| Ramie( Boehmeria nivea Gaud)is a perennial herbaceous plant of the Urticaceae family. Ramie bast fiber is a kind of fine plant texile materials. It is mainly planted in tropical and subtropical regions in China.It is difficult to achieve a breakthrough in breeding duo to its high heterozygotes, complex genetic background, long sexual hybridization breeding cycle. So development of a genetic transformation system to introduce desired genes into ramie could improve quality, develop new germplasm or cultivars. Several studies have successfully developed transgenic ramie by using A. tumefaciens-mediated transformation, but in most cases the transformation efficiency was low. All these studies emphasized on the feasibility of Agrobacterium-mediated transformation to generate transgenic ramie and the protocol has not been optimized.In this study, an efficient regeneration system for adventitious shoot induction was developed by using hypocotyls explants of ramie, either by shoot organogenes or multiple shoot formation from callus, and by direct regeneration of shoots without any intermediate callus phase. Transformation system of ramie was established via Agrobacterium tumefaciens-mediated method , particle bombardment and the floral dip method, transgenic plants integrated with an insect-resistance gene were produced.Main research results as follows:(1)The establishment of efficient protocols for plant regeneration from hypocotyls and cotyledonBased on pre-trial, we compared the responses of ramie hypocotyls segments on various TDZ concentrations in combination with different concentrations of BA to effect of direct shoot regeneration without an intermediate callus phase,a two-step procedure for efficient indirect regeneration by and multiple shoot formation. Based on the above study, two-step procedure for efficient indirect regeneration was chosen for the transformation system of ramie via Agrobacterium tumefaciens-mediated method due to its highest efficiency of shoot formation. Cotyledon and hypocotyl explants of var. Zhongzhu No.1 Zhongsizhu No.1 NC01was tested for feasibility of this regeneration protocol.(2)The establishment of Genetic transformation of ramie by Agrobacteriumtumefaciens-mediated method from hypocotyls and cotyledonBased on the above efficient protocols for plant regeneration, the effect of infection time, selection pressure , co-cultivation time, pre-cultivation time and concentration of cefotaxime on transient GUS expression and regeneration efficiency were evaluated and an efficient transformation system by Agrobacteriumtumefaciens-mediated was established. Cotyledon and hypocotyl explants of var. Zhongzhu No.1 Zhongsizhu No.1 NC01was tested for feasibility of this transformation protocol. GUS detection, PCR detection, RT-PCR and Southern blotting detection were used to detect putative transformed plants, while showed that foreign gene had been integrated into ramie genome.(3)The establishment of genetic transformation of ramie by particle bombardmentBased on research shoot-tip regeneration by our lab and the above efficient protocols for cotyledon regeneration, the effect of helium pressure, the vaccum pressure, target distance and times of bombardment.on transient GUS expression and were evaluated and an efficient transformation system by particle bombardment was established. Putative transformed plants were detected by GUS and PCR.(4)The establishment of genetic transformation of ramie by floral dip methodThe effect of concentration of Silwet L-77, infection of time, times of infection, drop-by-drop inoculation and submersion on the efficiency transformation and were evaluated and an efficient transformation system by floral dip method was established. T1 transformed plants was determined experimentally, by planting wild type plants on MS medium supplemented with a range of concentrations of the kanamycin. GUS detection, PCR detection, fluorescence quantitative PCR and Southern blotting detection were used to detect putative transformed plants, while showed that foreign gene had been integrated into ramie genome.
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