A Study On Introducing Of Glyphosat Tolerant Gene Into Castor (Ricinus Communis L.) By Non-Tissue-Culture Genetic Transformation Methods

Castor(Ricinus communis L.)is one of top-ten important oil crops of world,which is the number one crop for bioenergy.The occurrence of weeds is one of the main harms in the castor production.Using genetic engineering methods to breed for herbicide-resistant crop varieties can control the weeds in the castor field efficiently.Non-tissue-culture transformation method circumvents the lengthy and tedious tissue culture procedures and is simple and economical.In this study we used Agrobacterium-mediated transformation of germinating embryos in the castor for the first time,at the same time using the pollen tube pathway,carried the research of castor genetic transformation with glyphosate resistance gene.Optimal transformation systems for castor was established with the two methods,respectively,and the castor were transformed successfully with the glyphosate resistance gene.The main results are summarized as follows:For the first time,castor(Ricinus communis L.)was transformed,with a herbicide tolerant gene CP4-EPSPS by the method of Agrobacterium-mediated transformation with germinating embryos.The result showed that puncturing three stitches at the meristematic region of germinating seeds,ultra-sonication power of 800 W,and co-cultivating for 16 h with agrobacterium were the optimum transformation conditions.Five putative transgenic plants were detected from 800 randomly-selected treated seedlings before glyphosat screening,the average positive rate was 6.3 �.After 1.5�glyphosat screening,3 positive T0 seedlings were obtained out of 26 survivals by PCR and Southern blot analysis.If we take glyphosate tolerant plants as the base the transformation rate was 11.5%.This study demonstrated that the method of Agrobacteriuwm-transformation with germinating embryos could be used for castor genetic transformation.The study has provided a new thought and method for castor genetic engineering research.Three influencing factors,DNA concentration,DNA solvent and introducing time,which might affect the rate of seed set rate and conversion rates were analyzed by pollen tube pathway.The result showed that there was no significant difference on the seed set rate among of the three factors,but the DNA concentration was vital to the conversion rate.DNA concentration of 100ng/?L was the best in the three factors,and TE solvent was better than others.DNA addition time of 6-24h was suitable.3 PCR-positive seedlings were detected out of the 351 seedlings from the treated seeds.It preliminary proved that tolerant gene EPSPS has been successfully transferred into castor plant,which have provided some theory basis for improving the castor transformation rate by pollen tube pathway.