Construction And Analysis Of T-DNA Insertional Mutation Population Of Lilium Longiflorum

Lilies (Lilium spp.) are monocotyledonous ornamental plants belonging to the family Liliaceae and one of the three major flower bulbs in the commercial market. 1.8 billion seed bulbs were produced per year and costed 1.2 billion dollars in Netherlands. Substantial progress had been made in lily's traditional breeding. However, incompatibility and time-consuming became the main limiting factor in distant hybridization. In present the seed bulbs were mainly dependent on import in our country, which costed more money and resulted in variety degeneration. Although tissue culture could solve these problems, there existed barriers of serious contamination, low propagation coefficient , low propagation speed and low survival rate of plantlets after transplanted, which greatly limited the production of commercial seed bulbs. Aimed at solving those key problems in micropropagation and obtain valuable mutants of lily, a two-step explant method and T-DNA insertional mutagenesis by Agrobacterium had been developed in this study.An efficient micropropagation system of Lilium Longiflorum had been successfully established by a two-step explant method combined with embryogenic callus formation system and direct shoot formation system. Bulb scale segments were used as original explants and shoot sections excised from shoot clusters were used as the secondary explants. Our results indicated that the propagation coefficient through embryogenic callus formation system and direct shoot formation system could be as high as 67,500 and 54,000 times respectively . The whole progress of from bulb scale to new plant flowering need only 8-9 months.The medium supplemented with 2.0mg/L 2,4-D was most favorable for embryogenic calli formation and the MS with 1.0mg/L 6-BA was most effective for shoots induction and elongation.The medium of sand: hortensis soil: coconut bran=1:1:1 was optimum for the survival of plantlets. Moreover, the short basal stem was the best secondary explant for shoots and calli formation and different treatment time at 4℃had little effect on flowering.Based on the efficient micropropagation system, T-DNA insertional mutagenesis technique was introduced to construct a mutation pool. About 5000 of neomycin phosphotransferase II -resistant (NPTII) plantlets were regenerated from calli or directly from the shoot segments. Most of these plantlets were verified to be transgenic by PCR analysis . The callus regeneration method showed a PCR positive percentage of 37.5%, which is little higher than the particle bombardment method tested in this species(34.5%). Direct shoot formation method had even higher transformation efficiency, fewer escaped non- transgenic plants on the geneticin medium(64%). At the same time, different infection styles, temperature, infection time, co-cultivation time and the material number in each Petri dish had different effects on the transformation efficiency. The results suggested that the method which after 3 days of pre-cultivation, the segments of secondary explants had...