Optimizing flow cytometric analysis for nuclear DNA in Musa sp.
2000
Azhar M. | Rusli I.
Musa sp. cv. Berangan was used in the optimization of flow cytometric (FCM) analysis prior to the determination of their ploidy level and DNA content. Using Glycine max. var. palmetto (soy bean) as an internal standard, parameters studied were leaf storage, staining duration, sample portion and leaf ratio of sample and standard. All leaves were harvested, wrapped inwet filter paper, sealed in petri disk and kept under dark condition at 4°C prior FCM analysis. The leaves were chopped in LBO 1 buffer supplemented with 50ug Propidium Iodide stain and 50ug RNAse. DNA content and ploidy level were measured using Cell Counter Analyzer Cytometer (FCM Model Partec CCA-II) based on the relative fluorescence intensity of the stained nuclear DNA to obtain histogram which in relation to cells in G1 or G2 phases of the cell cycle. The coefficient of variation (CV) of the peaks of the histogram and the ratio between sample and standard were analyzed. Leaves from tissue culture sample and cigar leaves from matured plant gave the CV of 1.674 ± 0.38 and 1.760 ± 0.12 respectively after 24 hours of incubation at 4°C under dark condition. In addition, the CV and debris increased with time for stained homogenate nuclei after 24 hours incubation. At 96 hours after storage, CV for tissue culture leaf was shown exceeding 3%. The results suggested that the ratio of 2:1 for banana leaf to the standard give a balance histogram peak and high precision of the FCM analysis. DNA content of Pisang Berangan was determined to be 1.8717 ±0.007 pg in relation to triploid banana with AAA genome. The method described here is applicable to any other plant species to be established as a first procedure in optimizing the parameters of FCM analysis before determining the ploidy level and the DNA content.
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