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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
E-Journal Menara Perkebunan Vol 76, No 2: Desember 2008
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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.

Pertumbuhan dan perkembangan kalus embriogenik dan embrio somatik kelapa sawit (Elaeis guineensis Jacq.) pada sistem perendaman sesaat Growth and differentiation of embryogenic callus and somatic embryos of oil palm (Elaeis guineensis Jacq.) in a temporary immersion system . SUMARYONO; Imron RIYADI; Pauline D. KAS; Gale GINTING
E-Journal Menara Perkebunan Vol 75, No 1: Juni 2007
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

SummaryIn temporary immersion system (TIS),plant materials are exposed to the medium fora short time, therefore they are more exposedto the air and a lack of oxygen frequentlyexperienced by a liquid culture can be avoided.This experiment was conducted to determinethe procedure for callus proliferation up tosomatic embryo germination of oil palm(Elaeis guineensis Jacq.) in TIS culture.Embryogenic calli of oil palm clone MK 638from Marihat Research Institute were culturedon solid medium in the dark culture room andthen used as materials for TIS. Immersion timefor all cultures was three minutes every sixhours. Callus proliferation was conducted inDF liquid culture with 5 mg/L 2,4-D and0.1 mg/L kinetin with transfer interval of 4, 6and 8 weeks. The treatments for somaticembryo maturation were kinetin and ABA,whereas for somatic embryo germination wasIBA, kinetin and GA 3 . The results show thatthe best transfer interval for callus proli-feration was four weeks. In this treatment therelative growth rate of callus was0.38 g/g/week. Somatic embryo initiation fromthe callus was done in DF mediumsupplemented with 1 mg/L 2,4-D and 0.1 mg/Lkinetin. The percentage of somatic embryowas 80% based on biomass fresh weight afterthe fourth subculture. The addition of 0.5 mg/Lkinetin and 0.05 mg/L ABA improved somaticembryo maturation of oil palm; the averagenumber of somatic embryos at advanced stages(torpedo and cotyledonary) was 16.3 embryosper flask. The addition of 2 mg/L IBA and0.5 mg/L kinetin in DF medium with half-strength macro-salt enhanced significantly thegermi-nation of somatic embryos. GA 3 at0.1 mg/L increased the total number ofgerminants.RingkasanPada sistem perendaman sesaat (SPS),bahan tanam hanya terpapar sebentar dalammedium sehingga paparan dengan udara lebihlama dan kekurangan oksigen yang seringterjadi pada kultur cair dapat diatasi. Penelitianini bertujuan menetapkan prosedur untukperbanyakan kalus embriogenik sampai denganperkecambahan embrio somatik kelapa sawit(Elaeis guineensis Jacq.) dalam kultur SPS.Kalus embriogenik kelapa sawit klon MK 638yang diperoleh dari Balai Penelitian Marihatdiperbanyak pada medium padat di ruang gelapyang kemudian digunakan sebagai bahan untukkultur cair SPS. Lama perendaman semuakultur di SPS diatur tiga menit denganfrekuensi setiap enam jam. Perbanyakan kalusdalam medium cair DF dengan 2,4-D 5 mg/Ldan kinetin 0,1 mg/L dilaksanakan denganinterval subkultur 4, 6 dan 8 minggu.Perlakuan pematangan embrio somatik adalahkinetin dan ABA sedangkan perlakuan untukperkecambahan embrio somatik adalah IBA,kinetin dan GA 3 . Hasil penelitian menunjuk-kan bahwa untuk proliferasi kalus embriogenikkelapa sawit, interval subkultur terbaik adalahempat minggu. Pada perlakuan ini laju tumbuhrelatif kalus mencapai 0,38 g/g/minggu.Inisiasi embrio somatik dari kalus dilakukanpada medium DF ditambah 2,4-D 1 mg/L dankinetin 0,1 mg/L. Persentase embrio somatikmencapai 80% dari total bobot basah biomassasetelah subkultur keempat. Penambahan kinetin0,5 mg/L dan ABA 0,05 mg/L meningkatkanpematangan embrio somatik kelapa sawit; rata-rata jumlah embrio somatik fase lanjut (torpedodan kotiledon) adalah 16,3 embrio per bejana.Penambahan IBA 2 mg/L dan kinetin 0,5 mg/Lpada medium DF dengan setengah garammakro meningkatkan perkecambahan embriosomatik secara nyata. GA 3 0,1 mg/L mening-katkan jumlah kecambah yang terbentuk.

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
Menara Perkebunan Vol. 76 No. 2: 76 (2), 2008
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v76i2.81

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.

Pertumbuhan dan perkembangan kalus embriogenik dan embrio somatik kelapa sawit (Elaeis guineensis Jacq.) pada sistem perendaman sesaat Growth and differentiation of embryogenic callus and somatic embryos of oil palm (Elaeis guineensis Jacq.) in a temporary immersion system . SUMARYONO; Imron RIYADI; Pauline D. KAS; Gale GINTING
Menara Perkebunan Vol. 75 No. 1: 75 (1), 2007
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v75i1.152

SummaryIn temporary immersion system (TIS),plant materials are exposed to the medium fora short time, therefore they are more exposedto the air and a lack of oxygen frequentlyexperienced by a liquid culture can be avoided.This experiment was conducted to determinethe procedure for callus proliferation up tosomatic embryo germination of oil palm(Elaeis guineensis Jacq.) in TIS culture.Embryogenic calli of oil palm clone MK 638from Marihat Research Institute were culturedon solid medium in the dark culture room andthen used as materials for TIS. Immersion timefor all cultures was three minutes every sixhours. Callus proliferation was conducted inDF liquid culture with 5 mg/L 2,4-D and0.1 mg/L kinetin with transfer interval of 4, 6and 8 weeks. The treatments for somaticembryo maturation were kinetin and ABA,whereas for somatic embryo germination wasIBA, kinetin and GA 3 . The results show thatthe best transfer interval for callus proli-feration was four weeks. In this treatment therelative growth rate of callus was0.38 g/g/week. Somatic embryo initiation fromthe callus was done in DF mediumsupplemented with 1 mg/L 2,4-D and 0.1 mg/Lkinetin. The percentage of somatic embryowas 80% based on biomass fresh weight afterthe fourth subculture. The addition of 0.5 mg/Lkinetin and 0.05 mg/L ABA improved somaticembryo maturation of oil palm; the averagenumber of somatic embryos at advanced stages(torpedo and cotyledonary) was 16.3 embryosper flask. The addition of 2 mg/L IBA and0.5 mg/L kinetin in DF medium with half-strength macro-salt enhanced significantly thegermi-nation of somatic embryos. GA 3 at0.1 mg/L increased the total number ofgerminants.RingkasanPada sistem perendaman sesaat (SPS),bahan tanam hanya terpapar sebentar dalammedium sehingga paparan dengan udara lebihlama dan kekurangan oksigen yang seringterjadi pada kultur cair dapat diatasi. Penelitianini bertujuan menetapkan prosedur untukperbanyakan kalus embriogenik sampai denganperkecambahan embrio somatik kelapa sawit(Elaeis guineensis Jacq.) dalam kultur SPS.Kalus embriogenik kelapa sawit klon MK 638yang diperoleh dari Balai Penelitian Marihatdiperbanyak pada medium padat di ruang gelapyang kemudian digunakan sebagai bahan untukkultur cair SPS. Lama perendaman semuakultur di SPS diatur tiga menit denganfrekuensi setiap enam jam. Perbanyakan kalusdalam medium cair DF dengan 2,4-D 5 mg/Ldan kinetin 0,1 mg/L dilaksanakan denganinterval subkultur 4, 6 dan 8 minggu.Perlakuan pematangan embrio somatik adalahkinetin dan ABA sedangkan perlakuan untukperkecambahan embrio somatik adalah IBA,kinetin dan GA 3 . Hasil penelitian menunjuk-kan bahwa untuk proliferasi kalus embriogenikkelapa sawit, interval subkultur terbaik adalahempat minggu. Pada perlakuan ini laju tumbuhrelatif kalus mencapai 0,38 g/g/minggu.Inisiasi embrio somatik dari kalus dilakukanpada medium DF ditambah 2,4-D 1 mg/L dankinetin 0,1 mg/L. Persentase embrio somatikmencapai 80% dari total bobot basah biomassasetelah subkultur keempat. Penambahan kinetin0,5 mg/L dan ABA 0,05 mg/L meningkatkanpematangan embrio somatik kelapa sawit; rata-rata jumlah embrio somatik fase lanjut (torpedodan kotiledon) adalah 16,3 embrio per bejana.Penambahan IBA 2 mg/L dan kinetin 0,5 mg/Lpada medium DF dengan setengah garammakro meningkatkan perkecambahan embriosomatik secara nyata. GA 3 0,1 mg/L mening-katkan jumlah kecambah yang terbentuk.

Karakterisasi Morfologi Bunga dan Buah Abnormal Kelapa Sawit (Elaeis guineensis Jacq) Hasil Kultur Jaringan Helen Hetharie; Gustav A. Wattimena; Maggy Thenawidjaya S.; Hajrial Aswidinnoor; Nurita Toruan-Mathius; Gale Ginting
Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) Vol. 35 No. 1 (2007): Buletin Agronomi
Publisher : Indonesia Society of Agronomy (PERAGI) and Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Bogor, Indonesia

Tissue culture-derived plants of oil palm (Elaeis guineensis Jacq) can produce abnormal female dan male flowers, especially female, which develop into mantled fruit. This abnormality have caused yield lost up to 40%. The objectives of this research were to study the stages of flower development in which the abnormality started to appear, to characterize the morphology of the abnormal flower and fruit, and to determine the degree of abnormality. Plant materials used in this research were MK152, MK 176, MK 203, MK163, MK104, MK 212 and MK 209 clones obtained from the collection of "Badan Pengkajian dan Penerapan Teknologi (BPPT)" in Ciampea, Bogor. Characterization of abnormality at the flowering stage was conducted based on morphological performance at several stages of flower development, while the degree of abnormality at fruiting stage was determined based on the condition of carpel, mesocarp and seed. Normal flower and fruit of the same clone were used as the control. The results showed that the abnormality started at primordial stage of flower organ formation. Supplement carpel was found surrounding gynoecium for the abnormal female flower, which was composed of three to six carpels present at separate whorl of flower. Abnormal male flower had no additional whorl of flower, instead stamens were converted into carpel-like structures. In this case, number of carpels structure depends on the degree of abnormality. There were three levels of abnormality, i.e. light abnormality (AbR), heavy abnormality (AbB) and severe abnormality (AbSB). The severe abnormal fruit was seedless, with soft mesocarp (AbSB1) and woody mesocarp (AbSB2). Key words : oil palm, mantled fruit, supplement carpel, flower whorl, tissue culture

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