Roles of cell culture regeneration system, Vir genes, and physiological conditions in T-DNA expression in kenaf (Vir genes).

摘要

Kenaf (Hibiscus cannabinus L.) is a fast growing annual that belongs to the Malvaceae family. Its stem fiber is an attractive source for many uses including ropes, textiles, and paper. Commercial production of kenaf is hindered by weed competition, diseases, insect pests and nematodes. Development of effective plant regeneration and transformation technique will provide opportunities for improvement of kenaf. This study focused on different plant cell culture regeneration systems, as well as physiological factors that have been reported to influence transformation efficiency. Attempts to develop a regeneration protocol for kenaf via somatic embryogenesis were unsuccessful. Embryogenic calli were obtained from hypocotyl and cotyledon explants of 3 kenaf cultivars. Globular embryos were produced and maintained on MS basal medium with 4.0 mg/1 2,4-D and 0.1 mg/l kinetin, but they failed to develop to advanced stages. Variations in cell inoculation density, and modification of the maturation media by adding cytokinins, silver nitrate, charcoal, trans-cinnamic acid or TritonX-100 did not enhance globular embryo development. In contrast, normal plants were regenerated in the shoot apex culture system. A high frequency of shoots elongated on MS with 0.1 mg/l BA. Multiple shoots were obtained on MS with 0.22 mg/l TDZ. Shoots rooted within 2 w.; Experiments evaluating conditions suitable for kenaf shoot apex transformation indicated the importance of multiple factor interactions. Additional virG/virE genes have been shown to enhance transformation efficiency in tobacco. However, this construct required the presence of TDZ in the co-cultivation. medium to increase transient transformation in kenaf. Sonicated shoots showed a marked increase in transient expression. Preculture (of the shoot apices for 2 d) or 200 μM acetosyringone (added into Agrobacterium culture 2 hr prior to co-cultivation) alone did not enhance transformation. Sonication of precultured shoot apices for 5 s and co-cultivation with LBA4404 in combination with 200 μM acetosyringone at room temperature (ca. 25°C significantly enhanced T-DNA transfer and transient expression in the kenaf shoot apex. This study is a foundation for further research to obtain transgenic kenaf with stable gene integration and expression.