Journal of Taiwan Agricultural Research

Author:Chi-Ni Hsia*, Jian-Tan Huang, Uei-Chern Chen, Chin-Yi Tsao, Shu-Hui Liang, and Hsin-Sheng Tsay

Abstract:

    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 M) of colchicine, oryzalin and trifluralin in a liquid culture for two weeks, and the highest polyploidy rate was found at 400 M 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.

Key words:Anoectochilus formosanus, Micropropagation, Colchicine, Polyploidy, Flow cytometry

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