Regenerarion Of Alfalfa And Transformation Of LEA₃ Gene By Particle Bombardment

Alfalfa (Medicago sativa) is one of the most important legume forages in the world. Because of its rich food value, drought and salinity tolerance, deep roots, soil holding ability etc., it is widely cultivated throughout the world. Unfortunately, due to increasingly widespread droughts, all pasture crops, including alfalfa, reach tolerance limits which restrict their spread and application. Gene engineering methods to improve drought and salt tolerances are the most economical and effective means to improve alfalfa.Seven species, which are widely used and well suited to the conditions of China, were screened. They are "Zhong Mu No.1", "Empress", "Chinese alfalfa", "American alfalfa", "Canadian alfalfa", "Setil" and "Derby". "Zhong Mu No.l" was outstanding among those seven species in many respects, such as high seed germination rate, plant POD activity, the growth rate under saline conditions and easiest to be induced and cultured in the tissue culture laboratory. Therefore, the embryogenic calli receptor system came from "Zhong Mu No1" are adopted.Hypocotyl, stem, and leaf tissue were taken as explants, induced them on MS with various plant hormones. The results indicated that MS+2,4-D1.0mg/L+6-BA0.5mg/L produced the optimum calli. The best explant was hypocotyl and the inducement rate approached 100%. To shorten the generation time, which also reduced the browning rate of calli, the concentration of 2,4-D was decreased and the concentration of 6-BA or KT was increased. Calli redifferentiate through somatic embryogenesis. Regenerated plants were got from MS+GA₃ 1.0mg/L and the redifferentiation rate reached 68.7%. The regenerated plants with plump, bright white roots and stems greater than 8cm in length were choosed. These tiny plants were grown on in garden soil and vermiculite (1:1), in pots, in the greenhouse. More than 90% of the plantlets thrived.For gene bombardment, somatic embryo calli of "Zhong Mu No.l" were taken. The E.coli plasmid obtained through the generosity of Cornell University which contained lea3 gene banded to bar gene was used. The plasmid was delivered to the calli by the particle delivery system. Control calli were bombarded with uncovered particles. All bombarded calliwere grown on MS+2,4—D1.0mg/L+6-BA0.5mg/L for 10 days, then transferred to an identical medium, except now also containing 8mg/L PPT. After two months' screening, all the controls had died. Of the transgenic calli, most of them died but some still alive. Those alive were transferred to regeneration medium. One month later, roots, stems and leaves are regenerated from the calli. Leaf samples were taken for genome extraction. The result of PCR showed 2 lines have attained the Iea3 gene, the transformation rate was nearly 10%.