An efficient protocol for Agrobacterium-mediated transformation of the biofuel plant Jatropha curcas by optimizing kanamycin concentration and duration of delayed selection

Abstract

<i>Jatropha curcas</i> is considered a potential biodiesel feedstock crop. Currently, the value of <i>J. curcas</i> is limited because its seed yield is generally low. Transgenic modification is a promising approach to improve the seed yield of <i>J. curcas.</i> Although <i>Agrobacterium</i>-mediated genetic transformation of <i>J. curcas</i> has been pursued for several years, the transformation efficiency remains unsatisfying. Therefore, a highly efficient and simple <i>Agrobacterium</i>-mediated genetic transformation method for <i>J. curcas</i> should be developed. We examined and optimized several key factors that affect genetic transformation of <i>J. curcas</i> in this study. The results showed that the EHA105 strain was superior to the other three <i>Agrobacterium tumefaciens</i> strains for infecting <i>J. curcas</i> cotyledons, and the supplementation of 100 mM acetosyringone slightly increased the transient transformation frequency. Use of the appropriate inoculation method, optimal kanamycin concentration and appropriate duration of delayed selection also improved the efficiency of stable genetic transformation of <i>J. curcas</i>. The percentage of β-glucuronidase positive <i>J. curcas</i> shoots reached as high as 56.0 %, and 1.70 transformants per explant were obtained with this protocol. Furthermore, we optimized the root-inducing medium to achieve a rooting rate of 84.9 %. Stable integration of the T-DNA into the genomes of putative transgenic lines was confirmed by PCR and Southern blot analysis. Using this improved protocol, a large number of transgenic <i>J. curcas</i> plantlets can be routinely obtained within approximately 4 months. The detailed information provided here for each step of <i>J. curcas</i> transformation should enable successful implementation of this transgenic technology in other laboratories.