Effect of Growth Factor In Callus Induction and Bioactive Compounds In Seed Explant of Kaffir Lime (Citrus hystrix DC.)

Abstract

Our previous study showed that kaffir lime leaf extracts may have anticancer properties. However, production of bioactive compounds is affected by environmental factors. Here, we present a method to control environmental conditions using in vitro culture techniques such as callus induction. Calluses were induced from seed embryo explants of kaffir lime on MS medium with combinations of 2,4-D and BAP at concentrations 1:0.5; 1:1; and 2:1, respectively. Fourty and 60 days-old calluses were extracted using chloroform and ethyl acetate and analyzed by GC-MS. Results showed all combinations of 2,4-D and BAP were able to induce callogenesis from seed embryo explants of kaffir lime with no significant differences of callus initiation time, biomass, morphology and growth rates. However differences were detected in the bioactive compound profiles. In kaffir lime callus, both fatty acids and secondary metabolites were detected. Specifically, in 40 daysold calluses (exponential growth phase) we detected α-pinene and 1.8cineole in plants treated with 2,4-D: BAP at concentration 1:0.5 and 2:1. In 60 days-old calluses (stationary phase) we detected a number of compounds in plants treated with 2,4-D:BAP at concentrations of 1:0.5 and 2:1, including

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

“…Hence, in this study, we focused only on three concentrations of 2,4-D: BAP namely 1:0.5, 2:0 and 5:0 ppm. Also, we revealed that without the addition of growth regulators (MS medium only), kaffir lime seeds will germinate and callus did not form (Tunjung et al, 2020). Thus, in this study, we did not culture callus without growth regulators.…”

“…This basal medium was added with a combination of various concentrations of 2,4-Dichlorophenoxyacetic (Sigma-Aldrich):BAP (Benzylaminopurine) (Sigma-Aldrich) hormones. The concentration of growth regulators was based on previous studies, in which we tried several concentrations of 2,4-D and BAP (Tunjung et al, 2020;Tunjung et al, 2018) and several concentrations of 2,4-D (Damayanti et al, 2020). Hence, in this study, we focused only on three concentrations of 2,4-D: BAP namely 1:0.5, 2:0 and 5:0 ppm.…”

“…Thus, in this study, we did not culture callus without growth regulators. The seeds were sterilized by soaking them in 5.25% NaClO (Sigma-Aldrich) while being shaken gently for 5 minutes under aseptic conditions so that the seeds did not become contaminated (Tunjung et al, 2020). The seeds were sliced to create a wound on the surface and then sowed in a culture bottle that contained solid MS medium.…”

“…Secondary metabolites in kaffir lime leaves are used as antibacterial agents (Tunjung et al, 2018), and secondary metabolites of kaffir lime are a potential resource to be used on a large scale in the pharmaceutical industry (Tunjung et al, 2020). However, the production of secondary metabolites with natural raw materials is less effective because many samples are required and the process is dependent on natural conditions.…”

“…Previously, we established a callus induction method for kaffir lime from seed explant. However, this study was conducted on generation 0 (G0) only (Tunjung et al, 2020, Tunjung et al, 2018. Kaffir lime callus can be used as raw material to produce cell suspension.…”