Browsing by Author "Sisharmini ...[at al], Atmitri"

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    Analisis Molekuler Integrasi Gen PinII pada Ubi Jalar
    (Balai Penelitian Bioteknologi dan Sumberdaya Genetik Pertanian, 2003-12) Sisharmini ...[at al], Atmitri; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
    Konfirmasi awal keberadaan atau integrasi gen-gen target pada tanaman putatif transgenik akan sangat berguna, karena tanaman yang tidak mengandung gen yang diintroduksi dapat langsung diketahui, sehingga hanya tanaman yang mempunyai gen-gen yang diinginkan yang digunakan untuk penelitian selanjut-nya. Identifikasi terjadinya integrasi gen pada tanaman transgenik dapat dilaku-kan dengan teknik PCR (Polymerase Chain Reaction). Untuk dapat mendeteksi ada tidaknya gen target yang tersisip dalam genom tanaman ubi jalar transgenik dengan teknik PCR, diperlukan DNA terekstrak dengan kualitas dan kuantitas yang baik. Pada tahun 2002 telah dihasilkan 26 tanaman ubi jalar putatif transgenik yang diduga mengandung gen pinII dan 9 tanaman dengan gen CP-SPFMV, yang akan dianalisis secara molekuler untuk mengkonfirmasi terjadi-nya integrasi gen pinII atau CP-SPFMV dalam genom ubi jalar. DNA diisolasi dari daun ubi jalar putatif transgenik menggunakan metode CTAB dari Tanaka dan Nakatani (2001). DNA ubi jalar yang dihasilkan mempunyai kemurnian 1,4-2,26 dengan konsentrasi 0,009-2,5 g/l. Analisis PCR dari 35 tanaman putatif transgenik menunjukkan bahwa tidak ada tanaman yang positif mengandung gen pinII maupun CP-SPFMV.
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    Construction and Introduction of OsAER1::LeAlaAT Cassette to Improve the Nitrogen Use Efficiency in Rice cv. Mekongga
    (Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian, 2022-01-19) Sisharmini ...[at al], Atmitri; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian
    Abstract. Nitrogen use efficiency (NUE) in rice can be improved by developing a variety that absorbs and use nitrogen (N) more efficiently. We have successfully isolated the Lycopersicon esculentum alanine aminotransferase (LeAlaAT) gene from tomato, which is responsible for improved efficiency of N use in plants, and Oryza sativa alkenal reductase (OsAER1) promoter from rice cv. Awan Kuning. These gene and promoter can be used to develop NUE rice through the genetic engineering approach. The purposes of this study were to construct an OsAER1::LeAlaAT expression plasmid cassette, to introduce the constructed plasmid cassette into genome of rice cv. Mekongga, and to test the transgenic rice lines at several levels of N concentration. The cassette was constructed by replacing the β-glucuronidase (GUS) gene derived from the pCAMBIA1300_OsAER1::GUS cassette with LeAlaAT gene. The recombinant plasmid was then transformed into Agrobacterium tumefaciens vector strain LBA4404 and introduced into rice cv. Mekongga. The plant transformation technique was carried out at with immature embryos as explants. Molecular analysis was performed using PCR to evaluate the presence of targeted gene construct introgression in the rice lines and the efficacy test of NUE was carried out hydroponically to determine the performance of lines at several levels of N concentrations. The results showed that the OsAER1 promoter and LeAlaAT gene were successfully constructed in pCAMBIA1300 vectors and introduced to Agrobacterium vector. Rice transformation cv. Mekongga resulted in 50 transgenic rice lines (T0). Hydroponic rapid screening of NUE in 50 T1 transgenic rice lines using Yoshida solution with ¼ strength of N, resulted in five selected rice lines based on superior biomass dry weight compared to control, namely M40, M41, M45, M50, and M54 lines. The T1 generation of rice lines was tested hydroponically to evaluate the response and plant performance at several N concentrations treatment. Compared to the control, the transgenic lines showed the best performance at ¼ strength of N concentration. Further NUE testing is required to determine the performance of selected rice lines in greenhouses and confined field trials.
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    Respon Genotipe Padi Indonesia terhadap Efisiensi Regenerasi dan Transformasi Genetik melalui Agrobacterium tumefaciens
    (KOMISI NASIONAL SUMBER DAYA GENETIK, 2021-09-15) Sisharmini ...[at al], Atmitri; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
    Biotechnology-based rice breeding, especially genetic engineering, can support the assembly program or improvement of high-yielding varieties. However, controlling the plant regeneration and transformation system is an essential step in the rice genetic engineering process. In addition, the genetic background of rice for genetic engineering also plays a vital role in the efficiency of plant regeneration and transformation. Previous research has shown that Nitrite reductase (NiR) and Glucose dehygrogenase (Gluc) genes affect plant regeneration ability. The purpose of this study was to determine the response of Indonesian rice genotypes to genetic transformation through Agrobacterium tumefaciens and to determine the expression profile of NiR and Gluc genes. The research material used several genotypes of Indonesian rice, namely Fatmawati, Inpari-6, Situ Patenggang, Situ Bagendit, Sarinah, Mekongga, Inpari-32 and Nipponbare varieties as controls. The method used is transformation through Agrobacterium tumefaciens using immature embryos as explants. The results showed that the Nipponbare model rice variety had a transformation efficiency of 53.1%. Among the tested genotypes, Fatmawati and Situ Patenggang were the most efficiently transformed genotypes with an efficiency of 19.5%. At the same time, the genotype of rice that has the lowest transformation efficiency is Inpari-32 at 0.8%. The NiR gene expression profile in the tested rice genotypes showed the same expression level between genotypes, except that in Nipponbare there was an amplicon associated with a high regeneration response. Meanwhile, the Gluc gene expression profile showed different expression levels between genotypes, but it was not related to its regeneration ability. This information is helpful as a reference and consideration in determining the genotype to be used for rice genetic engineering research in Indonesia.