Alteration of Spathoglottis eburnea Gagnep. Ploidy Level after Colchicine Treatments
Keywords:Mixoploid, flow cytometry, chromosome count, stomata analysis
Improvement of Spathoglottis eburnea Gagnep (Orchidaceae) with alterative morphology was performed and observed by polyploidy induction. Two millimeters long shoot tips were immersed and cultured in ½ MS liquid medium supplemented with 0, 0.25, 0.5, 0.75, and 1 % (w/v) of sterilized colchicine solutions for 1 d. After 3 months of culture, the highest polyploidy induction was obtained with 1 % (w/v) colchicine for 1 d, which gave a shoot survival rate of about 45 % and which regenerated into 40 % polyploid plantlets. Five polyploid plantlets were further maintained by subculturing every 2 months on ½ MS agar medium supplemented with 8 g L-1 activated charcoal. After the third subculture, all of them were morphological and cytological chimeras, which had alterations in leaf morphology. The variation of ploidy level was determined again by flow cytometry and chromosome counting. A flow cytrometry showed that tetraploid plantlets changed to mixoploid plants, where phenotype instability was further observed, in correlation with chromosome instability. The density of stomata and the stoma index were observed and showed no significant differences between mixoploids and diploids. However, the guard cell size of mixoploids was larger than those of diploids. In the light of our findings, we can consider polyploidy induction to be an effective technique for plant improvement; however, there is a necessity to study genetic stability between the clones after micropropagation.
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