Efficient regeneration system and Agrobacterium-mediated transformation of Vetiveria zizanioides (L.) Nash

Vetiver (Vetiveria zizanioides (L.) Nash) has been widely used in South China for erosion control and ecological restoration, but it cannot be used in North China due to poor resistance to cold. To better improve the cold resistance of vetiver, a method was established to enhance cold resistance of vetiver. 2,4-dichlorophenoxyacitic acid (2,4-D) and 6-benzyladenine (6-BA) at a concentration of 1.0 and 0.5 mg l(-1), respectively were most successful in inducing embryonic calli with an induction frequency up to 96.7%. Cytology observation proved that embryonic calli originated from epidermal cells and parenchyma cells of vetiver, and formed typical embryonic structure of monocotyledon. The regeneration ability of embryonic calli could be maintained for over two years, and the regeneration frequency was over 80% regardless of subculture times. Plant expression vector p 1301 UN-otsA was constructed by inserting the otsA gene digested with Sac I/Kpn I into Multiple Colony Site (MCS) of binary vector p1301UN. The freeze-thaw method was used to mobilize the recombinant plasmid into DH5a. Both Restriction analysis and DNA sequence analysis confirmed that the construction of plant expression vector p1301UN-otsA was successful. Moreover, an efficient genetic transformation system of vetiver was determined as follows: embryonic calli were infected with A. tumefaciens EHA105/p1301 (OD600=0.4-0.5) for 20 min., and then transferred to co-cultivation induction medium (CIM) in the dark at 25 degrees C for 4 days; thereafter the infected calli were selected-on screening induction medium (SIM) in the dark at 25 degrees C for 4 weeks. Using the optimized protocol, 18% of the infected calli were hygromycin B (Hyg B) resistant. Transient integration and expression were confirmed by GUS assay.