Studies On Blue-related Genetic Transformation Of Lilium ‘Robina’

Flower color, as the most visual impact aesthetic characteristics, is one of the important factors in determining the ornamental value of floricultural plants. Lilium is a monocotyledonous plants belonging to the family Liliaceae. It is also one of the world famous five cut flowers, and occupies very important share of the market. Due to the lack of delphinidin-based anthocyanins in Lilium, there are no purple or blue color innature. Therefore, the creation of blue flower becomes the target of many breeders.In this study, lily(Lilium spp.) ’Robina’ is a department of hybrids, we established the acceptor system of Agrobacteruim-mediated transfortmation. Then by using the suspension cells and small bulbs as the gene transformation accepotor material, transformed the Phalaenopsis F3’5’H vector(p1300-pPZP-F3’5’H-DFR). Optimized the transformation system, obtain lily hygromycin resistant plants. Through PCR detection, reverse transcription PCR assay, southetn blot analysis, indicating that the vectors was transformed into the genome of lily, and get six transgenic lines.The main results obtained are as follows:(1) In order to obtain embryonic callus, different sizes of bud filament, style and ovary, were used as explants, When bud size is 8 ~ 10 cm, the highest induction rate: filaments 35.77%, the ovary 22.22%, style 18.13%. Meanwhile bud size of 5 ~ 7 cm when the ovaries produce buds highest rate of 40.74%. Induction of embryonic callus and adventitious buds provide material for subsequent conversion.(2) we established the suspension culture system of lily. Seedling experiments were carried out in suspension culture and regeneration system of adventitious buds small scale experiments. For small-scale, to determine the most suitable concentration of NAA induced adventitious buds was 1.5 mg ? L-1. Early obtain lily transgenic seedlings were transplanted and acclimated culture through successfully height of 15 ~ 20 cm lily plants.(3) In order to optimize the process of genetic transformation, respectively, pre-culture time, bacterial concentration infection, co-culture time and concentration by AS were assessed. With embryogenic callus the pre-culture 3d, OD600 =0.6, infection time 10 min, co-cultured for 3d, 100μmol/L acetosyringone conditions, which the stable transformation rate was the highest 13.33%; however, pre-culture 2d, OD600= 0.8, infection time 10 min, co-cultured 3d, with 100μmol/L acetosyringone conditions, the stable transformation rate of regenerated bulb scales could reach to the highest 12.78%.(4) We assessmented the suspension cell mass and the small bulbs resistant concentration of hygromycin. When hygromycin concentration of 20mg·L-1, the small bulbs germination rate was 1.67%, the cell mass germination rate was 0.67%, when hygromycin concentration of 25mg·L-1, Whether small bulbs or suspension cell mass all browning death, determined hygromycin as 20 mg·L-1 was lethal concentration. Carbenicillin is able to achieve inhibitory effect, so determine the concentration of carbenicillin is 200 mg·L-1.(5) In this study, 187 hygromycin resistant plantlets were obtained, 80 hygromycin resistant plantlets were at random selected for PCR and got the 15 plants strip, reverse transcription PCR assay and southern blot analysis, which has 6 strains showed positive.