exploitation of cell and hairy root in vitro cultures for the bio synthesis of secondary metabolites...
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Exploitation of cell and hairy root Exploitation of cell and hairy root in vitro in vitro cultures for the biosynthesis of secondary cultures for the biosynthesis of secondary
metabolites in several metabolites in several SalviaSalvia speciesspecies
ExperimentalExperimental InstituteInstitute forfor FloricultureFloriculture Sanremo Sanremo ItalyItaly
Speaker: Annalisa Giovannini
Authors: Giovannini A., D’Adamo E., Farina S., Ruffoni B., Istituto Sperimentale per la Floricoltura di Sanremo, corso Inglesi 508, I-18038 Sanremo (Imperia)
Bertoli A., Pistelli L., Dipartimento di Chimica Bioorganica e Biofarmacia Universitàdi Pisa, via Bonanno 33, I-56126 Pisa
Romussi G., Bisio A., Dipartimento di Chimica e Tecnologie Farmaceutiche ed Alimentari (DICTFA) Università di Genova, via Brigata Salerno, I-16147 Genova
AIM of the AIM of the presentationpresentationto take a survey of theto take a survey of the current current applications of tissue culture applications of tissue culture
technology in technology in SalviaSalvia species for species for handling the production of bioactive handling the production of bioactive
plant metabolitesplant metabolites
Cell culturesCell cultures
� Organic substances are extractable from cell cultures� Precursor feeding, transformation methods and immobilization techniques can be employed in order toobtain high yields suitable for commercial exploitation� Automated control of cell growth and rationalregulation of metabolite processes would reduce of laborcosts and improve productivity
Undifferentiated plant cell cultures are capable of producing specific medicinal compounds at a rate similaror superior to that of intact plant. The biosynthetic activityof cultured cells can be enhanced by regulatingenvironmental factors, as well as by artificial selection of high producing lines
MAJOR ADVANTAGESMAJOR ADVANTAGES
Hairy root culturesHairy root cultures
• Hairy root cultures have an elevated biosyntheticpotential combined with genetic stability and high growthrates, moreover are able to biotransform naturalcompounds• Long-term aseptic hairy root cultures have beenestablished in more than 200 species of higher plants
• The roots induced by the soil-borne Gram negative pathogen Agrobacterium rhizogenes represent a trulyremarkable range of biosynthetic capabilities, with theirability to synthesize a wide diversity of secondarymetabolites and to adjust their metabolic activities in response to biotic and abiotic stress
Current applications in Current applications in SalviaSalvia sppspp..
Ge and Wu, ApplMicrobiolBiotechnol. 2005 68(2): 183-8
Yeast elicitor + BABA(amino acid beta-aminobutyricacid)
Hairy root cultures
Diterpenoidtanshinones
Salvia miltiorrhiza
Yan et al., Journal of Biotechnology 2005 Vol 119 (4): 416-424
Yeast elicitor + hydrophobic polymeric resin (X-5)
Hairy root cultures
Diterpenoidtanshinones
Salvia miltiorrhiza
Zhang et al., Planta Med. 2004 70(2): 147-51
Ag(+)Hairy root cultures, ATCC 15834
Diterpenoidtanshinones
Salvia miltiorrhiza
Chen et al., Enzyme MicrobTechnol. 2001 28(1): 100-105
Yeast elicitorHairy rootcultures, ATCC 15834
Cryptotanshinone, tanshinone I, tanshinone IIA and tanshinone IIB, rosmarinic acid and lithospermic acid B
Salvia miltiorrhiza
ReferencesElicitorCulture typeSecondarymetabolites
Plant name
Skala and WysokinskaNaturforsch [C]. 2005 60(7-8):583-6
Roots of micropropagated plants
Tanshinone I and IIA
Salvia przewalskii
Li et al., Phytochemistry2006 Jan 19
Hairy rootexpressing (CaMV) 35S
CHI gene
Apigenin, total flavonoids
Salvia involucrata
De Felice et al., Proc. XLVIII SIGA Congr. 2004
Cell and hairy root cultures (ATCC 15834)
Cytotoxic activity against colon and lung cancer and mammary carcinoma
Ortonaphtoquinone diterpensand novel compounds
Salvia sclarea
Fraga et al., J Agric Food Chem. 2005 29;53(13):5200-6
Hairy root
cultures
Antifeedant against insect pests Spodoptera littoralisand Leptinotarsadecemlineata, cytotoxic against mammalian cell
New diterpensSalvia broussonetii
ReferencesCulture type
ActivitySecondarymetabolites
Plant name
In the framework of the European INTERREG ALCOTRA Project �SALVIE�, for the valorisation of new germplasm, the research group of the Propagation Section, in collaboration with Pisa and Genoa Chemical Departments proposed to developed efficient protocols for in vitro tissue culture in some ornamental Salvia species grown in the �Riviera dei Fiori� with new pharmacologicalproperties
Micropropagated plantswere used as a source of
explants
The ProjectThe Project
CallusCallus inductioninductionCallus developed from leaf and stem explants of micropropagated plants on callus induction medium in:
S. cinnabarinaS. corrugataS. elegansS. jamensis “La siesta”S. somaliensisS. wagneriana
Callus induction medium containing basal salts and vitamines MS, 30 g/l sucrose and 8 g/l agar, with 2,4-D 0,5 mg/l and Kinetin 0,5 mg/l, pH 5,7
S. jamensis �La siesta�
S. elegans
Explants developing callus and explant viability after one monthof culture on callus induction medium in the light
01 02 03 04 05 06 07 08 09 0
1 0 0pe
rcen
tage
S . c in n a b a rin a S . co rru g a ta S . w a g n e ria n a
c a llu s
v ia b ility
S. somaliensisfriable callus
developed from leafexplant on callus
induction medium
CellCell culturescultures
S. cinnabarina, S. elegans and S. somaliensis cell cultures were established from callus. They were grown in 250 ml Erlhenmeyerflasks with 30 ml of callus induction liquid medium and cultured in the growth chamber, in the light with continuous rotary shaking (90 rpm). After 15 days of culture the callus masses were filtered through a 500 µm iron sieve. Once the cell cultures had been established, the medium was substituted weekly with fresh medium.
HairyHairy rootroot inductioninduction: basic : basic protocolprotocolPlantPlant material: material: leaf (30-40 mm²) and stem (20-30 mm) tissues of micropropagated plants
AgrobacteriumAgrobacterium rhizogenesrhizogenes wild wild typetype strainsstrains: : ATCC 15834, NCPPB 1855Bacterial solution: overnight-grown bacterial suspension was diluted 1: 10 (v/v) in sterile water (0.1 OD at 550 nm)
CoCo--cultivation: cultivation: explants were soaked for 20 min in the bacterial suspension; infected and control fragments were then placed in Petri dishes on a medium without growth regulators, containing basal saltsand vitamines MS, 30 g/l sucrose and 8 g/l agar (Basal Medium: BM)After three days all the explants were transferred to BM medium supplemented with 100 mg l-1 Cefotaxime (BM-CX)
MolecularMolecular analysisanalysis: : single hairy root, protruted from co-cultivatedexplants were isolated and screened by PCR amplification of genomicDNA with bacterial genes
S. S. wagnerianawagneriana hairyhairy rootrootinductioninduction fromfrom coco--cultivatedcultivated stemsstems
S. wagneriana hairyroot induction fromco-cultivated leaves
S. S. wagnerianawagneriana hairyhairy rootsroots protrutedprotruted fromfrom stemstemtissuetissue
S. S. cinnabarinacinnabarina control and control and coco--cultivatedcultivated leavesleavesafter one after one monthmonth of culture in the lightof culture in the light
Explants developing hairy roots after 60 days of co-cultivationof stem cuttings with two A. rhizogens strains
TransformationTransformation efficiencyefficiency
0
10
20
30
40
50
60
70
80
perc
enta
ge
S.cinnabarina
S. coccinea S. corrugata S. jamensis"La siesta"
S.wagneriana
ATCC 15834
NCPPB 1855
HairyHairy rootroot culturesculturesSeveral hairy root lines, obtained from a single transformation event, were selected for each species and cultivated in solid BM-CX medium. PCR amplification of A. rhizogenes rolC gene was detected in each line
HairyHairy rootrootselectedselected lineslines, ,
cultivatedcultivated in in solidsolidBMBM--CX mediumCX medium
S. cinnabarina 1855 hairy root line C1
S. wagneriana 15834hairy root line D3
S. S. jamensisjamensis ““La SiestaLa Siesta�� isolatedisolated hairyhairy rootrootlineline
S. cinnabarina hairy root line C1 and S. wagneriana 15834hairy root line D3 liquid cultures were established in 300 ml glass vases with 150 ml of BM-CX medium and cultured with continuous rotary shaking (78 rpm) in the dark. Culture medium was substituted at 10 day intervals with fresh medium
HairyHairy rootroot liquidliquid culturescultures
Handling Handling tissuetissue culture culture conditionsconditions
Jasmonic acid (6,6, 13,3 and 26,6 mg/l)
Casein hydrolysate (200 and 400 mg/l)
Cellulase (100mg/l)
Macerozyme (100mg/l)
ElicitorsElicitorswere added tothe liquidculture medium and after 10 days hairy rootswere taken forfurther analysis
S. wagneriana 15834 hairy root line D3 cultureconditions were modified:
light temperature (5°C for 12h and 24h) carbohydrate supply (Mannitol 20g/l and 40 g/l)
InvestigationInvestigation on on Salvia Salvia wagnerianawagnerianasecondarysecondary metabolitesmetabolites
Secondary metabolitesextracted from Salvia
wgneriana adult plants
Flavonoids, phenolicacids, triterpens,
diterpens:
quercetin, apigenin, luteolin, apigenin 7-
glucoside, caffeic acid, chlorogenic acid, ursolic
acid, betulinic acid, lupeol, β-sitosterol, and new unknown diterpens
Salvia wagnerianaSCREENING by TLC
Extracts
Diterpene stds
Triterpene stds
Hairy root culture extractsCORRESPONDENCE
Ursolic acidβ-sitosterol
CORRESPONDENCE
sw98, sw22 sw92, sw4
TLC procedureNormal phase: SiO2Mobile phase: C/M 10:0,5 Spray reagent: solphoric vanilline
No correspondencereference Flavonoids
(Normal/Reverse phaseSpecific spray reagents)
Results of the SCREENING by DIRECT INFUSION and LC-DAD-ESI-MS Salvia wagneriana hairy root culture extracts
Presence of ursolic acid, β-sitosterol
Presence of caffeic acid and other flavonoids different from the available reference material in the samples elicitatedwith casein hydrolysate (200 mg/l), jasmonic acid (6,6 mg/l) and conditioned with Mannitol (40 g/l). Evaluation of different extraction procedures has to be performed
Presence of diterpene SW 22, SW4 and SW23 especially in the samples elicitated with jasmonic acid (6,6 mg/l) and conditioned with Mannitol (40 g/l)
QUALITATIVE ANALYSIS
Investigation on the fragmentation pathways of unknown terpenoidic and flavonoidic compounds is in progress
ConclusionsConclusionsCell and hairy root cultures have been established in potentially useful Salvia species. Efforts are currently on the way to manage the extraction and the characterization of sage secondary metabolites from in vitro cultures and to monitor the effect of elicitation on culture growth and on the production of the bioactivecompounds
BIOACTIVE BIOACTIVE COMPOUNDSCOMPOUNDS
MyMy sincere sincere thanksthanks toto
Barbara Claudio and the StaffBarbara Claudio and the Staff
ForFor theirtheir special care and special care and efficiencyefficiency regardingregarding allall detailsdetails
toto the success of the success of thisthis SymposiumSymposium