| Alfalfa (Medicago sativa L) is one of the leguminous forage crops which is grown most extensive and had high economic benefit around the world. However, many abiotic stresses such as high salinity, drought and low temperature severely reduce the growth and yield of alfalfa, and result in further degradation of vegetation and ecosystems. Therefore, better understanding the adaptation mechanism of alfalfa to various abiotic stresses and improving stress-tolerance by genetic engineering technology have an important theoretical and practical significance. In view of this, six alfalfa varieties which are grown in Northwest region were used as the materials, and their stress-tolerance during both germination and seedling stage were comparatively analyzed by high salinity, drought, low temperature and methyl viologen (MV)-mediated oxidative stress treatments in this study. With the selected tolerant and sensitive alfalfa varieties, the responses of growth, cell membrane stability and antioxidant enzymes system to various stresses were further evaluated. In order to develop the alfalfa with enhanced tolerance, some factors influencing in vitro regeneration from hypocotyl and cotyledon, and Agrobacterium tumefaciens-mediated genetic transformation of alfalfa were examined. The excellent regeneration and transformation system were established for two stress-tolerant varieties, Xinjiang Daye and Xinmu No.1. Two stress-tolerance related genes, AtNDPK2 gene encoding Arabidopsis thaliana nucleoside diphosphate kinase 2 and codA gene encoding Arthrobacter globiformis choline oxidase, were introduced into alfalfa. The stress-tolerance of transgenic plants were also evaluated. The major results are as follows:1. High salinity, drought and low temperature stresses resulted in the postpone of germination time by 13 days, and the decline of germination percentage by 3.0643.01 %,1.7524.2 % and 014.73 %, respectively. Xinjiang Daye and Xinmu No.1 exhibited the strongest tolerance, whereas Northstar showed the weakest tolerance to various stresses. At seedling stage, high salinity and drought reduced the chlorophyll content, and depressed the primary photosynthetic energy conversion efficiency of PSⅡby 3.078.93 % and 8.4122.07 %, respectively. 10μmol·L-1 MV-mediated oxidative stress destroyed the integrality of membrane. Xinmu No.1 and Xinjiang Daye showed the strongest tolerance, whereas Northstar was the weakest one. Comparing all results in different developmental stages, the tolerance order of six alfalfa varieties to various stresses was: Xinmu No.1, Xinjiang Daye > Algonquin, Victor, Golden Empress > Northstar.2. High salinity, drought and low temperature inhibited the accumulation of seedling fresh weight of Xinmu No.1 (tolerant variety) and Northstar (sensitive variety). Although drought stimulated the root elongation, high salinity and low temperature restrained the elongation of shoots and roots. Xinmu No.1 experienced lower inhibition than Northstar. All stresses resulted in the increase of H2O2, MDA contents and relative membrane permeability, whereas all above index detected in Xinmu No.1 were lower than that in Northstar, which indicated that the seedlings encountered apparent oxidative stress and membrane injury, and the damage of tolerant variety was more mild than that of sensitive one. The antioxidant enzyme system showed a similar co-ordination response to various stresses. Stimulated by each stress, SOD activity increased, which indicated that O2-. scavenging capacity was enhanced. APX activity of shoots increased and this enzyme activity in roots showed different response pattern. Whatever in shoots or roots, the APX activity of Xinmu No.1 was higher than that of Northstar. CAT and POD activity exhibited obvious morphological differences. CAT activity in shoots was more higher than that in roots, while POD showed opposite trend. CAT activity in shoots and POD activity in roots of Xinmu No.1 were significant higher than that in Northstar (except for low temperature). The antioxidant enzyme co-ordination mechanisms were different in two kind of organ. ROS was mostly scavenged in shoots through SOD, APX and CAT, while the scavenging was mostly performed by SOD, APX and POD in roots. These results also indicated that the antioxidant enzyme system plays an important protective role in alfalfa seedling establishment.3. The isozymes responses of SOD, APX, CAT to various stresses were analyzed. The results showed that SOD-2 (Rf = 0.48) activity was the strongest one in all SOD isoforms, possibly indicating that this isozyme play an important role in SOD catalytic process. The isozyme activity of APX-1 (Rf = 0.64) was stronger than other APX isoforms, and its changes was basically consistent with the changes of total APX activity. The result revealed that APX-1 not only mostly contributed to APX total activity, but also closely associated with abiotic stress including high salinity, drought and low temperature. The isozymes activities of POD-1, POD-3 and POD-4 (Rf = 0.07, 0.13, 0.26) in roots were more higher than other POD isoforms, and those changes were also consistent with the changes of total POD activity. These results indicated that the three POD isozymes mostly contributed to the total POD activity, and they were more sensitive to various stresses.4. The effect of genotypes, explants, medium and hormones on callus induction, embryoes differentiation and root induction were studied The results showed that effect of genotype on callus induction was not significant. Callus induction and in vitro regeneration by hypocotyls, the highest regeneration rate being up to 54.96 %, was stronger than cotyledons, suggesting the former is better explant. 2,4-D combined with 0.2 mg·L-1 kinetin (KT) could act better for callus induction. The SH medium was a very effective medium for the callus induction compared to MS medium, whereas the latter was more advantageous to embryo induction than the former medium. As to Xinjiang Daye and Xinmu No.1, the applicable media for induction of callus, embryoes and roots were SH (containing 2 mg·L-1 2,4-D and 0.2 mg·L-1 KT), MS (containing 1.0 mg·L-1 BAP and 0.3 mg·L-1 NAA), and hormone-free 1/2 MS (containing 1 mg·L-1 IBA when propagated), respectively.5. The using concentration of kanamycin and cefotaxime, Agrobacterium infection methods and co-culture time during transformation were studied. The results showed that hypocotyls were sensitive to kanamycin, and 50 mg·L-1 kanamycin was adopted as the effective concentration for transformants selection. 250 mg·L-1 cefotaxime could effectively inhibited the growth of Agrobacterium tumefaciens EHA105, whereas did not significantly influence the induction of callus and embryoes. The excellent transformation system was established as follows. The Agrobacterium cultured to the concentration of A600=0.60.8 was suspended by four-fold volume SHO liquid medium. Hypocotyls were directly cutted in suspension and infected with shaking for 20 min. After co-culture three days on B5h medium, the remanent Agrobacterium were eliminated by 250 mg·L-1 cefotaxime reagent, and hypocotyls were selected cultured on corresponding medium containing 50 mg·L-1 Kanamycin and 250 mg·L-1 cefotaxime for plant regeneration.6. The transformation of AtNDPK2 and codA genes to Xinjiang Daye and Xinmu No.1 were performed, respectively. Twelve AtNDPK2 transgenic lines and ten codA transgenic lines were successfully regenerated. PCR and RT-PCR preliminary indicated that the target genes have been not only integrated into alfalfa genome but also transcribed to mRNA drived by SWPA2 promoter under oxidative stress for all transgenic lines. Under MV-mediated oxidative stress, the relative membrane permeability of transgenic plants was closely correlated with the target genes mRNA transcription level. The relative membrane permeability of selected transgenic lines significantly decreased than non-transgenic plants. The preliminary results indicated that the genetic transformation of AtNDPK2 and codA genes into alfalfa enhanced tolerance to oxidative stress. |
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