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