蝴蝶蘭誘導擬原球體與轉殖ictB (inorganic carbon transporter B)基因之研究

Abstract

Orchid is a high-economic value crop. Breeders always choose the better character to reduce production cost. However, using traditional breeding processes to improve a character could take a lot of time. The objects of this study are to establish a technological protocol of gene transfer into Phalaenopsis and Oncidium to raise their growth rates. Cultivars of Phal. amadinal Taida and Onc. Gower Ramsey were used in this study. Application of α-Naphthaleneacetic acid (NAA) and Benzylaminopurine (BAP) to induce protocorm-like body(PLB), from cutting root tips, leaves and shoot apex, Sixteen concentration combinations of NAA and BAP were tested. The result indicated that only with BAP 2 mg/L in leaves have the bigest PLB production. Instead of Thidiazuron (TDZ) to BAP, we found that TDZ have better efficiency than NAA with BAP. Moreover, it took less time and generated more PLBs. The best concentration of TDZ in PLBs induction was 1 mg/L, which has 80% induction efficiency. To test the Phalaenopsis PLBs regeneration efficiency, 1/2MS (Murashige & Skoog) medium，NDM (New Dogashima) medium，and HP2B (hyponex-potato bacto) medium were used. The result indicated that the best regeneration efficiency is using HP2B medium, which has 95% regenerate efficiency in Phalaenopsis and 50% regenerate efficiency in oncidium. Plasmid poe1515 which carried cyanobacterium ictB gene was transformed into Agrobacterium GV310. This strain was used to infecte PLBs for 7 days and selected with hygromycin 50 mg/L for one month. Transgenic plant has GUS activity which express in all parts of plant. Moreover, PCR analysis of genomic DNA showed two bands which is 662 bp for hptII and 507 bp for ictB, we checking the PCR products and BLAST search in the NCBI website, thas obtained 99% similarity with the origenal genes. The results demonstrated that have transferred ictB in Orchid successfully. Finalily, comparison the growth rate of transgenic and untransgenic plants, that transgenic plant has higher fresh weight than untransgenic plants after 3 months.The possibility to short vegetative period needs further study.