Studies On Silencing The AGAMOUS Gene Via RNA Interference In Lilium By Agrobacterium Tumefaciens

The Lily is the perennial bulbs monocotyledon plant for the Lilium,for its graceful posture,lily has be hold one of the five fresh cutting flower on the world. But its shape is unitary,the wild Lily stock is a simple flower.In recent years,all breeders have been improving the Lily shape so diligently.The researches in Arabidopsis thaliana indicated that,double-stranded RNA(dsRNA)-mediated interference with expression of the floral organ identity gene AGAMOUS(AG)which belongs to the C function gene,the AG strong mutant performance for the stamen petal,the pistil replaces by another flower,thus presents the double flower phenotype.In this study,we established the acceptor system by Agrobacteruim-mediated transformation on L.longiflorum "White Fox".Using lily bulb's scale segments as the gene transformation acceptor,the foreign gene LLAGMLIKE which cloned from L.longiflorum was built as an ihpRNA silencing vector by our own laboratory,and was transformed by Agrobacterium EHA105.This study aimed at suppression of the expression of AG gene in Lily by RNAi technology,and to obtain the double petals mutant.The main results were as followings:1.The optimal culture media for inducing shoots from L.longiflorum "White Fox" scale was MS + 6-BA 2.0mg/L+NAA0.1mg/L,and the frequency of induction reaches 95.5%,which offered solid technical foundation for genetic transformation.2.Determination of the optimal screening concentration of transformation:The selectable marker gene was HPT,so adopting hygromycin as selective agent in transformation.The screening concentration of hygromycin for the scales slice was 8～10 mg/L.For Lily scales slice's tolerant to hygromycin is very low,has beneficial effects on genetic transformation efficiency to decrease content in selection medium in steps.3.By the establishment of the efficient transformation system(on several factors affecting Agrobacterium-mediated transformation),the results were,showed that:(1)The scales of plantlets in vitro showed a higher transformation rate under 3 day's pre-cultured;(2)It was suitable for immersion with OD₆₀₀=0.8 Agrobactium concentration;(3)The duration of immersion was 25～35 minute; (4)Co-culture term was 3～4days;(5)The efficiency of immersion could be increase by supplying 150μmol/L AS in co-culture medium.4.Five regenerated plants with hygromycin resistance were obtained.And with PCR and Southern blot,we proved that the LLAGMLIKE RNA interference gene was integrated into 3 transgenic Lilies genome.The expression of this AG gene was depressed significantly based on the Northern blot upon the transgenic plants.In conclusions,the depress interference of genes related to the lily flower development by ihpRNA vector were observed this study.Based on the regeneration and transformation systems,we suggested an RNAi inheritable transformation system for lily.However,the RNAi technique in the improvement of modifying the flower's architecture of lilies is still need to be study intensively.