Integrasi t-DNA pembawa gen AtRKD4 pada tanaman transforman Coffea arabica L. klon AS2K

GERALD DAFA RHIZARDY, Prof. Dr. Endang Semiarti, M.S., M.Sc.

2024 | Skripsi | BIOLOGI

Kopi Arabika (Coffea arabica L.) klon AS2K merupakan salah satu komoditas perkebunan di Indonesia yang memiliki nilai ekonomi tinggi dan banyak diminati oleh banyak masyarakat. Perbanyakan kopi Arabika di Indonesia masih belum optimal dibandingkan kopi Robusta. Tujuan peneliti adalah untuk menghasilkan tanaman kopi Arabika unggul melalui rekayasa genetik dengan penyisipan gen kunci embriogenesis Arabidopsis thaliana RWP-RK Domain 4 (AtRKD4). Dilakukan metode transfer gen AtRKD4 ke sel-sel kalus melalui Agrobacterium tumefaciens strain EHA105 pembawa T-DNA dengan gen AtRKD4 dalam plasmid pta7002 dengan perendaman larutan agro OD 0.8 selama 1 jam. Konfirmasi integrasi T-DNA pembawa gen AtRKD4 pada genom C. arabica dilakukan dengan amplifikasi gen RKD4 menggunakan Polymerase Chain Reaction (PCR). PCR dilakukan dengan primer AtRKD4, HPT dan ACTIN sebagai kontrol positif serta DegRKD4 untuk mengetahui adanya gen homolog AtRKD4. Hasil penelitian menunjukkan sebanyak 44 dari 342 kalus hasil transformasi dapat bertahan pada medium seleksi dengan nilai frekuensi transformasi 11.26%. Morfologi kalus transforman yang diperoleh menunjukkan tekstur friable dan berwarna kuning serta karakterisitk anatomi yaitu sel-sel kecil, nukleus besar, dan sitoplasma tipis. Analisis molekuler dibuktikan dengan hasil amplifikasi DNA genom kopi yang ditandai munculnya fragmen DNA sepanjang 188 bp pada primer AtRKD4, 114 bp pada primer ACTIN, 545 bp pada primer HPT, dan 384 bp pada primer DegRKD4.

Arabica coffee (Coffea arabica L.) AS2K clone is one of the plantation commodities in Indonesia that has high economic value and is in great demand by many people. The propagation of Arabica coffee in Indonesia is still not optimal compared to Robusta coffee. The researcher's goal is to produce superior Arabica coffee plants through genetic engineering by inserting the key gene for Arabidopsis thaliana embryogenesis RWP-RK Domain 4 (AtRKD4). The AtRKD4 gene transfer method was carried out to callus cells through T-DNA carrying Agrobacterium tumefaciens EHA105 strain with the AtRKD4 gene in the pta7002 plasmid by soaking in agro OD 0.8 solution for 1 hour. Confirmation of the integration of the T-DNA carrying the AtRKD4 gene into the C. arabica genome was carried out by amplifying the RKD4 gene using Polymerase Chain Reaction (PCR). PCR was carried out with the primers AtRKD4, HPT and ACTIN as positive controls and DegRKD4 to determine the presence of the AtRKD4 homologous gene. The results showed that 43 out of 382 calli from the transformation could survive on the selection medium with a transformation frequency value of 11.26%. The morphology of the transformed calli obtained showed a friable texture and yellow, as well as anatomical characteristics, namely small cells, large nuclei, and thin cytoplasm. Molecular analysis was proven by the results of coffee genome DNA amplification which was marked by the emergence of DNA fragments of 188 bp in the AtRKD4 primer, 114 bp in the ACTIN primer, 545 bp in the HPT primer, and 384 bp in the DegRKD4 primer.

Kata Kunci : Coffea arabica L. klon AS2K, embriogenesis somatik, Agrobaterium tumefaciens, RKD4.