Research On Somaclonal Variation And Genetically Transformation Of Several Turfgrass

Transgenic Japanese lawngrass plants containing a codon-modified insecticidal Bacillus thuringiensic (Bt) cry1A(b) gene were obtained through Agrobacterium-mediated transformation, based on optimizations of regenerative and transformation system; somaclonal variation combined with mutation breeding technology were used to genetically improve tall fescue, based on the established regeneration system. The results showed as the follows: 1) The major factors for callus induction, differentiation of Japanese lawngrass and tall fescue were studied, using the mature seeds as the original material. The results indicated that the optimal culture medium for Japanese lawngrass callus induction is MS basal medium containing hormone combinations with 2,4-D 1mg/L and NAA 3mg/L; for subculture is MS basal medium containing hormone combinations with 2,4-D 0.5 mg/L and 6-BA 0.2 mg/L; for callus differentiation is MS basal medium containing hormone combinations with 6-BA 3mg/L and KT 3.5mg/L, and for root formation is MS basal medium containing 0.3mg/L NAA. The optimal culture medium for tall fescue callus induction is MS basal medium containing hormone combinations with 2,4-D 5 mg/L and 6-BA 0.2 mg/L; for callus differentiation is MS basal medium containing hormone combinations with 6-BA 3mg/L, and for root formation is 1/2 MS basal medium with hormone free. All of the solid mediums above were supplemented with 30mg/L sucrose and 3g/L phytagel. 2) Some Japanese lawngrass and tall fescue mutants with leaf shape, plant height and leaf color variation were obtained, after the embryogenic callus were collected and irradiated with γ radial. Among them, the yellow tall fescue was treated with space technique. The results showed that space technique had slight physiological damage in SP1 generation, and no chlorophyll-deficient mutation was detected at the SP2, however richer variations in plant morphology, leaf shape, slender leaf, late maturing, and thermo-tolerant mutants were isolated from the yellowed tall fescue. 3) The genetic transgenic system of Japanese lawngrass mediated by Agrobacterium and factors were investigated using the transient expression of gus gene and the optimal conditions were determined as follows: the embryogenic callus that had been subcultured once were transferred to pre-culture medium supplemented with 100μmol/L AS for 3-5 days; the callus were irradiation with 2Gy γ radial 24 hours before transformation; bacterial concentration OD600 0.2, infection time 5 min, appropriate negative pressure treatment, co-culture 3 days, and intervals of subculture 7-10 days. The effects of antibiotics on bacterial growth and in vitro response were comprehensively investigated. Carbenicillin with 250-500mg/L was optional to remove the bacterial cells after co-cultivation in the process of Agrobacterium-mediated transformation. To Japanese lawngrass embryogenic callus, higher sensitivity to hygromycin was observed than that to kanamycin. The preferable concentration of hygromycin for transformant screening is 200 mg/L. 4) The insecticidal Bacillus thuringiensis (Bt) cry1Ab gene was firstly transformed into the mature seeds-derived calluses of Japanese lawngras through Agrobacterium-mediated transformation, and 24 hygromycin resistant plantlets were obtained. Subsequent GUS histochemistry analysis, PCR and Southern hybridization assays revealed that the foreign genes had been stably integrated into Japanese lawngras genome. Bt protein determination confirmed that the Bt proteins were expressed in the most transgenic plants and the amounts of Bt proteins were variable in different transgenic plants.