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

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

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

Introduksi Konstruk Over-Ekspresi Kandidat Gen OsWRKY76 Melalui Agrobacterium Tumefaciens Pada Tanaman Padi Nipponbare

Delivering of Over-Expression Construct OsWRKY76Candidate Gene in Rice cv. Nipponbare throughAgrobacterium tumefaciens. Aniversari Apriana, AtmitriSisharmini, Wening Enggarini, Sudarsono, Nurul.Khumaida, and Kurniawan R. Trijatmiko. Plant geneticimprovement can be done through classical breeding orgenetic engineering. WRKY is a transcription factor involvedin regulating plant defense responses. OsWRKY76 gene islocated in a narrow segment of chromosome 9 which isidentified previously to be related to wide spectrumresistance in rice. A sequence of OsWRKY76 (+1.200 bp)has available in the gene bank and it makes possible toisolate, clone, and construct the gene into over-expressionvector. The aim of this research was to assemble an overexpressionconstruct of OsWRKY76 candidate gene andintroduce it into rice through Agrobacterium-mediatedtransformation. A construct of pCAMBIA-1301::35S::OsWRKY76 has been successfully assembled andtransformed into embryogenic calli of rice cv. Nipponbareusing A. tumefaciens strain Agl-1 and EHA 105. A number of126 independent lines has been produced, in which Agl-1showed 3.8 times more efficient than EHA 105. PCR analysisof randomly selected 25 independent lines showed that allof them positively contained hptII gene, a selectable markerused in the over-expression construct of the OsWRKY76candidate gene. Based on the result, it could be concludedthat the over-expression construct of OsWRKY76 candidategene have been successfully introduced into the tissue ofNipponbare

Pembentukan Genotipe Padi Berumur Sangat Genjah Melalui Kultur Antera

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