Study On The Agrobacterium-Mediated Transformation Of The SAP1 And GNA Gene In Turfgrass (Trifolium Repens Etc.)

Turf is the major component of the Urban greening.Dicotyledon turfgrass,such as Trifolium repens,Trifolium pretense,Lotus corniculatus Linn and Dichondra repens Forst.,has great value as ornamental turf.Plant genetic engineering can improve the quality and disease resistance of Dicotyledon turfgrass.Because of the method of lawn maintenance and planting pattern,the turfgrass easy to be harm by pathogen and pest. The main diseases are powdery mildew,southern blight and wilt disease.The main insect pest is cotton leafworm(Spodoptera litura),meadow moth(Loxostege sticticalis L.),leafhopper(Cicadellidae).etc.Agrobacterium-mediated transformation is one of the most intensive,common and mature methods in plant genetic engineering.It has many advantages such as simple operation,low price and high transformation rate.The establishment and improvement of the system of tissue culture and plantlets regeneration of Trifolium repens,Trifolium pretense and Dichondra repens Forst.was presented in this dissertation.Both the resistant SAP1 gene mediated by aagrobaterium LBA4404 and the insect-resistant GNA gene genetic transformation system of Trifolium repens were established.And transgenic plant of Trifolium repens was achieved in this dissertation.Here are the main research results:1.Effects of different Hormone Proportions on callus induction of Dichondra repens Forst.were different.Using the hypocotyl,cotyledon and seed as explant,the best hormone concentration of callus induction were 2,4-D 1mg/L+NAA 0.5mg/L+KT 0.1mg/L,2,4-D 2mg/L+6-BA 0.1mg/L and 2,4-D 0.5mg/L + NAA 0.1mg/L respectively. The qualities of the callus generated by different explants were different.The quality of the calluses,which use hypocotyl and cotyledon as explant,are more dense than the callus generated by seeds as explant.2.Using imbibed cotyledon of Trifolium repens,the multiple shoot was differentiated on the MS culture media with 6-BA 0.5 mg/L+NAA 0.05 mg/L,and differentiation rate was 75.00%.The multiple shoot was rooted on the original culture media.While using the cotyledon of Trifolium pretense as explant,it was directly differentiated on the MS culture media with 6-BA 1 mg/L +NAA 0.05 mg/L+1mg/L KT, and the differentiation rate was 73.75%.And the best rooted culture media was MS+NAA 0.5 mg/L.3.By inoculation of agrobacterium with GNA gene and SAP1 gene with imbibed cotyledon of Trifolium repen as explant.The bacterial liquid OD₆₀₀is 0.2 and 0.3,and the infection time is 35min and 25min respectively.Then co-cultured for 3 days,and cultured again in regeneration media MS+6-BA 0.5 mg/L+NAA 0.05 mg/L with 45mg/L kanamycin and 400mg/L carbenicillin for 28 days,the resistant buds were regenerated by cotyledon petiole,and the SAP1 and GNA gene convertion rate was 26.32%and 28.21%.4.After the selection of the resistant buds on the rooted media with 30mg/L kanamycin and 400mg/L carbenicillin,resistant root came out and the whole transformed plants were formed.By PCR screen with transformed plants,the positive rate of SAP1 gene was 84.84%,and the positive rate of GNA gene was 77.42%.