Studies On Embryogenic Callus Suspension Genetic Transformation Of Lily ’Robina’

Lily (Lilium spp.) is one of the five most famous cutting flowers in the world. Theblooming period is an important index to evaluate ornamental value. Lily belongs toethylene-climacteric flower. Down-regulation or knock-out of the key genes related toethylene biosynthesis (ACO gene) by RNA interference (RNAi) technology would delay theflower senescence and extend the vase life of cut flower.In this study, lily(Lilium spp.)’Robina’ is a department of hybrids, we established theacceptor system of Agrobacteruim-mediated transfortmation. Then by using the suspensioncells as the gene transformation accepotor material, transformed the intron-containing hairpinACO RNAi vector (pCAMBIA1301-35S-RNAi-ACO). Optimized the transformation system,obtain hygromycin resistant plants. Through GUS histochemical detection, PCR detection,southetn blot analysis, indicating that the ACO RNAi gene was transformed into the genomeof lily, and get four transgenic lines.The main results obtained are as follows:(1) Based on the preliminary studies, we established the suspension culture system of lily,microscopic observation of the suspension cell mass.The results showed that: throughsuspension culture of embryogenic callus3-4times, we can establish the size of cell mass in1-5mm, uniform cell morphology and speed propagation of suspension culture system.(2) We assessmented the suspension cell mass resistant concentration of hygromycin andcefotaxime. When hygromycin concentration of20mg·L-1, the cell mass mortality rate was89.44%, determined hygromycin as20mg·L-1was lethal concentration; cefotaxime was100mg·L-1, that si able to achieve inhibitory effect, and also does not inhibit the growth ofsusspension cell mass, so determine the concentration of cefotaxime is100mg·L-1.(3) In the Agrobacterium-mediated genetic transformation of lily suspension cell, weassessed the co-culture methods, bacterial concentration; and before the infection, let explantsfor cold treatment, the bacteria before infection adding1.5μM of spermidine and adding100μM L-glutamine in the co-culture medium. We evaluated the effect of three treatmentssingle or combination for transient expression rate, browning rate and stable expression. Theresults show that: the liquid co-culture can effectively enhance the transformation rate,transient expression rate was70.67%, significantly higher than solid culture. Liquidco-cultivation can effectively reduce the browning rate and increase the stability expression.When bacterial concentration OD600of0.8is optimal infection concentration. There have no effect in transient rate, bowning rate and stable expression when cold treatment alone process.Glutamine treatment alone had no effect on transient expression rate and browning rate, theimpact on stability transformation rate is not great. Three treatments have a synergistic effect,when three treatment processing jointly, the transient expression rate was93.33%,significantly higher than contrast, while three treatment combinations can effectively reducethe browning rate, it was37.77%, compared with the control group58.33%lower; stabletransformation rate was the highest37.22%, significantly higher than contrast18.33%.(4) In this study,237hygromycin resistant plantlets were obtained,132hygromycinresistant plantlets were at random selected for PCR and got the18plants strip, and southernblot analysis, which has4strains showed positive.