Embriogenesis somatik anggrek bulan (Phalaenopsis amabilis (L.) Blume

UTAMI, Edy Setiti Wida, Promotor Prof. Dr. Issirep Sumardi

2009 | Disertasi |

Penelitian ini bertujuan untuk (1) menentukan bahan eksplan dan konsentrasi NAA yang tepat untuk menginduksi pembentukan embrio somatik; (2) mengetahui pengaruh pemberian NAA + BA 1,0 mg/L dalam media dan waktu inkubasi terhadap maturasi embrio; (3) mengetahui struktur dan pola perkembangan embrio; (4) mengetahui struktur permukaan embrio dan (5) mengetahui profil protein daun anggrek bulan. Penelitian pertama disusun dalam rancangan faktorial dengan 2 faktor, yaitu: bahan eksplan terdiri dari 2 aras (daun bagian tengah - ujung dan daun bagian tengah - pangkal), konsentrasi NAA terdiri dari 6 aras ( 0,0; 0,1; 1,0; 2,0; 3,0 dan 4,0 mg/L). Lama waktu terbentuk embrio dilakukan setiap hari dan morfologi embrio somatik diamati menggunakan mikroskop cahaya. Pada penelitian kedua dihitung jumlah embrio globular dan embrio skutelar setiap 2 minggu selama 10 minggu. Penelitian ketiga, diamati struktur dan pola perkembangan embrio dengan membuat sayatan melintang dan membujur. Pembuatan preparat mikroskopis menggunakan metode Jensen. Struktur permukaan embrio dianalisis dengan Scanning Electron Microscope. Profil protein daun dianalisis dengan metode SDS-PAGE. Pembentukan embrio somatik tercepat teramati pada daun bagian tengah -pangkal yang dikultur pada media NP diberi 2,0 mg/L NAA. Embrio tampak kuning kehijauan, bulat, dan mengilap. Perlakuan NAA 0,5 mg/L + BA 1,0 mg/L dan waktu inkubasi 10 minggu meningkatkan jumlah embrio skutelar. Pada tahap perkembangan yang berbeda embrio somatik mempunyai struktur yang berbeda. Pola perkembangan embrio somatik anggrek bulan menyerupai pola perkembangan embrio zigotiknya. Struktur permukaan proembrio halus ditutup oleh material dinding sel sebagai matriks ekstraseluler. Embrio globular permukaannya menonjol halus tertutup oleh dinding sel yang baru.Tonjolan-tonjolan tersebut dihubungkan oleh mikrofibril selulose. Embrio matang (skutelar) struktur permukaannya stabil. Profil protein eksplan daun yang dikultur pada media NP diberi NAA berbeda dengan profil protein daun yang dikultur tanpa NAA. Pemberian NAA setelah 2 hari inkubasi dapat menginduksi sintesis protein baru dengan berat molekul 14 kDa. Protein tersebut diduga merupakan protein embrionik penanda awal terjadinya embriogenesis somatik.

This experiments were aimed (1) to determine the appropriate explants materials and NAA concentration to induct somatic embryo formation; (2) to know the effect of different NAA concentration + 1.0 mg/L BA and incubation time on the maturation of embryos; (3) the structure and development pattern of embryos; (4) the surface structure of embryos; and (5) the protein profile of moon orchid leaf. First experiments were arranged in two factor Factorial design. First factor was explants material, consists of two levels (center to upper end and center to lower end). Second factor was NAA concentration which consists of six levels (0.0; 0.1; 1.0; 2.0; 3.0; and 4.0 mg/L). Time (day) of the somatic embryo formation and somatic embryo morphology were observed using a light microscope. The second experiment evaluated the effect of NAA concentrations and incubation time to embryo maturation. The number of globular and scutelar embryos were counted every 2 weeks for 10 weeks incubation period. The anatomical structure of embryo was observed by longitudinal section embryo, while the development pattern of embryo was observed by cross section of embryo. Microscopic slides were carried out using Jensen method. The surface structure of the embryo was examined by Scanning Electron Microscope. Protein profile of leaf explants was observed by electrophoresis using SDS-PAGE method. The fastest embryo formation was observed at center to lower end of leaves at NP media supplemented with NAA 2.0 mg/L. Embryo seemed to be greenish yellow, round, and shiny. Treatment with NAA 0.5 mg/L + BA 1.0 mg/L in 10 weeks incubation time increased amount of scutelar embryos. In every different development stages, somatic embryo has different structure. The development pattern of moon orchid somatic embryo was similar to zygotic embryo. The surface of pro-embryo was smooth, covered by cell wall as extra cellular matrix. The surface of globular embryos was smooth protrusion, covered by new cell wall after the cell wall undergoes regeneration. The protrusion was connected by cellulose micro fibril. The surface of scutelar embryos did not undergo many changes. The protein profiles of leaves explants that were cultured in NP media supplemented with NAA were different from leaves that were cultured in NP media without NAA. NAA supplement after 2 days incubation could induce the synthesis of 14 kDa new protein. The protein is expected to be an early marker for somatic embryogenesis.

Kata Kunci : Embriogenesis somatik, Anggrek bulan, Kompetensi embriogeneik, Protein embrionik, somatic embryogenesis, orchid, embryogenic competence, embryonic protein