Secondary metabolite production from organ culture - case studies

1. Organ Culture for Secondary Metabolite
Production – Case Studies
A. Manivannan
Scientist (Genetics)
DMR, New Delhi

2. Case Study 1
Camptothecin production from
Camptotheca acuminata

4. "Cancer tree, Tree of Life “Faith Drop” (Camptotheca acuminata)

5. Pentacyclic quinolines camptothecin and 10-
hydroxycamptothecin through the monoterpene indole
alkaloid pathway.
Camptothecin inhibits DNA Topoisomerase I and is very
effective against cancer cells in culture.
Treatment of ovarian, colorectal and small-cell lung cancer
Inhibitory activity against
Fowl plague virus
Trypanosomes and Leishmania
Human Immune Deficiency Virus (HIV)
Insect chemosterilant and as plant growth regulator
Camptothecin (CPT) & 10-hydroxycamptothecin (HCPT)

6. Camptotheca acuminata is also considered to be over
harvesting in its natural habitat and the threat of
major fungal diseases such as leaf spots and root rot
could seriously limit the cultivation of Camptotheca, and
thereby greatly diminish the production of CPT
Secondary metabolite Production
@ In-vitro plant propagation methods on solid medium
@ Temporary Immersion System (TIS), Bioreactor
Prospects of Campthoeca Organ culture

7. 1. To examine the variations of CPT and HCPT
contents in in vitro organ cultures of
C.acuminata grown in different culture systems
2. Analysis of CPT content was performed
on cell suspension cultures, shoots grown on
solid medium and in liquid culture medium using
the TIS.
Objective of the study

8. Shoots, calli and embryos initiated from these seedlings were employed for CPT analysis
Four seedlings were randomly chosen from each sources for further cultivation
(Ch 1 = Bp31, Bp81, Bp101, Bp141), (Ch 2 = Mp27,Mp28,Mp35,Mp36), (Lou = Lp4,
Lp13b, Lp18, Lp45).
Embryogenic calli and somatic embryos were cultured on half strength MS medium
supplemented with 2 mg/l BAP plus 0.1 mg /l IAA.
Shoot tips were excised and subcultured for a period of 8 weeks in TIS and on solid
medium.
Explants were cultured in both the Dual-Vessel System (DVS) and RITA vessel as
previously described containing a full strength MS medium fortified with 0.5 mg/l BAP
plus 30 g/l sucrose
During the first 4 weeks 200 ml culture medium was used in DVS and thereafter 400
ml and for the RITA vessel a volume of 250 ml was used throughout this study
.
Production of Secondary metabolites through Organ culture

9. Seven shoots (20 mm in height) were cultured in each vessel.
Immersion cycles set to 1 min every 3 and 6 h, were controlled by
electronic timers for both DVS and RITA
.
All cultures were of the same age and were maintained under a
16-h photoperiod at 25 ± 1C.
Collected into a 50 ml centrifuge glass tube to which 5 ml of 61%
ethanol was added.
The extract was centrifuged at 15,000g for 10 min
and the supernatant was filtered through a filter into HPLC vials
for CPT analysis.

10. Suspension culture
Cell suspension culture (5–10 ml) was centrifuged for 10 min to separate
the cells from supernatant.
Supernatant was filtered directly into HPLC vials for analysis.
The remaining cell sediment was prepared for analysis similar to the
above described method.
TIS liquid culture medium were filtered through a PTFE 0.45 lm directly
into HPLC vials for CPT and HCPT analysis.
Determination of CPT by HPLC
All analyses were performed on an isocratic reverse-phase high
performance liquid chromatography system (RP-HPLC).

11. Comparison of CPT and HCPT concentrations

12. CPT content in the different developmental stages.

13. Variation of CPT content among the different genotypes

16. Case Study 2
Crocin production from
Crocus sativus

18. Saffron
Saffron is the most expensive and precious spice in the
world
Treatment of cerebrovascular and cardiovascular diseases
Crocin, picrocrocin and safranal
one hectare of Crocus sativus L. Produces only about 6 kg of
dried saffron from 900,000 flowers traditional cultivation
cannot meet the increasing need for it.
In vitro induction of stigma-like-structure (SLS) from
floral organs alternative way to solve crocin supply
problem

19. To determine the elicitors compounds (like L-
alanine, coconut milk or glutamine) to
increase frequency and crocin production
To optimize the carotenoid biosynthesis
precursors ( sodium acetate, serine and glycine)
Objective of the study

20. Petal, Stigma and Style : Explants- SLS induction
MS medium supplemented with 23.32 µM (5 mg/l ) kinetin and 21.5 µM (4 mg/ l ) NAA
was used as basal medium.
To evaluate the effect of different additives on SLS induction and crocin production
Sodium acetate (SA), serine and glycine were added
activated charcoal and PVP (anti browning agents) , All media contained 6% (w/v)
sugar and were solidified with agar (0.6%, w/v)
Culture was maintained at room temperature in the dark, Effect of light on
induction was also studied
.
Production of Secondary metabolites
SLS formed on the explants were excised and dried at 40⁰C for 8 h
Crocin extraction : methanol extraction
Crocin in all the extraction samples were quantified by the reverse-phase HPLC

24. Case Study 3
Naphthoquninone production from
Lithospermum canescens

26. Naphthoquinones are shikonin derivatives ( acetylshikonin (ACS) and
isobutyrylshikonin (IBS) from Lithospermum canesence
L. canescens transgenic roots has been performed and
hydroxyvalerylalkannin and isobutylalkannin have been reported as novel
metabolites within Lithospermum genus
Broad spectrum of activity of these naphthoquinones, significant from the
medicinal point of view, i.e. their antibacterial, antifungal, antiamoebic,
antitumour, wound-healing, anti-inflammatory and immuno-stimulating
properties.
The effects produced by naphthoquinones have been attributed to the
inhibition of topoisomerase I and II, protection from UV-radiation,
stimulation of peroxidase, and inhibition of microsomal monooxygenase
Shikonin Derivatives

27. Study of root growth and shikonin derivatives
accumulation in three transgenic root lines of L.
canescens in order to identify the best candidate for the
production of shikonin derivatives
To choose the optimal growth stage to achieve the
highest red pigment productivity.
Developing a satisfactory strategy leading to significant
enhancement of red naphthoquinones production in
hairy root cultures.
Objective of the study

29. Hair root Induction from leaf

30. Hairy root cultures of L. Canescens ( A. rhizogenes strains ATCC 15834, LBA
9402 and NCIB 8196)
.
The bacteria were grown on YEB solid medium 24 h at 24 C, in the dark.
single colonies were inoculated into 50 ml YEB liquid medium and cultured
72 h at 24⁰C in the dark, on a gyratory shaker at 120 rpm.
The bacterial cultures were diluted (1:4) with YEB liquid medium before trans-
formation.
The leaves and stems of 6-week-old shoots were directly wounded with sterile
needles containing bacterial suspension.
The infected shoots were placed on hormone- free LS medium with the
addition of sucrose (30 g/l ) and solidified with 8 g /l Phytagar .
The incubation was performed at 25⁰C and under light for 18 h per day
Transgenic root lines and culture conditions

31. The roots emerging from the infected sites were transferred to the liquid
hormone-free LS medium supplemented with 0.05% Claforan (autoclave)
Cultured individually at 25⁰C in the dark on a gyratory shaker at 120 rpm.
Three lines of transgenic roots: Lc1A, Lc1D and Lc1G resulted from
transformation with strain ATCC 15834, were chosen for further investigations
and stably maintained over successive subcultures.
Every 6 weeks the roots were transferred to 50 ml of hormone-free liquid LS
medium in 250 ml Erlenmeyer flasks.
The transgenic roots were incubated at 25⁰C in the dark on a shaker at 105
rpm.

32. A time course study of hairy root growth and shikonin
derivatives production were examined starting from day 4 up
to day 42 of culture.
The hairy roots were cultivated as described above. Every 3–4
days samples of each root line from two flasks were
harvested.
The biomass increase measured as dry weight, was
investigated. The collected roots were gently pressed on filter
paper to remove excess medium.
Then they were lyophilized and their dry weight was recorded.
The post-culture media were filtered and the pH of the media ,
their conductivity and sucrose levels were determined
Determination of growth and shikonin derivatives Accumulation

33. To improve shikonin derivatives accumulation, the transgenic roots
were moved from LS medium to M9 medium and cultivated in 250 ml
Erlenmeyer flasks containing 50 ml of liquid M9 medium (Fujita et al.
1981).
The culture conditions were as described above. The 25-day-old or
32-day-old transgenic roots of each root line were moved to the M9
medium. After 1, 2 and 3 weeks of culture, roots from two flasks were
taken.
To study the influence of methyl jasmonate (MJ) on shikonin
derivatives accumulation the 25-day or 32-day- old transgenic roots
were cultivated for 2 weeks in liquid M9 medium supplemented with
100 lM MJ, added to the
medium after autoclaving.
The collected roots were gently pressed on filter paper, lyophilized
and their dry weight was determined. The post-culture media were
filtered and subjected to chemical analysis.
Treatment for enhancement of shikonin derivatives production

35. The results reported here show how the medium exchange can stimulate the
shikonin derivatives accumulation in the transformed roots of L. canescens
which seem to be a very promising alternative source for naphthoquinone
pigment production.
The three L. cansecens transgenic root lines produced the highest levels of
shikonin derivatives when on day 32 they were transferred to M9 medium
and cultured for 3 weeks. In these conditions, the total ACS and IBS content of
the highest producing root line Lc1D (182.01 mg l -1 ) was increased over
eightfold.
4.7-fold increase in the total ACS and IBS yield compared to natural roots.
However, the addition of methyl jasmonate caused both the reduction
of biomass increase and shikonin derivatives yield.
Transgenic roots of L. canescens could be considered as a potent and
abundant source of compounds demonstrating potential anticancer
properties.
Conclusions