Effect of 2,4-D and BAP on Morphological Characters and Genetic Stability of Kaffir Lime (Citrus hystrix DC.) Callus Cultures Among Generations

Kaffir lime has medicinal properties for the treatment of many diseases. We used in vitro culture techniques to maintain quality and increase the production of active compounds in kaffir lime. The objective of this study is to determine the phenotype and genotype of kaffir lime callus cultures grown on media with the addition of 2,4-Dichlorophenoxyacetic (2,4-D) and Benzylaminopurine (BAP) on three generations (G0, G1, and G2). The callus was induced and subcultured on Murashige and Skoog medium added with 2,4-D: BAP at concentrations of 1:0.5; 2:0 and 5:0. Results showed no significant difference in callus initiation time in all treatment groups. Morphological characteristics, including color, texture, and biomass, varied among growth regulator concentrations and generation level. A high level of callus generation corresponds to a more friable texture and a more yellowish callus. Callus grown with the addition of 2,4-D and BAP 2:0 and 5:0 showed a more friable structure and yellowish color than 1:0.5. Growth regulator concentrations in each generation did not affect the callus growth curves, but the length of each phase between generations was different. The exponential phases of G1 and G2 were faster than that of G0. Despite slight differences in phenotype, the DNA profiles of callus suggest the same pattern between treatment groups, thereby indicating that our kaffir lime callus has genetic stability and that the callus can be used as raw material for medicinal purposes. Keywords: ISSR markers, Kaffir lime callus, Generations, Genotype, Growth factor, Phenotype