Judul: Utilization of enhanced random amplified polymorphic DNA (E-RAPD) for geneticc variability analysis on plantlets of smooth cayenne pineapple, Ananas comosus L. Merr.
Abstrak: Recently, RAPD markers have been widely used in speeding up and assisting practical plant breeding, crop improvement or plant characterization. RAPD is known as in-expensive and simple technique with a number of advantages: (1) universal set of oligonucleotide primers can be used for all plant species, (2) a large number of samples may be easily and rapidly characterized, (3) automatically process. However, RAPD those commonly use 10-base oligonucleotides of primers amplify target DNA sequences randomly and have low reproducibility. In this research, 11-base oligonucleotides of primers have been utilized as enhanced-RAPD (e-RAPD). Utilization of this e-RAPD has been evaluated on Smooth Cayenne pineapple plantlets obtained from tissue culture as target DNA materials. The e-RAPD has been expected increase specificity of DNA amplified products than RAPD's, result in easier to analyze and more reproducible. Beside of those, it has been expected amplifiable on this DNA genome of Smooth Cayenne pineapple plantlets and can be used to evaluate genetic variability among the plantlets. The DNA sample materials from plantlets of 20 combination treatments were isolated base on CTAB methods with some of modifications, and precipitated by phenol chloroform isoamyl alcohol. Primer 11-mers as enhancer of RAPD markers were selected on four single primers (SOB-1, SOB-2, SOB-3, and SOB-4) and its six combinations. Total volume of each amplification reaction was 25 μl which consisted of buffer 2.5 µl, MgCl 2 ul, dNTP 1 ul, primer 2 ul, DNA sample 1.5 ul. Taq polymerase 0.2 µl, and sterilized deionized water 15.8 µl. The PCR selected profile programmed pre-denaturation at 94 °C; 1.5", 45 cyles with each denaturation 94 °C; 0.5", annnealing 37 °C; 1', and elongation 72°C; 2", then final elongation at 72°C; 4. In evaluating the e-RAPD's products, the data of DNA amplified products on five treatment samples obtained from RAPD reaction of primer 10-mers (OPE-7) has been compared in analysis process. The e-RAPD results using four selected primers (SOB-1, SOB-1-2, SOB-2-3, SOB-3-4) has successfully amplified DNA genome of Smooth Cayenne pineapple plantlets. However it is not optimal yet. The best results of e-RAPD's Cabe obtained by precisely optimization of PCR program and the optimal concentration of all reaction reagents, such as optimization on the annealing temperature, the amount of DNA samples, and concentrations of dNTP, MgCl2, and Tag polymerase. The e-RAPD produced 57 total fragments which all 100% showed as polymorphic DNA. The dendogram clustering perform the high variability among all treatments. Treatment of BA 13.32 µM combined with NAA 2 µM show the shortest genetic distance to control treatment without exogenous hormones. This variability which refers to somaclonal variations may occur after tissue culturing. It can be caused of some posible factors, such as the using of exogenous hormones, subculture frequency, explant type, micropropagation system, and other technical defects.
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