Establishment Of Transgenic System Of Alfalfa And Effects Of Transformation Of Citrate Synthase Genes On Aluminum Tolerance Of The Cell

Alfalfa (Medicago sativa L.) is the world's most important forage legume, often called the "Queen of the forages" because of its importance as a high quality perennial forage crop cultivated on worldwide. In acidic soils, however, the performance of alfalfa is very poor due to its low degree of tolerance to the aluminum toxicity. This disadvantage has limited its wide cultivation and further exploitation in these regions. How to improve its tolerance to aluminum toxicity and broaden the area of cultivation is an important research subject confront with the field of alfalfa production. Development and application of transgenic technique opened up a new way for breeding cultivars of alfalfa that can establish and be productive on acid soils.To solve this problem, some important factors which influence the in vitro plant regeneration and transformation of alfalfa were investigated in this study. On the base of this, we cloned citrate synthesis genes and delivered them into alfalfa using Agrobacterium tumefaciens-mediate transformation technology, so as to study the effect of this gene overexpressed in the cell on aluminum tolerance of alfalfa. The main results are shown as follows:1. The induction of somatic embryogenesis and plant regeneration of alfalfa To optimize the growth condition of callus, effect of four different base medium (MS, SH, SHO, MSHO) on the proliferation of callus derived from both cotyledon and hypocotyl of four-days-old seedlings were tested. The results indicated that the average fresh-weight of the cotyledon-derived callus was significantly higher than that the hypocotyl-derived in all tested media, and that MSHO was the optimal medium for the proliferation of callus compared with other three media (P<0.05); The highest average number of somatic embryos (5.7 per callus) and the induction embryogenic callus (78.3%) was obtained on MSHO medium supplemented with 2 mg/l 2,4-D and 0.05 mg/l KN (P<0.05); After culture for ten days, a large number of secondary somatic embryo (1-30 per somatic embryo) could be induced on growth-regulator-free MSHO medium through separation and subculture of both globular or torpedo stage primary somatic embryos; Somatic embryos at late-torpedo or cotyledonary stage were separated and transferred to plant growth-regulator-free half-MS medium, over 96.6% of somatic embryos could germinate into complete plantlets with both well-developed shoots and roots; Great genotype variation are existed among different cultivars in their ability to form somatic embryos using this protocol (0-76.6%).2. The induction of adventitious shoots and plant regeneration of alfalfa For direct adventitious shoot induction of alfalfa, the cotyledon nodes excised from the mature somatic of alfalfa were cultured on improved MS (containing inorganic elements of MS and organic elements of B5) medium supplement with different concentrations of TDZ. The results indicated that many adventitious shoot buds near axillary meristems could be induced from explants after two axillary shoots were removed. With the growth of adventitious buds, unfortunately, most of them turned into vitreous shoots. To overcome this problem, further experiments were made in our study. The results showed that both changing the culture methods (inserting the hypocotyl of explants vertically into media instead of transversely putting them on the media) and reducing TDZ concentrations from 0.05 to 0.025 mg/1, could significantly reduce the percentage of vitreous shoots (P<0.05), respectively; Appending 3.0 mg/1 AgNO3, a potent inhibitor of ethylene action, into the medium at the same TDZ level, there were no positive effects on the alleviation of vitrification, nevertheless, the results showed that the addition of AgNO3 was obviously beneficial to adventitious shoots formation(P<0.05); The highest frequency of root induction (83.3%) were achieved by dipping the lower part of shoots in 1.0 mg/1 NAA solution for 2 min before transferring them to hormone free 1/2 MS medium; This regeneration system was also successfully applied to eight different cultivars, plant regeneration frequencies, expressed as percentage of explants producing adventitious shoots, ranged from 63.8%-82.5% for all tested cultivars of alfalfa.3. Study on the system of genetic transformation of alfalfa To improve the efficiency of Agrobacterium tumefaciens-mediated transformation system of alfalfa, some important factors which influence the efficiency of transformation were tested in this study. The results indicated that Agrobacterium tumefaciens LBA4404 had stronger ability to infect the cells of alfalfa than EHA105, and the efficiency of transformation could be increased by adding 10 mg/1 AS in the solution used for the infection; The efficiency of transformation was highly relied on the in vitro regeneration system used, thereinto, the system plant regenerated via somatic embryogenesis was more suitable for the transformation of alfalfa, because it has a higher efficiency of transformation (10.6%) than the direct adventitious shoot regeneration system (2.26%); The somatic embryogenesis of the callus derived from cotyledon could be entirely suppressed when the medium containing 10 g/1 sugar and 20 g/1 mannose, but to the hypocotyl-derived callus it needed a higher concentration of mannose to suppressed the somatic embryogenesis; Compared with the transgenic system using HPT gene as selection marker, the system employing PMI gene as the selection marker are more suitable for the regeneration of transformed plant. In addition, the efficiency of transformation was highly relied on the type of explants selected for transformation, and that cotyledon was more suitable as the material for transformation, because it has a higher efficiency of transformation (39.6%) than the direct adventitious shoot regeneration system (29.2%)(P<0.05).4. Effects of CS genes transformed in the alfalfa on aluminum tolerance of the cell Two different CS genes were cloned from E.coli (CSI) and Oryza sativa L (CSII), respectively, and then delivered CSI gene alone or in combination with the gene CSII at one time to the cells of alfalfa using transgenic technique. The results indicated that the expression of the CS gene in the transformed cells could increase the activity of both ATPase and transformed cells(P<0.05), and that cells transformed with two CS genes (CSI and CSII) at one time showed a higher activity than the cell transformed with CSI alone(P<0.05).