Studies On Regeneration System Establishment And FPPS Cloning Of Lilium Spp.

Lily is a bulbous herbaceous perennial which belongs to the genus Lilium. It is mainly used as ornamental flower, cut flower, potted flower and applying directly in the landscape architecture for decoration. The bulb of lily has edible and medicinal values. China is one of the natural distribution centers of wild lilies, and the lilies are distributed widely in China, especially in the central and southwestern areas.In this study, we have investigated and analyzed the resources of wild lily in the middle reaches of Yangtze River. At the same time, complete regeneration systems of Lilium leucanthum and L. tigrinum were both established, which could build the basis for their tissue culture multiplication, resource conservation and genetic engineering research. In order to investigate the mechanism of biosynthesis and regulation of farnesyl pyrophosphate (FPP) of L. longiflorum, farnesyl pyrophosphate synthase(LIFPPS) gene were cloned from the petals of L. longiflorum and its expression patterns and regulation mechanism were studied.The main results were as follows:1. Resources investigation of wild lilies in the middle reaches of Yangtze River.There were wild lilies resources 14 species and 3 varieties in the middle reaches of Yangtze River, including Lilium brownii F. E. Brown ex Miellez, L. brownii F. E. Brown ex Miellez var. viridulum Baker, L. leucanthum (Baker) Baker, L. concolor Salisb. var. concolor, L. concolor Salisb.var.pulchellum (Fisch.)Regel, L. bakerianum Coll. et Hemsl, L. taliense Franch., L. speciosum Thunb. var. gloriosoides Baker, L. henryi Baker, L. rosthornii Diels, L. duchartrei Franch., L. pumilum DC., L. davidii Duchartre, L. callosum Sieb. et Zucc., L. papilliferum Franch., L.fargesii Franch. and L. tigrinum Ker Gawler. And distribution situation, ornamental characteristics and exploitation values of wild lilies in the middle reaches of Yangtze River were analyzed, which would lay the foundation for new cultivar breeding, and supply worthy suggestions for the wild resources conservation and suitable exploration of wild lilies.2. Regeneration system establishment of L. leucanthum.(1) Bulblets regeneration was tested on MS medium supplemented with different concentrations of BA and NAA. The highest frequency of regeneration (96.7%) and the largest mean number of regenerated bulblets per scale (3.07) were obtained on MS medium containing 0.5 mg/L BA and 0.1 mg/L NAA. In vitro cultured scale explants showed great ability to induce callus, followed by in vitro cultured petioles and leaves.(2) MS medium with 1.0 mg/L BA and 1.0 mg/L 2,4-D was found to be optimum for callus induction from in vitro leaves and petioles with the highest induction percentages of 79.6% and 83.3% respectively. MS medium containing 0.5 mg/L BA and 1.0-3.0 mg/L 2, 4-D was found best for callus induction from scales with the highest induction percentage (98.3%). The largest number of bulblets was regenerated (23.9) from 1.0g of fresh callus cultured on MS supplemented with 0.5 mg/L BA and 0.2 mg/L NAA.(3) The number of roots per plantlet was fewer and the length of roots was greater on the rooting media with AC compared to the media without AC, half-strength MS medium with 0.05 mg/L NAA was more suitable for rooting.3. In vitro regeneration of L. tigrinum.(1) Bulblets regeneration was tested on MS medium supplemented with different concentrations of BA and NAA. The highest survival rate (97.8%) and the largest mean number of regenerated bulblets per scale (3.1) were obtained on MS medium containing 0.3 mg/L BA and 0.5 mg/L NAA. MS medium containing 1.0 mg/L BA and 0.3 mg/L NAA is suitable for bulblets regeneration from in vitro cultured petioles, survival rate of petioles is up to 72.9%, and the mean number of regenerated bulblets per scale is 1. In vitro cultured scale explants showed great ability to regenerate bulblets, followed by in vitro cultured petioles. In vitro leaves almost die on all the media, few bulblets were regenerated.(2) It was hard to induce callus from scales of L. tigrinum on the media supplemented with different concentrations of BA, ZT, KT and 2,4-D.(3) MS medium containing 1.0 mg/L BA and 0.2 mg/L NAA is suitable for bulblets prolification culture, coefficiency of multiplication is up to 2.5.(4) Rooting of bulblets was carried out on MS medium supplemented with 0.1 mg/L NAA and 0.05%AC. White strong roots produced after 15 days, and can be transplanted until the length of roots is up to 1-2 cm.4. Molecular clone and functional analysis of LIFPPS gene.(1) The full-length cDNA sequence of FPPS was isolated from the petals of L. longiflorum (designated LIFPPS) using RT-PCR and RACE techniques, with GeneBank accession number JF273657, which consisted of 1267 nucleotides. It encodes an ORF of 1056 nucleotides corresponding to a protein of 351 amino acids. Sequence analysis shows that LIFPPS exhibits the features of prenyltransferase and other plant FPPS, including two high conserved Asn-rich motifs (FARM, DDXX(XX)D and SARM, DDXXD). Thus, it is most likely that we have isolated the L. longiflorum farnesyl pyrophosphate synthase.(2) Real-time RT-PCR analysis showed that expression levels of LIFPPS was high at the stage of bud. There was a small peak of expression level from the early flowering to pollination, then expression level dropped by degrees. The expression pattern of LIFPPS was investigated in healthy leaves and different parts of flower. There was no significant difference among the levels of LIFPPS expression of petals, ovary, stigma and stamens, and the level of LIFPPS expression is basically the same as the leaves.(3) The plant expression vector for LIFPPS gene was constructed. The LIFPPS was cloned into plant expression vector pBI121, removing the report gene gus. The expression vector was transformed into Escherichia coli DH5a for propagation. Then the plasmid was extracted and transformed into Agrobaterium tumefactions EHA105 which can be used for transformation.