Study On Genetic Transformation Into Gladiolus Hybridus With CBF1 Gene By Agrobacterium-mediated

Gladiolus (Gladiolus hybridus) is a perennial flower bulbs plant of lridaceae Gladiolu family. It's one of the four famous cutting-flowers all over the world. It takes a very important position in the field of cutting flower production and consumption. Although Gladiolus grows vigorously and healthily, the coldhardness was bad, easy to receive the frost bite, and the growth critical low temperature is only 3℃.The low temperature becomes the factor to limit distribution and production of Gladiolus hybridus.The modern Gladiolus hybridus variety is mostly Summer flowers variety, in the winter temperature low area, the field culture in the spring and summer festival, its cultivation scope has only received the very big limit, and it simultaneously gets sick so it causes the adult plant growth maldevelopment coldly, reduces the seedball and the cuting flower quality, which has affected Gladiolus hybridus's ornamental quality and the economical quality greatly, therefore it needs to cultivate the cold resistant variety. Duplication activation factor CBF1 can induce the low temperature adjustment protein (the COR protein) the expression, causes not to have certain anti-frozen strength after the low temperature domestication adult plant, therefore through transforming the CBF1 gene, it may enhance Gladiolus's winter resistance, and raises the Gladiolus output and the improvement quality efficient path.This article presents God Field as tries material and takes the corm bud as outside, studied regeneration process of injuried organization by the somatic embryo and established the high frequency somatic embryo regeneration system; Based on this and take the Gladiolus somatic embryo as the genetic transformation acceptor, it will lie between through the agricultural bacillus which leads the law to copy activation factor CBF1 to change over to Gladiolus, and established the stable heredity regeneration system;it obtains the masculine adult plant after the PCR examination, which will have our country proprietary intellectual property rights for the cultivation to resist the cold bears the counter variety to provide the theory and the experimental basis, the findings is as follows:1. Establishment of Gladiolus somatic embryo regeneration system: Plants the body take the corm bud slice as outside, the embryo induce medium is MS+2,4-D4mg/L+6-BA0.5mg/L+sugar30g/L+ + agar 7.5g/L, the determination best somatic embryo induction medium is MS+2,4-D0.5mg/L +TDZ0.3mg/L + sugar30g/L+mannitol 30g/L + agar 7.5g/L; the somatic embryo produced first vaccinates in MS+ sugar30g/L+mannitol 30g/L + agar 7.5g/L to cause its full growth, afterward changes over to MS+6-BA1.0mg/L+ sugar30g/L+mannitol 30g/L + agar 7.5g/L medium to induce germination, and changes over to MS+IBA1.5 mg/L+sugar30g/L+ agar 7.5g/L medium to induce rooting, finally domesticates transplants causes it to form the complete adult plant.2. Establishment of Gladiolus somatic embryo genetic transformation system: Take the somatic embryo as the genetic transformation acceptor, definite somatic embryo germination stage's grass Gan Lin (PPT) the selection pressure is 1.4mg/L, and spore mildew element (Cef) eliminates the fungus density is 350mg/L; The best genetic transformation system is: pre-culture 4d+ the bacterial concentration of OD₆₀₀ value 0.5+ infection time 25min+ co-cultivation 3d (ferulic acid 150mg/L) + delayed 2 weeks for selecting.3. PCR examination of transformed adult plant: we gained 26 resistant plants. Altogether 26 resistant adult plant was obtained by the somatic embryo regeneration way, take Gladiolus total DNA as the pattern plate, the CBF1 gene both sides sequence synthesis directs the thing, the antagonism adult plant carries on the PCR examination. Three of them is PCR positive plant, positive percentage is 11.6%.