KARAKTER FENOTIPIK DAN MOLEKULER PADI HITAM TRANSGENIK (Oryza sativa L. "Cempo Ireng") PEMBAWA GEN OsRKD4

PUTRI WIJAYANTI, Tri Rini Nuringtyas, M.Sc., Ph.D., Dr. Yekti Asih Purwestri, M.Si.

2018 | Tesis | MAGISTER BIOLOGI

Abstrak Penyisipan gen OsRKD4 untuk menginduksi pembentukan embrio somatik dengan konstruk 35S::GAL4::OsRKD4::GR pada kalus padi hitam telah berhasil dilakukan untuk mengatasi kegagalan regenerasi pada transformasi gen Hd3a. Saat ini terdapat 3 galur tanaman transgenik pembawa gen OsRKD4 generasi T0 yaitu OS1, OS2, dan OS3. Tujuan penelitian ini adalah mengetahui pola segegrasi dan ekspresi gen sisipan OsRKD4 pada padi hitam transgenik tersebut dalam menginduksi embriogenesis somatik. Pada penelitian ini, resistensi biji terhadap agen penyeleksi berupa antibiotik higromisin 20 ppm pada medium induksi kalus digunakan untuk mengetahui pola segregasi gen sisipan. Induksi ekspresi gen sisipan dilakukan dengan subkultur kalus pada medium dengan 20 ���µM/L dexamethasone. Setelah dua hari perlakuan dexamethasone 20 ���µM/L, kalus disubkultur pada dex free medium (MS0) dan dilakukan isolasi RNA untuk sintesis cDNA. Keberadaan gen sisipan diamati dengan amplifikasi serta elektroforesis gen HPT (455 bp) sedangkan kuantifikasi ekspresi gen OsRKD4 (191 bp) menggunakan Real-Time PCR. Fenotip berupa pembentukan embrio somatik diamati dengan pewarnaan menggunakan Sudan Red 7B serta preparat irisan kalus berumur 10 hari. Hasilnya, OS1 diketahui mengikuti pola segregasi 3:1 dengan nilai X2 dF 1 pada 1% sebesar 4,267 berdasarkan inokulasi yang dilakukan terhadap 500 biji padi, sedangkan gen HPT berukuran 455 bp pada terekspresi pada semua tanaman transgenik. Gen OsRKD4 berukuran 191 bp terdeteksi pada tanaman transgenik maupun nontransgenik. OS1-D mengekspresikan gen OsRKD4 paling tinggi dengan nilai 2,9 fold. Fenotip multi embrio somatik juga teramati berdasarkan pewarnaan Sudan Red 7B serta pengamatan preparat irisan.

The insertion of the OsRKD4 gene to induce somatic embryo formation with construct of 35S::GAL4::OsRKD4::GR to black rice callus has been successfully conducted to overcome the constraint of regeneration after Hd3a gene transformation. Currently there are 3 lines of transgenic plant harbouring the gene, which are OS1, OS2, and OS3. The aims of this research are to know the pattern of segregation and expression of OsRKD4 gene on the transgenic black rice I inducing somatic embryogenesis. In this study, seed resistance to hygromicine antibiotic 20 ppm on the callus induction medium was used to determine the pattern of segregation of insertion genes. Induction of the expression was performed by callus subculture on medium with 20 ���µM/L dexamethasone. After two days of dexamethasone treatment, callus sub cultured to free medium (MS0) and RNA isolation was conducted for cDNA synthesis. The presence of the inserted gene was observed by amplification and electrophoresis of the selectable marker HPT gene (455 bp) while the quantification of OsRKD4 gene expression (191 bp) is detected by using Real-Time PCR. The phenotype of somatic embryo formation was observed with Sudan Red 7B staining while callus slice were conducted by using 10 days old callus. As a result, OS2 is known to follow 3:1 segregation pattern with a value of X2 dF 1 at 1% is 4,267 after inoculation of 500 rice seeds, while the transgenic plants showed 455 bp HPT gene. The 191 bp OsRKD4 gene is detected in both transgenic and non-transgenic plants. OS1-D has the highest expression level with a value of 2,9 fold. The multi somatic embryos phenotype was also observed based on the Sudan Red 7B staining and also sliced preparation. K

Kata Kunci : Padi Hitam, OsRKD4, Transgenik, Embrio Somatik/Black Rice, OsRKD4, Transgenic, Somatic Embryo