Study On The Agrobacterium-Mediated Transformation Of The GNA Gene In Gramineae Turfgrass

Cynodon dactylon(L.)Pars.,Zoysia japonica Steud and Festuca arundinacea are the major gramineous turfgrasses which are world-widely utilized.Because of the method of lawn maintenance and planting pattern,the turfgrass easy to be harm by pest,and the insect pest is easy to spread rapidly.The improvement of its resistance to pest by genetic engineering is of momentous significance to the defense of the pest, reduction the use of pesticide,environment protection and saving the maintenance fee of turfgrasses.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.According to the research of plant regeneration and Agrobacterium-Mediated trandformation system,Galanthus nivalis agglutinin gene- a kind of important insect-resistant gene was incorporated into the genome of these grasses to obtain transgenic plants with increased resistance to pest.Below is the main research result:1.Callus was induced using seeds as explant on Cynodon dactylon(L.)Pars., Zoysia japonica Steud and Festuca arundinacea.The best culture media of Cynodon dactylon(L.)Pars.and Zoysia japonica Steud is N6+2,4-D 2mg/L and 4mg/L,and the best callus induction frequency were 74.67%and 86.67%respectively.For Festuca arundinacea,MS is the most suitable culture media.The best callus induction frequency 73.33%was achieved when the concentration of 2,4-D is 11mg/L.2.Callus induction of Cynodon dactylon(L.)Pars.can be differentiated on the culture media 1/2 MS +6-BA 2mg/L+ NAA 0.5mg/L,and the differential rate is 28.13%.The bud differentiation were rooted in the culture media 1/2 MS.For the callus induction of Zoysia japonica Steud,the differentiation media are 1/2 N6+0.1 mg/L 2,4-D while the differential rate is 46.88%,and the rooted culture media is MS+NAA 0.5mg/L+KT 0.5mg/L.MS+1 mg/L 2,4-D+6-BA 2mg/L is the differentiation media for Festuca arundinacea with the differential rate of 77.78%, and the rooted culture media is the same as the differentiation media.3.Adding a certain concentration of Chitosan showed promoting effects on callus induction and differentiation of Cynodon dactylon(L.)Pars.and Zoysia japonica Steud.By adding the Chitosan,the callus induction frequency were improved,and the highest callus induction frequency of Cynodon dactylon(L.)Pars. and Zoysia japonica Steud are 86.68%and 98.33%respectively,while the differential rates are 35.00%and 78.13%.4.In the genetic transformation system of Cynodon dactylon(L.)Pars.,the optimum concentration of agrobacterium is OD₆₀₀≈0.4,and the infection time is 30min;the situation about Zoysia japonica Steud is same to Cynodon dactylon(L.) Pars.'s.The optimum condition of Festuca arundinacea is OD₆₀₀≈0.5 and 20min. These calluses were cultured on the regeneration media with additional antibiotics after 3 days,and resistant buds are obtained from the callus.After the selection of the resistant buds on the rooted media with antibiotics,resistant root came out and the whole transformed plants were formed.By PCR screen with transformed plants,the positive rate are all more than 70%.