Browsing by Author "Apriana ...[at al], Aniversari"

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    Konstruksi dan Ekspresi Transien Kaset Kimera yang Mengandung Fusi Promotor CaMV 35S atau OsAER1 dan Gen GUS pada Tembakau
    (BB Biogen, 2018) Apriana ...[at al], Aniversari; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
    Uji gen pelapor umumnya digunakan untuk mempelajari pola ekspresi gen dan aktivitas promotor. Tujuan penelitian ini adalah merakit konstruk gen kimera yang terdiri atas promotor konstitutif CaMV 35S atau promotor gen OsAER1 yang disambungkan dengan gen pelapor GUS yang menyandi enzim β-glucuronidase dan mendapatkan metode transformasi transien yang efisien pada kecambah tembakau utuh dengan bantuan Agrobacterium tumefaciens. Fragmen promotor CaMV 35S yang diisolasi dari vektor biner pCAMBIA1301 dan promotor gen OsAER1 yang diamplifikasi dari DNA padi cv. Awan Kuning masingmasing diligasikan ke pCAMBIA1300int::gus::tNOS untuk menghasilkan vektor biner pCAMBIA1300int::p35S::gus::tNOS dan pCAMBIA 1300int::prOsAER1::gus::tNOS. Kedua konstruk vektor ini digunakan untuk transformasi transien kecambah tembakau berumur 5 hari. Transformasi transien tembakau dilakukan dengan menggunakan dua kerapatan bakteri (OD600 = 0,5 dan OD600 = 1,0) yang dikombinasikan dengan perlakuan vakum selama 15 menit dan 30 menit. Kecambah tembakau yang ditransformasi dengan pCAMBIA 1300int::p35S::gus::tNOS menunjukkan efisiensi transformasi yang lebih tinggi dibanding dengan yang ditransformasi dengan pCAMBIA1300int::prOsAER1::gus::tNOS. Efisiensi transformasi yang lebih tinggi diperoleh pada transformasi menggunakan bakteri dengan kerapatan OD600 = 0,5 dikombinasikan dengan perlakuan vakum selama 30 menit. Ekspresi gen GUS pada kecambah tembakau yang ditransformasi dengan konstruk promotor CaMV 35S diamati di seluruh organ kecambah tembakau (akar, hipokotil, kotiledon, dan daun), sedangkan ekspresi gen GUS pada kecambah tembakau yang ditransformasi dengan konstruk promotor gen OsAER1 terjadi di bagian akar, hipokotil, dan kotiledon, tetapi tidak di daun. Hasil ini menunjukkan bahwa transformasi transien merupakan metode yang cepat dan sederhana untuk pengujian konstruk gen kimera.
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    Observasi Fenotipik dan Stabilitas Genetik Mutasi Gen GA20ox-2 pada Padi Mutan CRISPR/Cas9 Turunan Inpari HDB
    (KOMISI NASIONAL SUMBER DAYA GENETIK, 2021-09-15) Apriana ...[at al], Aniversari; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
    Plant height is one of the important characters for target selection in rice plants. Rice plants that have a relatively high posture can cause lodging if blown by strong winds or heavy rain. The lodging of the rice plant can affect the yield loss. The semidwarf nature of the IR8 rice variety is caused by a mutation in the SD1 (GA20ox-2) gene that encodes the GA20 oxidoreductase-2 enzyme. A precise mutation method with genomic editing technology-CRISPR/cas9 targeted for the GA20ox-2 gene has resulted in the Kitaake (K-15) mutant rice line and has been used for crossbreeding with the medium stature elite Inpari HDB rice variety. The purpose of this study was to observe the phenotype and analyze the stability of the GA20ox-2 gene mutation in the F3 generation mutant rice lines derived from Inpari HDB. Phenotypic observations were carried out on 25 mutant rice lines, by observing the agronomic characters and yield components. Analysis of the genetic stability of the GA20ox-2 gene mutation in the mutant rice lines was carried out using the sequencing method. The results of phenotypic observations showed that the plant height of these mutant lines in general had a shorter stature than the female parents (125.73 cm), which ranged from 67.23 to 112.68 cm, and could reduce the height of up to 10,38-46,53%. In addition, mutant rice lines in general also have earlier flowering times than their female parents. Of the 25 lines, there are 6 lines that are phenotypically homogeneous and have been confirmed to carry the mutant allele of the GA20ox-2 gene. This combination of genome editing and conventional breeding techniques can be used to improve the character of plant height in rice plants.
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    Phenotypic and Genetic Stability Evaluation of the Targeted GA20ox-2 Gene Mutation in CRISPR/Cas9 Mutant Rice Derived from Mentong Cultivar
    (Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian, 2022-01-19) Apriana ...[at al], Aniversari; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian
    Abstract. Lodging is a major constraint in cereal crops which lead to reduction of yield and grain quality. Therefore, lodging tolerant varieties have led to remarkable harvest index increases in cereal crops. Gibberellin (GA) is important endogenous hormones responsible to plant height trait and its deficiency in plants may cause dwarfism. In this study, a set of semidwarf F3 mutant rice population generated from a cross between Mentong, an Indonesian local swamp rice cultivar (female parent exhibits tall plant height), and K-15, a CRISPR/Cas9-target mutant of GA20ox-2 in Kitaake (donor parent), was subjected to assessment for their phenotypic and genotypic stability. A total of 19 mutant rice lines along with parents were planted in the greenhouse and some important agronomic traits (plant height, tiller number, and heading date) were observed to assess stability of the phenotype. The genetic or hereditary stability of the mutated sites on GA20ox-2 gene was further confirmed by sequence comparison between targeted gene in mutant rice lines and their parents. Based on phenotype observation, all lines generally showed reduction in plant height (20.6–53.8%) compared to their female parent and had more tiller number (14.33–26.50) compared to their parents. Of these, three lines were phenotypically homogeneous, and were molecularly confirmed conferring the allele of the GA20ox-2 which were homozygous. Our findings suggest that development of mutant rice using genome editing technology approach combined with conventional breeding would be useful to facilitate rapid and precise crop improvement in plant breeding program.