Variation of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration on kaffir lime callus growth as raw material for cell suspension

Abstract

Our previous study showed that kaffir lime leaf has anti-cancer compounds. Elicitation in kaffir lime cell suspension could increase the amount of the anti-cancer compounds. In order to develop cell suspension, a friable callus as a raw material is needed. Therefore, the objective of this study was to determine the best concentration of 2,4-D to produce the best kaffir lime friable callus and make the cell suspension culture from the best callus. In this study, we used Murashige and Skoog (MS) media with various 2,4-D concentrations (0 ppm, 2 ppm, 5 ppm, and 10 ppm). Calli were incubated for 50 days. The parameters measured were biomass, color, and texture. Criteria of the best callus were good growth, green-yellowish color, wet and crumb textured. The best callus grew in media containing certain 2,4-D concentrations, then used as raw material for cell suspension. Cell suspension used the same media as the best callus induction media. The parameters such as cell growth and sugar content of suspension media were observed using the spectrophotometric method. The criteria to succeed cell suspension was having a good growth curve. The results showed that 2 ppm of 2,4-D was the best concentration to produce friable callus. The lag phase for callus induction started from 0-10 days, exponential phase occurred on days 10-30 whereas day 30-40 was the stationary phase for callus. A callus that attained the age of 30 days (initial step of stationary phase) were used as raw material for cell suspension. According to the reduction of sugar levels in the media, cell suspension had a good growth curve because the sugar level showed an energy source for cell division. The lag phase occurred on days 0-2, and the exponential phase occurred on days 2-16 whereas days 16-18 were the stationary phase for cell suspension.