectly differentiate from the leaf disc. The result in our research are as follows:The best method for sterilization of the explant:It was eluted with detergent (3g/L) for5-6min and washed with flowing water for4h then agitating in a solution of0.1%HgCl2for5-6min,and finally rinsed five times with sterile distilled water.The best basic medium for adventitious bud differentiation is1/2MS,and the sutiable growth hormone combination and density is1/2MS+6-BA0.5mg/L+IB A0.8mg/L,the bud can grow well and accelerate the multiplication, the differentiation rate is83%.The best subculture medium is MS+6-BA0.5mg/L+IBA0.8mg/L,the propagation coefficient is4.7.The rooting medium is1/2MS+IBA0.5mg/L and can induce root after30d culture,the rooting rate is98%.The best culture results could be achieved with the direction of perlite and humus soil with a ratio of2:1,the transplanting survival rate is91%.(2) Agrobacterium-mediated Transformation system of the Begonia semperflorensIn the transformation system mediated by Agrobacterium tumefaciens EHA105(containing the plasmid pCA-Cor413),the selective concentration of Kanamycin (Km) for adventitious bud differentiation and rooting were50mg/L and100mg/L respectively. The inhibition of EHA105by Carbenicillin (Carb)was better than Cephalothin (Cef) and the selective concentration was200mg/L. The optimal method of transformation was as follows:The leaf was cut into the size of1cm×1cm and pre-cultured in the medium1/2MS+6-BA0.5mg/L+IBA0.8mg/L for3days. Then the leaf disc that had been pre-cultured was put into the resuspended bacterial liquid (OD600=0.2) to be infected for10min in the shaking bed. These leaf dies were transferred into the medium1/2MS+6-BA0.5mg/L+IBA0.8mg/L+AS100μmol/L in the darkness for3days. Later, it was time to wash bacteria off on the surface of leaf disc in the liquid MS+6-BA0.5mg/L+IBA0.5mg/L+Km50mg/L+Carb200mg/L. Then the plants were transferred to MS+6-BA0.5mg/L+IBA0.5mg/L+Km100mg/L+Carb200mg/L when the shoots formed.Finally,32transgenic plantlets were obtained, in which the transgenic lines were identified by GUS((3-glucuronidase) histochemical staining, PCR and RT-PCR analysis.3. Measurement of physiological indexesof transgenic plantlets under low temperature.13plantles of transgenic Begonia semperflorens and4control(wild type) were treated under4℃respectively. After3days treatment,the contents of chlorophyll, proline,MDA and SOD before and after treatment in leaf.The results showed that the changed extent of content of chlorophyll, proline and MD A were little betewt transgenic and non-transgenic plantlets.We found that the content of SOD in the non-transgenic plants was almost unchanged after the cold treatment,but the SOD content of transgenic Begonia semperflorens increased obviously.