Studies On Agrobacterium-Mediated Transformation Of Lily With ACO Gene RNAi Expression Vector

Asiatic lily is one of the main cut-flowers in the world. But compared with the Easter lily it has shorter flower period, which influences its ornamental value and vase life seriously. Therefore producing novel Asiatic lily varieties with longer flowering period is particularly important and urgent. The past results showed that Lily is a type of Ethylene releasing flower, the flowers were blooming along with the releasing of ethylene and decaying. ACO is a key enzyme which catalytics ethylene production in the final step, it controls rate of ethylene production. Therefore, through RNA interference technology the mRNA transcripted by endogenous ACO gene will be degraded, ACO gene will be silenced efficiently, ethylene production will be inhibited, and the florescence will be relayed.With the Asiatic hybrids, Lilium pollyanna as the research material, a direct differentiation acceptor system and genetically transformation system were established. And transgenic plants were achieved by Agrobacterium-mediated transformation of Asiatic lily with ACO gene RNAi expression vector, which provide a basis for genetic transformation of lily. The main results of the study:1. A direct differentiation acceptor system was established(1)The optimum culture medium for the scales and the scale leaves of the test-tube plantlets to differentiate adventitious buds were MS+BA2.0 mg/L+NAA0.2 mg/L;(2)The suitable culture medium for the petioles of the test-tube plantlets to differentiate adventitious buds was MS+BA1.0 mg/L +NAA0.3 mg/L;(3)The culture medium for bulblet swelling of the test-tube plantlets was 1/2MS+IBA0.5 mg/L +90gSucrose+Light culture after dark culture 1month;(4)The appropriate medium for rooting was 1/2MS+IBA0.5 mg/L +0.1%AC;(5)The results of kannamycin resistant selection showed that 150mg/L is suitable for the scales, and 100mg/L is suitable for the scale leaves。2. A genetically transformation system was established.(1)The best instrusion time and agrobacterium concentration for scale leaves and scales were:8min with OD600=0.3 for scale leaves, 30min with OD600=0.8 for scales,;(2)The efficiency of intrusion could be increased by supplying 20 mg/L AS, activity culture of Agrobacterium for 2h after AS was filled in MS liquid and solid co-cultivation medium either ; (3) None preculture could decrease contamination rate after instrusion by Agrobacterium which is better for the transformation of Lilium;(4) Using NH4NO3-free medium for co-cultivation could improve the transformation rate a time.3.106 of Kanamycin resistant plants were achieved, and 55 was identified that we have get 4 PCR positive plantlets。...