Study On Genetic Transformation Of EgFT Gene In Eustoma Grandiflorum

Lisianthus (Eustoma grandiflorum) is a perennial herb of Gentianaceae family, quickly ranked in the top ten cut flowers worldwide due to its lovely flower. The regeneration system was established first. Based on the GUS staining and PCR detection, we studied about many factors, such as concentration of kanamycin and cefotaxime, pre-culture time, infection time, co-culture time, AS concentration at infection and co-culture period and later-selection time to establish steady and efficient transformation system. The results showed that EgFT gene had been steadily transferred to the lisianthus, and 35 transgenic lines had been gained. Conclusions are as follows:1. Establishment of the regeneration systems in E. grandiflorum ’Tiramisu’ and’Arena’Leaves of two lisianthus cultivars (’Tiramisu’ and ’Arena’) were used as explants. The different kinds of medium type, such as bud induction, strong seedling, proliferation, rooting, were optimization. Results indicated that the most appropriate medium for bud induction was MS+6-BA 0.5mg/L+NAA 0.05mg/L; shoot proliferation medium was MS+IBA 0.2mg/L+GA 0.3mg/L+6-BA 0.2mg/L; shoot strengthening medium was MS; and rooting medium was 1/2MS+IBA 0.5mg/L.2. Establishment of the transformation systems in E. grandiflorum ’Tiramisu’ and’Arena’Leaves of two lisianthus cultivars (’Tiramisu’ and ’Arena’) were used as explants. Effects of kanamycin, cefotaxime, pre-culture time, infection time, co-culture time, AS concentration at infection and co-culture period and later-selection time on the transformation system of lisianthus were studied. The results showed that both of two cultivars of lisianthus were very sensitive to kanamycin, and the regeneration of non-transformed leaves was completely inhibited at the concentration of 10mg/L. Meanwhile leaves of two kinds of lisianthus cultivars were insensitive to cefotamime, and the agrobacterium overgrowth was completely inhibited at concentration of 300mg/L, and the regeneration of leaves did not affect at all. The vitrification of seedlings in ’Tiramisu’ happened much more often than’Arena’. Thus finally selected the efficient transformation system for lisianthus ’Arena’:10mg/L kanamycin,300mg/L cefotaxime, non- pre-culture,15 minutes’ infection,3 days’ co-culture,50μmol/L AS used at infection and non-AS used at co-culture period,8 days’ later-selection.3. The GUS staining and PCR detection of the resistant seedlingsWe selected 9 resistant seedlings obtained from E. grandiflorum ’Arena’ genotype and extracted DNA by CTAB, then tested the DNA by PCR. The results showed that the 5 resistant plants were PCR positive plants. GUS staining was used to test the leaves of the resistant seedlings; we found that they had blue reaction. Therefore, it indicated that the GUS gene had been steadily transferred to the E. grandiflorum ’Arena’ genotype.4. Introductions of EgFTgene into E. grandiflorum ’Arena’We transformed the EgFT genes into Eustoma grandiflorum ’Arena’ by Agrobacterium mediated and got 40 resistance seedlings.The results of PCR analysis showed that 35 positive individuals transformed with EgFT were obtained, which indicated EgFT gene possibly had been integrated to the genome of transgenic plantlets.