| Summary: | 碩士 === 中興大學 === 農藝學系所 === 95 === The objectives of this study were to investigate the effects of colchicine treatments on the polyploidy induction of in vitro nodal and rhizome explants of Anoectochilus formosanus Hayata. First, three anti-microtubule chemicals colchicines, oryzalin, trifluralin at concentration of 25, 100 and 400 μM were added on proliferation medium to treat nodal explants for 2 weeks. Explants were transfered to the acclimatization medium after treatment and data of shoot proliftration and shoot length were collected 4 and 8 weeks after subculturing. The results indicated that all three chemicals inhibited shoot proliferation and growth and among them colchicines was the most effective one. The best polyploidy was induced from 400 μM of colchicines with 67% tetraploids and 33% octoploid, respectively.
Various concentrations of colchicine in 0, 0.625, 1.25, 2.5 and 6.25 mM were added into proliferation medium. Nodal explants were treated with colchicine for 3 days before subculturing into an acclimatization medium and the data was collected ten weeks after treatment. The results showed that shoot proliferation, shoot length as well as survival rate of explants decreased along with increasing of colchichine concentrations. The highest percentage of tetraploid was obtained from 2.5 mM colchicine treatment with 80%.
Various duration of 0, 3, 6 and 9 days for treatment with 2.5 mM colchicine in proliferation medium was compared. Shoot proliferation rate, shoot length and survival rate of nodal explants were all decrease as the increasing of treatment duration. The highest percentage of tetraploid(50%) was found in the treatment duration of 3 days.
Rhizome explants age at 10, 11 and 14 weeks were used for polyploidy induction. Colchicine concentrations of 0, 0.025, 0.25 and 2.5 mM were added into medium for 3 days culturing. After colchine treatment, explants were subculture into a fresh medium for eight-weeks acclimation. Among three age explants, it was found that 10-week-rhizome had the hightst tetraploid induction rates from 10-50% in various concentration of colchicine. Moreover, The survival rates of rhizome explants were found decreasing as colchine concentration increased.
Age of 13-week-rhizome explants were cultured in a medium containing with 0, 0.025, 0.25 and 2.5 mM colchicines for one week before subculturing into a solid fresh culture medium for eight weeks acclimation. The results showed that as concentration increasing to 0.25 and 2.5 mM, survival rates were decrease to 32.7 and 27.2%, respetively. However, a higher percentage of tetraploids, 30 and 20% was obtained from the 0.25 and 2.5 mM colchicine treatment.
Colchicine of 0, 0.025, 0.25 and 2.5 mM were added into a solid or liquid culture medium for polyploidy induction using 12-week-rhizome explants. Treatment duration was last two weeks before subculturing into a fresh solid culture medium without colchicines for eight weeks acclimation. The results showed higher survival rates (21-35%) and percentages of tetraploids (5-30%) in liquid medium than that of in solid medium.
Various treatment duration of 16, 24, 48 and 72 hr was compared using 10-week-rhizome culturing in a liquid medium containing 2.5 mM colchicine. The survival rates of explants decreased as the treatment duration increased. Treatment duration as short as 16 hr was found capable to induce tetraploid plants with 10% induction rate.
Measurements of stoma size and its distribution density in leaves of polyploidy plants induced from anti-microtubule chemical treatment were conducted. A positive correlation between stoma size and ploidy level was obtained. Furthermore, the decreasing in stoma distribution density along with higher polyploidy level was also found.
In conclusion the polyploidy induction experiments of in vitro A. formosanus Hayata in this study, using nodal explants culturing in a medium containing 2.5 mM colchicine for 3 days had the best result. The polyploidy level of regenerated plantlets was determined use young leaves by flow cytometry. Leaf stoma parameters observed by microscopy could also use for assistance polyploidy level detection.
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