25 research outputs found
Induksi Kalus dan Regenerasi Beberapa Genotipe Gandum (Triticum aestivum L.) secara In Vitro
No full text<p>Callus Induction and In Vitro Plant Regeneration of<br />Wheat Genotypes (Triticum aestivum L.). Atmitri<br />Sisharmini, Aniversari Apriana, and Sustiprijatno. Development<br />of a reliable in vitro plant regeneration procedure for<br />wheat is a prerequisite for its improvement by genetic transformation.<br />The purpose of this study was to obtain methods<br />of callus induction and regeneration of wheat genotypes.<br />This experiment was conducted at ICABIOGRAD. Immature<br />embryos from four wheat genotypes, ie Perdix, Naxos Wew,<br />Combi and Fasan were used to induce callus formation and<br />regeneration rate of callus. For the preparation of callus<br />induction medium, MS-L7 basal medium was supplemented<br />with combination of growth regulators 2,4 dichlorophenoxy<br />acetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid<br />(picloram). While, plant regeneration medium was prepared<br />using MS basal medium supplemented with combination of<br />three growth regulators i.e. IAA, BAP and kinetin. The results<br />showed that genotype, in vitro culture medium and growth<br />regulators played a dominant role in callus induction and<br />plantlet regeneration. All the 4 genotypes responded positively<br />to callus induction, however, variability was observed<br />not only among the genotypes but also within callus<br />induction medium used. The best induction medium was<br />the MS-L7 basal medium supplemented with combination of<br />phytohormon 4 mg/l 2,4-D + 2 mg/l picloram (GIK-3) which<br />showed 100% callus induction frequency. Whereas, the best<br />regeneration medium was shown by MS basal medium with<br />combination of phytohormon 1.5 mg/l BAP dan 0.5 mg/l<br />kinetin (RG3). Regarding plant regeneration, Perdix was the<br />most responsive genotype to be regenerated with regeneration<br />frequency of 57.33%. The successfully acclimatized<br />planlets in greenhouse were obtained from Perdix and<br />Naxos Wew genotypes. These results will potentially facilitate<br />genetic transformation research of wheat in Indonesia.</p></jats:p
Introduksi Konstruk Over-Ekspresi Kandidat Gen OsWRKY76 melalui Agrobacterium tumefaciens pada Tanaman Padi Nipponbare
No full text<p>Delivering of Over-Expression Construct OsWRKY76<br />Candidate Gene in Rice cv. Nipponbare through<br />Agrobacterium tumefaciens. Aniversari Apriana, Atmitri<br />Sisharmini, Wening Enggarini, Sudarsono, Nurul.<br />Khumaida, and Kurniawan R. Trijatmiko. Plant genetic<br />improvement can be done through classical breeding or<br />genetic engineering. WRKY is a transcription factor involved<br />in regulating plant defense responses. OsWRKY76 gene is<br />located in a narrow segment of chromosome 9 which is<br />identified previously to be related to wide spectrum<br />resistance in rice. A sequence of OsWRKY76 (+1.200 bp)<br />has available in the gene bank and it makes possible to<br />isolate, clone, and construct the gene into over-expression<br />vector. The aim of this research was to assemble an overexpression<br />construct of OsWRKY76 candidate gene and<br />introduce it into rice through Agrobacterium-mediated<br />transformation. A construct of pCAMBIA-<br />1301::35S::OsWRKY76 has been successfully assembled and<br />transformed into embryogenic calli of rice cv. Nipponbare<br />using A. tumefaciens strain Agl-1 and EHA 105. A number of<br />126 independent lines has been produced, in which Agl-1<br />showed 3.8 times more efficient than EHA 105. PCR analysis<br />of randomly selected 25 independent lines showed that all<br />of them positively contained hptII gene, a selectable marker<br />used in the over-expression construct of the OsWRKY76<br />candidate gene. Based on the result, it could be concluded<br />that the over-expression construct of OsWRKY76 candidate<br />gene have been successfully introduced into the tissue of<br />Nipponbare.</p></jats:p
Spektrum Ketahanan Galur Haploid Ganda Turunan IR64 dan Oryza rufipogon yang mengandung QTL Ketahanan terhadap Penyakit Blas (Pir)
No full text<p>Resistance Spectrum of Double Haploid Lines Derived<br />from IR64 and Wild Rice Species, Oryza rufipogon<br />Contained the Blast Resistance QTL (Pir). Dwinita W.<br />Utami, A. Dinar Ambarwati, Aniversari Apriana, Atmitri<br />Sisharmini, Ida Hanarida, Didier Tharreau, and Santosa.<br />This study was initiated to determine the spectrum resistance<br />of the candidate durable blast resistance variety contained<br />the QTL (quantitative trait locus), Pir1 and 2. This QTL<br />was mapped on chromosome 2 detected using the advanced<br />backcross population (BC5) from the wild rice species<br />Oryza rufipogon to IR64. Pir (1 and 2) also established<br />on double haploid (DH) population derived from the selected<br />lines of BC2F3 population, progenies from the same<br />parents. The DH lines were developed to speed up the fixation<br />process of the recessive alleles in the selected lines.<br />Near isogenic lines with different blast resistance genes and<br />combination were used in this study comparing to the DH<br />population on their resistance spectrum using the known<br />avr gene isolates both on green house and field screening.<br />The determination of the resistance spectrum will useful on<br />the prediction of durability of blast resistance gene in DH<br />population. The results of spectrum resistance test in green<br />house and field showed that Pir1and Pir2 segregated on 1 : 1<br />proportion related with specific respond to blast avr gene<br />PH14 and CM28 resistance. Pir1 was identic to Pi33 or Pi25<br />and Pir2 to Pitq5 on spectrum resistance.<br /><br /></p></jats:p
Pembentukan Genotipe Padi Berumur Sangat Genjah melalui Kultur Antera
No full text<p>Development of Very Early Maturing Rice Genotypes through Anther Culture. Iswari S. Dewi, A. Dinar Ambarwati, Aniversari Apriana, Atmitri Sisharmini, Ida H. Somantri, Bambang Suprihatno, and Iman Ridwan. Rice is the most important food crop in Indonesia. Increase in production is needed due to population increase. Rice production in rainfed area is contributed the second after irrigated area. Rainfed condition requiring very early maturity (90-104 days) varieties. Rice anther culture can be applied to accelerate obtainment of doubled haploids (DHs) or pure lines needed in rice breeding. The experiment was aimed to obtain pure lines for developing very early maturing and high yielding rice varieties. Materials used for anther culture were F1s of Fatmawati/Kinamase, Inpari 1/Kinamase, Fatmawati/ Waseaikoku, Inpari 1/Waseaikoku, Fatmawati/IR71146, Inpari 1/IR71146, OM4495/Silugonggo, IR7146/Dodokan, and IR71730/OM1490. Anther culture media were N6 + NAA 2,0 mg/l + kinetin 0,5 mg/l for callus induction, MS+ NAA 0,5 mg/l + kinetin 2,0 mg/l for plantlet regeneration, and MS + 0,5 mg/l IBA for rooting. Putrescine 10-3 M was added to callus induction and regeneration media. The results shown that calli forming green plantlet (CFGP) were ranged from 0.25 to 83.33%. Fatmawati/Kinamase gave the highest CFGP (245 calli), followed by Inpari 1/Kinamase (78 calli) and Fatmawati/ Waseaikoku (68 calli). Total green plantlets obtained were 2.038 plantlets. After plantlet acclimatization and greenhouse grow-out, we obtained 507 DHs. The evaluation of 100 DHs at farmer field (Ciranjang District in Cianjur), based on their 50% heading date of 65 days, resulted in 33 lines cathegorized as very early maturing lines (+100 days). They were 18 lines from Fatmawati/Kinamase, 5 lines from Inpari 1/Kinamase, 8 lines from Fatmawati/Waseaikoku, and 2 lines from Inpari 1/ Waseaikoku.</p><p> </p><p><strong>Abstrak</strong></p><p>Padi (Oryza sativa L.) merupakan komoditi pangan terpenting di Indonesia. Peningkatan produksi diperlukan seiring dengan peningkatan jumlah penduduk. Lahan sawah tadah hujan merupakan lumbung padi kedua setelah sawah irigasi. Kondisi lahan sawah tadah hujan memerlukan varietas-varietas padi berumur sangat genjah (90-104 hari). Teknik kultur antera dapat digunakan untuk mempercepat perolehan tanaman dihaploid (DH) atau galur murni dalam pemuliaan padi. Penelitian ini bertujuan untuk mendapatkan galur-galur murni yang akan digunakan dalam perakitan padi berdaya hasil tinggi dan berumur sangat genjah. Bahan tanaman yang digunakan untuk kultur antera adalah malai dari tanaman F1 hasil persilangan Fatmawati/Kinamase, Inpari 1/Kinamase, Fatmawati/Waseaikoku, Inpari 1/Waseaikoku, Fatmawati/IR71146, Inpari 1/ IR71146, OM4495/Silugonggo, IR7146/Dodokan, dan IR71730/OM1490. Media kultur antera adalah N6 + NAA 2,0 mg/l + kinetin 0,5 mg/l untuk media induksi kalus, MS+ NAA 0,5 mg/l + kinetin 2,0 mg/l untuk media regenerasi, dan MS + 0,5 mg/l IBA untuk media perakaran. Putresine 10-3 M ditambahkan pada media induksi kalus dan regenerasi. Hasil penelitian menunjukkan bahwa kalus yang menghasilkan tanaman hijau (KMTH) berkisar antara 0,25-83,33%. Persilangan Fatmawati/ Kinamase memberikan KMTH tertinggi (245 kalus), diikuti oleh Inpari 1/Kinamase (78 kalus) dan Fatmawati/ Waseaikoku (68 kalus). Total tanaman hijau yang diperoleh adalah 2.038 planlet dihaploid, namun diperoleh 507 tanaman setelah planlet diaklimatisasi dan tanaman ditumbuhkan di rumah kaca. Evaluasi terhadap 100 DH dilakukan di lahan petani Ciranjang, Cianjur. Berdasarkan hari berbunga 50% (65 hari setelah semai), diperoleh 33 galur yang termasuk kategori sangat genjah (dipanen +100 hari). Galur-galur tersebut adalah 18 galur dari persilangan Fatmawati/Kinamase, 5 galur dari persilangan Inpari 1/Kinamase, 8 galur dari persilangan Fatmawati/ Waseaikoku, dan 2 galur dari persilangan Inpari 1/ Waseaikoku.</p></jats:p
Construction of the CRISPR/Cas9 module and genetic transformation of aromatic rice cv. Mentik Wangi for developing bacterial leaf blight resistance
No full text. Rifhani NF, Apriana A, Sisharmini A, Santoso TJ, Trijatmiko KR, Slamet-Loedin IH, Yunus A. 2023. Construction of the CRISPR/Cas9 module and genetic transformation of aromatic rice cv. Mentik Wangi for developing bacterial leaf blight resistance. Biodiversitas 24: 3258-3268.Rice cv. Mentik Wangi is a local aromatic rice that is susceptible to pests and diseases, such as bacterial leaf blight (BLB) caused by Xanthomonas oryzae(Xoo). This bacteria can cause damage to plants thereby reducing crop yields. This study aimed to obtain the CRISPR/Cas9 module construct as well as introducing the construct into rice cv. Mentik Wangi for developing BLB resistance. The manufacture of the CRISPR/Cas9 module was carried out using the Golden Gate method, and the introduction of that construct into rice cv. Mentik Wangi was carried out using Agrobacterium tumefaciens. Constructing a CRISPR/Cas9 module with multiple gRNA of OsSWEET11 and OsSWEET14 genes has succeeded, 129 putative transformant lines of T0 generation produced with the regeneration and transformation efficiency values ??are respectively 9.4% and 9.8%. The results showed that 36 lines were positive for the hpt gene, indicating that the CRISPR/Cas9-gRNAOsSWEET module construct was successfully entered into rice cv. Mentik Wangi. Further analysis is needed to identify mutagenesis in the target gene region of TI generation transgenic plants as well as phenotypic tests for BLB resistance
Pengembangan Populasi Mutan Penanda Aktivasi: I. Transformasi Padi Japonica Tropis Lokal Sulawesi cv. Asemandi dengan bantuan Agrobacterium tumefaciens
No full text<p>The rice transformation technology<br />is not only provides valuable methods for the introduction<br />of useful genes into rice plant to improve important<br />agronomic traits, but also helps in studying gene function<br />and regulation based on rice genome sequence information.<br />Knockout of genes by insertional mutagenesis is a straightforward<br />method to identify gene functions. One of the<br />methods to develop rice mutants is through genetic transformation<br />mediated by Agrobacterium using activation<br />tagging by Ac-Ds system. A study was done with an objective<br />to obtain mutant rice of local tropical japonica cv. Asemandi<br />through genetic trans-formation mediated by Agrobacterium<br />tumefaciens. The transformation was conducted using<br />Agrobacterium vector with the strain of Agl-1 containing<br />activation tag construct. The result of experiment showed<br />that it has been obtained 17 independent line (304 plants)<br />transgenic Asemandi containing activation tag construct.<br />These starter lines will be used as materials to develop<br />several generations of stabil rice mutant through selfing.</p></jats:p
Identifikasi Galur dan Gen-gen Terkait Toleran Kekeringan pada Padi Transgenik cv. T309 yang Mengandung Vektor Penanda Aktivasi
No full text<p>Activation tagging is an efficient tool for<br />functional analysis of the rice genes. We have developed a<br />number of transgenic rice lines (Oryza sativa L. ssp.<br />japonica cv. Taipei 309) containing activation tagging vector.<br />However, the phenotypes and genotypes of these lines, in<br />relation to the drought stress, have not been analyzed. The<br />objectives of this research were to identify transgenic rice<br />lines that showed tolerance to the drought stress and to<br />identify the genes that may be associated with the drought<br />stress. The drought stress tolerance in transgenic rice lines<br />was identified by testing their tolerance to the drought stress<br />and also by detecting the presence of bar and nptII genes.<br />The result showed that 56 out of 59 rice lines were resistant<br />to Basta herbicide and three of them showed tolerance to<br />drought stress, namely PA.T-1.2, PA.T-4.1, and PA.T-5.1 lines.<br />PCR analysis showed that PA.T-1.2 and PA.T-4.1 contained<br />both hptII and bar genes, while the PA.T-5.1 line contained<br />bar gene only. Thermal Asymetric Interlaced-PCR (TAILPCR)<br />analysis showed that two genes may be asssociated<br />with the drought stress tolerance. Those genes are<br />OSJNBa0004120.14 that produces uridylate putative kinase<br />and OsPPCK2L that produces phosphoenolpyruvate<br />carboxylase kinase.</p></jats:p
Optimasi Konsentrasi Asetosiringon dan Higromisin dalam Transformasi Genetik Padi Fatmawati dengan Perantaraan Agrobacterium tumefaciens
Get PDFProtocols for genetic transformation of rice have been widely developed, however the protocols are not universal and inapplicable for all types of rice plants directly. Transformation protocol on rice cv. Fatmawati needs to be developed to generate transgenic lines. The present research was carried out to optimize genetic transformation protocol in rice cv. Fatmawati mediated by Agrobacterium tumefaciens harboring pCambia1301 construct using immature embryo as an explant. The experiment was arranged in a completely randomized design. Factors influencing efficiency of transformation, i.e., sensitivity of callus to hygromycin antibiotic, acetosyringone concentration used in cultivation medium, hygromycin concentration for transformant selection were optimized. The results showed that genetic transformation of rice cv. Fatmawati mediated by A. tumefaciens using immature embryos have been successfully carried out with several parameters. Addition of 100 µM acetosyringone in co-cultivation medium and 30 mg L-1 hygromycin for transformant callus selection were optimal for genetic transformation of rice cv. Fatmawati mediated by A. tumefaciens. Transformation efficiency was found to be 7.84% based on the lines carrying the hpt gene. This result would be a valuable reference in genetic transformation of rice cv. Fatmawati using target genes.Keywords: immature embryo, Oryza sativa, pCambia1301, transformation efficienc
Strategy to select the CRISPR/Cas9 target sequences of the
No full textBacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. Allii, is a newly identified pathogen that infects shallots. This disease can lead to yield losses ranging from 20% to 100%. The genome editing system via CRISPR/Cas-9 is a technology that can be used to modify susceptibility genes accurately and precisely. The target gene for CRISPR/Cas-9 genome editing system to develop a BLB-resistant shallot is the sucrose-phosphate synthase (SPS). One of the critical factors in the CRSPR/Cas-9 genome editing includes the preparation of single guide RNA (sgRNA) design and construction of it into an expression plasmid vector. The study aimed to develop strategies for selecting the SPS gene’s CRISPR/Cas9 target sequences to assemble an efficient gRNA spacer in shallot. The gRNA design of the SPS gene was carried out using the software at http://crispor.tefor.net/. The efficiency of sgRNAs is then filtered and predicted based on GC contents, the last four nucleotides, secondary RNA structure, and stem loops. The construction of the CRISPR/Cas9 module was carried out using the Golden Gate method. The results showed that based on the selection of target gRNA sequences in the SPS gene, one of the best gRNAs, gRNA-54/forw, has been produced for use in SPS gene editing research. The best-selected gRNA of the SPS gene was then successfully inserted into the CRISPR/Cas9 pRGEB32 cassette vector after verification using DNA sequencing analysis. Based on this result, the pRGEB32_gRNA-AcSPS construct is ready to be introduced into shallot to develop a BLB-resistant shallot variety
Pengembangan Populasi Mutan Penanda Aktivasi: I. Transformasi Padi Japonica Tropis Lokal Sulawesi CV. Asemandi Dengan Bantuan Agrobacterium Tumefaciens
Get PDFThe rice transformation technologyis not only provides valuable methods for the introductionof useful genes into rice plant to improve importantagronomic traits, but also helps in studying gene functionand regulation based on rice genome sequence information.Knockout of genes by insertional mutagenesis is a straightforwardmethod to identify gene functions. One of themethods to develop rice mutants is through genetic transformationmediated by Agrobacterium using activationtagging by Ac-Ds system. A study was done with an objectiveto obtain mutant rice of local tropical japonica cv. Asemandithrough genetic trans-formation mediated by Agrobacteriumtumefaciens. The transformation was conducted usingAgrobacterium vector with the strain of Agl-1 containingactivation tag construct. The result of experiment showedthat it has been obtained 17 independent line (304 plants)transgenic Asemandi containing activation tag construct.These starter lines will be used as materials to developseveral generations of stabil rice mutant through selfing
