利用莖節培養誘導多倍體台灣金線連之研究

In Vitro Induction of Polyploidy from Nodal Explants of Anoectochilus formosanus

夏奇鈮(Chi-Ni Hsia) ； 黃健覃(Jian-Tan Huang) ； 陳威臣(Uei-Chern Chen) ； 曹進義(Chin-Yi Tsao) ； 梁淑惠(Shu-Hui Liang) ； 蔡新聲(Hsin-Sheng Tsay)

《台灣農業研究》 59卷3期 (2010/09) Pp. 165-176

https://doi.org/10.6156/JTAR/2010.05903.03

摘要

台灣金線連(Anoectochilus formosanus Hayata)為台灣特有種，亦為本土重要之草藥。為育種之目的，本研究以微體繁殖之瓶苗莖節為培植體，評估不同抗微管藥劑(antimicrotubule chemicals)、秋水仙素濃度與處理時間、以及秋水仙素與細胞分裂素BA處理配合的程序對多倍體誘導的影響，處理後長出之芽體並以流式細胞儀檢測其染色體之倍體數。莖節培植體在含有秋水仙素、歐拉靈及三福林25 μM、100 μM及400 μM三種濃度之液體增殖培養基中培養2週，發現多倍體誘導比率以400 μM秋水仙素最高，但存活之植株數最少。莖節培植體在含有0.625-6.25 mM秋水仙素之液體增殖培養基中培養3天，結果顯示除6.25 mM濃度處理之芽體生長受抑制外，各濃度皆可誘導多倍體的產生，其中以2.5 mM誘導率最高，達80%。以2.5 mM秋水仙素測試處理時間3-9天之影響，顯示處理時間長於3天，嚴重抑制培植體的生長，芽體平均成活率皆低於30%。將莖節進一步區分為頂芽及頂芽下一節之節芽作為培植體，以2.5 mM秋水仙素縮短處理時間為1-3天，發現節芽的生長在不同處理天數 皆受到抑制，但頂芽處理2天組多倍體誘導率可達100%。將處理時間固定為3天，以0.625-6.25 mM濃度之秋水仙素分別處理頂芽及節芽，結果顯示以1.25 mM處理之頂芽多倍體誘導率最高，達88.9%；濃度0.625 mM處理之頂芽雖然誘導率較低(66.2%)，但芽體存活率較高，獲得之多倍體株數亦最高。比較培植體在施用秋水仙素前，先以含有BA的增殖培養基進行預培養；或在施用秋水仙素之後，再於含有BA之增殖培養基中培養。兩種處理程序中，培植體於秋水仙素處理及增殖培養基中，培養的時間皆相同，但前者之嵌合體比例僅6.4%，而後者為23%。綜合本研究之結果，以微體繁殖台灣金線連之莖節進行多倍體誘導，以頂芽較節芽為佳；以較低之秋水仙素0.625濃度處理頂芽3天，可保持芽體之再生力，多倍體的誘導率最高可達161% (129株多倍體/80個頂芽)，是一種高效率的多倍體誘導方式。

關鍵字

金線連 ； 抗微管藥劑 ； 秋水仙素 ； 倍數體 ； 流式細胞儀

並列摘要

Anoectochilus formosanus is an important ethnic as well as a medicinal herb in Taiwan. Investigations of antimicrotubule chemicals, colchicine concentration, colchicine exposure time and colchicine combination with BA treatment on polyploidy induction using in vitro A. formosanus nodal explants were conducted in this study. Ploidy levels of shoots regenerated from antimicrotubule chemical treatments were analyzed using flow cytometry. Nodal segments were exposed to different concentration (25-400 μM) of colchicine, oryzalin and trifluralin in a liquid culture for two weeks, and the highest polyploidy rate was found at 400 μM colchicine with minimum survival. In order to find the optimum concentration for polyploidy induction, nodal explants were exposed to colchicine concentration range from 0.625 to 6.25 mM for three days. Polyploids were able to induce from all tested concentrations except of 6.25 mM which had severe toxicity on explants and the highest induction rate of 80% was found at 2.5 mM colchicine. Nodal explants were further exposed to 2.5 mM colchicine for 3-9 days, and minimum survival rates (＜ 30%) were found on prolonging exposure time longer than 3 days. Nodal explants were further defined into shoot tip and nodal segment as explants culturing at 2.5 mM colchicine for 1 to 3 days. No polyploidy was found from nodal segment regardless exposure times however a polyploidy induction rate up to 100% was obtained using shoot tip as explants for 2-day exposure. Extending colchicine concentration from 0.625 to 6.25 mM for 3-day exposure using both nodal segment and shoot tip as explants was conducted subsequently. Although the highest polyploidy induction rate 88.9% was found at 1.25 mM colchicine, the highest numbers of polyploids were obtained at lower concentration of 0.625 mM from shoot tip explants. BA treatment applied before or after colchicine exposure was compared using shoot tip explants. A chimera rate 23% observed from explants continuously exposed to BA-containing medium after the colchicine treatment, in contrast only 6.4% chimera observed in explants which were exposed to BA-containing medium only before the colchicine treatment. The present study demonstrates that polyploidy can be induced from in vitro grown nodal segments successfully with different antimicrotubule chemicals. An efficient polyploidy induction method has been established culturing shoot tip in a liquid medium containing 0.625 concentrations of colchicine for 3-day exposure with polyploidy induction rate up to 161% (129 polyploids/80 shoot tip explant) in this study.

並列關鍵字

Anoectochilus formosanus ； Micropropagation ； Colchicine ； Polyploidy ； Flow cytometry