Induksi Kalus Dan Regenerasi Beberapa Genotipe Gandum (Triticum Aestivum L.) Secara in Vitro

Callus Induction and In Vitro Plant Regeneration ofWheat Genotypes (Triticum aestivum L.). AtmitriSisharmini, Aniversari Apriana, and Sustiprijatno. Developmentof a reliable in vitro plant regeneration procedure forwheat is a prerequisite for its improvement by genetic transformation.The purpose of this study was to obtain methodsof callus induction and regeneration of wheat genotypes.This experiment was conducted at ICABIOGRAD. Immatureembryos from four wheat genotypes, ie Perdix, Naxos Wew,Combi and Fasan were used to induce callus formation andregeneration rate of callus. For the preparation of callusinduction medium, MS-L7 basal medium was supplementedwith combination of growth regulators 2,4 dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid(picloram). While, plant regeneration medium was preparedusing MS basal medium supplemented with combination ofthree growth regulators i.e. IAA, BAP and kinetin. The resultsshowed that genotype, in vitro culture medium and growthregulators played a dominant role in callus induction andplantlet regeneration. All the 4 genotypes responded positivelyto callus induction, however, variability was observednot only among the genotypes but also within callusinduction medium used. The best induction medium wasthe MS-L7 basal medium supplemented with combination ofphytohormon 4 mg/l 2,4-D + 2 mg/l picloram (GIK-3) whichshowed 100% callus induction frequency. Whereas, the bestregeneration medium was shown by MS basal medium withcombination of phytohormon 1.5 mg/l BAP dan 0.5 mg/lkinetin (RG3). Regarding plant regeneration, Perdix was themost responsive genotype to be regenerated with regenerationfrequency of 57.33%. The successfully acclimatizedplanlets in greenhouse were obtained from Perdix andNaxos Wew genotypes. These results will potentially facilitategenetic transformation research of wheat in Indonesia

Optimasi Sistem Regenerasi Dan Transformasi Padi Varietas Elit Indonesia

New ricevariety can be generated by means of transgenic approach.Transgenic rice researches have been conducted in manyinstitutions worldwide using Japonica, Indica, and Javanicavarieties. The most cultivated rice in Indonesia is Indica.Indica type is known to have low responsive tissues inculture and transformation media when compared toJaponica rice. This research activity is aimed to optimizeregeneration and transformation systems of the Indonesianelite rice varieties, so that good method can be achieved tobe used in the generation of transgenic elite rice varieties ofIndica type. The research consisted of two activities:regeneration and transformation optimizations in varieties ofDodokan (upland rice) and Inpari 6 (irrigated rice).Immature embryo was used as the explant in this research.The optimization studies used 2 types of media, NBH (N6salts and vitamins, cassamino acid 0.5 g/l, L-proline 0.5 g/l,sucrose 20 g/l, D-glucose 10 g/l, 2.4-D 2 mg/l, NAA 1 mg/l, BA1 mg/l, agarose Type I 5.5 g/l) and NBH-M (N6 macro salts,B5 micro salts, and vitamins, 0.3 g/l cassamino acid, 3 g/l Lproline,20 g/l sucrose, 3 mg/l 2.4-D, 1 mg/l NAA, 1 mg/l BAP,5.5 agarose Type I), 2 types of regeneration media, R1 (MSbase media and vitamis, 0.3 g/l glutamine, 30 g/l sucrose, 2mg/l kinetin, 1 mg/l NAA, 3 g/l phytagel) and R2 (MS basemedia and vitamins, 2 g/l cassamino acid, 20 g/l sucrose, 30mg/l sorbitol, 2.5 mg/l kinetin, 0.25 mg/l NAA, 3 g/l phytagel).Optimization transformation of Indonesian elite rice varietiesused developed an empty plasmid pCAMBIA 1301 containinghpt gene. The transformation was conducted using twotypes of co-cultivation media, K1 (N6 macro salts, B5 microsalts, and vitamins, 0.5 g/l cassamino acid, 0.5 g/l L-proline,20 g/l sucrose, 10 g/l glucose, 2 mg/l 2.4-D, 1 mg/l NAA, 1mg/l BAP, 0.1 mM acetosyringone) and K2 (N6 macro salts,B5 micro salts, and vitamins, 0.5 g/l cassamino acid, 0.5 g/l Lproline,20 g/l sucrose, 10 g/l glucose, 2 mg/l 2.4-D, 1 mg/lNAA, 1 mg/l BAP, 0.2 mM acetosyringone). The resultsshowed that Inpari 6 could form embryonic calli in NBHmedia and further regenerated well in R1 media (13.8%).The co-cultivation media K1 generated more selected calliwhich then generated green plant of young embryocompared to K2. Inpari 6 showed higher regeneration ratesafter transformation (3.6%) compared to Dodokan (0%).Molecular analysis showed that all 11 transformants (Inpari6) tested contained the hpt gene. These results are expectedto support the development of transgenic Indica ricegeneration in Indonesia

Regenerasi Empat Genotipe Gandum (Triticum Aestivum L.) Pasca Transformasi Melalui Agrobacterium Tumefaciens

Genetic transformation ofwheat mediated by Agrobacterium tumefaciens has notbeen established yet. This research aimed to obtain themost responsive wheat genotype to be transformed, toselect the most effective combination of growth regulator forregenerating transforman calli, and to determine thetransformation efficiency. Transformation mediated by A.tumefaciens was performed on four genotypes of wheat,namely Combi, Fasan, Perdix, and Naxos-Wew.Transformed calli with green spots in selection media weretransferred to regeneration media containing 25 mg/lhygromycin, i.e. R1H25 and R2H25. The obtained plantletswere analyzed by PCR using specific primers for hygromycinphosphotransferase gene. The results showed that Fasanwas the most responsive genotype in callus formation(95.47%) and regeneration both in R1H25 (27%) and R2H25(28.6%) media. Fourteen plantlets were succesfullyacclimatized and PCR analysis showed that there were fourpositive transformants containing the hpt gene. The resultsare expected to provide information on the development oftransgenic wheat in Indonesia, specifically in the success ofcallus formation and regeneration of wheat aftertransformation using A. tumefaciens

Pengembangan Populasi Mutan Penanda Aktivasi: I. Transformasi Padi Japonica Tropis Lokal Sulawesi CV. Asemandi Dengan Bantuan Agrobacterium Tumefaciens

The 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

Respon Padi Transgenik CV. Nipponbare Generasi T1 Yang Mengandung Gen Oryza Sativa Dehydration-response Element Binding 1a (Osdreb1a) Terhadap Cekaman Salinitas [Response of T1 Generation Transgenic Rice CV. Nipponbare Containing an Oryza Sativa Dehydration-response Element Binding 1a (Osdreb1a) Gene to Salinity Stress]

Salinity is one of the abiotic constraints in the cultivation of rice crop. One of the reasons agricultural land becomes saline is due to the intrusion of seawater into the mainland as a result of global climate change. Dehydration-responsive element binding (DREB) gene is a plant -specific transcription factor gene that have important role in regulating plant responses to abiotic stresses, including high salinity. Transgenic rice plants cv. Nipponbare carrying OsDREB1A gene have been generated. However, study of the response of putative transgenic plants to salinity has not been done. The research objective is to study the response of T1 generation Nipponbare-OsDREB1A transgenic rice plants to salinity stress. The result showed that the response of putative transgenic rice Nipponbare-OsDREB1A to salinity stress 25 mM and 150 mM NaCl indicated a level of tolerance varies from highly sensitive to highly tolerance. These variations were possibly occurred because of the segregation state of the T1 generation transgenic rice. Based on damage symptom scoring and PCR analysis provided information that transgenic rice plant cv. Nipponbare-OsDREB1A which showed positive PCR had a very high tolerance to salinity stress 150 mM compared with non-transgenic rice cv. Nipponbare

Construction and Transformation of OsERA1 Gene Into Expression Vector and Response of Nipponbare-OsERA1 Transgenic Rice to Drought Stress

Drought stress is a major constrain which could influence rice productivity. Enhanced Response to ABA1 (ERA1) gene encoding a β-subunit farnesyltransferase enzyme plays a role to control sensitivity of the guard cells to abscisic acid (ABA), hence regulating drought stress response in plant species including rice. This study aimed to clone the OsERA1 gene into expression vector, introduce it into rice plant, and confirm the positive OsERA1-rice plants conferring drought tolerance. This study was initiated by isolation of the OsERA1 gene from rice cDNAs and cloned it to an expression vector cassette, pCAMBIA1301. The cassette harboring OsERA1 gene was introduced into rice plant cv. Nipponbare mediated by Agrobacterium tumefaciens strain LBA4404.Putative transgenic lines were detected using PCR and Southern blot analyses to confirm the inserted transgene and the positive lines were assayed their tolerance to drought. The OsERA1 gene was successfully isolated and constructed into expression vector to generate pCAMBIA1301-OsERA1. Introduction of the gene into Nipponbare has produced nine putative transgenic rice lines, of which, six lines harbored OsERA1 gene. Southern blot analysis of sixteen T2 plants from two PCR-positive transgenic lines revealed1–3 copies of transgene were integrated into rice genome of transgenic lines. Five transgenic lines of Nipponbare-OsERA1 showed better response to drought at vegetative phase compared to control in term of recovery ability. At generative phase, the five transgenic lines yielded less unfilled grains compared to control. Overall, the transgenic lines obtained from this study could bepotential candidates for developing rice varieties tolerant to drought

Keragaan Sifat Tahan Penyakit Blas dan Agronomi Populasi Silang Balik dan Haploid Ganda Turunan IR64 dan Oryza Rufipogon

Perakitan varietas tahan blas sebagai galur harapan, merupakan salah satu prioritas dalam program pemuliaan padi. Dalam rangka mendukung program tersebut, telah dilakukan pembentukan populasi haploid ganda (HG) dan silang Balik (BC) dengan IR64 sebagai tetua berulang dan Oryza rufipogon (No. aksesi IRGC 105491) sebagai tetua donor gen tahan penyakit blas. Penelitian ini bertujuan menganalisis keragaan tingkat ketahanan galur-galur haploid ganda (HG_I, HG_II, dan HG_III) dan galur-galur silang Balik (BC2, BC3, dan BC5) terhadap penyakit blas di rumah kaca dan lapang, sehingga diperoleh kandidat galur harapan. Hasil pengujian beberapa populasi HG dan BC menunjukan bahwa terdapat variasi keragaan yang berbeda-beda. Variasi paling kecil terdapat pada populasi HG_III. Hasil yang sama juga diperoleh pada populasi silang Balik (BC2-BC5). Variasi paling kecil terdapat pada populasi BC5. Bila dibandingkan antar populasi HG dan BC, tingkat variasi pada populasi HG_III lebih kecil dibandingkan dengan tingkat variasi pada populasi BC5. Hal ini menunjukkan bahwa tingkat homosigositas paling tinggi terdapat pada populasi HG_III. Berdasarkan evaluasi penampilan agronomis beberapa galur HG_III terpilih, diperoleh tiga galur kandidat galur harapan Bio1, Bio2, dan Bio8

Rice Anther Culture to Develop Double Haploid Population and Blast Resistant Lines

Penyakit blas pada padi yang disebabkanoleh cendawan Pyricularia grisea, merupakan salah satukendala dalam produksi beras. Sumber gen ketahanan terhadappenyakit blas dijumpai pada spesies padi liar Oryzarufipogon. Populasi silang ganda (BC2F3) turunan IR64 danO. rufipogon mempunyai QTL untuk sifat ketahanan terhadappenyakit blas. Untuk mempercepat perolehan tanamanhomosigot dari populasi tersebut, dilakukan kultur anterpada dua media induksi kalus: I1 (N6 + NAA 2 mg/l + kinetin0,5 mg/l + sukrosa 60 g/l + putresin 0,16 g/l) dan I2 (N6 +2,4-D 2 mg/l + sukrosa 50 g/l) dan dua media regenerasi: R1(MS + NAA 0,5 mg/l + kinetin 2 mg/l + sukrosa 40 g/l +putresin 0,16 g/l) dan R2 (MS + NAA 1 mg/l + kinetin 2 mg/l+ sukrosa 30 g/l). Kultur anter dilakukan pada sembilan genotipe,di mana tiga genotipe (149-16, 343, 337-13) memberikanrespon terbaik dalam produksi planlet hijau setelahdikulturkan pada media regenerasi R1. Dari 208 planlet hasilregenerasi diperoleh 42 planlet haploid ganda dari genotipe149-16, 11 planlet haploid ganda dari genotipe 343, dan 44planlet haploid ganda dari genotipe 337-13. Skrining ketahananblas di rumah kaca pada populasi haploid gandamenghasilkan 46 tanaman tahan terhadap ras 001, 33 tanamantahan terhadap ras 033, dan 79 tanaman tahan terhadapras 173. Sebanyak 28 tanaman bersifat tahan, baik terhadapras 001, 033, maupun 173 seperti halnya O. rufipogon.Galur-galur homosigot ini akan diuji di lapang untuk ketahanannyaterhadap penyakit blas dan karakter agronominya