Study On Genetic Transformation Into Oriental Hybrid Lily Lilium Cv. Siberia With DREB Gene By Agrobacterium-mediated

Lily (Lilium spp.) is one of perennial and monocotyledonous bulbous flowers which belonging to the family Lilium of Liliaceae. Lily is one of the most important cut flowers in the world. Oriental hybrid lily is the best Lily species as cut flowers product because of its hard and standing stem, large and beautiful flower and it smells good. It is very popular for many people. However, the Oriental hybrid Lily likes growing on acid soil and needs high level management. It cannot over-winter in open field except for planting in green house and which are high cost. The condition of Drought, high salt and low temperature, which are common stress in North of China, is adversely affectting plant growth and application of Oriental hybrid Lily as cut flower and garden plant materials. So it is necessary to cultivate strong stress resistance lilies to rich landscape plants in the northern region. However, it is difficult to abtain ideal resist abilities through traditional breeding technique and moleculars marker aided selection and transformation of single functional gene into plants. Recently, scientists pay attention to transcription factor because it can control the expression of many genes involved in correlative character, so plants obtain all-around resist ability. Such as transcription factor DREB can act on DRE cis-acting element of correlative gene and regulate the expression of them under all above stress and improve stress tolerance ability of plant. According to above-mentioned facts, this study took Oriental hybrid Lily Lilium cv. Siberia. as material to establish regeneration system and genetic transformation system. The DREB gene which was regulated respectively by constitutive expression promoter E12 and inducible expression promoter rd29A were transformed into Lily by Agrobacterium-mediated with transcription factor DREB gene and obtained PCR positive and PCR-Southern positive plants.The research work will provide the function for cultivating transgenic variety that resist to osmotic stress and studying osmotic stress resist machanism.The main results were summarized as follows:1. Establishment of direct regeneration system of Lilium cv. Siberia: we used the bulbs of lily as the initial explants to obtain leaflets.The adventitious buds inducing medium of bulbs is MS+NAA1.0mg/L+ 6-BA0.5mg/L;The adventitious buds inducing medium of leaves which were sliced from leaflets is MS+NAA1.0 mg/L+6-BA 0.5 mg/L+KT0.1mg/L;The medium of bud proliferation is MS+ 60g/L Suc;induced roots on the culture medium of MS. The suitable ratio of transplanting medium for soil:Vermiculite is 1:1.2. Establishment of callus regeneration system of Lilium cv. Siberia: Took the leaves which were sliced from leaflets as the explants, the callus inducing medium is MS+NAA0.1mg/L+ 6BA1.0mg/L+2,4-D 0.1mg/L;the optimize concentration of callus inducing by Picloram is 2.0 mg/L;3. Leaves sliced from leaflets were taken as explants, The suitable transformation condition was determined as followed: pre-culture 3d, the bacterial concentration reached 0.5-0.6(OD600) with 20mg/l acetosyingone,15 minutes infection time,using10.3mmol/lNH4NO3 medium for co-cultivation 3 days, 200mg/L Cef for removing bacterium cultivation,delayed 12 days for selecting by 30mg/l kanamycin on regeneration medium and 110mg/l kanamycin for rooting selection.4. Callus was taken as explant, the suitable transformation condition was determined as followed: pre-culture 3d, the bacterial concentration reached 0.5-0.6(OD600) with 60mg/l acetosyingone, 15 minutes infection time, using 10.3mmol/lNH4NO3 medium for co-cultivation 6 days ,200mg/L Cef for removing bacterium cultivation and 110mg/l kanamycin for rooting selection.4. 19 and 21 resistant plants which were transformed respectively with LBA4404(pBcE12) and LBA4404(pBc29A) were obtained,.The rate of resistant buds are 10.3% and 9.7% respe -ctively. We obtained 11 PCR positive plants from 21 resistant plants, which were transformed with LBA4404(pBc29A). The rate of PCR positive is 52.4% .We also obtained 11 PCR positive plants from 19 resistant plants, which were transformed with LBA4404 (pBcE12).The rate of PCR positive is 57.9%; The eleven PCR positive plants which were transformed with LBA4404 (pBcE12) were all PCR-Southern positive plants.Eight of the eleven PCR positive plants which were transformed with LBA4404 (pBc29A) were PCR-Southern positive plants.