Agrobacterium tumefaciens-mediated approach is an effectively and widely used to introduce foreign DNA into plants. However, there were few reports published on ramie transformation mediated by Agrobacterium tumefaciens up to date. In the present study, transgenic ramie containing Bt gene were obtained by our highly efficient Agrobacterium-mediated transformation system using ramie Luzhuqing. Three T0 plants of transgenic ramie were chosen for further experiments and were transplanted into the fields. Laboratory assay of artificial infestation and the survey on agronomic traits and quality traits of T0 plants were conducted. The results showed that T0 transgenic plants exhibited more excellent pest resistance than the controls, and some were significantly superior to the controls. In addition, T0 transgenic plants maintained the main agronomic traits, such as plant height, stem diameter, rate of effective tiller, raw fiber weight, fiber production ratio from stem and fiber production ratio from phloem, and quality traits, such as strength, filament breaking tenacity, fiber fineness, fineness uniformity and glue content of raw fiber in Luzhuqing. PCR and Southern blotting analysis showed that T1 plants contained Bt gene, which indicated that Bt gene could be stably inherited. T1 plants in the fields performed highly pest resistance compared with non-transgenic plants.

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5-0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20 degrees C, in comparison to 15, 25 and 28 degrees C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie.

The factors influencing SRAP analysis, including 10×PCR Buffer, the concentration of DNA template, Mg2+, dNTP, primers, Taq polymerase, thermal cycles and annealing temperature in ramie were studied. PCR system for SRAP in ramie has been found: in 20μl reaction solution, contained 0.3μmol/L primers, 60~100ng DNA template, 2.0mmol Mg2+, 0.2mmol dNTP, 1U Taq polymerase. Amplification was carried out in two steps: in the first 5 cycles, the annealing temperature was 33℃, and during the following 30 cycles, the annealing temperature was 52℃. In all the cycles, denaturation was carried out for 1min at 94℃, annealing for 1min, and amplification for 1min at 72℃. After all the cycles, amplification was done for 1min at 72℃. It was shown that SRAP technology is an useful molecular marker system for mapping and gene tagging in ramie.