| Lilies(Lilium spp.), perennial bulbous plant, bulbiferous, are one of fabulous cultivated flower varieties of great commercial value. They are popular cause their nutritional and medicinal value, as well as ornamental. To get the higher genetic transformation frequency and obtain Lilium germplasm with good stress resistance, in this study, the scales of Oriental hybrid ’Sorbonne’ were used as the gene receptors of exogenous gene AtCKX3 to estibalish the corresponding efficient co-transformation systems with security selection marker based on spray plant reactor (an open cultivation system), tissue culture and pollen-tube pathway(combined with cell penetrating peptide). Finally, the genetic plants were got by using the new method of the open cultivation system. This study could provide a reference for stress resistance and transgenic research for Lily. And what’s more, main research results were obtained as follows:1. Efficient co-transformation systems based on spray plant reactor (an open cultivation system):(1)After sprayed the different concentration phosphinothricin (PPT) on’Sobonne’ seedling, results showed that PPT were used as a suitable selective marker with a concentration of 8.5 mg·L-1.(2)For the first time, a new genetic transformation system to combine of a spay plant reactor and agrobacterium mediated gene transformation was used. Research results showed that the spray reactor had the advantages of simple operation, lower cost, lower operation requirements, and easier to achieve a large number of exogenous gene transformation work. Analyses of the putatively transformed plantlets were carried out by PCR and PCR-southern blotting. The results demonstrate that open culture is available to get higher genetic transformed frequency which was about 0.29%, and the transformed transplanting survival rate was higher to 75%. So the feasibility of this kind of genetic transformation system was proved successfully.2. Efficient co-transformation systems based on tissue culture:(1)By comparing with the differentiation efficiency and growth status of lily bulb induced by two diffrent media(MS+0.01mg·L-1 TDZ+0.2mg·L-1NAA; MS+2.0mg·L-16-BA+0.2 mg·L-1NAA), the scales cultured on MS media with 0.01mg·L-1TDZ and 0.2mg·L-1/’NAA showed higher regeneration efficiency and directly induced bulb seeding. Hence the combination was selected to establish the transformation system.(2)The ’Sorbonne’ regeneration scales respectively corresponding screened for carbenicillin (Cb) concentration and herbicide phosphinothricin (PPT) concentration in vitro. The results showed that the tolerance concentration of tissue culture scale against agents of Cb was 500mg·L-1, the concentration of PPT 2.5 mg·L-1 was suitable for screnning.(3) The transformation system of lily scales mediated by Agrobacterium in vitro was established. Transformation plants were screened. Analyses of the putatively transformed plantlets were carried out by PCR and results of PCR proved preliminarily that the AtCKX3 gene had integrated into Oriental lily ’Sorbonne’.3. Efficient co-transformation systems based on pollen-tube pathway(combined with cell penetrating peptide).The genetic transformation system based on pollen-tube pathway(combined with DNA cell penetrating peptide injection) was also tested. The flowes of four Oriental Hybrids cultivated variety and L. longiflorum (Leishan No.l) were used as material. The results showed that hybridized combination L. longifiorumx Sorbonne had high rate of capsules and seed embryo, which were 94.74%,18.31% respectively. In additon, spreading or the combination of spreading and injection were more suitable to inject the DAN cell penetrating peptide solution with less damage to plants. |
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