Study On Transfering Leaf Senescence-inhibition Gene To Wheat Via Microprojecticle Bombardment

The wheat leaf is the main organ for photosynthesis. The decline of assimilation induced by leaf senescence may limit yield potential. Cytokine has the characteristic of delaying plant cell and tissue senescence. The leaf senescence -inhibition gene PSAG12-IPT that was designed by Gan and Amasino make using genetic transformation methods to modify early senescence of wheat. In this study, positive wheat plants of Xiaoyan22 and Ningchun16 with PSAG12-IPT gene were obtained by micro-projectile bombardment. The main result showed as follows:1. The mature embryos and the immature embryos as explants didn't have obviously different ability in callus induction. High concentration of 2,4-D was effective on mature embryos'induction, but during immature embryo culture the effect of 2,4-D concentration was related to varieties. Both callus differentiation rates of two explants were closely related to the concentration proportion of KT/IAA and high concentration of hormone was not good for the differentiation of mature embryos. Differentiation rate of callus induced on the medium with 2,4-D of low concentration was higher than that from the medium with high concentration. There were obvious interactions between each kind of medium and varieties so that the best group of medium for the culture of Xiaoyan22'mature embryos was MSD+3.0mg/L 2,4-D and MSD+0.5mg/L IAA+1.0mg/L KT and for immature embryos was MSD+4.0mg/L 2,4-D and MSD+0.5mg/L IAA+1.0mg/L KT. To Ningchun16 MSD+4.0mg/L 2 , 4-D and MSD+1.0mg/L IAA+1.0mg/L KT and MSD+1.0mg/L 2 , 4-D and MSD+2.0mg/L IAA+2.0mg/L KT were the best group respectively for mature embryos and for immature.2. The amount of gold particle had obvious effect on regeneration of immature embryo and callus after bombardment. The result show that the amount of gold particle between 0～250ug/gun had no action on the regeneration of immature embryo but between 400～600ug/gun the capacity of regeneration went down obviously. The effect of the amount of gold particle was related to genotype. The amount of gold particle had no effect on the regeneration of Ningchun16 callus, but when it was 600ug/gun it made the rate of regeneration of Xiaoyan22 callus down.3. The different amount of gold particle had different action on transformation frequency. 120～250ug/gun was preferable amount of gold particle of bombardment, and the transformation frequency of callus was 2.5% at 250ug/gun which was obvious higher than the others. 4. The callus was better genetic transformation acceptor than the immature embryo. The transformation frequency of immature embryo was 0.39% and the callus was 0.75%. Meanwhile Ningchun16 showed stronger capability to accept foreign gene. The best bombardment condition for callus was 1ug DNA per gun, 250ug gold particle per gun, 1100psi bombardment pressure, 9cm distance.5. Hygromycin was preferable for selecting transformast and we established Ningchun16's efficient selection system. Adding 80mg/L hygromycin to the subculture media was efficient to exclude the callus which was not transformed successfully.6. We first transferred Leaf senescence-inhibition Gene to Ningchun16 and Xiaoyan22 via microprojecticle bombardment, and 11 positive plants was found by PCR. Among them there were 2 Xiaoyan22 plants and 9 Ningchun16 plants. The total transformation frequency was 0.611%. The transformation frequency of Ningchuan16 callus and immature embryo were 1% and Xiaoyan22 was 0.222%.