The effectiveness of 2,4-Dichlorophenoksiacetic acid (2,4-D) and Kinetin on Induction and Chlorophyll and Carotenoid Content of Alfalfa (Medicago sativa L.) Callus.

Abstract

Alfalfa (M. sativa L.) is a plant type of legume, which is used in the manufacture of health products, because they contain chlorophyll and carotenoids is quite high. Both pigments can be obtained through tissue culture techniques one with callus cultures. One of the factors that determine the success of callus cultures is growth regulator used. The purpose of this study was to determine the effectiveness of plant growth regulator 2,4-dichlorophenoksiacetic acid (2,4-D) and kinetin on callus growth, total chlorophyll, and carotenoids alfalfa callus. The study was conducted at the Laboratory of Plant Tissue Culture, Plant BSF, Department of Biology, Science Faculty UNDIP in January-December 2010. This research used Completely Randomized Design (CRD) with factorial pattern 2x4 or 8 treatments. The first factor is 2,4-D (0 mg/L, 1 mg/L), while the second factor is kinetin (0 mg/L, 1,5 mg/L, 2 mg/L, 2,5 mg/L) with 5 replications. Explants used were leaves that were grown for 30 days in medium Murashige and Skoog (MS) with 2 kinds of plant growth regulator treatment. The variables measured were the time of callus initiation, callus fresh weight, total chlorophyll and carotenoids. Data were analyzed by using Analysis of Variance (ANOVA) test followed by Duncan Multiple Range Test (DMRT) at the test level of 95%. The results showed that the fastest time of callus initiation (5,4 days) obtained in the treatment of 1 mg/L 2,4-D with 2 mg/L kinetin. The highest callus fresh weight (1,398 g) contained in the treatment of 1 mg/L 2,4-D with 2 mg/L kinetin, while the highest chlorophyll content (4,148 mg/L) and the highest carotenoid content (108,329 µmol/L) obtained in treatment 1 mg/L 2,4-D with 2,5 mg/L kinetin. Growth regulator 2,4-D is absolutely desirable to induce of callus, while kinetin would support the role of 2,4-D to optimal growth of callus. Giving both ZPT combination in MS medium, effectively increase the content of chlorophyll and carotenoids in alfalfa callus.