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.

Ramie [Boehmeria nivea (L.) Gaud] is one of the most important perennial fiber crops in China. In vitro tissue culture of ramie could serve as an important means for its improvement through genetic transformation. To improve the regeneration capacity of ramie, the effects on plant regeneration of donor plant age, basal medium, plant growth regulators, and culture conditions were evaluated using explants derived from the cotyledon, hypocotyl, leaf, petiole, and stem of ramie seedlings. Cotyledons and hypocotyls excised from 4-d-old seedlings and leaves and petioles and stems from 15-d-old seedlings were optimal explants. The highest regeneration efficiency was obtained on Murashige and Skoog salts with Gamborg’s B5 vitamins basal medium containing 2.27 μM thidiazuron (TDZ) and 0.054 μM naphthaleneacetic acid (NAA) for the five explant types tested. A photoperiod of 16:8 h (light/dark) was found to be superior than continuous darkness for regeneration of ramie using TDZ. The regenerated shoots were transferred to hormone-free medium for shoot elongation and successfully rooted on half-strength Murashige and Skoog supplemented with 0.134 μM NAA. The rooted plantlets with four to five leaves were transplanted to greenhouse for further growth.

An efficient culture system for direct adventitious shoot regeneration of plantlet from cotyledon of ramie (Boehmeria nivea Gaud) was established and factors affecting shoot regeneration efficiencies were evaluated. Cotyledons excised from 4-day-old seedlings showed the highest regeneration capacity. Various concentrations of thidiazuron (TDZ) and indoleacetic acid (IAA) gave rise to adventitious shoots with different efficiencies. The cultures of cotyledons from 4-day-old ramie seedlings on MS medium supplemented with 2.27 mu M TDZ and 0.057 mu M IAA were shown to have the highest efficiency (83.6%) of shoot regeneration among the four auxin supplements (2,4-D; IAA; IBA; NAA). Regeneration of shoot in six cultivars was genotype-dependent, '5041-3' being the most responsive one. The regenerated shoots were transferred to hormone-free medium for shoot elongation, after successfully rooted on half-strength MS medium supplemented with 0.27 mu M NAA. The rooted plantlets with 4-5 leaves were transplanted to greenhouse for further growth. To our knowledge, this is the first report on efficient regeneration of plantlet from cotyledon of ramie, which will be of value for genetic improvement in the near future.

The effect of kanamycin,carbenicilin,cefotaxine on shoot regeneration from cotyledon explants of ramie and the effect of kanamycin on seed germination and growth were evaluated.The results showed that cotyledons of ramie were highly sensitive to kanamycin and no shoots could be regenerated from cotyledon explants on medium supplemented on 20 to 25mg/L kanamycin.Carbenicilin significantly inhibited plant regeneration from cotyledon explants,while cefotaxine under 500mg/L has no significant effect on plant regeneration from cotyledon explants of ramie.Furthermore,ramie seed was sensitive to kanamycin and seed cultured on medium supplemented with 100mg/L kanamycin for 30 days became completely yellow or white.

Recently, inter-simple sequence repeat (ISSR ) markers have emerged as an alternative system with reliability and advantages of microsatellites (SSR). ISSR markers are inherited in Mendelian mode and segregated as dominant markers. This technique has been widely used in the studies of cultivar identification, genetic mapping, gene tagging, genetic diversity, evolution and molecular ecology. In this paper, some factors influencing ISSR analysis, including 10×PCR Buffer ,the concentration of DNA template, Mg2+, dNTP, Primers, Taq polymerase ,thermal cycles and annealing temperature in Ramie inbred lines were studied. PCR system for ISSR in Ramie has been found: in 20ul reaction solution, contained 1.0umol/L primers, 60-100ng of genomic DNA template, 2.0mmol/LMg2+, 0.2 mmol/L dNTP , 1U Taq polymerase . The suitable procedure is one cycle 94℃ for 5min; denaturing at 94℃ for 30sec; primer annealing at 56℃ for 45sec , extension at 72℃ for 90sec,34cycles; extension at 72℃ for 7min at last and then remaing at 4℃.