Selection Of Elite Cell Lines, Optimization And Elicitation Of Cell Suspension Culture Of Misai Kucing (Orthosiphon Stamineus Benth.) For The Production Of Rosmarinic Acid
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Date
2014-06
Authors
Bordbar, Laleh
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Plants from tropical rain forests have incredible abilities to produce biological
and chemical defense resources, which make them suitable leads for the synthesis of
new drugs and pharmaceutical products. Orthosiphon stamineus Benth., commonly
known as Misai Kucing is a very important medicinal plant that has been used for the
treatment of many ailments, especially as a remedy for removal of kidney stones.
Many chemical compounds were successfully isolated from O. stamineus and
rosmarinic acid (RA) is one of the major secondary metabolites. However, the
production of most secondary metabolites from plants varies due to the location and
environmental factors. Hence, in vitro culture techniques could be used as the
alternative for consistent production of these useful medicinal compounds. In the
present study, attempts were made to produce rosmarinic acid from cell suspension
culture of O. stamineu. Two callus lines of O. stamineus were induced from the in
vitro plants. The induced callus lines with another eight callus lines maintained from
the previous studies were selected and categorized based on the growth index into
three groups of fast, intermediate and slow growing lines. The factors influencing cell
biomass and secondary metabolite production which include, agitation, sucrose
concentration, medium pH, photoperiod and temperature, were studied on the
categorized cell lines. Effect of the MS medium nutrients (macronutrients and
micronutrients) was also studied in which five macronutrients of MS were
individually studied and the micronutrients were tested in different concentrations of
MS strengths (0, ½ MS, MS, 1.5MS and 2MS strengths). The impact of different
elicitors including silver nitrate, salicylic acid and methyl jasmonate and also the
effects of flask size (100, 250, 500 and 1000 mL) on cell biomass and RA production
were evaluated. The results indicated that the slow and fast growing cell lines
produced high fresh cell biomass when agitated at 120 rpm. However, the
intermediate growing cell line produced no significantly different cell biomass when
grown with agitation at 100 and 120 rpm. The fast and intermediate growing cell lines
produced higher fresh cell biomass when cultured in cell proliferation medium with
30 g/L sucrose compared to 45 g/L sucrose except it was not significantly different in
the slow growing cell line. The most suitable medium pH was 5.65 - 5.8 for cell
biomass and RA production of the three cell lines. O. stamineus cell lines showed
differences in terms of temperature and photoperiod requirment. Based on the
medium modification results two media were formulated, Bio-Mod and RA-mod
media. When the O. stamineus cells were cultured in Bio-Mod and RA-Mod media
for the production of cell biomass and RA respectively, it was found that the cells
cultured in the cell proliferation medium (MS + 1 mg/L NAA and 1 mg/L 2,4-D)
produced higher cell biomass and RA content compared to the two modified media
(Bio-mod and RA-mod). In the elicitation study,
Description
Keywords
Culture of Misai kucing , production of rosmarinic acid