Optimasi Metode Transformasi Genetik melalui Agrobacterium tumefaciens pada Tanaman Kosmos (Cosmos sulphureus Cav.)

DEVI SATYA PUDJA, Dr. Ir. Aziz Purwantoro, M.Sc ; Widhi Dyah Sawitri, S.Si., M.Agr., Ph.D

2021 | Skripsi | S1 AGRONOMI

Kosmos kuning (Cosmos sulphureus Cav.) merupakan tanaman hias yang banyak di budidayakan karena keindahan warna bunganya. Keberagaman warna bunga kosmos dapat ditingkatkan dengan rekayasa genetika melalui teknik transformasi genetik. Namun belum ada laporan mengenai transformasi genetik secara in vitro pada tanaman kosmos kuning. Oleh karena itu penelitian dilakukan dengan tujuan untuk memproleh protokol metode transformasi genetik secara in vitro melalui Agrobacterium tumefaciens pada kosmos kuning (Cosmos sulphureus Cav.). Kultur Agrobacterium tumefaciens strain LBA4404 dengan vektor plasmid pRI101AN membawa gen nptII sebagai marker seleksi. Prosedur yang dilakukan antara lain penyediaan dan pemilihan eksplan serta komposisi media induksi kalus, optimasi waktu infeksi Agrobacterium tumefaciens, kombinasi kepadatan bakteri (OD) dengan konsentrasi asetosiringon, kemudian skrining pada medium seleksi, dan dilakukan analisis molekuler dengan PCR untuk melihat keberhasilan integrasi gen nptII ke dalam genom tanaman. Pemilihan sumber eksplan dan media induksi kalus dilakukan sebelum transformasi genetik dengan membandingkan persentase pembentukan kalus pada eksplan kotiledon dan eksplan daun planlet in vitro yang diberi perlakuan 2 media induksi kalus 1,5 mg/L BAP + 1 mg/L NAA dan 2 mg/L 2,4-D. Tahapan transformasi genetik pada penelitian ini meliputi inokulasi, ko-kultivasi, induksi kalus, resting, seleksi, deteksi dini dengan PCR, dan regenerasi. Percobaan yang dilakukan pada metode transformasi antara lain perlakuan waktu infeksi eksplan selama 5, 10, dan 15 menit dan perlakuan kombinasi kerapatan bakteri (OD600 = 0.25, 0.50, 0.75) dan konsentrasi asetosiringon (10, 20, 30 mg/L). Hasil kalus yang lolos seleksi dibuat persentasenya dan kalus putatif transforman dikonfirmasi dengan analisis PCR. Hasil yang didapatkan eksplan kotiledon memiliki persentase pembentukan kalus mencapai 100% dan media induksi kalus dengan penambahan 1,5 mg/L BAP + 1 mg/L NAA mampu membentuk kalus dalam waktu 6 hari, berwarna hijau muda segar dan diameter kalus 1-1,8 cm. Waktu infeksi terbaik ditemukan pada perendaman 10 menit dengan persentase 26,25%. Perlakuan kombinasi terbaik didapatkan pada kepadatan bakteri (OD) 0.25 dan konsentrasi asetosiringon 30 mg/L dengan efisiensi transformasi sebesar 60%.

Yellow cosmos (Cosmos sulphureus Cav.) is an ornamental plant that is widely cultivated because of its beautiful flower color. The color diversity of cosmos flowers can be increased by genetic engineering through genetic transformation techniques. However, there have been no reports of genetic transformation in vitro of yellow cosmos plants. Therefore, this study was conducted with the aim of obtaining a protocol for in vitro genetic transformation method using Agrobacterium tumefaciens in yellow cosmos (Cosmos sulphureus Cav.). A culture of Agrobacterium tumefaciens strain LBA4404 with the plasmid vector pRI101AN carried the nptII gene as a selection marker. The procedures carried out included the provision and selection of explants and the composition of callus induction media, optimization of Agrobacterium tumefaciens infection time, combination of bacterial density (OD) with acetosyringone concentration, then screening on the selection medium, and conducting molecular analysis with PCR to see the success of the integration of the nptII gene into in the plant genome. The selection of explant sources and callus induction media was carried out before genetic transformation by comparing the percentage of callus formation in cotyledon explants and plantlet leaf explants in vitro treated with 2 callus induction media 1.5 mg/L BAP + 1 mg/L NAA and 2 mg/L 2,4-D. The method for this genetic transformation consist of inoculation, co-cultivation, callus induction, resting, selection, early detection by PCR, and regeneration. Experiments carried out on the transformation method included treatment of explant infection time for 5, 10, and 15 minutes and combination treatment of bacterial density (OD600 = 0.25, 0.50, 0.75) and concentration of acetosyringone (10, 20, 30 mg/L). The percentage of callus that passed the selection was determined and the transformant putative callus was confirmed by PCR analysis. The results obtained were cotyledon explants had a callus formation percentage of 100% and callus induction media with the addition of 1.5 mg/L BAP + 1 mg/L NAA were able to form callus within 6 days, fresh light green color and callus diameter 1-1, 8 cm. The best infection time was found in 10 minutes of infection with a percentage of 26.25%. The best combination treatment was found at a bacterial density (OD) of 0.25 and an acetosyringone concentration of 30 mg/L with a transformation efficiency of 60%.

Kata Kunci : Kosmos, transformasi genetik, in vitro, waktu infeksi, kepadatan bakteri, asetosiringon/Cosmos, genetic transformation, in vitro, infection time, bacterial density, acetosyringone