Deteksi metilasi DNA genom Elaeis guineensis Jacq hasil kultur jaringan dengan teknik Randomly Amplified Fingerprint (RAF) DNA dan Reverse Phase HPLC (RP-HPLC) Analysis DNA genom methylation of Elaeis guineensis Jacq from tissue culture by Randomly Amplified Fingerprint DNA (RAF) and Reverse Phase HPLC (RP-HPLC) - Nurita TORUAN-MATHIUS; Nesti F SIANIPAR; G A WATTIMENA; Hajrial ASWIDINNOOR; Maggy THENAWIDJAYA-SUHARTONO; Gale GINTING (INDONESIAN OIL PALM RESEARCH INSTITUTE, 2008)
Abstract Embryo somatic (ES) abnormalities of oil palm were probably caused by numbers and location of DNA genom cytosin methylation. Quantity of methylation could be determined by Reverse phase HPLC (RP-HPLC) techniques, while location of DNA cytosin methylation was detected by Random Amplified Fingerprint DNA (RAF-DNA) technique. The objective of this research was to determine numbers and pattern of DNA cytosin methylation of normal or abnormal ES and normal ortet as a standard. DNA genomic of samples were cut by HpaII and MspI enzymes at CCGG site, and amplified by RAF. HpaII cut at mCCGG sequences, but if second C were methylated the sequences can not be cut by HpaII.While Msp1 will cut if internal of cytosine was methylated (CmCGG). The results showed that AB16, AE11, AO12 and AP12 primers could detect the changes of methylation site on normal ortet and abnormal ES cotyledone. RP-HPLC analyses showed that DNA cytosin methylation content between ES globular and ES cotyledone, both normal and abnormal and also normal plantlet and ortet were unsignificantly different. DNA methyl content was around 0.25 – 2.72 %. Internal and fully methylation was found on 124 – 457 bp. Abnormal ES of MK638 clone showed the hipomethylation pattern. It was concluded that methylation cytosine content was very low and it seems that DNA methylation undirectly affects on process of morphology abnormalities. Abnormalities of ES globular and cotyledone might be caused by the change of DNA genom sequences. Abstrak Abnormalitas pada embrio somatik (ES) tanaman kelapa sawit diduga disebabkan oleh kandungan serta lokasi terjadinya metilasi sitosin DNA genom. Kandungan metilasi dapat ditetapkan dengan teknik Reverse Phase HPLC (RP-HPLC), sedang lokasi terjadinya metilasi sitosin DNA genom ES dapat dideteksi dengan teknik Random Amplified Fingerprint DNA (RAF-DNA). Tujuan penelitian ini adalah untuk menetapkan pola metilasi sitosin DNA genom ES kotiledon normal dan abnormal, sebagai pembanding adalah ortet yang normal. DNA genom contoh dipotong dengan enzim HpaII dan MspI yang mengenali situs CCGG, selanjutnya diamplifikasi dengan RAF. Enzim HpaII memotong sekuen mCCGG tetapi jika C kedua mengalami metilasi sekuen tersebut tidak terpotong. Msp1 akan memotong apabila sitosin internal termetilasi (CmCGG). Hasil yang diperoleh menunjukkan bahwa terjadi perubahan situs metilasi antara ortet normal dan ES kotiledon abnormal. Perubahan situs metilasi sitosin dapat dibedakan dengan primer RAF, yaitu AB16, AE11, AO12 dan AP12. Hasil analisis RP-HPLC menunjukkan bahwa perbedaan kandungan metilasi sitosin DNA ES globular maupun kotiledon masing-masing antara yang normal dan abnormal, serta antara planlet dan ortet normal sangat kecil. Kandungan metil sitosin berkisar antara 0,25 – 2,72 %. Tampak bahwa pada 124 - 457 pb terjadi metilasi internal, eksternal maupun metilasi penuh. Pada ES abnormal klon MK638 menunjukkan terjadi hipometilasi sitosin. Perbedaan kandungan metilasi sitosin yang sangat kecil diduga tidak berpengaruh langsung terhadap proses abnormalitas. Abnormalitas yang terjadi pada ES globular dan kotiledon kemungkinan akibat terjadinya perubahan sekuens DNA genom.