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Isolation and characterization of Dehydrin promoter region from sugarcane (Saccharum officinarum L.) Hayati MINARSIH; Sonny SUHANDONO; Anissa K FUADI; Tati KRISTIANTI; Riza A PUTRANTO; Deden SUKMAJDAYA; . SUSTIPRIJATNO
E-Journal Menara Perkebunan Vol 88, No 1 (2020): April, 2020
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

The development of molecular biology techniques nowadays has enabled to engineer drought tolerant sugarcane by genetic engineering to accelerate the breeding program. Dehydrin (DHN) is known to have an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). While plant tissues are subjected to drought stress (dehydration), DHN protein is accumulated to high content throughout all vegetative or generative tissues. The research aimed to isolate and characterize the DHN promoter from sugarcane that can be used as transformation material in generating drought tolerant sugarcane. Specific primers for DHN promoter amplification were designed and DHN promoter region was successfully isolated by PCR cloning method. Two putative promoter sequences were identified namely Pr-1DHNSo and Pr-2DHNSo. In silicoanalyses were carried out and cis-regulatory elements motifs that play a role in adaptation on abiotic stress as well as biotic stress including ABRE, MBS, CGTCA-motif, TGACG-motif, GARE-motif, P-box TCA-element and Box-W1 were identified. The promoter Pr-1DHNSo was then cloned into pBI121 expression vector by Overlap Extention PCR (OE-PCR) for further characterization. Functional test of the promoter construct pBI- Pr-1DHNSo was conducted through Agrobacterium transformation into sugarcane calli. GUS assay and PCR analysis showed that the DHN promoter was transformed and expressed in the sugarcane calli.

Isolasi dan karakterisasi gen dehydrin dari tebu (Saccharum officinarumL.) yang terlibat dalam respon toleransi cekaman kekeringan (Isolation and characterization of dehydrin gene from sugarcane (Saccharum officinarum L.) involved in drought tolerance response) Hayati MINARSIH; . FANIAR; Tati KRISTANTI; Dian M AMANAH; . SUSTIPRIJATNO; Sony SUHANDONO
E-Journal Menara Perkebunan Vol 86, No 2 (2018): Oktober 2018
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Nowadays,the development of molecular biology techniques has enabled to engineer drought tolerant sugarcane to accelerate thebreeding program. Dehydrin(DHN)that belong to the group II late embryogenesis abundant (LEA) family is known to havean important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). Literature study and bioinformatics analysis reported that DHN1gene on sugarcane showed high homology sequences with sorghum DHN. The expression of DHN1gene on sugarcane var. PSJT 941 treated with various periodof drought stress had been conducted using semi-quantitative reverse transcriptase (RT)-PCR method. The results showed that the expressionlevel of DHN1 geneincreased along withthe increased period of the treatment. The highest expression level of DHN1 gene was resulted from plants that had been subjected to drought for 25 days. Amplification of DHN1gene from plants withthe highest gene expression, resulted an amplicon with a size of 465 bp which representsa full length coding sequence (CDS) of DHN1. Identification using Blast analysis showed that DHN1sequences from sugarcane var. PSJT 941 shared high homology with DHN gene on sugarcane and sorghum. The alignment results also revealed a conserved motif that characterized DHN genes.[Key words: drought stress, dehydrin, DHN1gene, sugarcane]Abstrak Dengan berkembangnya teknik biologi molekuler saat ini, maka perakitan tanaman tebu yang toleran kekeringan lebih diarahkan melalui teknik rekayasa genetika untuk mempercepat program pemuliaan tanaman. Protein dehydrin (DHN) yang termasuk ke dalam kelompok II famili LEA (Late Embryogenesis Abundant)diketahui berperan penting dalam respon dan adaptasi tanaman terhadap cekaman abiotik (kekeringan, salinitas tinggi, suhu dingin, panas, dll). Studi literatur dan analisis bioinformatika menunjukkan bahwa gen DHN1pada tanaman tebu memiliki homologi yang tinggi dengan gen DHNpada sorghum. Analisis ekspresi gen DHN1pada tanaman tebu varietasPSJT 941yang diberi cekaman kekeringan telah dilakukan menggunakan semi-kuantitatifreverse transcriptase (RT)-PCR dan terlihat bahwa ekspresi gen DHN1meningkat secara nyata sejalan dengan semakin lamanya waktu pemberian cekaman. Tingkat ekspresi gen DHN1paling tinggi diperoleh dari tanaman yang mengalami cekaman kekeringan selama 25 hari. Amplifikasi gen DHN1pada tanaman dengan tingkat ekspresi yang paling tinggi menunjukkan pita dengan ukuran 465 bp yang merepresentasikan full coding sequence(CDS) gen DHN1. Identifikasi menggunakan analisis Blast menunjukkan bahwa sekuen gen DHN1dari tanaman tebu varietas PSJT 941yang diperoleh memiliki homologi yang tinggi dengan gen DHNpada tanaman tebu dan sorghum. Hasil penjajaran sekuen protein juga menunjukkan adanya motif lestari yang mencirikan gen DHN. [Kata kunci: cekaman kekeringan, dehydrin, gen DHN1, tebu]

Pengaruh jumlah subkultur dan media sub-optimal terhadap pertumbuhan dan kemampuan regenerasi kalus tebu (Saccharum officinarum L.) (Effect of repeated subculture and suboptimum media on the growth of sugarcane calli (Saccharum officinarum L.)) Hayati MINARSIH; . Suharyo; Imron RIYADI; Diah RATNADEWI
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Sugarcane (Saccharum officinarum L.) is an important crop for sugar production. One attempt to increase sugarcane productivity is through micropropagation and quality improvement of sugarcane seedlings in vitro. This research aimed to study the effect of repeated subcultures on callus capacity for regeneration and plant survival in acclimatization phase, as well as the influence of suboptimum media on the recovery capability of sugarcane callus to proliferate in vitro. Fourth subcultured sugarcane callus derived from young leaves were used as material in this research. Basic medium of Murashige and Skoog (MS) added with 3 mg/L 2,4-D, 10% coconut water, and 3% sucrose was used for callus initiation. For callus regeneration, the MS medium was supplemented with 2 mg/L BAP, 0.2 mg/L IAA, 10% coconut water, and 3% sucrose. Study on the effect of subculture numbers consisted of three stages, i.e. initiation, regeneration, and acclimatization, while the study on resting phase or the use of sub-optimal media included six treatment media and two pathways. Results showed that the fifth subcultures produced embryoid callus (91%), the highest non mucilaginous callus (97%), and the highest abnormality rate (6%). Results from the suboptimum media treatment, showed that B pathway (4 week resting phase) was better than the A pathway (8 week resting phase), based on fresh weight and callus abnormality percentage. A and B pathways indicated that the growth of callus can be recovered when it was grown back to the normal media and 1.5D-MS treatment of the resting phase showed the best growth and appearance.

Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium-mediated transformation Asmini BUDIANI; Imam Bagus NUGROHO; Hayati MINARSIH; Imron RIYADI
E-Journal Menara Perkebunan Vol 87, No 2 (2019): OKTOBER, 2019
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

AbstrakCekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]Abstract Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS (Δ1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]

Deteksi Ganoderma secara molekuler pada kebun kelapa sawit yang diberi perlakuan biofungisida Ganor (Molecular detection of Ganoderma on oil palm plantation treated with Ganor biofungicide) Hayati MINARSIH; Happy WIDIASTUTI; Djoko SANTOSO
E-Journal Menara Perkebunan Vol 86, No 1 (2018): April, 2018
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

AbstractGanor organic fungicide potentially reduces Ganoderma, a pathogenic fungus causing basal stem rot disease. Application of Ganor on oil palm trees in the plantation attacked Ganoderma, inhibits the growth of Ganoderma fruiting bodies, improves rooting and stimulates the opening of the spear leaf. This study aims to identify molecularly the presence of Ganoderma in oil palm trees that have been attacked by Ganoderma routinely treated with Ganor for three months. Molecular analysis was performed by PCR using Ganoderma specific primers. The analysis results of sample from trunks and roots of oil palm, indicating that the Ganoderma infected oil palm which has been treated with Ganor, were relatively free (96.4%) of Ganoderma. Of the 28 samples examined of treated plants, 27 samples did not indicate the presence of Ganoderma specific DNA band. On the other hand, the untreated oil palm trees infected by Ganoderma were still detected by the appearence of DNA bands specific to Ganoderma. The results of molecular analysis indicated that Ganor treatments can effectively reduce the attack rate of Ganoderma in oil palm trees in the plantation infected by Ganoderma. However, the use of the molecular technique for early detection needs to be further tested to evaluate its consistency prior to introduction to the commercial growers. The reproducibility can be confirmed by repeating the experiment using more samples. Ganor effectiveness in curing oil palm trees infected by Ganoderma, maybe indicated by the ability of the reproductive organs to develop, particularly female flowers. The sex ratio of Ganor treated oil palms was clearly higher than that of control palms in 10 to 12 weeks after the treatment.[Keywords: organic fungicides, stem rot, molecular analysis, Elais guinensis Jack.] AbstrakFungisida organik Ganor berpotensi mengurangi serangan Ganoderma, cendawan patogenik penyebab penyakit busuk pangkal batang. Aplikasi Ganor pada tanaman kelapa sawit di kebun yang terserang Ganoderma, menghambat pertumbuhan tubuh buah Ganoderma, memper-baiki perakaran dan merangsang pembukaan daun tombak. Penelitian ini bertujuan untuk mengidentifikasi secara molekuler adanya Ganoderma pada tanaman kelapa sawit terserang Ganoderma yang telah mendapat perlakuan Ganor secara rutin selama tiga bulan. Analisis molekuler dilakukan dengan teknik PCR menggunakan primer DNA spesifik Ganoderma. Hasil analisis sampel batang dan akar tanaman kelapa sawit, menunjukkan bahwa tanaman Perlakuan, yaitu kelapa sawit terserang Ganoderma yang telah mendapat perlakuan Ganor, 96,4% bebas Ganoderma. Dari 28 sampel tanaman Perlakuan yang diperiksa, 27 sampel tidak menunjukkan adanya pita DNA spesfik Ganoderma. Sementara itu pada tanaman Kontrol, yaitu tanaman kelapa sawit terserang Ganoderma dan tidak mendapat perlakuan Ganor, 100% masih terdeteksi adanya Ganoderma. Dari 7 sampel tanaman kontrol yang diperiksa semuanya menunjukkan adanya pita DNA spesifik Ganoderma. Hasil analisis molekuler ini mengindikasikan bahwa perlakuan Ganor efektif mengurangi tingkat serangan Ganoderma pada tanaman kelapa sawit di kebun yang terinfeksi Ganoderma. Namun demikian, untuk lebih meyakinkan praktisi perkebunan, penggunakan teknik molekuler ini masih perlu diuji lebih lanjut terkait konsistensinya. Reprodusibilitas dapat dikonfirmasi dengan mengulangi percobaan menggunakan lebih banyak sampel. Efektivitas Ganor dalam menyehatkan tanaman kelapa sawit terserang Ganoderma ini, terindikasi juga dari perkembangan organ reproduktifnya. Sex ratio meningkat dalam waktu 10 hingga 12 minggu setelah perlakuan.[Kata Kunci: fungisida organik, busuk pangkal batang, analisis molekuler, Elais guinensis Jack. ]

Pengaruh jumlah subkultur dan media sub-optimal terhadap pertumbuhan dan kemampuan regenerasi kalus tebu (Saccharum officinarum L.) (Effect of repeated subculture and suboptimum media on the growth of sugarcane calli (Saccharum officinarum L.)) Hayati MINARSIH; . Suharyo; Imron RIYADI; Diah RATNADEWI
Menara Perkebunan Vol. 84 No. 1 (2016): 84 (1), 2016
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v84i1.219

Sugarcane (Saccharum officinarum L.) is an important crop for sugar production. One attempt to increase sugarcane productivity is through micropropagation and quality improvement of sugarcane seedlings in vitro. This research aimed to study the effect of repeated subcultures on callus capacity for regeneration and plant survival in acclimatization phase, as well as the influence of suboptimum media on the recovery capability of sugarcane callus to proliferate in vitro. Fourth subcultured sugarcane callus derived from young leaves were used as material in this research. Basic medium of Murashige and Skoog (MS) added with 3 mg/L 2,4-D, 10% coconut water, and 3% sucrose was used for callus initiation. For callus regeneration, the MS medium was supplemented with 2 mg/L BAP, 0.2 mg/L IAA, 10% coconut water, and 3% sucrose. Study on the effect of subculture numbers consisted of three stages, i.e. initiation, regeneration, and acclimatization, while the study on resting phase or the use of sub-optimal media included six treatment media and two pathways. Results showed that the fifth subcultures produced embryoid callus (91%), the highest non mucilaginous callus (97%), and the highest abnormality rate (6%). Results from the suboptimum media treatment, showed that B pathway (4 week resting phase) was better than the A pathway (8 week resting phase), based on fresh weight and callus abnormality percentage. A and B pathways indicated that the growth of callus can be recovered when it was grown back to the normal media and 1.5D-MS treatment of the resting phase showed the best growth and appearance.

Deteksi Ganoderma secara molekuler pada kebun kelapa sawit yang diberi perlakuan biofungisida Ganor (Molecular detection of Ganoderma on oil palm plantation treated with Ganor biofungicide) Hayati MINARSIH; Happy WIDIASTUTI; Djoko SANTOSO
Menara Perkebunan Vol. 86 No. 1 (2018): 86 (1), 2018
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v1i1.289

AbstractGanor organic fungicide potentially reduces Ganoderma, a pathogenic fungus causing basal stem rot disease. Application of Ganor on oil palm trees in the plantation attacked Ganoderma, inhibits the growth of Ganoderma fruiting bodies, improves rooting and stimulates the opening of the spear leaf. This study aims to identify molecularly the presence of Ganoderma in oil palm trees that have been attacked by Ganoderma routinely treated with Ganor for three months. Molecular analysis was performed by PCR using Ganoderma specific primers. The analysis results of sample from trunks and roots of oil palm, indicating that the Ganoderma infected oil palm which has been treated with Ganor, were relatively free (96.4%) of Ganoderma. Of the 28 samples examined of treated plants, 27 samples did not indicate the presence of Ganoderma specific DNA band. On the other hand, the untreated oil palm trees infected by Ganoderma were still detected by the appearence of DNA bands specific to Ganoderma. The results of molecular analysis indicated that Ganor treatments can effectively reduce the attack rate of Ganoderma in oil palm trees in the plantation infected by Ganoderma. However, the use of the molecular technique for early detection needs to be further tested to evaluate its consistency prior to introduction to the commercial growers. The reproducibility can be confirmed by repeating the experiment using more samples. Ganor effectiveness in curing oil palm trees infected by Ganoderma, maybe indicated by the ability of the reproductive organs to develop, particularly female flowers. The sex ratio of Ganor treated oil palms was clearly higher than that of control palms in 10 to 12 weeks after the treatment.[Keywords: organic fungicides, stem rot, molecular analysis, Elais guinensis Jack.] AbstrakFungisida organik Ganor berpotensi mengurangi serangan Ganoderma, cendawan patogenik penyebab penyakit busuk pangkal batang. Aplikasi Ganor pada tanaman kelapa sawit di kebun yang terserang Ganoderma, menghambat pertumbuhan tubuh buah Ganoderma, memper-baiki perakaran dan merangsang pembukaan daun tombak. Penelitian ini bertujuan untuk mengidentifikasi secara molekuler adanya Ganoderma pada tanaman kelapa sawit terserang Ganoderma yang telah mendapat perlakuan Ganor secara rutin selama tiga bulan. Analisis molekuler dilakukan dengan teknik PCR menggunakan primer DNA spesifik Ganoderma. Hasil analisis sampel batang dan akar tanaman kelapa sawit, menunjukkan bahwa tanaman Perlakuan, yaitu kelapa sawit terserang Ganoderma yang telah mendapat perlakuan Ganor, 96,4% bebas Ganoderma. Dari 28 sampel tanaman Perlakuan yang diperiksa, 27 sampel tidak menunjukkan adanya pita DNA spesfik Ganoderma. Sementara itu pada tanaman Kontrol, yaitu tanaman kelapa sawit terserang Ganoderma dan tidak mendapat perlakuan Ganor, 100% masih terdeteksi adanya Ganoderma. Dari 7 sampel tanaman kontrol yang diperiksa semuanya menunjukkan adanya pita DNA spesifik Ganoderma. Hasil analisis molekuler ini mengindikasikan bahwa perlakuan Ganor efektif mengurangi tingkat serangan Ganoderma pada tanaman kelapa sawit di kebun yang terinfeksi Ganoderma. Namun demikian, untuk lebih meyakinkan praktisi perkebunan, penggunakan teknik molekuler ini masih perlu diuji lebih lanjut terkait konsistensinya. Reprodusibilitas dapat dikonfirmasi dengan mengulangi percobaan menggunakan lebih banyak sampel. Efektivitas Ganor dalam menyehatkan tanaman kelapa sawit terserang Ganoderma ini, terindikasi juga dari perkembangan organ reproduktifnya. Sex ratio meningkat dalam waktu 10 hingga 12 minggu setelah perlakuan.[Kata Kunci: fungisida organik, busuk pangkal batang, analisis molekuler, Elais guinensis Jack. ]

Isolasi dan karakterisasi gen dehydrin dari tebu (Saccharum officinarumL.) yang terlibat dalam respon toleransi cekaman kekeringan (Isolation and characterization of dehydrin gene from sugarcane (Saccharum officinarum L.) involved in drought tolerance response) Hayati MINARSIH; . FANIAR; Tati KRISTANTI; Dian M AMANAH; . SUSTIPRIJATNO; Sony SUHANDONO
Menara Perkebunan Vol. 86 No. 2 (2018): 86 (2), 2018
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v86i2.300

Nowadays,the development of molecular biology techniques has enabled to engineer drought tolerant sugarcane to accelerate thebreeding program. Dehydrin(DHN)that belong to the group II late embryogenesis abundant (LEA) family is known to havean important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). Literature study and bioinformatics analysis reported that DHN1gene on sugarcane showed high homology sequences with sorghum DHN. The expression of DHN1gene on sugarcane var. PSJT 941 treated with various periodof drought stress had been conducted using semi-quantitative reverse transcriptase (RT)-PCR method. The results showed that the expressionlevel of DHN1 geneincreased along withthe increased period of the treatment. The highest expression level of DHN1 gene was resulted from plants that had been subjected to drought for 25 days. Amplification of DHN1gene from plants withthe highest gene expression, resulted an amplicon with a size of 465 bp which representsa full length coding sequence (CDS) of DHN1. Identification using Blast analysis showed that DHN1sequences from sugarcane var. PSJT 941 shared high homology with DHN gene on sugarcane and sorghum. The alignment results also revealed a conserved motif that characterized DHN genes.[Key words: drought stress, dehydrin, DHN1gene, sugarcane]Abstrak Dengan berkembangnya teknik biologi molekuler saat ini, maka perakitan tanaman tebu yang toleran kekeringan lebih diarahkan melalui teknik rekayasa genetika untuk mempercepat program pemuliaan tanaman. Protein dehydrin (DHN) yang termasuk ke dalam kelompok II famili LEA (Late Embryogenesis Abundant)diketahui berperan penting dalam respon dan adaptasi tanaman terhadap cekaman abiotik (kekeringan, salinitas tinggi, suhu dingin, panas, dll). Studi literatur dan analisis bioinformatika menunjukkan bahwa gen DHN1pada tanaman tebu memiliki homologi yang tinggi dengan gen DHNpada sorghum. Analisis ekspresi gen DHN1pada tanaman tebu varietasPSJT 941yang diberi cekaman kekeringan telah dilakukan menggunakan semi-kuantitatifreverse transcriptase (RT)-PCR dan terlihat bahwa ekspresi gen DHN1meningkat secara nyata sejalan dengan semakin lamanya waktu pemberian cekaman. Tingkat ekspresi gen DHN1paling tinggi diperoleh dari tanaman yang mengalami cekaman kekeringan selama 25 hari. Amplifikasi gen DHN1pada tanaman dengan tingkat ekspresi yang paling tinggi menunjukkan pita dengan ukuran 465 bp yang merepresentasikan full coding sequence(CDS) gen DHN1. Identifikasi menggunakan analisis Blast menunjukkan bahwa sekuen gen DHN1dari tanaman tebu varietas PSJT 941yang diperoleh memiliki homologi yang tinggi dengan gen DHNpada tanaman tebu dan sorghum. Hasil penjajaran sekuen protein juga menunjukkan adanya motif lestari yang mencirikan gen DHN. [Kata kunci: cekaman kekeringan, dehydrin, gen DHN1, tebu]

Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium-mediated transformation Asmini BUDIANI; Imam Bagus NUGROHO; Hayati MINARSIH; Imron RIYADI
Menara Perkebunan Vol. 87 No. 2 (2019): 87 (2), 2019
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v87i2.336

AbstrakCekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]Abstract Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS (Δ1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]

Isolation and characterization of Dehydrin promoter region from sugarcane (Saccharum officinarum L.) Hayati MINARSIH; Sonny SUHANDONO; Anissa K FUADI; Tati KRISTIANTI; Riza A PUTRANTO; Deden SUKMAJDAYA; . SUSTIPRIJATNO
Menara Perkebunan Vol. 88 No. 1 (2020): 88 (1), 2020
Publisher : INDONESIAN OIL PALM RESEARCH INSTITUTE

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v88i1.363

The development of molecular biology techniques nowadays has enabled to engineer drought tolerant sugarcane by genetic engineering to accelerate the breeding program. Dehydrin (DHN) is known to have an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). While plant tissues are subjected to drought stress (dehydration), DHN protein is accumulated to high content throughout all vegetative or generative tissues. The research aimed to isolate and characterize the DHN promoter from sugarcane that can be used as transformation material in generating drought tolerant sugarcane. Specific primers for DHN promoter amplification were designed and DHN promoter region was successfully isolated by PCR cloning method. Two putative promoter sequences were identified namely Pr-1DHNSo and Pr-2DHNSo. In silicoanalyses were carried out and cis-regulatory elements motifs that play a role in adaptation on abiotic stress as well as biotic stress including ABRE, MBS, CGTCA-motif, TGACG-motif, GARE-motif, P-box TCA-element and Box-W1 were identified. The promoter Pr-1DHNSo was then cloned into pBI121 expression vector by Overlap Extention PCR (OE-PCR) for further characterization. Functional test of the promoter construct pBI- Pr-1DHNSo was conducted through Agrobacterium transformation into sugarcane calli. GUS assay and PCR analysis showed that the DHN promoter was transformed and expressed in the sugarcane calli.

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