Study On Agrobacterium-Mediated Gladiolus Transformation Of Chitinase Gene

gladiolus(Gladiolus hybridus) is one of perennial and monocotyledonous bulbous flowers,which belongs to the gladiolus of iridaceous and is one of the four world-famous cut flowers.Cut flower production of gladiolus plays an important role in Chinese folwer industry.The main problem in the production of gladiolus is that diseases and insect pests are so much, especially,the fungi disease is very serious. Using the disease-resistant varieties is the best way for preventing and curing the plant diseases.But the disease-resistant resource of gladiolus is so little and the cycle of the general breeding is so long that the disease-resistant variety is far from meeting the demand of the gladiolus production.With the rapid development of biotechnology, transferring new disease-resistant gene of other sources into the gladiolus by using genetic engineering means brings new hope in the disease-resistant breeding of gladiolus. The disease-resistant gene (chitinase gene) has been transferred into gladiolus mediated by the agrobacterium.Seted up high-frequency regeneration system and genetic transformation system,gained positive plant by PCR.Stablished the foundation for cultivation of new disease-resistant gladiolus variety , Offered the theoretical foundation for genetic transformation of the monocotyledon.The main content and result of experiment are as follows: 1.Established direct differentiation and callus differertiation regeneration system.Taking bulbs as explants , direct differentiated adventitious bud on the culture medium of MS+NAA0.2mg/L+6BA2.0mg/L+KT0.8mg/L,induced roots on the culture medium of MS+ IBA0.5mg/L.Taking young buds as explants,induced callus on the culture medium of MS+2,4-D4.0mg/L+6BA0.5mg/L,differentiated on the culture medium of MS+NAA0.2mg/L +6BA2.0mg/L+KT0.8mg/L and induced roots on the culture medium of MS. 2.Determined the select pressure of antibiotics.The select and sign gene NPTâ…¡on the carrier,which can make the trangenic plants have the resistance to kanamycin. The select pressure of kanamycin was determined as 110mg/L. 3.Studied four factors effect on genetic transformation which included pre-culture time,infect time,co-culture time and co-culture PH.For direct differentiation regeneration system,the best combination is:pre-culture 0d + infect 15min + co-culture 2d + co-culture pH 5.6.For callus differertiation regeneration system,the best combination is:pre-culture 2 d + infect 15min + co-culture 3d + co-culture pH5.2. 4.Detection of the transformed plant:For direct differentiation regeneration system, we gained 44 resistant plants.One of them is PCR positive plant,positive percentage is 2.3% and transformation percentage is 0.07%.For callus differentiation regeneration system,we gained 31 resistant plants.Three of them are PCR positive plants,positive percentage is 9.7% and transformation percentage is 2.0%.