Study On The Agrobacterium-Mediated Transformation Of The Herbicide Tolerance Gene In Turfgrass

Turfgrasses are playing a more and more important role in our daily life, it is an important task to select and breed new good turfgrass variety. For a long time, many new varieties have been selected through introduction and breeding, such as cool tolerance, heat tolerance, disease resistance and so on. Transgenic technology has many advantages comparing to traditional breeding method, such as shortening breeding period, breaking genetic linkage, getting exotic gene easily and so on. So it is necessary to initiate the turfgrass transgenic research.Together the most main crops, such as rice, corn and wheat, weeds had the biggest bane in construct and seed production of turf, and usually waste a flood of manpower material resources, but results are not effective. With the genetic engineering breeding the herbicide tolerance turfgrasses, can save the expenses of large quantity, easy to mechanization and extend efficiently with the application of the extremely high effect herbicide species, resolve the problem that herbicide remain, is the most economic now effectively of a kind of means. Tissue culture and plantlet regeneration of two turfgrasses, tall fescue (Festuca arundinacea Schreb.) and creeping dichondra (Dichondra repens Forst.) were studied in the second part of thesis. The method of Agrobacterium-mediated transformation was adopted to establish the genetic transformation systems for creeping dichondra. The main results were as follows: 1 High frequency plant regeneration from mature embryos in vitro culture of tall fescueThe effects of different growth regulators, different genetypes (Finelawn, Dynasty, Mustang II, Bingo, Crossfire II, Barlexas) and different media on seed sprout and callus induction were studied with the uniform design, and the mature seeds of tall fescue were used as explants. The results indicated that 2,4-D had significant effect on seed-derived callus induction of tall fescue, the genetype had little inflection on callus induction, however, no effect of medium upon callus induction, and the seed-derived callus could differentiate immediately on medium without growth regulator. The optimum medium supplemented with growth regulators for callus induction of tall fescue was N6 + 18 mg/L2,4-D, the explants was the mature seeds of "Dynasty" of tall fescue, and the induction frequency was up to 95.60%, and the differentiation frequency reached 100%.2 High frequency plant regeneration from explants in vitro culture of creeping dichondraThe effect of four different growth regulators (2,4-D, 6-BA, KT and a-NAA) on callus induction of cotyledon, young leaf, petiole and hypocotyls in creeping dichondra with the orthogonal design. The results indicated that a mass of calli could be induced from different explants in vitro culture on induction medium supplemented with different growth regulators, and the callus induction frequency are from 43.3% to 100%. The growth regulator was an important factor on callus induction, and 2,4-D was essential to callus induction. The optimum medium supplemented with growth regulators for callus induction of creeping dichondra was MS + 1.0 mg/L 2,4-D + 0.2 mg/L 6-BA + 0.2 mg/L KT + 1.0 mg/L a-NAA. The differentiation from the young leaf of creeping dichondra was studied, and the optimum differentiation medium was MS + 5.0 mg/L 6-BA +3.0 mg/L ZT + 100 mg/L casein hydrolysate, the differentiation frequency could reach 74.80%.3 Established the Agrobacterium-medi&ted transformation systems of creeping dichondraWe used callus from the young leaf of creeping dichondra as target tissue for Agrobacterium-mediaXed transformation. The effects of time, OD600 and pH are studied with the orthogonal design. The genetic transformation systems for creeping dichondra are established, and Bar gene was transferred into creeping dichondra respectively by Agrobacterium-mediated, and many transgenic plants resistant to herbicide had been acquired. The optimum parameter was: the time was 10 min, OD6oo=0.6-0.8, pH5.4. The selection medium was MS + 1.0 mg/L 2,4-D + 0.2 mg/L 6-BA + 0.2 mg/L KT + 1.0 mg/L a-NAA + 250 mg/L Cef + 10 mg/L PPT, and the frequency of anti callus cultures could reach 85.35%., The best differentiation medium was MS + 5.0 mg/L 6-BA + 3.0 mg/L ZT + 100 mg/L casein hydrolysate + 250 mg/L Cef + 10 mg/L PPT.Postive frequency was 97.25% by PCR analysis for Bar gene in selective randomly resistance callus of creeping dichondra, and 100% in regenerated creeping dichondra. In daubing with herbicide experiments, the results showed that transgenic plants had more resistance than plants control did.