An in Vitro Grafting Technique for Enhancing the Growth of Isolated Buds and Shoots in Jatropha Curcas

In most Agrobacterium-mediated Jatropha curcas transformation systems, antibiotics are used for suppressing the growth of the bacteria. However, these antibiotics could also hinder largely the further growth of the resulted buds. To tackle this problem, grafting methods were tested by using J. curcas buds and shoots isolated from seedlings as scions and the lower part of J. curcas seedlings as stocks. It was found that the most suitable stocks were those prepared from five days old seedlings and had the upper part radical. In later experiments, significant enhancement on the growth of buds in culture was confirmed in 1/2 MS medium containing0.3 mg/l indole-3-butyric acid (IBA). With the help of above established grafting methods by grafting the buds to the stocks, the growth of the grafted buds could be effectively enhanced. Introduction Jatropha curcas L. is the common name for physic nut and belongs to the Euphorbiaceae family.J. curcasis a type of woody plant widely distributed in the tropical and sub-tropical areas. The latex from J. curcas contains alkaloids such as Jatrophine and Jatropham, which are reported to have medicinal properties and can be used for extracting pharmaceutical compounds[1,2]and insecticides[3]. However, this tree is most famous for the high oil content (40% to 60%) in its seeds, andthere has been a surge of interest in J. curcascultivation around the world since studies showed that its methyl ester could yield biodiesel[4,5]. The primary limitation in its large-scale cultivation as an energy crop is the low and inconstant seed yield due to heterozygous nature of J. curcas plants. In order to breed advanced J. curcas varieties for large scale cultivation and produce more seeds, genetic transformation of the species might be a possible solution. Agrobacterium-mediating transformation method has been considered as an advanced strategy for breeding new varieties with improved traits. Since then, Agrobacterium-mediating method has been used widely in J. curcas transformation [6-8]. In a protocol of Agrobacterium-mediated transformation, a vector containing a target gene linked with a marker gene is constructed and a certain kind of antibiotics or herbicide is applied to screening the transformants, since the transformants contain the marker gene that can detoxificate the antibiotic or herbicide and by this means the transformants can be saved. However, the suppression effects of antibiotics or herbicide applied in the system on the growth of the transgenic buds and shoot is still evident, resulting in the recovery of only very poor health plants and a several months long growing period before transplanting from in vitro to soil of the transformants[9,10]. In vitro grafting is a method primarily used for recovering virus-free clones from virus infected plants[11]. Because in vitro grafting can effectively improve the growth of scion by replacing to the scion with a more suitable nutrient source (i.e. a stock), this method has also been used successfully in many cases rather than eradicating the virus infection [12,13]. In order to shorten the cycle of transformation and improve the health status of the transformants in J. curcas, we have recently evaluated the efficiency of J. curcas in vitro grafting method by comparison of several main factors in the in vitro culture system. Materials and Methods Plant Source Mature seeds were collected from cloned J. curcas trees coded M-19 in Hainan Island, China. Mature dry seeds were kept under 4 before used in the present studies. Medium Composition and Culture Conditions All the media contained myo-inositol 100mg/l, sucrose 3%, were adjusted to pH 5.8-6.0 with 1mol/l NaOH, and gelled with 0.6% agar prior to autoclaving under a temperature of 121 for 15 min, except those otherwise indicated. Whensodium nitroprusside (SNP), cefolexin andcarbenicillin were used, theywere filter-sterilized and then added to hot (not yet gelled) medium after autoclaving. All culture treatments were kept at 25±1°C. A 12-h photoperiod of cool-white light of 60-80 μ mol m-2s-1 intensity was provided. Preparation of Scion and Stock The shells of seeds were removed and the embryos were surface-sterilized by 2% sodium hypochlorite (NaClO) for 5 min and rinsed 5 times in sterile distilled water.The sterilized embryos were inoculated on hormone-free MS basal medium [14]. Bud scions of about 5 mm without the leaves were obtained from 5 days old germinated seeds and shoot scions of about 10 mm including the leaves were obtained from 15 days old seedlings. Stocks of different ages were obtained from seedlings by cutting away the upper part of the seedlings at about 3 mm distance under the cotyledons. Three types of stock were prepared: hypocotyls with intact radical, hypocotyls with 0.2 cmradical and hypocotyls without radical. Grafting Methods The upper surface of the hypocotyls stocks was cut across at the middle to about 5 mm depth, and then one scion was cut to have a “V” shape base and inserted into the cutting at the middle. In Vitro Growth and Transplantation to the Soil of the Grafted Seedlings Grafted seedlings were first cultured in vitro for 20 days under 12h photoperiod light conditions as mentioned above, then taken out of the culture bottles, cleaned to remove residual medium on the roots and dipped in tape water for one days. These cleaned seedlings were transplanted to autoclaved soil in pots and covered with transparent plastic film for keeping the humidity high. Ten days later, these seedlings were subjected to common cultivation conditions. Data Analysis All experiments were set up in a completely randomized factorial design and repeated three times with 20-30 replicates per treatment. Statistical analysis of the data was carried out using SPSS 17.0 soft ware, and significance of differences among means was determined by Duncan’s multiple range testes at p ≤ 5%. The results were expressed as means±SD (standard deviation) of three independent experiments. Results Influence of Stock Age on the Growth of Grated Seedlings The surface-sterilized embryos of seeds were inoculated on hormone free MS medium for various days and hypocotyls stocks with intact radical were prepared from these seedlings. From the results summarized in table 1, it is clear that the influence of age of the stocks on the growing parameters was all significant, with stocks from 5 days old seedling yielded the best results.