Organ Culture for Secondary Metabolite Production – Case Studies A. Manivannan Scientist (Genetics) DMR, New Delhi
Case Study 1 Camptothecin production from Camptotheca acuminata
"Cancer tree, Tree of Life “Faith Drop” (Camptotheca acuminata)
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 De?ciency Virus (HIV) Insect chemosterilant and as plant growth regulator Camptothecin (CPT) & 10-hydroxycamptothecin (HCPT)
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
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
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 forti?ed with 0.5 mg/l BAP plus 30 g/l sucrose During the ?rst 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
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 ?ltered through a ?lter into HPLC vials for CPT analysis.
Suspension culture Cell suspension culture (5–10 ml) was centrifuged for 10 min to separate the cells from supernatant. Supernatant was ?ltered 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 ?ltered 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).
Comparison of CPT and HCPT concentrations
CPT content in the different developmental stages.
Variation of CPT content among the different genotypes
Case Study 2 Crocin production from Crocus sativus
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 ?owers traditional cultivation cannot meet the increasing need for it. In vitro induction of stigma-like-structure (SLS) from ?oral organs alternative way to solve crocin supply problem
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
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 solidi?ed 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 40?C for 8 h Crocin extraction : methanol extraction Crocin in all the extraction samples were quantified by the reverse-phase HPLC
Case Study 3 Naphthoquninone production from Lithospermum canescens
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, signi?cant from the medicinal point of view, i.e. their antibacterial, antifungal, antiamoebic, antitumour, wound-healing, anti-in?ammatory 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
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 signi?cant enhancement of red naphthoquinones production in hairy root cultures. Objective of the study
Hair root Induction from leaf
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 24C, in the dark. single colonies were inoculated into 50 ml YEB liquid medium and cultured 72 h at 24?C 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 solidi?ed with 8 g /l Phytagar . The incubation was performed at 25?C and under light for 18 h per day Transgenic root lines and culture conditions
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 25?C 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 ?asks. The transgenic roots were incubated at 25?C in the dark on a shaker at 105 rpm.
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 ?asks were harvested. The biomass increase measured as dry weight, was investigated. The collected roots were gently pressed on ?lter paper to remove excess medium. Then they were lyophilized and their dry weight was recorded. The post-culture media were ?ltered and the pH of the media , their conductivity and sucrose levels were determined Determination of growth and shikonin derivatives Accumulation
To improve shikonin derivatives accumulation, the transgenic roots were moved from LS medium to M9 medium and cultivated in 250 ml Erlenmeyer ?asks 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 ?asks were taken. To study the in?uence 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 ?lter paper, lyophilized and their dry weight was determined. The post-culture media were ?ltered and subjected to chemical analysis. Treatment for enhancement of shikonin derivatives production
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