Secondary metabolite from Plant organ culture

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Secondary metabolite production from organ culture - case studies

Transcript of Secondary metabolite from Plant organ culture

  • Organ Culture for Secondary Metabolite Production Case Studies 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)"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 Deciency Virus (HIV) Insect chemosterilant and as plant growth regulator Camptothecin (CPT) & 10-hydroxycamptothecin (HCPT)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 cultureProspects 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 studyObjective 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 fortied 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 cultureProduction 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 (510 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 concentrationsComparison of CPT and HCPT concentrations
  • CPT content in the different developmental stages.CPT content in the different developmental stages.
  • Variation of CPT content among the different genotypesVariation of CPT content among the different genotypes
  • Case Study 2 Crocin production from Crocus sativus
  • SaffronSaffron 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 studyObjective of the study
  • Petal, Stigma and Style : Explants- SLS induction MS medium supplemented with 23.32 M (5 mg/l ) kinetin and 21.5 M (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 solidied 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 metabolitesProduction of Secondary metabolites SLS formed on the explants were excised and dried at 40C 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, signicant from the medicinal point of view, i.e. their antibacterial, antifungal, antiamoebic, antitumour, wound-healing, anti-inammatory 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 signicant enhancement of red naphthoquinones production in hairy root cultures. Objective of the studyObjective 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 24C 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 solidied with 8 g /l Phytagar . The incubation was performed at 25C 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 25C 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 25C 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 34 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 press