BackgroundRice can absorb less than 40% of applied nitrogen fertilizer, whereas the unabsorbed nitrogen fertilizer may cause environmental problems, such as algal blooms in freshwater and increased production of nitrous oxide, a greenhouse gas which is 300 times more potent than carbon dioxide. Development of nitrogen use efficient (NUE) rice is essential for more environmentally friendly rice production. Recently, NUE rice has been developed by root-specific expression of alanine aminotransferase (AlaAT) gene from barley, a monocot plant. Therefore, we tested the efficacy of AlaAT gene from cucumber in transgenic rice, aiming to provide evidence for the conservation of AlaAT gene function in monocot and dicot.ResultsAlaAT gene from cucumber (CsAlaAT2) has been successfully cloned and constructed on pCAMBIA1300 plant expression vectors under the control of tissue-specific promoter OsAnt1. Agrobacterium tumefaciens-mediated transformation of Indonesian rice cv. Fatmawati using this construct produced 14 transgenic events. Pre-screening of T1 seedlings grown in the agar medium containing low nitrogen concentration identified selected events that were superior in the root dry weight. Southern hybridization confirmed the integration of T-DNA in the selected event genomes, each of them carried 1, 2, or 3 T-DNA insertions. Efficacy assay of three lead events in the greenhouse showed that in general transgenic events had increased biomass, tiller number, nitrogen content, and grain yield compared to WT. One event, i.e., FAM13, showed an increase in yield as much as 27.9% and higher plant biomass as much as 27.4% compared to WT under the low nitrogen condition. The lead events also showed higher absorption NUE, agronomical NUE, and grain NUE as compared to WT under the low nitrogen condition.ConclusionsThe results of this study showed that root-specific expression of cucumber alanine aminotransferase2 gene improved nitrogen use efficiency in transgenic rice, which indicate the conservation of function of this gene in monocot and dicot.

Delivering of Over-Expression Construct OsWRKY76Candidate Gene in Rice cv. Nipponbare through Agrobacterium tumefaciens. Aniversari Apriana, Atmitri Sisharmini, Wening Enggarini, Sudarsono, Nurul. Khumaida, and Kurniawan R. Trijatmiko. Plant genetic improvement can be done through classical breeding or genetic engineering. WRKY is a transcription factor involved in regulating plant defense responses. OsWRKY76 gene is located in a narrow segment of chromosome 9 which is identified previously to be related to wide spectrum resistance in rice. A sequence of OsWRKY76 (+1.200 bp) has available in the gene bank and it makes possible to isolate, clone, and construct the gene into over-expression vector. The aim of this research was to assemble an overexpression construct of OsWRKY76 candidate gene and introduce it into rice through Agrobacterium-mediated transformation.A construct of pCAMBIA-1301::35S::OsWRKY76 has been successfully assembled and transformed into embryogenic calli of rice cv. Nipponbare using A. tumefaciens strain Agl-1 and EHA 105. A number of 126 independent lines has been produced, in which Agl-1 showed 3.8 times more efficient than EHA 105. PCR analysis of randomly selected 25 independent lines showed that all of them positively contained hptII gene, a selectable marker used in the over-expression construct of the OsWRKY76 candidate gene. Based on the result, it could be concluded that the over-expression construct of OsWRKY76 candidate gene have been successfully introduced into the tissue of Nipponbare.

Development of Activation Tagging Mutants Population: I. Agrobacterium-mediated Transform-ation of Tropical Japonica Rice of Local Sulawesi cv. Asemandi. Atmitri Sisharmini, Aniversari Apriana, Wening Enggarini, dan Kurniawan R. Trijatmiko. The rice transformation technology is not only provides valuable methods for the introduction of useful genes into rice plant to improve important agronomic traits, but also helps in studying gene function and regulation based on rice genome sequence information. Knockout of genes by insertional mutagenesis is a straightforward method to identify gene functions. One of the methods to develop rice mutants is through genetic transformation mediated by Agrobacterium using activation tagging by Ac-Ds system. A study was done with an objective to obtain mutant rice of local tropical japonica cv. Asemandi through genetic trans-formation mediated by Agrobacterium tumefaciens. The transformation was conducted using Agrobacterium vector with the strain of Agl-1 containing activation tag construct. The result of experiment showed that it has been obtained 17 independent line (304 plants) transgenic Asemandi containing activation tag construct. These starter lines will be used as materials to develop several generations of stabil rice mutant through selfing. PENDAHULUANPadi (Oryza sativa L.) adalah salah satu tanaman budi daya penting bagi masyarakat Indonesia dan merupakan sumber nutrisi utama bagi 40% populasi dunia (Hiei dan Komari 2006). Untuk tujuan penelitian biologi molekuler, padi juga merupakan salah satu tanaman model yang ideal untuk penelitian genomik. Hal ini disebabkan karena padi mempunyai ukuran genom yang relatif kecil dan informasi molekulernya telah dikarakterisasi dengan baik. Di samping itu, dalam penelitian rekayasa genetik, padi merupakan tanaman yang relatif efisien untuk transformasi genetik (Hirochika et al. 2004). Transformasi genetik dengan bantuan vektor Agrobacterium secara rutin telah digunakan pada beberapa tanaman monokotil, termasuk padi. Transformasi genetik pada padi, di samping meHak Cipta © 2009, BB-Biogen nyediakan teknologi dan metode yang efisien untuk mengintroduksikan gen-gen dengan sifat-sifat agronomi penting, tetapi juga dapat digunakan untuk mempelajari fungsi dan regulasi gen berdasarkan informasi sekuen genom padi (Roy et al. 2000). Pendekatan knockout gen atau transposon mutagenesis (pembuatan mutan berdasarkan transposon) dapat digunakan untuk mengungkap fungsi dari gen-gen penting pada padi (Hirochika et al. 2004).Sistem transposon mutagenesis telah dikembangkan pada tanaman padi dengan tujuan untuk mengetahui fungsi gen dengan menggunakan strategi forward dan reverse genetics. Pendekatan forward genetics mempelajari fungsi gen yang bergerak dari suatu mutan menuju ke sekuen gen (Bouchez dan Hofte 1998). Jadi pada pendekatan ini diawali dengan menguji perbedaan fenotipe dari mutan-mutan dibandingkan dengan tipe liar, setelah itu diidentifikasi sekuen gen yang bertanggung jawab terhadap perbedaan tersebut. Termasuk dalam pendek...

Moeljopawiro. Penyakit blas pada padi yang disebabkan oleh cendawan Pyricularia grisea, merupakan salah satu kendala dalam produksi beras. Sumber gen ketahanan terhadap penyakit blas dijumpai pada spesies padi liar Oryza rufipogon. Populasi silang ganda (BC 2 F 3 ) turunan IR64 dan O. rufipogon mempunyai QTL untuk sifat ketahanan terhadap penyakit blas. Untuk mempercepat perolehan tanaman homosigot dari populasi tersebut, dilakukan kultur anter pada dua media induksi kalus: I 1 (N6 + NAA 2 mg/l + kinetin 0,5 mg/l + sukrosa 60 g/l + putresin 0,16 g/l) dan I 2 (N6 + 2,4-D 2 mg/l + sukrosa 50 g/l) dan dua media regenerasi: R 1 (MS + NAA 0,5 mg/l + kinetin 2 mg/l + sukrosa 40 g/l + putresin 0,16 g/l) dan R 2 (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) memberikan respon terbaik dalam produksi planlet hijau setelah dikulturkan pada media regenerasi R 1 . Dari 208 planlet hasil regenerasi diperoleh 42 planlet haploid ganda dari genotipe 149-16, 11 planlet haploid ganda dari genotipe 343, dan 44 planlet haploid ganda dari genotipe 337-13. Skrining ketahanan blas di rumah kaca pada populasi haploid ganda menghasilkan 46 tanaman tahan terhadap ras 001, 33 tanaman tahan terhadap ras 033, dan 79 tanaman tahan terhadap ras 173. Sebanyak 28 tanaman bersifat tahan, baik terhadap ras 001, 033, maupun 173 seperti halnya O. rufipogon. Galur-galur homosigot ini akan diuji di lapang untuk ketahanannya terhadap penyakit blas dan karakter agronominya.Kata kunci: Kultur anter, IR64, Oryza rufipogon, ketahanan blas.

Root-specific promoters are useful in plant genetic engineering, primarily to improve water and nutrient absorption. The aim of this study was to clone and characterise the promoter of the Oryza sativa L. alkenal reductase (OsAER1) gene encoding 2-alkenal reductase, an NADPH-dependent oxidoreductase. Expression analysis using quantitative real-time PCR confirmed the root-specific expression of the OsAER1 gene. Subsequently, a 3082-bp fragment of the OsAER1 promoter was isolated from a local Indonesian rice cultivar, Awan Kuning. Sequencing and further nucleotide sequence analysis of the 3082-bp promoter fragment (PA-5) revealed the presence of at least 10 root-specific cis-regulatory elements putatively responsible for OsAER1 root-specific expression. Using the 3082-bp promoter fragment to drive the expression of the GUS reporter transgene confirmed that the OsAER1 promoter is root-specific. Further, the analysis indicated that OsAER1 promoter activity was absent in leaves, petioles and shoots during sprouting, vegetative, booting and generative stages of rice development. In contrast, the promoter activity was present in anthers and aleurone layers of immature seeds 7–20 days after anthesis. Moreover, there was no promoter activity observed in the aleurone layers of mature seeds. The OsAER1 promoter activity is induced by Al-toxicity, NaCl and submergence stresses, indicating the OsAER1 promoter activity is induced by those stresses. Exogenous treatments of transgenic plants carrying the PA-5 promoter construct with abscisic acid and indoleacetic acid also induced expression of the GUS reporter transgene, indicating the role of plant growth regulators in controlling OsAER1 promoter activity. Promoter deletion analysis was conducted to identify the cis-acting elements of the promoter responsible for controlling root-specific expression. The GUS reporter gene was fused with various deletion fragments of the OsAER1 promoter and the resulting constructs were transformed in rice plants to generate transgenic plants. The results of this analysis indicated that cis-acting elements controlling root-specific expression are located between −1562 to −1026bp of the OsAER1 CDS. Here we discusses the results of the conducted analyses, the possible role of OsAER1 in rice growth and development, possible contributions and the potential usage of these findings in future plant research.

Evaluation of resistance of double haploid population of crosses between IR64 and Oryza rufipogon against Bacterial Leaf Blight (BLB) at seedling stage was conducted during dry season 2005/2006 in the screen house, at Rice Centre Research at Sukamandi. Inoculum was prepared by isolating BLB infected leaf in laboratory using Wakimoto's media. Seeds were germinated in petri dish for 48 hours, and then were sown in the plastic boxes size of 40 cm x 30 cm, each family was planted in 10 cm long row. TN1, IRBB, Code, Angke, dan O. rufipogon were used as control. Leaf inoculation of isolates of Xanthomonas oryzae pv. oryzae (XOO) ras III, IV, and VIII with concentration of 10 8 cell/ml, were applied to the plants at 18-21 day old plants by cutting method. Fertilizer application as recommended. Pest and weed control were based on necessity. Observation of disease severity was carried out after a sensitive control, TN1, was a severely affected. Observation method based on SES IRRI (1996) which are 1 for plant showed 0-3% of leaf damage, 2(4-6%), 3(7-12%), 4(13-25%), 5(26-50%), 6(51-75%), 7(7-87%), 8(88-94%), and 9 for plant with 95-100% of leaf damage. Result showed that Bio50-ACBlas/BLB03, Bio59-AC-BLB05 and Bio67-AC-BLB05 lines were resistant to phato-type III, 11 lines showed moderate resistant to phato-type IV, and Bio46-AC-Blas/BLB03, Bio47-AC-BLB05, and Bio48-AC-BLB05 lines were resistant to phato-type VIII. Apart of those, there were 2 lines, Bio38-AC-BLB05, and Bio63-AC-Blas/BLB03 showed moderately resistance to three phatotypes tested.

Blast Resistance Performance of Promising Lines Derived from Backcross and Double Haploid Population Between IR64 and Oryza rufipogon. Developing blast resistance varieties with superior agronomical performance has been the one of the important priorities in rice breeding program. Based on the purpose of this study the double haploid and backcross populations were developed using the most popular cultivar IR64 as recurrent parent and wild rice species Oryza rufipogon (Acc. IRGC 105491) as blast resistance donor parent. This study was initiated to analyze the blast resistance and agronomical performance of double haploid populations (DH_I, DH_II and DH_III) and backcross populations (BC 2 , BC 3 , and BC 5 ), based on the green house and field screening tests. The results of statistical analysis showed that the blast resistance performance of DH population were diverse among DH_I, DH_II and DH_III. The smallest diversity was on the DH_III population. The same results were also detected on BC populations. The smallest diversity was on BC 5 population. The diversity comparison between DH and BC population showed that DH_III population had smaller variation than BC 5 . Indicated that DH_III population has the most fixed population. The agronomic performance evaluation of DH_III population selected lines showed that Bio1, Bio2, and Bio8 qualitified as the candidate of promising lines. Keywords PENDAHULUANKetersedian keragaman genetik merupakan salah satu faktor penting untuk menunjang program pemuliaan padi. Spesies tanaman liar merupakan salah satu alternatif sumber keragaman genetik. Pemanfaatan spesies liar dalam program pemuliaan tanaman padi telah banyak dilakukan. Dalam rangka perakitan varietas tahan penyakit blas dengan penampilan agronomis yang sesuai harapan, telah dilakukan pembentukan populasi haploid ganda (HG) dan silang balik (BC) menggunakan tetua IR64 dan spesies padi liar Oryza rufipogon (No. aksesi IRGC 105491) sebagai tetua donor gen ketahanan terhadap penyakit blas. Spesies padi liar ini berpotensi untuk program perbaikan kultivar IR64 sebagai kultivar padi terpopuler di Asia dan Indonesia. Spesies padi liar O. rufipogon diketahui memiliki gen Pir4 yang merupakan gen ketahanan terhadap penyakit blas yang berspektrum luas .Penyakit blas (disebabkan oleh cendawan patogen Pyricularia grisea, Sacc; sinonim dengan Pyricularia oryzae Cavara) (Roosman et al., 1990) merupakan penyakit penting pada pertanaman padi.

Genetic engineering is one of the strategies for developing nitrogen (N)-use-efficient rice (Oryza sativa) varieties. One gene that plays an indirect role in N metabolism is alanine aminotransferase (AlaAT). It can efficiently increase N content and crop yield. In a previous study, the tomato AlaAT gene (LeAlaAT) was successfully isolated and introduced into ‘Mekongga’ rice. The present research was conducted during 2018 and 2019 at the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor, Indonesia. The objectives of the present study were to perform the molecular characterization of LeAlaAT ‘Mekongga’ rice lines on the basis of the hpt marker gene, the direct PCR of the LeAlaAT fragment, and the phenotypic evaluation of the selected LeAlaAT T1 ‘Mekongga’ rice lines in response to different N fertilizer rates (0 kg ha−1 [control] and 60, 90, and 120 kg ha−1). This research involved three activities, namely (1) Southern blot analysis, (2) direct PCR, and (3) N use efficiency (NUE) test of ‘Mekongga’ transgenic lines. Southern blot analysis revealed that in T0 transgenic lines, the copy number of the hpt marker gene varied from 1 to 3. Direct PCR confirmed the presence of the AlaAT fragment in the T1 generation of five ‘Mekongga’ transgenic lines. The five transgenic lines showed high panicle number, biomass weight, shoot dry weight, and total grain weight under 120 kg ha−1 nitrogen. The high agronomical NUE of transgenic lines under 120 kg ha−1 N implied that the transgenic rice lines have the potential for efficient N use at a certain minimum level of N (120 kg ha−1 of nitrogen) and should be further evaluated at high N levels.