Establishment of Callus and Cell Suspension Cultures for Biomass Production and Phytochemical Characterization of Vernonia amygdalina Delile

Kong, Eveline Yee Yan (2017) Establishment of Callus and Cell Suspension Cultures for Biomass Production and Phytochemical Characterization of Vernonia amygdalina Delile. Masters thesis, Universiti Malaysia Sarawak.

PDF (Please get the password by email to repository@unimas.my , or call ext: 082-583914/3942/3933) Eveline.pdf Restricted to Registered users only Download (5MB)

Abstract

Vernonia amygdalina was small tree or shrub commonly known as South Africa leaf or ‘Pokok Bismillah’ in Malaysia. It can be considered as a potential medicinal plant as it possessed various phytochemicals which were responsible for a multitude of biological activities. As the production of phytochemicals would varied according to physiological, geographical as well as environmental conditions, plant cell culture technique was a potential alternative to ensure stable production of phytochemicals. Therefore, the study began with surface sterilization of nodal explants in order to obtain aseptic cultures. Clorox® and mercuric chloride were used as sterilizing agents prior to obtain in vitro plantlets. However, mercuric chloride was not suitable due to its phytotoxic effect. Therefore, the explants were surface-sterilized with 15% Clorox® for 15 min. Next, the study was carried out to establish leaf-derived callus culture and cell suspension cultures of Vernonia amygdalina. The callus induction frequency (CIF) value of in vitro leaf explants cultured in MS media containing 0.1 to 2.0 mg/L 2, 4-D was higher (93.3 ± 6.7 % to 100.0 ± 0.0 %) than 0.5 to 4.0 mg/L picloram (73.3 ± 19.4 % to 100.0 ± 0.0 %). Combination of cytokinins (TDZ or kinetin) and auxins (2, 4-D or picloram) could not increase the callus biomass. However, callus proliferated in media supplemented with 0.5 mg/L picloram alone achieved a maximum dried weight of 159.6 ± 16.0 mg. Callus growth under continuous darkness was significantly better than in light condition. Therefore, callus was induced in media containing 2, 4-D and proliferated in media containing picloram under continuous darkness. Subculture cycle of six to eight weeks was essential to maintain callus growth and development. Callus proliferation media was found to be suitable for establishing cell suspension culture. However, the study on direct and indirect shoot organogenesis by manipulating different types and concentrations of PGRs such as BAP, TDZ, kinetin, NAA, and IBA was unsuccessful. Lastly, the effect of elicitation on the phytochemical content (alkaloids, total phenols, saponins, tannins, and flavonoids) of V. amygdalina cells by using yeast extract (YE), methyl jasmonate (MeJA) and salicylic acid (SA) was studied. Cell biomass was reduced when high concentrations of elicitors (YE and MeJA) was applied. However, cells treated with low concentration of elicitors (YE and MeJA) had comparable biomass as control. Elicitation with 0.5 µM methyl jasmonate successfully enhance alkaloid content (11.7 ± 1.1%) and total phenols (36.2 ± 0.3 mg of GA/ g of extract) present in V. amygdalina cells. Further study on the effect of elicitors on the bioactivities of V. amygdalina cells could be carried out.

Actions (For repository members only: login required)

View Item