Studies On Establishment Of Regeneration System And Transformation Of BADH Gene Of Fraxinus Velutina Torr.

Fraxinus Velutina Torr., introduced from East of Northern American to northern of Mexico and droughty area of Middle American. It is widely planted due to its high adaptability, salt-resistant, drought-resistant, pollution-resistant, disease & pest-resistant and landscaping function. In this program, the Agrobacterium tumefaciens mediated transformation of BADH gene of Fraxinus Velutina Torr. has been studied, for the purpose of improving resistance to soline-alkali and drought of the ash; of enlarging its arable land; of fully making benefits of its social and ecological value.In this research, seeds, which selected from a salt-resistant variety, have regenerated vitro culture and transformed the BADH gene by Agrobacterium mediated under establishment of the regeneration tissue culture technical system. The aim for this research was to achieve the salt-resistant plants of Fraxinus velutina Torr.. According to reference, this research was the first time to establish regeneration tissue culture and transformation gene technical system. The results could provide information and technology for later research and production practice. The main research results are following:1 Regeneration tissue culture system establishments and ability of salt-resistant: To different explants, the most effective media for inducing buds isn't same. The leaf explant could inducing buds on media MS supplemented with BA 8 mg ·L-1 and NAA 0.3 mg·L-1, but inducement rate low for average 3-4 buds on every explant. While cotyledon nodes produce average 7-8 buds on media MS containing TDZ 0.5 mg·L-1, BA 0.5 mg·L-1 and IBA 0.2 mg·L-1.Then buds were transferred to secondary MS medium containing BA 4.0 mg·L-1, IBA1.0 mg·L-1 and LH500 mg·L-1.The plants formed through buds developed well. More than 94% of adventitious shoots rooted if 1/2WPM medium were used with 1.0 mg·L-1 IBA and 20g·L-1 sucrose containing Before being planted into outdoor soil, the tube-plants should be acclimated in green house for about 15d firstly. The transplant-surviving rate could up to100%. So we regard it is a good tissue culture regeneration system with cotyledon nodes as explants.2 BADH gene transformation system establishment: 400mg·L-1 Cef was used as bacteriophage to control and wipe off the agrobacterium for selective culture, but had the disadvantage effect on buds inducing. We selected AgNO3 0.5 mg· L-1 and decreased Cef to 300 mg·L-1 during selective culture, and got good effects on wiping-off and regeneration. Fraxinus velutina Torr. is very sensitive to Kan, because there was no bud regenerated under 5mg·L-1 Kan. So we adopt delayed selection combined with gradings selection, it was the first time to adopt this combined way, and got a good result. According to regenerative rate and number of Kan-resistant buds, we established transformation system: cotyledon nodes as infective explants, 3 days preculture, OD0.3-0.5 agrobacterial density, 10-15 minutes infection time and 3 days coculture. After more than 90 days selection 14 regenerated Kan-resistant plantlets were abtained.