Construction Of Genetic Transformation System Of The Cbf1 Genes Into Phalaenopsis By Pollen-Tube Pathway

Phalaenopsis(Phalaenopsis SSP.)is called "Orchid Queen",tropical orchid treasures,with high ornamental value and economic value,occupies an important position in the world flower industry.At present,Phalaenopsis breeding methods are mainly cross breeding,but the breeding cycle is long and descendants choose is complicated.In order to create a special way to obtain new germplasm,transfer of the cfb1 gene,into Phalaenopsis by pollen-tube pathway was studied.The main results are as follows:(1)Studies on the pollen viability and stig ma fertility of Phalaenopsis showed that Pollen mother cell meiosis process is different with other plants,the reproductive development process is more special,male gamete development complete in the bud stage,Phalaenopsis pollen mother cells have entered the obvious tetrad stages,germ cells and nutrient nuclei have been formed.The pollen viability rate(dyeing rate)was as follows: 1d <bud stage <bud stage,the highest pollen activity was during the bud development period,then increasing over time,the activity gradually decreased.The results showed that the stigma could maintain a high activity in 10 d ~ 30 d,and it could be higher in 10 d ~ 15 d.The pollen was pollinated for blooming period of pollen to 15 days after the third day of budding,and the success rate of hybridization was obtained.(2)Study on the gene transformation system of Phalaenopsis' s Pollen Tube pathway.The optimal medium for the formation of protocorms from nonsymbiotic germination of Phalaenopsis' s seeds is Hyponex(3g/L)+agar7.5g/L+banana(100g/L)+agar7.5g/L+banana mud 100g/L+sucrose 25g/L+peptone2.0g/L+100g/L+sucrose 25g/L+peptone1.5g/L+activated carbon 2.0g/L+NAA1.0mg/L+6-BA10.0mg/L;The proliferation medium of protocorm is hyponex(3g/L)+agar7.5g/L+banana mud100g/L+sucrose 25g/L+peptone 2.0g/L+NAA0.01mg/L+6-BA0.01mg/L;The optimal medium for plant regeneration from protocorms was 1/2MS+agar 7.0g/L+banana mud 100g/L+peptone 2.0g/L+ activated carbon 2.0g/L+sucrose 25g/L;The antibiotic susceptibility test showed that the growth is slightly inhibited when the kanamycin selection pressure of Phalaenopsis protocorms and regenerated plants is 0~200mg/L,the growth is significantly inhibited when the kanamycin selection pressure is 300~400mg/L,the survival rate is 0 when kanamycin selection pressure is higher than 500mg/L.So,500mg/L of Kan can be used to the selection of transformed positive plant selection pressure.(3)The p BI121 plasmid carrying the target gene cbf 1 and the agrobacterium liquid transformed with cbf 1 expression vector were used to carry out genetic transformation using pollen granule and ovary injection respectively.The results showed that pollen-carrying method had higher seed setting rate than ovary injection method,but the conversion rate of ovary injection method was higher.The seed setting rate obtained by plasmid infection was higher than that of agrobacterium liqiud.The optimal concentration of plasmid was 100 ng/?L,and agrobacterium was OD600 = 1.0.PCR analysis of the positive plants screened by Kan showed that the exogenous cbf 1 gene had integrated into the genome of the Kan+.