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.

Key words: Limau madu, cell suspension, sucrose, sorbitol, galactose.