Efficient somatic embryo production of Limau madu (Citrus suhuiensis Hort. ex Tanaka) in liquid culture

Abstract

Effects of N6-benzylaminopurine (BAP) concentration, initial cell density and carbon sources and concentrations for producing cell suspension and somatic embryos of Limau madu (Citrus suhuiensis Hort. ex Tanaka) were investigated using cell suspension culture. Cells were first inoculated into Murashige and Skoog (MS) medium (1962) supplemented with 4.4 to 13.3 µM BAP. Growth rate of cells was at its maximum (6.69 mg day-1) in media supplemented with a lower concentration of BAP (6.7 µM). Embryogenic cell at 2 mg ml-1 was found to be the most effective inoculum size for the highest growth rate (3.35 mg day-1) of cell proliferation within a period of 15 to 30 days after inoculation (DAI). This inoculum size resulted in 31.75% faster embryo growth than those with inoculum densities of 4 to 6 mg ml-1. Sucrose (88, 117 and 146 mM), glycerol (16, 22 and 27 mM) and combinations of sorbitol and galactose (146:0, 110:36, 73:73, 36:110 and 0:146 mM) were tested for their effects on embryogenic cell proliferation and somatic embryo induction. Results indicates that sucrose at 146 mM induced cell proliferation (7.65 mg day-1) and produced a higher quantity of cells than glycerol at 27 mM (2.33 mg day-1) and a combination of sorbitol and galactose at 73:73 mM (4.64 mg day-1), but failed to induce somatic embryos. Glycerol in different concentrations was ineffective in cell proliferation and somatic embryo induction. At optimal BAP concentration (6.7 µM), a small amount of embryogenic cells (100 mg in 50 ml) can be multiplied profusely in sucrose-containing medium. A large number of somatic embryos (951) were induced in a medium containing 110 mM sorbitol and 36 mM galactose as the most effective carbon source for inducing somatic embryos without BAP.

Effect of Cell Density and Benzyl Amino Purine on the Growth of Somatic Embryo of Citrus Mandarin Batu 55 (Citrus reticulata Blanco.) in Liquid Culture

“…In general, low cell density causes low proliferation, but high cell density inhibit cell growth because of accelerate accumulation of toxic compound in liquid culture, resulted of an imbalance between medium and cell density in the medium [8]. The highest multiplication of somatic embryo of Citrus suhuinensis and Citrus kalamondin were obtained at cell denisty 4 -6 mgL -1 and 2 -6 mgL -1 , respectively [2]. Benzyl Amino Purine (BAP) is one of the cytokinin compounds to induce and stimulate the growth of citrus somatic embryos.…”

“…The higher concentrations of BAP in liquid culture had a negative effect on embryogenic cell growth. The addition of 3 mgL -1 BAP into liquid media descreased the growth of somatic embryo in C. suhuinensis [2]. Liquid culture system significantly accelerated multiplication of somatic embryo, thus it has a promising potential as micropropagation technique in citrus trought somatic embryogenesis.…”

“…Incubation period was also determined cell growth of somatic embryo in liquid media [2]. Culturing somatic embryo for too long period in liquid cultuer caused disturbance of osmotic process within the cell, therefore the water content of somatic embryo became very low.…”

“…is one of Indonesian fruit commodities that has high economic value and consumers demand [1]. Citrus mandarin high vitamin and sweet taste [2]. But, some problems faced in cultivation of citrus mandarin found are limited land provision, low seedlings available, and a difficulty to get well seeds for high quality crops.…”

Potensi Pemanfaatan Teknologi Embriogenesis Somatik in Vitro Dalam Perbanyakan Massal Benih Jeruk Bebas Penyakit

“…Inisiasi kalus relatif lebih cepat pada jaringan muda dan mengandung sel hidup dibandingkan pada jaringan tua. Bahan tanam yang umum digunakan pada perbanyakan tanaman jeruk melalui ES adalah nuselus yang berasal dari biji buah muda umur 1214 minggu setelah bunga mekar (Devy et al 1997;Agisimanto et al 2011;Wulansari et al 2015), ovul yang belum berkembang (El-Sawy et al 2006), jaringan endosperm (Kosmiatin et al 2014), tangkai + putik, benang sari dan jaringan vegetatif semaian biji, serta embrio yang ada dalam biji seperti pada tanaman buah lainnya (Sutanto dan Aziz 2006).…”