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Kloning parsial gen penyandi enoil-ACP reduktase dari mesokarp buah kelapa sawit (Elaeis guineensis Jacq.) Partial cloning of gene encoding enoyl-ACP reductase from mesocarp of oil palm (Elaeis guineensis Jacq.) Asmini BUDIANI; Djoko SANTOSO; Hajrial ASWIDINNOOR; Antonius SUWANTO
Menara Perkebunan Vol. 72 No. 1: 72 (1), 2004
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v72i1.126

Summary Enoyl-ACP reductase (ENR) is a component of fatty acid synthase (FAS) that is considered to play an important role in fatty acid elongation and oil accumulation of several plants. One of the proteins expressed coinciding with fruit development and oil accumulation in oil palm has been detected from the previous study and had homology with ENR. Therefore, as a part of genetic engineering program to improve oil content and quality in oil palm mesocarp, this research was aimed to clone cDNA conserved region of gene encoding enoyl-ACP reductase from oil palm mesocarp. Based on the amino acid sequence of the polypeptide that was homologous with ENR and combined with information of conserved region sequences of the same gene from other plant sources, primers were designed for amplifying conserved region of the ENR gene. Amplifi-cation was carried out by RT-PCR using total RNA as template, at several annealing temperatures and MgCl2 concentrations. Amplification product was cloned using pCR 2.1-TOPO, and the sequence was subjected into BlastN analysis. The results confirmed that the cloned cDNA fragment with 698 bp in size was the conserved region of the ENR gene. This sequence was highly homologous with the same gene from other plants such as Oryza sativa, Olea europaea, Brassica napus, Triticum aestivum and Arabidopsis thaliana with E-value 1e-96, 7e-77, 2e-64, 5e-41 and 3e-36, respectively. Based on this result, primers have been made and used to amplify the 5’- and 3’ ends of the ENR -cDNA of oil palm mesocarp. Ringkasan Enoil-ACP reduktase (ENR) merupakan salah satu komponen asam lemak sintase (FAS) yang berperan penting dalam pemanjangan asam lemak dan akumulasi minyak pada berbagai tanaman. Salah satu protein yang ter-ekspresi sejalan dengan tahapan perkembangan buah sawit dan akumulasi minyak pada penelitian sebelumnya diketahui mempunyai homologi dengan ENR. Oleh karena itu, sebagai salah satu bagian dari usaha rekayasa metabolisme minyak pada mesokarp buah sawit, penelitian ini bertujuan untuk mengklon cDNA daerah konservatif gen penyandi ENR dari mesokarp buah sawit. Berdasarkan sekuen asam amino polipeptida yang mempunyai homologi dengan ENR dan dikombinasikan dengan hasil penjajaran daerah konservatif gen tersebut dari berbagai anaman lain, dirancang primer untuk amplifikasi daerah konservatif ENR. Amplifikasi dilakukan dengan RT-PCR menggunakan templat RNA total pada berbagai suhu penempelan dan konsentrasi MgCl2. Hasil amplifikasi dimurnikan dari gel dan diklon menggunakan vektor kloning pCR2.1-TOPO serta dianalisis nya menggunakan BlastN. Hasilnya mengkonfirmasi fragmen cDNA terklon berukuran 698 pb sebagai daerah konservatif ENR tersebut mempunyai homologi tinggi dengan gen yang sama dari O. sativa, O. europaea, B. napus, T. aestivum dan A. thaliana masing-masing dengan E-value 1e-96, 7e-77, 2e-64, 5e-41 dan 3e-36. Berdasarkan hasil tersebut telah dibuat primer spesifik untuk amplifikasi cDNA daerah ujung 5’- dan 3’- gen ENR dari mesokarp kelapa

Nucleotide sequence of cryIA gene cloned from Btk isolate of Bacillus thuringiensis and comparison with cryIA(c) gene from B. thuringiensis subsp. kenyae Sekuen nukleotida gen cryIA dari B.thuringiensis isolat Btk dibandingkan dengan gen crylA(c) dari B. thuringiensis subsp. Kenyae Asmini BUDIANI; Djoko SANTOSO
Menara Perkebunan Vol. 68 No. 1: 68(1), 2000
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v68i1.134

Ringkasan Perakitan tanaman perkebunan yang toleran terhadap serangga hama dapat ditempuh melalui rekayasa genetika menggunakan gen cry. Gen cryIA merupakan gen cry yang paling banyak dipelajari di antara gen cry lainnya. Berdasarkan homology sekuen dan spesifisitas protein yang disandinya terhadap serangga sasaran, gen ini telah diklasifikasikan menjadi 10 subklas. Tulisan ini melaporkan hasil sekuensing (ragmen gen cryIA penyandi domain toksin yang diisolasi dengan teknik PCR dari Bacillus thuringiensis isolat Btk dan diklon menggunakan vektor pGEMT. Untuk menentukan sekuen gen cryIA yang berukuran sekitar 2 kb tersebut, dilakukan konstruksi satu seri mutan terdelesi searah dari ujung 5' menggunakan kit Erase-a-Base-System. Tiga DNA gen cryIA mutan dengan tingkat delesi yang sesuai dan satu nonmutan dipilih untuk sekuensing DNAnya. Sekuensing dilakukan dari satu arah menggunakan primer universal SP6 pada alat ABI 377A automatic DNA sequencer. Sekuen lengkap dari gen cryIA diperoleh dengan cara menggabungkan sekuen ketiga mutan dengan sekuen dari gen cryIA nonmutan secara manual. Untuk konfirmasi sekuen ujung 3', dilakukan sekuensing dari arah lainnya menggunakan primer universal T7. Sekuen lengkap dari fragmen tersebut mengandung 2021 nukleotida dan menyandi protein dengan 673 asam amino. Dibandingkan dengan sekuen gen crylA(c) dari B. thuringiensis subsp. kenyae, terlihat adanya sepuluh mutasi titik masing-masing pada nukleotida ke 444, 477, 1089, 1092, 1098, 1242, 1566, 1869, 1906 dan 1961. Tujuh mutasi titik pada nukleotida ke 444, 477, 1089, 1092, 1242, 1566, dan 1869 tidak merubah asam amino, sedangkan tiga mutasi lainnya mengakibatkan perubahan asam amino, yaitu pada nukleotida ke 1098 (kodon ke 366, yang menyebabkan perubahan dari Phe menjadi Leu), nukleotida ke 1906 (kodon ke 636, yang mengubah Val menjadi Leu) dan pada nukleotida ke 1961(kodon ke 654, yang mengubah Cys menjadi Tyr).Summary Estate crops tolerant to pests can be development through genetic engineering using cry gene. CryIA is the best studied among cry genes. Based on the sequence homology and specificity of their encoded proteins to the, targeted insect, these genes have been classified into 10 subclasses. This paper reports sequencing of cryIA gene fragment en-coding toxin domain isolated from Btk isolates of Bacillus thuringiensis using PCR technique and cloned with pGEM-T vector. To determine the full sequence of the 2-kb gene fragment, a series of mutants uni-directionally deleted at the 5'-end were constructed. Mutation was done using Erase-a Base-System kit. Three DNA mutants with appropriate degree of deletion and the un-mutated DNA were chosen for sequencing. Sequencing was conducted from one direction with SP6 universal primer using the ABI 377A automatic DNA sequencer. The full sequence of cryIA fragment was assembled manually using the sequences of DNA mutants and the non-mutant cryIA fragment. To confirm the sequence of the 3'-end, sequencing from the other direction was performed using the T7 universal primer.The completed sequence of the fragment contains 2021 nucleotides encoding a protein of 673 amino acids. Compares to the sequence of cryIA(c) from B. thuringiensis subsp. kenyae, it was shown that there were ten point mutations (nucleotides of 444, 477, 1089, 1092, 1098, 1242, 1566, 1869, 1906 and 1961), sevent of them (nucleotides of 444, 477, 1089, 1092, 1242, 1566 and 1869) were identified as silent mutations, while the other three substituted the amino acids, which are at the nucleotide 1089 (codon 366, substitution of Leu for Phe), nucleotide 1906 (codon 636, substitution of Leu for Val), and nucleotide 1961 (codon 654, substitution of Tyr for Cys).

Transformation of Coffee arabica using chitinase gene and regeneration of planlets from transformed-zygotic embryos Transformasi Coffea arabica menggunakan gen kitinase dan regenerasi planlet dari embrio zigotik-transforman Asmini BUDIANI; T CHAIDAMSARI; . PRIYONO; S MAWARDI; . SISWANTO
Menara Perkebunan Vol. 68 No. 2: 68 (2), 2000
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v68i2.138

RingkasanRekayasa genetika kopi arabika tahan penyakit cendawan dapat dilakukan dengan cara memasukkan gen kitinase (gen chi) ke dalam genom tanaman tersebut. Penelitian ini bertujuan untuk mengintroduksikan gen chi pada kopi arabika serta meregenerasi eksplan yang ditransformasi menjadi plantlet. Gen chi disubkloning dari pBS G11 ke dalam plasmid pCAMBIA2301. Melalui Agrobacterium tumefaciens, plasmid rekombinan pCAMBIA2301/35s-chi kemudian dimasukkan ke dalam eksplan daun dan embrio zigotik kopi arabika. Eksplan daun transforman ditumbuhkan pada media seleksi yang mengandung kanamisin untuk induksi kalus embriogenik . Beberapa kombinasi 2,4-D dan dicamba serta kinetin, BAP dan 2-iP diuji kemampuannya untuk menginduksi terbentuknya kalus embriogenik. Embrio zigotik transforman ditumbuhkan pada media MS modifikasi yang mengandung kanamisin. Hasil penelitian menunjukkan bahwa perbedaan tipe sitokinin dan kombinasinya dengan 2,4-D atau dicamba. menyebabkan terjadinya variasi persentase pembentukan kalus embriogenik tahan kanamisin. Penambahan 100 mg/L kanamisin dalam media seleksi cukup efektif untuk menghambat pertumbuhan eksplan daun nontransforman. Persentase tertinggi induksi kalus embriogenik pada eksplan daun non transforman maupun transforman diperoleh pada media yang mengandung 5 mM 2,4- D dengan 5 mM of kinetin atau 5 mg/L dicamba dengan 5 mM BAP. Sedangkan dalam media dengan penambahan 5 mM kinetin, 100 mg/L asam sitrat dan 100 ppm asam askorbat, jumlah eksplan yang membentuk kalus mencapai optimum pada konsentrasi 0 dan 1 ppm dicamba untuk eksplan transforman dan 10 mg/L dicamba untuk non transforman. Pada eksplan embrio zigotik transforman, peningkatan konsentrasi kanamisin dari 100 mg/L hingga 500 ppm menurunkan persentase pengecambahan embrio dari 80.5 % menjadi 49%, persentase perakaran, dari 34 % menjadi 16%, jumlah akar, panjang akar dan tinggi tunas dari 7 mm menjadi 4 mm. Pada semua perlakuan kanamisin, embrio zigotik non transforman tidak membentuk akar dan pada umur kultur yang sama tunas yang dihasilkan lebih pendek dibandingkan dengan embrio-zigotik transforman. Hasil tersebut membuktikan bahwa gen ketahanan terhadap kanamisin (NPTII) telah terinsersi dan terekspresi dengan baik pada plantlet kopi arabika yang berasal dari eksplan embrio-zigotik transforman. Karena gen chi dikonstruksi dalam satu vektor dengan NPTII, maka diharapkan gen tersebut juga telah terinsersi ke dalam genom tanaman kopi.SummaryGenetic engineering of arabica coffee resistant to fungal diseases might be done by introducing a chitinase-encoding gene (chi) into genome of this plant. This research was aimed to introduce chi construct into arabica coffee and regenerate plantlets from the transformed explants. The chi gene was previously subcloned from pBS G11 into pCAMBIA2301 plasmid. With Agrobacterium tumefaciens,the recombinant plasmid pCAMBIA2301/35s-chi was then introduced into leaf and zygotic embryos explants of arabica coffee. The transformed leaf explants were cultured on the selection media containing kanamycin in the presence of several combinations of 2,4-D and dicamba with kinetin, BAP and 2-iP to induce the formation of embryogenic callus. The transformed zygotic embryos were cultured on the media of modified MS containing kanamycin. The results showed that the several types of cytokinin used in combination with 2,4-D or dicamba caused the percentage of kanamycin resistant-embryogenic calli was varied. The addition of 100 mg/L kanamycin in the selection media was effective for inhibiting the growth of untransformed explants. Among the several combinations of auxin and cytokinin tested, the highest percentage of embryogenisis for untransformed and transformed leaf explants were achieved on the media containing 5 mM 2,4-D and 5 mM kinetin or 5 mg/L dicamba and 5 mM BAP. However in the presence of 5 mM kinetin together with antioxidants of 100 mg/L citric acid and 100 mg/L ascorbic acid, the explants calluses was optimum at 0 - 1 mg/L dicamba for transformed explants and 10 mg/L dicamba for untransformed explants. In the explants of transformed-zygotic embryos, increasing kanamycin from 100 mg/L up to 500 mg/L decreases the percentage of embryo germination from 80.5 % to 49%, rooted-shoots from 34 % to 16%, number of roots, root length and shoot length from 7 mm to 4 mm. At all the kanamycin treatments, root was not developed from the untransformed-zygotic embryos and the lenght of shoots were shorter compared to the transformed-zygotic embryos. This result demonstrates that the kanamycin-resistant gene (NPTII) has been inserted and well expressed in the plantlets of arabica coffee derived from transformed-zygotic embryos. Since the chi gene was constructed in one vector with NPTII, this gene might also been inserted in the genome of coffee.

Keragaman sekuen DNA fragmen gen penyandi ACCase subunit BCCP dari tiga tipe kelapa sawit Variability of DNA sequence of gene fragment encoding BCCP subunit of ACCase from three types of oil palm Asmini BUDIANI; Djoko SANTOSO; A.R. PURBA PURBA
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.v75i1.149

SummaryHeteromeric acetyl-CoA carboxylase (ht-ACCase) is one of key enzymes in palm oilbiosynthesis. Isolation and characterization ofthe gene is an important step in metabolicengineering to increase palm oil content andquality. The objective of this research was toisolate DNA fragment of gene encoding biotincarboxyl carrier protein (BCCP) subunit of ht-ACCase from three different oil palm types(Simalungun, Hibrida and Backcross) andinvestigate the variation of its DNA sequence.Total RNA was isolated from the mesocarp ofoil palm. DNA fragment encoding BCCP wasamplified by means of Reverse TranscriptasePolymerase Chain Reaction (RT-PCR) usingspecific primers with total RNA as a template.The products of RT-PCR were then purifiedfrom the gel, cloned and sequenced. The DNAsequences were analyzed for their homologiesto BCCP gene using BlastN and aligned todetect the sequence variability using ClustalWprogram from BioEdit. The results show thatone of the two RT-PCR products at about 300bp was highly homologous with the geneencoding BCCP from Glycine max, Brassicanapus and Arabidopsis thaliana. Nucleotidesequences of that BCCP fragments from thethree types of oil palm displayed some degreesof variability. Further investigation is neededto analyze the variability of the DNA sequencesof the full-length gene in relation with oilcontent or other characterRingkasanAsetil-CoA karboksilase heteromerik (ht-ACCase) merupakan salah satu enzim kuncidalam biosintesis minyak sawit. Isolasi dankarakterisasi gen tersebut merupakan langkahpenting dalam upaya rekayasa metabolismeuntuk peningkatan rendemen dan kualitasminyak sawit. Penelitian ini bertujuan untukmengisolasi fragmen DNA penyandi subunitbiotin carboxyl carrier protein (BCCP) dari ht-ACCase dari tiga tipe kelapa sawit yang ber-beda (Simalungun, Hibrida dan Backcross)dan mempelajari keragaman susunan nukleo-tidanya. RNA total diisolasi dari mesokarpbuah sawit. Fragmen gen penyandi BCCPdiamplifikasi dengan Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) meng-gunakan primer spesifik dan templat RNA total.Fragmen hasil RT-PCR dimurnikan dari gel,diklon kemudian disekuen. Sekuen DNA yangdiperoleh dianalisis homologinya dengan genBCCP menggunakan BlastN dan disejajarkanuntuk mengetahui keragamannya mengguna-kan program ClustalW dari BioEdit. Hasilnyamenunjukkan bahwa satu dari dua fragmenhasil RT-PCR yang berukuran sekitar 300 pbmemiliki homologi yang tinggi denganfragmen gen penyandi BCCP dari Glycine max,Brassica napus dan Arabidopsis thaliana.Urutan nukleotida fragmen BCCP dari ketigatipe kelapa sawit menunjukkan keragaman.Perlu analisis lebih lanjut mengenai keragamansekuen DNA dari gen lengkapnya dan dikajihubungannya dengan akumulasi minyak ataukarakter lain

Embriogenesis somatik langsung dan regenerasi planlet kopi arabika (Coffea arabica) dari berbagai eksplan Direct somatic embryogenesis and regeneration of arabica coffee plantlets (Coffea arabica) from different explants Fetrina OKTAVIA; . SWANTO; Asmini BUDIANI
Menara Perkebunan Vol. 71 No. 2: 71 (2), 2003
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v71i2.161

SummaryTissue culture technique for arabica coffeefaces some problems, mainly in plantletsregeneration from cultured explants. Theobjectives of this experiment were to examine theeffect 2,4-D and 2-ip combinations on somaticembryogenesis and regeneration of arabicacoffee from several different explants. Basalmedium used in this experiment was MS mediumwith ½ concentration of macro and micro salts.Experiment to induce primary somatic embryos(SE) was arranged in factorial randomizedcomplete design with 10 repeats. The first factorwas the type of explants, leaf, epicotyl, hipocotyland root explants. The second factor was plantgrowth regulator i.e. combination of 1  M 2,4-Dwith 5, 10, 15, 20  M and combination of 5  M2,4-D with 5, 10, 15 and 20  M 2-ip. To multiplySE, secondary SE was induced from primary SEon medium containing combination of 0.6  MIAA and 13.3; 17.8 and 22.2  M BAP.Cotyledonary SE were germinated on mediacontaining GA 3 (0, 5, 10 and 15  M), and thenregenerated on medium free of growth regulator.Plantlets with 4-5 leaf pairs were transfered intothe soil medium for acclimatization. The resultsshow that primary SE can be induced from allexplants with the highest frequency on mediumcontaining 1  M 2,4-D and 15  M 2-ip.Induction of primary SE, in leaf explant wasmore effective than other explants. Mediumcontaining 0.6  M IAA and 22.2  M BAP gavethe highest percentage of SE multiplication i.e.52.6% with average SE number of 6.25. Plantletsregeneration can be conducted by culturing SEon maturation medium free of growth regulatorfor one month followed by germinating onmedium containing GA 3 , and then culturing onmedium free of growth regulator again. Thehighest percentage of germinated embryos wasobtained after three weeks and six weekscultured in the medium containing 5  M GA 3 , i.e49% and 90.15 respectively. From total plantletsobtained, 75% of them were normal. Sixtypercents of the young plants grew well in thegreenhouse.RingkasanTeknik kultur jaringan tanaman kopi arabikamasih menghadapi beberapa kendala terutamapada tingkat regenerasi planlet dari eksplan yangdikulturkan. Penelitian ini bertujuan untukmengetahui pengaruh kombinasi 2,4-D dan 2-ipterhadap embriogenesis somatik dan regenerasikopi arabika dari berbagai eksplan. Media dasaryang digunakan adalah medium MS ½konsentrasi garam makro dan mikro. Percobaaninduksi embrio somatik (ES) primer disusunmenurut rancangan acak lengkap faktorial dengan10 ulangan. Faktor pertama adalah jenis eksplan,erdiri atas daun, epikotil, hipokotil dan akar invitro. Faktor kedua adalah zat pengatur tumbuh,yaitu kombinasi 1 M 2,4-D dengan 5, 10, 15dan 20M 2-ip, serta kombinasi 5 M 2,4-Ddengan 5, 10, 15 dan 20 M 2-ip. Untuk mem-perbanyak jumlah ES yang didapatkan, dilakukaninduksi ES sekunder dari ES primer pada mediumyang mengandung kombinasi 0,6 M IAA dan13,3; 17,8 dan 22,2 M BAP. ES fase kotiledonkemudian dikecambahkan pada medium yangmengandung GA 3 (0, 5, 10 dan 15 M) danselanjutnya diregenerasikan pada medium tanpazat pengatur tumbuh. Planlet yang mempunyai4-5 pasang daun dipindahkan ke medium tanahuntuk aklimatisasi. Hasil yang diperolehmenunjukkan bahwa ES primer dapat diinduksipada semua eksplan yang digunakan denganfrekuensi tertinggi pada medium yang me-ngandung 1 M 2,4-D dan 15 M 2-ip. InduksiES primer pada eksplan daun lebih efektifdibandingkan eksplan lainnya. Untuk per-banyakan ES, medium yang mengandung IAA0,6 M dan BAP 22,2 M memberikanpersentase tertinggi pembentukan ES sekunderyaitu 52,6% dengan rata-rata jumlah ES 6,25.Regenerasi planlet dapat dilakukan denganmengkulturkan ES pada medium maturasi tanpazat pengatur tumbuh selama satu bulan, kemudiandikecambahkan dalam medium yang mengan-dung GA 3 , dan selanjutnya dipindah ke mediumtanpa zat pengatur tumbuh kembali.Perkecambahan ES tertinggi diperoleh padamedium dengan penambahan GA 3 5 M yaitu40,9% setelah tiga minggu dan 90,1% setelahenam minggu. Dari total planlet diperoleh 75%planlet normal. Hasil aklimatisasi menunjukkanbahwa 60% bibit mampu bertahan di rumah kaca.

Transformasi kopi robusta (Coffea canephora) dengan gen kitinase melalui Agrobagterium tumefaciens LBA4404 Transformation of robusta coffee (Coffea canephora) with chitinase gene mediated by Agrobacterium tumefaciens LBA4404 . SISWANTO; Fetrina OKTAVIA; Asmini BUDIANI; . , SUDARSONO; . PRIYONO; Surip MAWARDI
Menara Perkebunan Vol. 71 No. 2: 71 (2), 2003
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v71i2.162

SummaryGenetic engineering of robusta coffee forresistance to pathogenic fungi is considered to beone of the potential approaches to overcome theproblem at robusta coffee plantation caused bypathogenic fungi. This research was aimed tointroduce chitinase (CHI) gene into embryogeniccalli of robusta coffee and regenerate theplantlets. Embryogenic calli were co-cultivatedwith Agrobacterium tumefaciens LBA4404harboring pCAMBIA1301 which containschitinase gene under 35S promoter. In thisresearch four concentrations (0, 50, 100 and150 mg/L) of acetosyringone (AC) were used inthe co-cultivation medium. Selection fortransformed calli was conducted by graduallyincreasing the concentration of hygromicin from5 to 25 mg/L. Somatic embryo (SE) was inducedfrom callus on the medium containing acombination of BAP 5 mg/L and IAA (0, 0.25 or0.50 mg/L). Integration CHI in plant genome wasexamined by GUS assay and PCR. The resultrevealed that among the four AC concentrationstested, 100 mg/L gave the highest percentage ofcalli growing on the selection medium (42.5%).BAP concentration of 5 mg/L alone was the mosteffective for inducing of SE from transformedcalli with the highest percentage of 43.1% andaverage number SE of 8.8 ± 3. The strongestGUS expression on the calli at 3 days aftertransformation and the calli grown on selectionmedium containing 150 mg/L AC, which were56.5% and 40% respectivelly. PCR analysisshowed that 7 out of 12 plantlets tested,contained CHI gene. From this research 28transgenic plantlets of robusta coffee wereobtainedRingkasanRekayasa genetika untuk merakit tanamankopi robusta tahan jamur pathogen dipandangmerupakan salah satu pendekatan alternatif yangpotensial untuk mengatasi masalah padaperkebunan kopi robusta akibat serangan jamurpatogen. Penelitian ini bertujuan untuk meng-introduksikan gen kitinase (CHI) ke dalam kalusembriogenik kopi robusta dan regenerasinyamenjadi planlet, sebagai upaya untuk merakittanaman kopi robusta tahan serangan jamur.Kalus embriogenik diko-kultivasi denganAgrobacterium tumefaciens LBA4404 pembawapCAMBIA1301 yang mengandung gen kitinasedi bawah kontrol promotor 35S. Pada percobaanini, empat konsentrasi asetosiringon (AS) (0, 50,100 dan 150 mg/L) digunakan dalam medium ko-kultivasi. Seleksi kalus hasil transformasidilakukan dengan peningkatan konsentrasi higro-misin secara bertahap dari 5 mg/L sampai25 mg/L. ES diinduksi dari kalus pada mediumyang mengandung BAP 5 mg/L dan IAA (0; 0,25dan 0,50 mg/L). Integrasi gen CHI ke dalamgenom tanaman dianalisis melalui uji GUS danPCR. Hasil penelitian menunjukkan bahwa darikeempat konsentrasi AS yang diuji, AS 100 mg/Lternyata menghasilkan persentase tertinggi kalusyang tumbuh pada medium seleksi (42,5%).Konsentrasi BAP 5 mg/L tanpa penambahan IAAefektif menginduksi ES dari kalus hasiltransformasi dengan persentase tertinggi 43,1%dan rata-rata jumlah ES 8,8±3. Ekspresi GUStertinggi dideteksi pada kalus tiga hari setelahtransformasi dan kalus yang tumbuh di mediumseleksi yang mengandung AS 150 mg/L,masing-masing 56,5% dan 40,0 %. Analisis PCRmenunjukkan bahwa 7 planlet dari 12 planletyang diuji, membawa gen CHI. Dari penelitianini dihasilkan 28 planlet kopi robusta transgenik.

Physiological responses and P5CS gene expression of transgenic oil palm plantlet induced by drought stress Turhadi TURHADI; Hayati MINARSIH; Imron RIYADI; . PRIYONO; Asmini BUDIANI
Menara Perkebunan Vol. 88 No. 2 (2020): 88 (2), 2020
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v88i2.386

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.

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