Genetic Transformation Of Sugarcane Mediation By Agrobacterium Tumefacien With Bt Gene

In various methods of plant genetic transformation nowadays, Ti and Ri plasmid of Agrodacterium tumefaciens-mediated is applied earliest and widest .Sugarcane is a monocotyledonous plant and a very important sugar-yielding crop from Gramineae family. During the growth period, sugarcane is apt to attack by insects of Lepidoptera and Homoptera ect., insecticidal genetic engineering is a basic method to sugarcane pest control. The plasmid containing Bt gene and kanamycin-resistant gene was transformed into sugarcane by the method of treating callus with Agrodacterium tumefaciens-mediating and A. tumefaciens-mediating combining negative pressure. By comparing research, all the factors were found out that influence Agrodacterium tumefacien transformation efficiency, which are choosing right physiological state of receptor tissue, blowing to dry the receptor tissue before transformation, suitable infection time, the number of co-culturing days, concentration of antibiotics, negative pressure, and AS. TheⅡ-type calli, blowing 30~60 min before transformation, infecting 45 min, co-culturing 3 days, the concentration of ceftaxine(cef) is below 500mg/L and AS is 100mg/L, negative pressure range is -0.05MP that can get an efficient transformation of sugarcane mediated by Agrobacterium. Culture tender leaves in culture medium of MS+2,4-D1.5mg/L+30g/L suger+0.7% agar pH5.8 for 20 days in darkness at 25℃, and then subculture to induced Ⅱ-type calli. Use forceps cutting the tissue to nubble with 2mm2, and put the tissue into Agrodacterium tumefaciens LBA4404 liquid supplemented with AS 100mg/L,then, co-culture 3 days, resume 7 days in MS+2,4-D1.5mg/L+30g/L suger+500mg/L cef, take to MS+2,4-D1.5mg/L+30g/L suger+100mg/L cef+10.0mg/L kanamycin(kn) culture 20 days in darkness. After that to make it polarize in MS+30g/L suger+100mg/L cef+10.0mg/L km. The percentage of km resistant callus was reached max after infection for 45 min, the average is 29.66%. Simultaneity, tender leave callus are infected 5 min by A. tumefaciens liquid in different negative pressure, and kept on 15 min in Agrodacterium tumefaciens liquid without negative pressure. Then screen out resistant callus and obtain transgenic plants. When the negative pressure is -0.05MP the percentage of km resistant callus was reached max, the average rate is 37.5%. The resistant callus was shifted to MS culture medium supplemented with 2,4-D1.5mg/L+30g/L suger+100mg/L cef+10.0mg/L km for plant regeneration, then in 1/2MS+NAA1.0+15g/L suger+9.0mg/L km for rooting. When sprouts grow 5cm, the transplant seedlings were transformed in field. After growing for 5 months, spires were used for isolate total DNA. PCR was used to detect the transforming plantlets, 20 transforming plantlets were obtained from 118 plantlets. Insect bioassay showed that some transgenic plants could resistance the damage of Diatraea sacchararlis. The result indicated primarily that the Bt gene was integrated into transgenic plants genome.