Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22696
標題: 蝴蝶蘭誘導擬原球體與轉殖ictB (inorganic carbon transporter B)基因之研究
Studies on protocorm-like body (PLB) induction and genetic tansformation ictB (inorganic carbon transporter B) of Phalaenopsis
作者: 陳俞廷
Chen, Yu-Tin
關鍵字: Phalaenopsis
蝴蝶蘭
PLB
genetic transformation
擬原球體
基因轉殖
出版社: 生命科學系所
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摘要: 蘭花是一種高經濟價值的作物,可是栽培過程費時費力,成本高昂,栽培業者不斷嘗試栽培方式,希望能夠降低生產成本,縮短生長期,可是傳統的育種方法無法達成此目標,本研究擬利用基因轉殖方法,轉殖inorganic carbon transporter B (ictB)基因進入蝴蝶蘭與文心蘭中,期能提高蘭花生長效率。選用蝴蝶蘭品系Phal. amadinal "Taida"及文心蘭品系 Onc. Gower Ramsey 為實驗材料。首先是誘導蝴蝶蘭PLB (protocorm-like body)的形成,利用α-Naphthaleneacetic acid (NAA)與Benzylaminopurine (BAP)組合共16 種分別以蝴蝶蘭根尖、小葉及生長點進行誘導,結果發現小葉僅使用BAP 2 mg/L 的效果最好;接著改以Thidiazuron (TDZ)進行相同實驗,發現TDZ 效果更佳,誘導時間短且PLB 數量多,其中以TDZ 1 mg/L 的效果最好,PLB的誘導產率高達85%。其次是PLB 的再生培養條件的探討,利用1/2MS (Murashige & Skoog)培養基、NDM (New Dogashima)培養基、以及HP2B(hyponex-potato bacto)培養基等三種培養基。結果發現以HP2B 培養對蝴蝶蘭的再生效果最好,可以達到95%的再生率,HP2B 培養基同時對文心蘭的PLB 也有50%的再生效率。農桿菌基因轉殖,蝴蝶蘭PLB 及文心蘭PLB為材料,載體是帶有cyanobacterium ictB gene 的Poe1515 (11kb)。農桿菌感染七天後移入含有hygromycin 50 mg/L 的培養基進行一個月的篩選培養, 所得到的轉殖株分別進行GUS 組織染色、萃取genomic DNA 與PCR 分析。結果發現GUS 活性在植株根莖葉都有表達,表示ictB 的活性為全株表現,進一步萃取genomic DNA 進行PCR (polymeraise chain reaction)反應,電泳後可以得到預期大小的hptII 產物662 bp 及ictB 產物507 bp。再將PCR 產物純化並進行定序,定序結果於NCBI BLAST 上分析,結果得到99%的相似度,證明轉殖的外源基因確實進入轉植株中。分析轉殖株生長情形,經過三個月的培養後,轉殖株鮮重比未轉植株高,顯示轉殖ictB 確實可以提高蘭花的生長效率。至於是否能縮短蘭花幼年期則有待近一步觀察。
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.
URI: http://hdl.handle.net/11455/22696
其他識別: U0005-1901200923034800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1901200923034800
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