High-frequency Plant Regeneration And Genetic Transformation Of Jatropha Curcas L.

Jatropha curcas L. is an important biofuel-producing plant, but its narrow cultivate range and low yield has limited large-scale cropping and commercialization as an energy plant. Some traits of Jatropha curcas L, such as resistance, yield and oil quality, can be enhanced through genetic engineering to expand it's distribution range, increase it's yield, and improve it's oil contend and quality. However genetic improvement of Jatropha curcas L requires a high-frequency plant regeneration and transformation system. In this study a high-frequency plant regeneration of Jatropha curcas L. from leaf-discs has been developed, and Agrobacteriuim-mediated leaf disc transformation studied.The leaves of Jatropha seedlings in the greenhouse as explants were used for adventitious shoots regeneration. Shoot induction was carried out on MS medium with varying concentrations of BA and IBA and cultures were maintained in the dark. The results showed that the optimal concentrations for adventitious shoots induction with BA and IBA were 5mg-L-1 and 0.5mg-L-1 respectively. To further increase the frequency and amount of the adventitious shoots, the effect of gibberellic acid (GA3) on the regeneration of adventitious shoots was investigated. The regeneration frequency of leaf-discs could reach 90.9% and the average number of up to 4.6 adventitious shoots could be obtained when the concentration of GA3 was 0.05 mg·L-1. Based on these improvements, a high-frequency indirect regeneration system of Jatropha curcas L. from leaf-discs was established.Although higher frequency regeneration can be obtained by indirect regeneration, it could lead to greater plant genetic variation due to dedifferentiation as well as longer plant regeneration period when compared to direct regeneration. Genetic stability can be maintained and foreign gene can be inherited by direct plant regeneration. In this study, the leaves of Jatropha seedlings from the greenhouse were used as explants for adventitious shoots regeneration. The results of orthogonal experiment showed that the optimal concentrations for adventitious shoots induction with TDZ, BA and IBA were 1mg·L-1,0.5mg·L-1 and 1.5mg·L-1, respectively. To further increase the frequency, the effect of Sodium Nitroprussid (SNP) on the regeneration of adventitious shoots was investigated. When the concentration of SNP was 2mg·L-1, the frequency of adventitious shoots was increased significantly. After further culture the average number of adventitious shoots induced was markedly higher than the control. It was also found that the fourth spread leaf produced the highest frequency of adventitious shoots induced. Under the above best condition, after only four weeks lots of adventitious shoots were observed and the frequency of adventitious shoots reached 88%. Based on these improvements, the high-frequency direct regeneration system of of Jatropha curcas L. from leaf-discs was established.Employing the high-frequency regeneration system of Jatropha curcas L. from leaf-discs, Agrobacteriuim-mediated leaf-disc transformation was studied. First, the effect of Kanamycin, Hygromycin, Glufosinate ammoniumon, and Cephalothin on callus induction was investigated. Then, the effect of different selection stress, pre-culture time, OD value and infection time on transformation was evaluated. Using the GUS staining method, blue reaction was observed in the center part of resistant callus, suggesting that the gus gene is transformed into and expressed in the cell. Lastly, Agrobacteriuim-mediated callus transformation was conducted, and resistant adventitious shoots were obtained.