Agrobacterium-Mediated Transformation Of Ramie (Boehmeria Nivea (L.) Gaud) By Male Sterility Gene (Barnase)

Ramie (Boehmeria nivea (L.) Gaud), one of the most important bast fiber crops, has been cultivated and used in China for a long time, but genetic engineering of ramie is at inchoate stage. The goal of this study is to provide the foundation for the widely use of genetic transformation on ramie. As ramie varieties (strain) "5041-3", "HuaZhu No. 4" and "HuaZhu No.5" for meterials, it was studied that the effection of different mediums, different basta and Km concentrations on explants regeneration, different factors on the efficiency of genetic transformation, and the Agrobacterium-mediated transformation of ramie (Boehmeria nivea (L.) Gaud) by male sterility gene (barnase). The major results are as follows:(1) By the combination between medium of MS, MSB (MS inorganic with B5 organic) and auxin of IAA, NAA, it was confirmed that IAA was suitable for the regeneration of cotyledon and NAA was suitable for the regeneration of leaf. In addition, choosing MS or MSB medium depended on the genotypes of explants.(2) The concentration of basta which effectively inhibited the regeneration of ramie explants was assured. It was enough for ramie transformation at the concentration of 0.5-0.7 mg/L, but it should make some appropriate adjustments referring to the physiological state of explants.(3) Futhermore, the concentration of kanamycin (Km), an antibiotic used as stringency of selection, should reach 25 mg/L at least when leaves were used for transformation. Genotype and physiological state had an impact on the appropriate concentration of Km. Therefore, to increase the positive rate, it was necessary to identify the appropriate concentration of antibiotics before the transformation of different explants.(4) As a reporter gene, GFP gene via Agrobacterium-mediated transformation was used to optimize the transformation system of ramie leaves from three parameters. Results showed that there would be a higher conversion rate at the condition that the incubation time was more than 24 h, AS concentration was between 50 and 100 mg/L, and the concentration of bacteria was in the OD₆₀₀ 0.2-1.5.(5) Transgenic plants containing male sterility gene were obtained using the transformation system of ramie cotyledon. PCR detection and Southern blotting detection were used to detect putative transformed plants, which showed that foreign gene had been intergrated into ramie genome.