Identifikasi penanda kelamin tanaman salak (Salacca zalaca [Gaertner] Voss) berdasar analisis kromosom dan DNA

PARJANTO, Promotor Prof.Dra. Sukarti Moeljopawiro, M.App.Sc.,Ph.D

2006 | Disertasi | S3 Ilmu Pertanian

Penanda kelamin (sex marker) untuk membedakan tanaman jantan dan betina pada stadia bibit (vegetatif) sangat diperlukan untuk mendukung studi genetika dan program pemuliaan pada tanaman salak (Salacca zalacca) yang bersifat berumah dua dan mencapai umur reproduktif lambat (3-4 tahun). Tujuan penelitian ini adalah mendapatkan penanda kelamin tanaman salak berdasar analisis variasi kromosom dan Deoxyribonucleic acid (DNA). Penelitian ini mempelajari: (1) perbedaan morfologi kromosom salak jantan, betina dan hermaprodit; (2) perbedaan pola pita CMA kromosom salak jantan, betina dan hermaprodit; dan (3) perbedaan susunan basa DNA salak jantan dan betina dengan teknik RAPD. Lima tanaman salak jantan dan 5 salak betina (hasil cangkok anakan dari tanaman salak petani di Turi, Sleman, Yogyakarta) serta 5 tanaman salak hermaprodit asal biji digunakan untuk pengamatan morfologi dan pola pita CMA kromosom. Jumlah dan morfologi (panjang dan bentuk) kromosom diamati dengan teknik pencet (squase) dan pewarnaan aceto orcein, pola pita CMA kromosom (jumlah dan posisi pita CMA) diamati dengan teknik pencet (squase) dan pewarnaan fluoeresn chromomycin A3 (CMA). Analisis perbedaan kromosom salak jantan, betina, dan hermaprodit dilakukan dengan membandingkan sifat-sifat morfologi dan pola pita CMA kromosom pada tiga jenis kelamin tanaman salak. Deoxyribonucleic acid (DNA) yang diekstraksi dari daun 5 tanaman salak jantan dan 5 salak betina (dari populasi campuran yang ditanam di Banguntapan, Yogyakarta) digunakan untuk analisis variasi susunan basa DNA tanaman salak jantan dan betina dengan teknik RAPD. Sebanyak 49 primer 10 mer acak (produksi Operon Technologies) digunakan untuk amplifikasi DNA tanaman salak jantan dan betina. Penanda RAPD untuk membedakan kelamin diidentifikasi berdasarkan adanya pita/fragmen DNA spesifik pada satu jenis kelamin dan tidak ada pada jenis kelamin yang lain. Hasil penelitian menunjukkan bahwa tidak terdapat perbedaan jumlah dan morfologi kromosom antara tanaman salak jantan, betina, dan hermaprodit. Ketiga jenis kelamin salak mempunyai rumus kariotip sama, yakni 2n = 28 = 11 m + 1 m (SAT) + 2 sm (terdiri atas 11 pasang kromosom metasentris, 1 pa-sang kromosom metasentris dengan satelit kromosom, dan 2 pasang kromosom submetasentris). Berdasar morfologi kromosom, tidak teridentifikasi adanya kromosom kelamin ataupun penanda kromosom utuk membedakan tanaman salak jantan dan betina. Terdapat perbedaan pola pita CMA kromosom salak jantan dan betina. Pola pita CMA kromosom nomor 1 pada salak jantan bersifat homomorfik, sedangkan salak betina bersifat heteromorfik. Diusulkan bahwa pasangan kromosom nomor 1 merupakan kromosom kelamin, tanaman jantan bersifat homogametik (ZZ),pasangan kromosom nomor 1 dapat digunakan sebagai penanda kelamin untuk membedakan tanaman salak jantan dan betina. Amplifikasi DNA genom tanaman salak dengan teknik RAPD menggunakan primer 10 mer OPP-08 (ACATCGCCCA) menghasilkan fragmen DNA berukuran 400 bp yang bersifat spesifik pada jenis kelamin jantan, pada jenis kelamin betina tidak dihasilkan fragmen tersebut. Fragmen DNA berukuran 400 bp hasil amplifikasi menggunakan primer OPP-08 (OPP-08400) dapat digunakan sebagai penanda kelamin untuk membedakan tanaman salak jantan dan betina sedangkan tanaman salak betina bersifat heterogametik (WZ). Pola pita CMA

Marker for early sex identification will be an essential tool for genetics and breeding program in the salak (Salacca zalacca) which is a dioecious mode (separate male and female individuals) and in the late initial reproductive age (3-4 years). The objectives of this research were to obtain the sex marker for differentiating male and female of salak based on chromosomes and deoxyribonucleic acid (DNA) analysis. Three researches were conducted, they were: (1) an analysis of variation of chromosome morphology of female, male, and hermaphrodite salak, (2) an analy-sis of variation of chromosome CMA banding pattern of female, male, and hermaphrodite salak, and (3) an analysis of DNA variation of female and male salak using RAPD technique. Five male and 5 female plants of salak (cloned from farmer’s salak plant in Sleman, Yogyakarta) and 5 seedling hermaphrodite plants of salak were analyzed to study the chromosomes morphology and the chromosomes CMA banding pattern. Chromosomes morphology were observed using squase and aceto orcein staining technique. CMA banding pattern were observed using squase and chromomycin A3 (CMA) fluorescent staining technique. Variation of chromo-somes morphology of male, female, and hermaphrodite salak were analyzed according to number, length size, and shape of chromosomes. Variation of chromosomes CMA banding pattern were analyzed according to number and position of CMA band of chromosomes. Deoxyribonucleic acid (DNA) extracted from 5 male and 5 female salak leafes (from bulk population of salak grown in Banguntapan, Yogyakarta) were used to distinguish the variation of DNA sequence of female and male salak using RAPD technique. Forty nine random primers (produced by Operon Technologies) were used to amplify the genom DNA of male and female salak. RAPD marker for sex differentiating was identify by the presence of one or more bands in one sex and absent in the other sex. The results of the analysis showed that there were no differences in chromosome number and chromosome morphology among male, female, and hermaphrodite salak. All sex showed the equal karyotipe formula, that were 2n = 28 = 11 m + 1 m (SAT) + 2 sm (consists of 11 pairs of metacentric chromosomes, 1 pair of metacentric chromosome bearing satellite, and 2 pairs of submetacentric chromosomes). Cytologycal sex marker or the heteromorphic sex chromosome based on the chromosomes morphology were not identified. There was difference of chromosome CMA banding pattern in male and female salak, that is in the first chromosome pair. The male was homomorphic and female was heteromorphic. It was suggested that the first chromosome pair appeared as the sex chromosome, male salak was homogametic (ZZ) and female salak was heterogametic (WZ). The CMA banding pattern in the first chromosome pair could be used as sex marker for differentiating male and female plants of salak. DNA amplification with primer OPP-08 (ACATCGCCCA) using RAPD technique was producing a 400 bp band that was present only in male plants and absent in the female plants of salak. It was concluded that this band (OPP-08400, a 400 bp band produced using OPP-08 primer) is linked to the gene (s) that control sex determination in salak. Thus, this band (OPP-08400) could be used as sex marker for differentiating male and female plants of salak.

Kata Kunci : Tanaman Salak,Penanda Kelamin,Tanaman Salak Jantan dan Betina, chromosome, karyotipe, CMA banding, RAPD, sex, salak