Preparation Of Cell Suspension Culture Of Artemisia Annua L. For The Production Of Artemisinin
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Date
2005-04
Authors
Singaram, Nallammai
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Abstract
Artemisia annua from the Asteraceae family contained artemisinin that had
proven to have anti-malarial properties. Elite clones of Artemisia annua were selected
based on plant height from the population of eight-week old in vitro plantlets. The
selected elite clones were maintained via nodal cuttings on growth regulator free solid
MS medium + 3% (w/v) sucrose. The selected elite clones were found to contain
artemisinin in the range of 90 - 330 ~g/g (OW) in the aerial portion of the plant. The
leaves and roots of A. annua were found to be better explants for the production of
friable type of callus when cultured on MS medium + 0.5 mg/L SA + 0.5 mg/L NAA and
produced higher amount of artemisinin compare to the stem as an explant. Yellowish
white calli derived from leaves were friable and fast growing but contained lower
artemisinin (1.7 J..lg/g OW) compared to the green compact calli (3. 7 J..!glg OW). The MS
medium supplemented with 0.25 mg/L 2,4-0 and 0.25 mg/L SA induced the most
friable and fine cells but with a very low cell biomass. The cells induced on picloram
supplemented MS medium were soft, fine and friable but browning of the cells occurred
after culturing ·in this medium. The addition of IAA or ISA into the MS liquid medium
induced root formation from the A. annua cells after two weeks of culture. However, the
production of artemisinin (8.2 J..lg/g OW) was high in cells cultured in culture medium
supplemented with 0.25 mg/L IAA + 0.25 mg/L SA. The growth curve of A. annua cell
suspension in Y2MS medium + 0.25 mg/L SA + 0.25 ·mg/L NAA showed an optimum
increase in cell biomass at the late exponential stage (15th day of culture). The optimum
production of artemisinin was seen in the early exponential (9th day of culture) and late
stationary stage (21 51 day of culture). The addition of casein hydrolysate (0.1 or 0.5 giL)
stimulated the growth of the A. annua cells and the friability of the cells but did not
stimulate an increase in the production of artemisinin. While, the addition of 2 mg/L GA3
to the liquid culture medium containing ~MS + 0.25 mg/L BA + 0.25 mg/L NAA did not
stimulate the growth of the A. annua cells but increased the production of artemisinin to
50 ~g/g OW. The A. annua cells were found to gro·..v well in ~MS medium + 0.25 mg/L
BA + 0.25 mg/L NAA supplemented with 10 - 30 g/L of sucrose and higher
concentration of sucrose (50 - 120 g/L) inhibited the increase in cell biomass and did
not stimulate the production of artemisinin. The amount of macronutrient (KH2P04,
MgS04.7H20, and KN03) in the ~MS medium had a positive effect on the A. annua
cell biomass as well as the artemisinin content. The presence of more than 3- mM of
CaCI2.2H20 did not affect the A. annua cell biomass. The nitrogen ions provided in the
form of potassium nitrate (KN03) was found to be more suitable than ammonium nitrate
(NH4N03). The addition of NH4N03 to the culture medium reduced both the artemisinin
content and the cell biomass. The optimized medium consisted of 0.67 mM KH2P04,
2.04 mM MgS04.7H20. 3 mM CaCI2.2H20, 7.5 mM KN03 and 2.3 mM NH4N03 in Y2MS
medium supplemented with 0.5 g/L casein hydrolysate + 30 g/L sucrose + 0.25 mg/L
BA + 0.25 mg/L NAA with the pH adjusted to 5.7 induced a homogenous A. annua cells
with a high cell biomass. A 2.5 fold increase in cell biomass was obtained for a 0.5 g of
fresh cells/ 50 ml medium cultured in the optimized medium for seven days. A largestale
production of A. annua cells showed no decrease in the growth index while the
artemisinin content in the cells increased from 4 ~g/g (OW) to 12 ~g/g (OW). However,
the amount of artemisinin obtained in the cell suspension culture was lower than that in
the in-vitro plantlets.
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Keywords
Artemisia Annua L