PENGEMBANGAN ANGGREK VANDA HIBRIDA (Vanda limbata Blume X Vanda tricolor Lindl. var. suavis) DENGAN PERLAKUAN KOLKISIN SECARA IN VITRO

Mustika Tuwo, Dr.rer.nat. Ari Indrianto, S.U

2014 | Tesis | S2 Biologi

Pengembangan tanaman anggrek dapat dilakukan melalui peningkatan kualitas dan kuantitas. Peningkatan kualitas dapat diperoleh dari kegiatan persilangan dan mutasi kimia dengan menggunakan kolkisin, sedangkan peningkatan kuantitas dengan perbanyakan melalui kultur in vitro. Setiap jenis tanaman mempunyai respon yang berbeda terhadap kolkisin sehingga diperlukan metode untuk mengetahui konsentrasi dan lama perendaman optimum. Penelitian ini bertujuan untuk mengevaluasi respon tanaman anggrek Vanda hibrida (Vanda limbata Blume X Vanda tricolor Lindl. var. suavis) pada fase pertumbuhan in vitro akibat perlakuan kolkisin, menetapkan konsentrasi kolkisin dan lama perendaman yang efektif dalam menginduksi poliploidisasi dan menentukan fase pertumbuhan anggrek Vanda hibrida yang menghasilkan multiple shoot akibat perlakuan kolkisin. Penelitian ini menggunakan variasi konsentrasi kolkisin 0,01%; 0,05%; 0,1%; 0,5%; 1% serta 0% dengan lama perendaman 6 jam; 12 jam; 18 jam; 24 jam; dan 4 hari secara aseptis menggunakan anggrek Vanda hibrida pada berbagai fase pertumbuhan in vitro. Setelah perendaman, tanaman anggrek ditanam kembali ke medium Vacint and Went (VW) ditambah air kelapa 150 ml/l. Setelah 4 bulan kemudian disubkultur ke medium VW ditambah air kelapa dan ekstrak pisang. Parameter penelitian meliputi karakter morfologis (jumlah dan panjang akar; jumlah, panjang, dan lebar daun), anatomis (panjang dan lebar stomata, dan indeks stomata), sitologis (preparasi kromosom dengan pewarna DAPI (4,6-diamidino-2-phenylindole dihydrochloride) dan penentuan tingkat ploidi melalui analisis flow cytometry). Analisis data dengan ANOVA (Analysis of Variance) dan untuk mengetahui letak beda nyata antar perlakuan dilakukan pengujian lebih lanjut dengan DMRT (Duncan’s Multiple Range Test). Hasil penelitian menunjukkan bahwa anggrek Vanda hibrida tidak toleran terhadap perlakuan kolkisin konsentrasi 0,1; 0,5 dan 1% dengan kematian protokorm lebih dari 50%. Meskipun mengalami kematian jaringan, protokorm mampu menghasilkan tunas yang lebih banyak dibanding kontrol yang membuktikan bahwa kolkisin mampu menginduksi terjadinya multiple shoot. Karakter morfologi plantlet tetraploid memiliki rata-rata jumlah dan panjang akar; jumlah, panjang, dan lebar daun yang lebih rendah dibanding kontrol. Karakter anatomi tanaman tetraploid menunjukkan ukuran stomata yang lebih besar dan memiliki indeks stomata yang kecil dibanding kontrol. Hal ini menunjukkan bahwa terdapat korelasi negatif antara indeks stomata dan tingkat ploidi. Analisis sitologi dengan flow cytometry menunjukkan tanaman tetraploid (2n=4x=76), pengecatan DAPI memperlihatkan jumlah kromosom lebih banyak dibandingkan dengan kontrol (2n=2x=38)

Improvement of orchid plants can be done through an increase in quality and quantity. Quality improvement can be achieved by crossing and chemical mutation using colchicine, while quantity improvement can be done by in vitro culture. Each plant species has a different response to colchicine so it is needed a multiplication method to determine the optimum concentration and treatment duration. The aim of this research was to evaluate the growth phase of orchid Vanda hybrid (Vanda limbata Blume X Vanda tricolor Lindl. var. suavis) response with colchicines treatment, to determine precise effective concentration of colchicines and duration of treatment to induce polyploid and to determine growth phase of orchid Vanda hybrid that generate the most multiple shoot with colchicine treatments. This research used variation of colchicines concentration which were 0.01%; 0.05%; 0.1%; 0.5%; 1% and 0% and incubation for 6 hours; 12 hours; 18 hours; 24 hours; and 4 days. Treatment was done in aseptic condition using orchid Vanda hybrid at various phase of growth in vitro. After treatments, plants were planted on Vacint and Went (VW) medium supplemented with of 150 ml/l coconut water. After four months, they were subcultured in medium VW + 150 ml/l + 150g/l banana extract. Parameters observed were morphological characters (number, and length of root; number, length, and width of leaves), anatomical characters (length and width of stomata, and index of stomata), cytological characters (chromosome preparation with DAPI (4,6- diamidino-2-phenylindole dihydrochloride) solution, and determination of ploidy level by flowcytometric analysis). Data were analyzed using ANOVA (Analysis of Variance) and tested by DMRT (Duncan's Multiple Range Test). The results showed that the orchid Vanda hybrid was intolerant to colchicine concentration of 0.1; 0.5 and 1% by death protocorm was more than 50%. Although the protocorm tissue died, protocorm in treated plant was able to produce more shoots than the control which proved that colchicine was able to induce multiple shoots. Morphological characters of tetraploid plantlets had lower average number and length of roots, the number, length, and width of the leaves than those of control. Anatomical characters of the tetraploid plantlets revealed the highest stomatal size and had smaller of stomatal index than controls. This evident indicated a negative correlation between stomatal index and ploidy level. Cytological analysis with flow cytometry revealed that tetraploid plantlets (2n=4x=76) stained with DAPI showed the chromosome number more than those of control (2n=2x=38).

Kata Kunci : poliploidisasi, kolkisin, anggrek vanda, jumlah kromosom