Construction Of Overexpressional Vectors With F3' 5' H Gene And Elementary Research About Transformantion Of Chrysanthemum

Chrysanthemum cultivars have a rich range of color variations, including yellow, white, violet, red, pink, and lilac. More exotic colorations can also be found, yet nobody has successfully created a blue chrysanthemum cultivar. Research indicates the accumulation of delphinidin is a key factor in the formation of blue flowers, thus the delphinidin encoding gene F3'5'H is known as a "Blue Gene". Inserting F3'5'H into the gene sequences of chrysanthemum cultivars may therefore yield blue flowers. The main results of the examination:We extracted F3'5'H gene sequences from the flowers of Petunia hybrida, Delphinium grandiflorum and Cineraria cruenta, using PCR and RT PCR techniques. We also obtained clones of difF, a gene that facilitates F3'5'H expression and encodes cytochrome b5, from Petunia hybrida. These clones were incorporated into an overexpressing vector, which was introduced into Agrobacterium tumefaciens LBA4404 and GV3101. The modified LBA4404 was used to induce a transgenic chrysanthemum while the transformed GV3101 was used on Arabidopsis thaliana.We chose field-grown chrysanthemum cultivars jianliuxiang and hongju, as well as tissue cultured chrysanthemum cultivars fenqiu and fanyuncaigui, as targets of infection. Three growth media of different hormonal combinations (6-BA, NAA and 2,4-D) were used. We established a rapid and efficient regeneration system.Agrobacterium-mediated genetic modification of chrysanthemum was successfully attempted. The explants were precultured for 3 days, dipped in solutions of agrobacterium LBA4404 (OD600=0.5) for 8-20 minutes, co-culutured with Agrobacterium tumefaciens for 3 days, grown in a medium of the composition MS + 6-BA + NAA/2,4-D + 250mg/L Carb for another 3 days, and then transferred into Kan-containing media. The calluses were cultured in a medium of the composition MS + 6-BA + NAA + 250mg/L Carb + 10mg/L Kan for resistance selection. The resistant calluses selected were in a medium of the composition MS + 6-BA + NAA + 100mg/L Carb + 10mg/L Kan, and then in a medium of the composition MS + NAA + 5mg/L Kan for shooting selection.In this study, we also attempted to use calluses induced from highly differentiated petals as explants. The results suggest that this method is superior to the conventional approach.Using 4 chrysanthemum cultivars and 3 Agrobacterium tumefaciens strains each carrying a F3'5'H homolog (petunia Hf1, Hf2, and cineraria F3'5'H), we obtained 12 experimental combinations. All but fanyuncaigui infected with LBA4404-Hf1 showed positive results of genetic modification. However, only fenqiu infected with LBA4404-F3'5'H was able to yield genetically modified seedlings. Of the 21 seedlings, 14 contained the target gene.Blue-violet calluses were observed during the tissue culture process. We used PCR techniques to examine these calluses, extracted pigments from them, measured absorption spectra of the pigments, and conducted acid tests on the pigments. The results suggest that these calluses were caused by the accumulation of delphinidin produced by transgenic tissues.Meanwhile, in order to provide reference for F3'5'H gene function study, we also have done some experiments with Arabidopsis thaliana mediated by Agrobacterium tumefaciens.