| Tall fescue (Festuca arundinacea Schreb.) is one of the major cool-season turfgrasses widely utilized at home and abroad, and improvement of its stress tolerances by genetic engineering is of momentous significance to maintaining of perennial evergreen turf, saving of water resource, enlargement of establishment area, and especially, to melioration of ecological environments in western regions of China. In this dissertation, influences of various factors on plant regeneration and Agrobacterium-mediated transformation of tall fescue embryogenic calli were systematically studied, and thereafter, stress tolerance-related CBF1 gene guided by constituent promoter CaMV 35S was incorporated into genome of this grass to obtain transgenic plants with increased stress tolerances. The main results are as follows:(1) Plant regeneration system of tall fescue embryogenic calli was established according to studies on impacts of manifold factors on callus induction from mature seeds and subculture production and differentiation of embryogenic calli. The results showed that high concentration of 2,4-D was required for callus induction from mature seeds of tall fescue, and combination of 8mg/L 2,4-D with 2mg/L ABA gave best induction effects. By slicing sterilized seeds longitudinally or cutting embryos, callus induction frequency was profoundly increased over intact seeds from one and half to eight times. Adoption of MS basal medium and supplementation of 0.5g/L casamino acids and 0.5g/L glutamine in medium were found to help to facilitate callus induction. It was also noted that irradiation treatment with low dose (10Gy) of y rays exerted slight stimulating effects on callus induction and formation of embryogenic calli in particular. Production of embryogenic calli was obviously promoted by addition of 0.1~0.2mg/L BAP or 2.5mg/L CuSO4 5H2O, or enhancement of sucrose concentration to 60g/L in subculture medium. BAP performed more important function than KT in differentiation of tall fescue embryogenic calli, but better results could be achieved with combination of 2mg/L BAP and 0.5mg/L KT. At this cytokinin level, 0.5mg/L NAA was recommended to obtain the highest callus regeneration frequency. Plant regeneration could be evidently boosted when embryogenic calli were pre-differentiated on high-osmoticum medium with 60g/L sucrose, and/or when the pre-differentiated compact calli were differentiated on differentiation medium solidified with l0g/L agar. Callus regeneration frequency was decreased but numbers of regenerated albino plantlets slightly reduced on differentiation medium supplemented with L-proline. In addition, it was observed that distinct differences in culture response existed among the tested cultivars, indicating the necessity of cultivar selection.(2) Based on transient expression of gus gene, the optimal conditions for Agrobacterium-mediated transformation of tall fescue embryogenic calli were determined as follows: addition of 0.5mg/L BAP and 0.5mg/L NAA to callus pre-culture medium, pre-culture time of 3~5d, bacterial suspension OD600 of 0.5-0.7, infection time of 10~20min, co-culture medium pH of 5.2-5.6, co-culture temperatureof 23-25 , co-culture time of 3d, and supplementation of 100 mol/L acetosyringone to Agrobacterium pre-cultivation and suspension media and co-culture medium. The results also revealed that it was a reasonable choice to use carbenicillin as the antibiotics of inhibiting growth of remnant Agrobacterium after co-culture, and when hygromycin was used as the selective agent, continuous selection at low concentrations (50~150mg/L) produced the highest numbers of transgenic plants without escapes. On the basis of the above studies, Agrobacterium-mediated transformation system of tall fescue embryogenic calli has been set up.(3) By adopting the established Agrobacterium-mediated transformation system, stress tolerance-related CBF1 gene was incorporated into genomes of four tall fescue cultivars for the first time in the world and 112 independent transgenic plants were obtaine...
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