VARIASI MORFOLOGIS, FISIOLOGIS, DAN MOLEKULAR TANAMAN ANGGREK Spathoglottis plicata Blume HASIL IRRADIASI SINAR-X

SUYITNO ALOYSIUS, Dr. Endang Semiarti, MS, M.Sc.; Dr. Aziz Purwantoro, M.Sc.; Dr. Kumala Dewi, M.Sc.

2017 | Disertasi | S3 Ilmu Biologi

Pengembangan bibit anggrek Spathoglottis plicata melalui irradiasi biji dengan sinar-X penting dilakukan karena keragamannya yang masih rendah. Penelitian ini bertujuan untuk meningkatkan keragaman genetik dengan irradiasi sinar-X dan untuk mengetahui pengaruh sinar-X terhadap pertumbuhan dan perkembangan tanaman. Penelitian dilakukan dalam tiga tahap meliputi : 1) Irradiasi biji dengan sinar-X, 2) uji perkecambahan biji, analisis pertumbuhan dan perkembangan embrio, 3) identifikasi variasi morfologis dan deteksi perubahan genetik pada struktur DNA : DNA genom dan DNA transkrip (cDNA) POH1 homolog pada planlet mutan dan tanaman aklimatisasi. Pada uji perkecambahan, biji S. plicata umur 30-32 hari hasil penyerbukan sendiri diradiasi dengan sinar-X pada dosis 0, 6, 12, 18, dan 24 rad. Biji dikultur pada medium 1/2 MS sampai menjadi protokorm. Jumlah biji berkecambah, serta pertumbuhan panjang dan lebar embrio diukur setiap minggu, pada 6 minggu pertama menggunaan optilab yang dikalibrasi dengan mikrometer. Protokorm umur 8 minggu disubkultur pada medium New Phalaenopsis-Shoot Induction Medium (NP-SIM) yang merupakan suplementasi medium NP dengan 5 mikromolar 2iP dan 0,15 mikromolar NAA yang dimasukkan ke dalam cawan petri untuk menumbuhkan protokorm menjadi planlet. Planlet umur 5 bulan dipindah-tanam ke botol sampai umur 10 bulan. Variasi morfologis planlet diamati. Pencapaian pertumbuhan planlet diukur pada planlet umur 10 bulan pada saat akan diaklimatisasi. Deteksi perubahan genetik planlet dilakukan dengan Polymerase chain reaction (PCR) mengunakan Random Amplified Polymorphic DNA (PCR-RAPD). Sembilan macam primer (OPA1, OPA2, OPA11, OPA12, OPA14, OPB1, OPB4, OPD12, OPD14) digunakan berdasar hasil skrining 22 primer pada tiga sampel (1 WT dan 2 mutan) DNA genom. Hasil RAPD dinyatakan dalam data biner dengan memberi skor 0 atau 1 jika tidak ada atau jika ada fragmen DNA. Data dianalisis menggunakan GenAlex 6.1 untuk mendapatkan jarak genetik dan persentase polimorfisme DNA. Program NTSYS ver.2 digunakan untuk pembuatan dendrogram klaster dengan algoritma UPGMA (Unweighted Pair Group Method with Arithmethic Averages). Deteksi perubahan genetik juga dilakukan dengan membandingkan nukleotida genom DNA dan cDNA homolog POH1 dari empat planlet mutan pucuk dengan planlet WT. Semua planlet WT dan WT-L dari berbagai dosis irradiasi umur 10 bulan yang diperoleh diaklimatisasi. Variasi morfologis diamati pada ciri daun, ukuran rumpun, umur pertama berbunga dan morfologi bunganya. RT-PCR digunakan untuk analisis perubahan genetik pada transkrip DNA (cDNA) homolog POH1. Hasil penelitian menunjukkan bahwa irradiasi sinar-X dosis 12-18 rad menghambat pertumbuhan dan perkembangan embrio dan planlet serta memicu terjadinya mutasi bentuk dan warna daun, akar, pucuk dan pembungaan dini. Hasil analisis GenAlex, jarak genetik dan persentase polimorfisme DNA kelompok planlet mutan semakin besar sehingga menguatkan terjadinya mutasi DNA secara random. Perbandingan nukleotida putative cDNA homolog POH1 planlet mutan dan planlet WT (cDNA 'POH1') menunjukkan adanya insersi dan substitusi nukleotida pada beberapa titik pada cDNA mutan yang membuktikan terjadinya mutasi pada planlet hasil irradiasi biji. Tetapi perubahan nukleotida pada tanaman mutan ini tidak diikuti dengan perubahan morfologis yang jelas. Pada tanaman aklimatisasi, variasi morfologis kelompok tanaman mutan tidak dapat dibedakan dengan tanaman WT.

The diversity of Spathoglottis plicata is relatively low and one of techniques that can improve genetic diversity is through X-rays irradiation. This study was aimed to increase the genetic diversity obtained through X-ray irradiation of S. plicata seeds and to determine the effects of X-ray irradiation on growth and development of S. plicata seedlings. This study was conducted in three stages including: 1) X-ray irradiation of orchid seeds, 2) analyses of the germination, growth and development of the embryo, 3) identification of morphological variation and detection of genetic changes on DNA structure : genomic DNA and homologous POH1 cDNA on plantlets and acclimatized plants. In the germination test, seeds of S. plicata age of 30-32 days produced by self-pollination was irradiated with X-rays at doses of 0, 6, 12, 18 or 24 rad. Seeds were cultured on 1/2 MS medium to get protocorms. The percentage of germinated seeds was calculated and its development was observed. The measurement of lengths and widths of embryo was conducted every week for the first 6 weeks. Eight weeks old protocorms were subcultured onto New Phalaenopsis - Shoot Induction Medium (NP-SIM) that consists of NP medium suplemented with 5 micromolar 2iP and 0,15 micromolar NAA, in a petridish to promote growth of protocorm into plantlets. Five months old plantlets were then transfered into new medium until plantlets were large enough (10 months old). Morphological variations of plantlet were observed. Plantlets growth performance was measured at the age of 10 months at the time to be acclimatized. Genetic changes of the plantlets were detected by Polymerase Chain Reaction (PCR) using Random Amplified Polymorphic DNA (PCR-RAPD). Nine kinds of primer (OPA1, OPA2, OPA11, OPA12, OPA14, OPB1, OPB4, OPD12, OPD14) were used based on the results of screening 22 primer on three samples (1 wild type and 2 mutants) of genomic DNA. Results of RAPD were expressed in binary data by giving a score of 0 or 1 if there is no or if there is DNA fragment. Data were analyzed using 6.1 GenAlex to obtain genetic distance and the percentage of DNA polymorphism. Ver.2 NTSYS program was used for making a cluster dendrogram with UPGMA algorithm. Genetic changes were also detected by comparing of POH1 homologous cDNA of four shoots mutants with WT plantlet. All WT and WT-L plantlets age of 10 months obtained from various doses irradiation were acclimatized gradually. Morphological variation observed were leaf characteristics, size clumps, age at first flowering and morphology of flower. RT-PCR was used for the analysis of some changes in DNA transcripts POH1. The results showed that the X-ray irradiation dose of 12-18 rad suppress the growth and development of embryos and plantlets, that trigger the mutations in the shape and color of the leaves, roots, shoots and early flowering. Based on GenAlex analysis, genetic distance and the percentage of mutants DNA polymorphism were higher than WT plantlet that amplifies the occurrence of random DNA mutations. Comparison of 'POH1' cDNA nucleotides between mutant and WT plantlets showed a high variation of nucleotide polymorphisms in mutants group. Additionally, insertions or substitution of nucleotides occurring at some points indicated mutations were occurred in plantlets from seed irradiation results. In acclimatized plants, morphological variation in mutant groups could not be distinguished to the characteristics of WT plants.

Kata Kunci : Kata kunci : Biji irradiasi, keragaman genetik, mutasi, pertumbuhan, Spathoglottis plicata, sinar-X