Mass production of Artemisinin by Cultivation of Artemisia annua hairy roots in

a Nutrient Mist Bioreactor

Document By: Bharadwaj
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Abstract: Artemisia annua an herb native to Asia is known Ior producing the anti malarial drug
artemisinin. Artemisinin content varies Irom 0.01 to 0.4 in A. annua but some varieties may
produce up to 1 artemisinin. TransIormed (hairy) roots Iormed by inIection oI the plant
using Agrobacterium rhi:ogenes, have recently been explored Ior obtaining artemisinin. Hairy
roots are genetically stable and reasonably higher artemisinin content is Iound in them as
compared to non-transIormed roots. This makes them a suitable system Ior mass production
studies. To become biotechnologically useIul hairy roots need to be scaled up to bioreactor
level. Bioreactors used Ior hairy root culture are gas-phase liquid phase or two-phase reactors.
In the present study hairy roots were generated Irom apical meristem explants. Ten Iast growing
hairy root lines were selected and artemisinin content was estimated in them using HPLC. The
root line with high growth and maximum artemisinin content was selected. The growth index
substrate utilization and artemisinin content in shake Ilasks were also determined Ior the selected
hairy root. A 4-litre Nutrient Mist Bioreactor was designed Ior mass scale root cultivation. Mist
bioreactor is a gas-phase reactor in which the culture is exposed to a mist containing medium
micro-particles and gas. This reactor has distinct advantage over liquid phase reactors which
invariably provide thin liquid layer as additional barrier Ior nutrient transIer during growth. Mist
reactors provide better nutrient and gas absorption by hairy roots. Hydrodynamic stress in mist
bioreactor is also relatively less compared to shake Ilasks (liquid-phase growth). Hairy roots
grow as a dense mass oI entangled Iilaments. This causes non-uniIorm growth characteristics and
diIIiculty in harvesting and sampling. The details oI design will be discussed in the paper.
eywords: Artemisinin Agrobacterium rhi:ogenes Nutrient mist Bioreactor hairy roots
HPLC.
ntroduction
Artemisinin is a useIul drug which is very eIIective against multi-drug resistant !asmodium
faciparum malaria. The drug is isolated Irom annual herb calledArtemisia annua. Chemical
synthesis oI Artemisinin gives low yield |1 2|. Recently genetically engineered Saccharomyces
cerevisiae have been used Ior the production oI precursors oI Artemisinin |3|. Hairy root
research is another useIul technique Ior improving the production oI Artemisinin on large scale.
Hairy roots are generated by inIecting plant explants using Agrobacterium rhi:ogenes. The
transIormed roots are genetically stable and can be easily grown in hormone-Iree basal medium.
Hairy roots produce plant metabolites equivalent to parent plants. Hairy root culture can be
scaled up to bioreactors Ior the production oI useIul secondary metabolites |4|.
*Author for Correspondence (Fax: +91-11-26582282; E-mail:ashokiitd@hotmail.com)

Materials and methods
Plant material and induction of hairy roots
Seeds and plantlets oI Artemisia annua were obtained Irom Jamia Hamdard herbal garden
(Delhi). Induction oI hairy roots was done according to the method given by Waraporn |5| with
slight modiIications as explained below. Apical meristem explants oI Artemisia annua were
surIace sterilized in 10 sodium hypochlorite Ior 5 minutes. AIter being washed three times
with sterelized water the explants were immersed in 70 ethanol Ior 1 minute. The explants
were incubated in hormone-Iree MS medium containing 3 sucrose at 25 degree Celsius
overnight under white light. AIter overnight incubation in MS media the explants were inIected
withAgrobacterium rhi:ogenes strains (Table 1) and cultured on MS at 25 deg Celsius Ior 2 days
in dark and then transIerred to MS with CeIotaxime 500 mg/l. InIection was done by dipping the
cut ends oI apical meristem explants in saturated Agrobacterium culture Ior 20 minutes. The
bacteria Iree hairy roots were subcultured in MS media every three weeks.
%able 1: Agrobacterium strains used for induction of hairy roots. Strains which caused
hairy root formation and transformed callus formation are shown in bold and italics,
respectively.
S. No. Name of strain
1. A%CC 15834
2. BA 301
3. LBA 9402
4. MTCC 2364
5. A4
6. % 532
7. LBA 920
8. B-193 NRRL
9. 5140 NCM
10. LBA 063
11. %# 105
12. LBA 406
13. ATCC 43057

Selection of hairy roots
Hairy root lines generated Irom diIIerent explants are genetically diIIerent so the most desirable
hairy root line was selected. Selection was made on the basis oI growth (as Growth Index (G.I):
(Final Iresh weight- Initial Iresh weight)/ Initial Iresh weight) and Artemisinin content ( Iresh
weight). Ten Iast growing hairy root lines were chosen and the hairy root line showing maximum
Artemisinin content was selected Ior Iurther studies.
Morphological characterisation and isolation of hairy roots
The roots generated aIter transIormation subculturing Ior a number oI generations and selection
were characterised to be hairy roots on the basis oI morphology. The roots showed Iast growth
plagiotropism and high Artemisinin content. Normal roots seldom produce Artemisinin.
Molecular characterisation was attempted using Polymerase chain reaction (PCR) Ior presence
oI ro A gene (root locus gene) in the transIormed roots |6|. Suitable primers are being designed
Ior conIirmation oI transIormed nature oI the obtained hairy roots.
Selection of Media
MS media and B5 are two commonly used and tested basal media Ior tissue culture oI hairy
roots. Roots were subcultured in both the media and growth was observed in them. Little
variation was observed in the two diIIerent media. MS media was used Ior growth oI roots as it
was reported to be better Ior growth and least polyphenol production in hairy roots |7|.
Polyphenol accumulation in media can adversely aIIect root growth and more number oI
subculture cycles may be needed.
Estimation of Artemisinin content
1 gm oI roots (blotted Iresh weight) was taken to extract and assay the content oI artemisinin.
Extraction was perIormed as done by Smith |8| by grinding the roots in 2 ml Toluene (HPLC
grade) in a mortar pestle. The extract made was centriIuged at 5000 rpm Ior 30 minutes. The
supernatant was dried under nitrogen and the dried toluene extract was stored at -20 degrees till
HPLC analysis. The dried toluene extract was resolubilised in 0.1 ml methanol and mixed with
0.4 ml 0.2 NaOH and incubated at 50 degrees in a water bath Ior 30 minutes then cooled in
tap water to room temperature. A 0.1 ml 100 methanol and 0.4 ml 0.05 M acetic acid were
added to the reaction system and then Iiltered through 0.2 micron membrane to remove grains
and turbid material. HPLC analysis was perIormed using Agilent 1200 controller and assayed at
260 nm using C18 column. The injected volume was 20 ul. The mobile phase used was 0.01 M
pH 7.0 phosphate buIIer: 100 Methanol (50:50) at a Ilow rate oI 1 ml/min. The retention time
Ior artemisinin was 6.9 7.1 minutes at 260 nm.
inetic studies in shake flasks
Growth kinetic studies were perIormed in shake Ilasks (liquid-phase culture) as previously done
by S. Srivastava |6| in our lab. Growth index artemisinin content overall growth product
Iormation and substrate utilization kinetics were studied at an interval oI 5 days up to 25 days.
Design of a Mist Bioreactor
A Mist Bioreactor was designed Ior the batch cultivation oI hairy roots |9 10|. The schematic oI
the same is shown in Figure 1.






Figure 1: Schematic of Mist Bioreactor

#esult and Discussion

Genetic transformation of Artemisia annua
Seeds and plantlets oI Artemisia annua were obtained Irom Jamia Hamdard herbal garden
(Delhi). The plantlets were planted in pots in a glasshouse and reached 6-7 Ieet in height in Iour
months. Apical meristem explants obtained Irom them were sterelised using 10 sodium
hypochlorite solution under sterile conditions and inIected with 14 Agrobacterium
rhi:ogenes strains. (Table 1).
TransIormed hairy roots were obtained Irom explants within seven days using the strains: LBA
301 LBA 063 TR 105 and ATCC 15384. The transIormation eIIiciency was calculated using
the Iormula:
TransIormation eIIiciency ((number oI explants transIormed) X 100)/ (number oI
explants used Ior transIormation)


Maximum transIormation eIIiciency was obtained using the strain LBA 301 (46.34 ). When
the hairy roots were 2 cm long they were excised Irom the parent explants and subcultured as
independent root lines in 50 ml solid MS medium. The root lines were subcultured at an interval
oI 20 days. The hairy roots turned green in presence oI light (Figure 3).



Figure 2: Generation of hairy roots from field grown plants. A: Field grown plants of A.
annua, B: Generation of hairy roots from cut ends of infected explants, C: Hairy roots, D:
Hairy roots excised from mother explants.



Medium Ior growth oI hairy roots is crucial. MS medium and B5 medium were tried Ior growth
oI hairy roots. No signiIicant diIIerence was observed in growth oI hairy roots in the two media.
The role oI solidiIying agent was also studied Ior the growth oI hairy roots. 7.6 Agar and 0.05
Phytagel were used as solidiIying agent in hairy root growth medium. Hairy roots showed
better growth in terms oI root length in Phytagel containing MS medium. This may be due to
better penetration oI light and roots in transparent Phytagel as compared to opaque agar
containing medium.
Analysis of artemisinin in A. annua hairy roots using #P-HPC
The chromatographic separation was perIormed on a C18 column using Phosphate buIIer (0.01
M pH 7): Methanol (50:50) as mobile phase. Artemisinin standard (Sigma) was detected at 260
nm at a retention time oI 6.9-7.1 minutes and a good linear relationship over the investigated
concentration range (0.05 mg/ml 0.3 mg/ml) was observed with #
2
higher than 0.99 Ior those
cases. It was Iurther successIully applied to the time-course characterization oI the production oI
Artemisinin in genetically transIormed root cultures oI Artemisia annua.



Figure 3: Artemisinin standard (Sigma) concentration used: 0.2 mg/ml




Figure 4: HPC profile of extract from Artemisia annua (Artemisinin estimated: 0.024
Mg/Gm fresh weight



The artemisinin content was estimated to be 0.024 mg/gm Iresh weight oI hairy roots.



Conclusion
Artemisia annua hairy roots can be used Ior large scale commercial production oI Artemisinin.
The induction oI hairy roots Irom explants was aIIected by various Iactors. Some strains
oI Agrobacterium caused higher transIormation eIIiciency in explants. The strain LBA 301
showed highest transIormation eIIiciency and appearance oI hairy roots was seen within 8 days.
Hairy roots oI A. annua are Iine and Iragile in morphology. MS medium was selected Ior
maintaining hairy root culture Ior successive generations. DiIIerent root lines have diIIerent
genetic make-up and thereIore diIIerent content oI Artemisinin. The rate oI growth was also
variable Ior diIIerent root lines.
The root lines generated were characterised to be transIormed in nature based on morphological
Ieatures like Iast growth rate lesser doubling time indeIinite capacity to grow in hormone-Iree
media and plagiotropism. They also produced high amount oI Artemisinin unlike normal (non-
transIormed) roots. Shake Ilask studies were done Ior optimisation oI growth conditions Ior
maximum Artemisinin production. A suitable nutrient mist bioreactor was also designed Ior
mass propagation oI the hairy roots.

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O Document By: Bharadwaj
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O www.Engineeringpapers.blogspot.com
O More Papers and Presentations available on above site
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