Pengembangan Metode Fusi Protoplas dengan Tingkat Keberhasilan Pembentukan Fusan 25% untuk Memperoleh Tanaman Triploid Jeruk Garut Tanpa Biji dengan Produktivitas 15 ton/ha pada Umur 8 Tahun: Isolasi Protoplas, Kultur Protoplas, dan Regenerasi Protoplas Hasil Fusi
Abstract
Tangerine is a citrus seed Indonesia and including a true species of the genus Citrus. This orange tastes sweet but relatively many seeds fruit seed. (15-23 seeds/fruit). Seedless citrus improvement can be done by manipulating the level ploidy. Plants with triploid ploidy level usually have sterile fruit (seedless). Somatic hybridization by fusion technique is a breakthrough technique that allows the plant generated by an increased level ploidy. The strategy chosen to assemble the triploid tangerine because the technique of protoplast isolation, fusion and regeneration of tangerine with diploid level has been mastered ploidy. The plant material used for the production of embryogenic haploid cell is a tangerine flower buds were taken from older plants (± 20 years) and grafting plants from old plants (± 8 years) and the haploid cell population tangerine of Batu 55 and Garut and the haploid cell population of Batu 55 and Garut tangerine. Haploid cell population repeatedly sub cultured every 4 weeks in a double layer media: solid media MT + picloram + malt extract and liquid media, and liquid medium that added is the basic of the MT with growth regulators BA (0-3 mg/l) and Picloram 0-20 mg/l and add malt extract 500 mg/l.The plant material used for the production of diploid callus is a population of diploid cells induced from embryonic nucellar tangerine. Callus induction conducted on MS medium MW + vitamin + BA 3 mg/l malt extract + 500mg/l. The population of cells/callus that formed were sub cultured every 4 weeks on MS or MW medium + vitamin + malt extract 500 mg/l. Flower bud explants derived from young plants (8 years) responded better than explants from older plants (> 30 years). Pre cold treatment for more than 3 days on the response forming callus from pretreatment less than 3 days. The best enzyme formulations for the isolation of protoplasts from diploid (callus and leaves in vitro) and protoplast Haploid (pollen) is macerozim 0.5% and 0.5% cellulase enzyme with incubation period of 16 hours (overnight). With the concentration of PEG of 20% gives fusan amounted to more than 4% PEG two binary fusion, fusion homo, and multi-fusion. Regeneration of cell wall formation and cell suspension fusan best media formulations obtained from MW+EM SOO mg /1 2.4 D 0.1 mg / I+BA 3 mg / l