2011. aranzales rondon. use of tissue culture techniques techniques to the conservation and exchange...

Use of Tissue Culture Techniques to the Use of Tissue Culture Techniques to the Conservation and Exchange of Cassava Conservation and Exchange of Cassava GermplasmGermplasmMaterial / Health Certification of Planting MaterialMaterial / Health Certification of Planting Material

Ericson Aranzales Rondon

[email protected]

Graciela MaflaGraciela Mafla

Maritza Cuervo

GENETIC RESOURCES PROGRAM GENETIC RESOURCES PROGRAM

“Advance Course in Modern Technologies for Cassava Production and Processing: Support to Sustainable Development of the Cassava Sector in the

Caribbean Region”

March 14, 2011

.... . .

CIAT

Cali

Colombia

.... .

. .. ...

Holding Holding GermplamGermplam in CIATin CIATRegister into the International TreatyRegister into the International Treaty

Crops Taxa( No.)

Accessions(No.)

Beans (Phaseolus) 36,407

Rank(No.)

1 44

Country oforigin (No.)

110

(Agreement signed with the Governing Body on October 16, 2006)

Cassava (Manihot)

Tropical forages

6,592

23,140

65,929

331

1 734

28

75

(No. 2: Beans, USDA 17,192; Cassava, EMBRAPA 3,902; Tropical forages, ILRI 18,661)

Germplasm materials as International Public Goods

811

- Manihot is a Neotropical genus, distributedin its natural habitat from the southern UnitedStates through Mesoamerica, South America,Brazil, Perú, Bolivia, Paraguay and Argentina.

- Cassava is the fourth most important supplierof food calories in the tropics.

CassavaCassava

MOCHICA-PERU CULTUREAMANO MUSEUM-LIMA. 1300 B.C.

of food calories in the tropics.

-The principal economic product is starchyroots, which are utilized in a wide range of enduses, most notably including human food,animal feed, and industrial products.

Landraces

Wild

5,301

883

INTERNATIONAL PUBLIC GOODSINTERNATIONAL PUBLIC GOODSStatus of Status of ManihotManihot germplasmgermplasm at CIATat CIAT-- GRPGRP

Breeding Material (CIAT)

Total

408

6,592

Accessions registered into the Multilateral System of the International Treaty

Source: CIAT-GRP, 2011

Composition of the Manihot esculenta collection

SOURCE REGIONS ACCESSIONS

Colombia 2,000

Brazil 1,281

Other countries South America (6) 1,127

Others, Central America and Caribbean (11) 490 Others, Central America and Caribbean (11) 490

Asia (7) 374

Other countries (2) 29

5,301 landraces (28 countries)5,301 landraces (28 countries)

Priorities for exploration: Bolivia, Brazil, Colombia, Haití, Nicaragua, Perú, Venezuela,

Paraguay ( Hershey, 2008).

Future acquisitions: Perú (600), Guyana (90), and Central America and Caribbean (40).

Source: GRP-CIAT, 2011

Holdings of cassava Holdings of cassava germplasmgermplasm at CIATat CIAT-- GRPGRP

Colombia 2,000 (38,5%)

Country of origin

Accessions(No.)

Brazil 1,281 (24,7%)

421 (8,1%)Peru

Venezuela 253 (4,8%)

208 (4,0%)Paraguay

Costa Rica 81 (1,5%)

Thailand 37 (0,7%)

Nigeria 19 (0,4%)

Pto. Rico

United States 10 (0,2%)

17 (0,3%)

Country of origin

Accessions(No.)

Honduras 27 (0,5%)

Jamaica 20 (0,4%)

Costa Rica 81 (1,5%)

Indonesia 253 (2,6%)

Argentina 122 (2,3%)

Ecuador 116 (2,2%)

Mexico

Guatemala

106 (2,0%)

92 (1,7%)

Cuba 82 (1,5%)

Malaysia 61 (1,1%)Panama 47 (0,9%)

Vietnam

Salvador

Bolivia

Fiji

Philippines

Dominican Rep

China

9 (0,2%)

10 (0,2%)

7 (0,1%)

6 (0,1%)

6 (0,1%)

2 (0,1%)

5 (0,1%)Nicaragua 3 (0,1%)

5,301 Landraces (28 countries) Source: GRP-CIAT, 2011

Species Genotypes(No.)

M. brachyloba 1 (0,1%)

54 (6,1%)M caerulescens

M. carthaginensis 148 (16,7%)

M. cecropiaefolia 6 (0,6%)

M. chlorosticta 6 (0,6%)

M. esc. flabellifolia 314 (35,5%)

M. filamentosa 5 (0,5%)

M. fruticulosa 1 (0,1%)

M. glaziovii 5 (0,5%)M. guaranitica 35 (3,9%)

M. pentaphylla 1 (0,1%)

M. peruviana 139 (15,7%) M. purpureo-costata 1 (0,1%)

M. pseudoglaziovii 1 (0,1%)



M. longepetiolata 6 (0,6%)

M. sparsifolia 2 (0,2%)

M. tristis 6 (0,7%)

Holdings of Holdings of ManihotManihot wild species at CIATwild species at CIAT-- GRUGRU

M. aesculifolia 4 (0,4%)

M. alutacea 8 (0,9%) 2 (0,2%)M. anomala

M. dichotoma 4 (0,4%)

M. epruinosa 27 (3,0%) M. filamentosa 5 (0,5%)

M. irwinii 2 (0,2%)

M. orbicularis 10 (1,1%)

M. peltata 1 (0,1%)

M. pilosa 2 (0,2%)

M. jacobinensis 13 (1,4%)

M. pseudoglaziovii 1 (0,1%)

21 (2,3%)M. janiphoides

M. rubricaulis 15 (1,7%)

M. quinquepartita 2 (0,2%)

M. triphylla 14 (1,6%)

M. hastatiloba 4 (0,4%)

M. violacea 23 (2,6%)

883 genotypes (33 species) Source: GRP-CIAT, 2011

SPECIES POTENTIAL BENEFITS

M. M. flabellifoliaflabellifolia PohlPohl Resistance to whitefly Aleurotrachelus socialis Bondar

M. M. alutaceaalutacea RogerRoger y y AppanAppan Resistance to mites

M. M. chlorostictachlorosticta StandleyStandley y y GolmanGolman Resistant to mosaic virus African and tolerant to drought

M. M. carthaginensiscarthaginensis MuellerMueller von von ArqauArqau Tolerant to drought and the high activity to pyruvate decarboxylase fosfoenol

Why conserve wild species?

M. filamentosa M. filamentosa PittierPittier High pontential as forages

M. M. glazioviiglaziovii MuellerMueller von von ArqauArqau Resistant to mosaic virus African

M. M. guaraniticaguaranitica ChodatChodat y y HasslerHassler Tolerance to low temperatures

M.M. longepetiolatalongepetiolata Roger y Roger y AppanAppan Lower length in plants

M. M. orbicularisorbicularis PohlPohl Tolerance to aluminum toxicity

M. M. peltatapeltata PohlPohl

M. M. TristisTristis MuellerMueller von von ArgauArgau

Tolerance to acid soils

High protein and starch content

Source: Chavez et al., 1987; Bellotti, 2008; Carabali, 2010

CONSERVATION ALTERNATIVES FOR CASSAVA CONSERVATION ALTERNATIVES FOR CASSAVA

FIELD FIELD

Collection have traditionally been maintained in field plots.

Technical simplicity and the availability of planting material for evaluations, breeding nurseries, or others uses.

Periodic renewal every one or two years is desirable to avoid problems of excessive vegetative growth, cumulative disease and insect problems.

Material is not available for international shipment.Material is not available for international shipment.

IN VITROIN VITRO

Certified against viruses of quarantine importance and ready for distribution.

Need periodic regeneration.

Material is available for international shipment.

CRYOPRESERVATIONCRYOPRESERVATION

Conservation could theoretically be carried out indefinitely with no

need for renewal. Long-term storage.

Inadequate for international distribution

Investment in personnel and equipment.

Need periodic regeneration, but not frequent.

Suitable for storage and distribution of species.

SEEDSEEDSEEDSEED

Long-term storage.

Manihot wild species have not been systematically tested

for seed storage behaviour.

Seed storage behaviour: orthodox?

Preserving genes, but not the specific combinations of

the parent clon(s).

Need periodic regeneration, but not frequentSuitable for storage and distribution of species


BONSAIBONSAI


Slow-growth system based on restricting root development in small planting pots. Plants occupy only small fraction of the space they would

occupy if allowed unlimited growth in the field.


POLLENPOLLEN

Cryopreserved pollen should be a good system of preserving populations of genes.

Allow long-term conservation.

It can be used almost immediately (Breeder´s perspective).

Material is available for international shipment.

Protocols not available – in process.

DNA BANKDNA BANK

Protocols not available – in process.

International distribution is permitted

Potential use in research

Limited use genomics studies

Cost effectiveness


Import permit issued by Import permit issued by Import permit issued by Import permit issued by Colombian Agricultural Institute Colombian Agricultural Institute

(ICA)(ICA)

PhytosanitaryPhytosanitary CertificateCertificatePhytosanitaryPhytosanitary CertificateCertificate

Donor countryDonor country

PROCEDURES FOR INTRODUTION OF CASSAVA GERMPLASM

Virus eradicationVirus eradication

In vitro In vitro conservation conservation laboratorylaboratory

INDEXING (GHL)

Inspection of plants to determine if Inspection of plants to determine if there is any plant health problem

(fungi and bacteria)

40oC/35oC

Thermotherapy

INDEXING

STAKES

Thermotherapy

INTRODUCTION OPERATIONSINTRODUCTION OPERATIONS

37oC/35oC

12 days e/o

EMBRIÓNEmbryo

INDEXING

(GHL)

IN VITRO PLANTS

SEEDS

Rescue embryo

Growth media: 4E, 17N

Light intensity: 18.5 µmol.m-2.s-1

Photoperiod: 12 h light/12 h dark

Temperature: 26 – 28 oC

PATHOGENS OF QUARANTINE IMPORTANCEPATHOGENS OF QUARANTINE IMPORTANCE

• Implementation of sanitation Implementation of sanitation techniques:techniques:

Thermotherapy followed by Thermotherapy followed by meristemsmeristems cultureculture

Thermotherapy Thermotherapy in vitro in vitro

• Indexation of clonesIndexation of clones

GermplasmGermplasm Health LaboratoryHealth Laboratory-- GHLGHL

••Propagation of healthy materialPropagation of healthy material

PATHOGENS OF QUARANTINE SIGNIFICANCE TO PATHOGENS OF QUARANTINE SIGNIFICANCE TO DETECT BY GHLDETECT BY GHL

COMMONCOMMON MOSAICMOSAIC VIRUSVIRUS ((CsCMVCsCMV))

•• PotexvirusPotexvirus GenusGenus•• MosaicMosaic symptomssymptoms andand chloroticchlorotic inin thethe leavesleaves..•• ThereThere hashas beenbeen reportedreported inin severalseveral countriescountries ofofSouthSouth AmericaAmerica (Brazil,(Brazil, Paraguay,Paraguay, México,México, NorthNorth CoastCoastofof Colombia),Colombia), ÁfricaÁfrica andand AsiaAsia.. (Costa(Costa andand KitajimaKitajima,,19721972))..•• YieldYield lostlost upup toto ofof 6060 %%..•• UnknownUnknown vectorvector agentagent..•• UnknownUnknown vectorvector agentagent..•• MechanicalMechanical transmissiontransmission..

•• PotexvirusPotexvirus genus.genus.

•• No symptoms on cassava. In No symptoms on cassava. In NicothianaNicothiana

benthamianabenthamiana presented a systemic infection.presented a systemic infection.

•• It is found in the North Coast of Colombia (It is found in the North Coast of Colombia (LennonLennonet. al., 1986)et. al., 1986)

CASSAVA X VIRUS (CSXV)CASSAVA X VIRUS (CSXV)

CASSAVA FROGSKIN DISEASE (CFD)CASSAVA FROGSKIN DISEASE (CFD)

•• ReportedReported inin 19711971 inin thethe DepartmentDepartment ofof thetheCaucaCauca (Pineda(Pineda yy otrosotros,, 19831983))..

•• FoundFound inin thethe majoritymajority ofof thethe ColombianColombianregionsregions andand hashas beenbeen spreadingspreading constantlyconstantly;; nownowitit isis foundfound inin Brazil,Brazil, CostaCosta Rica,Rica, Panama,Panama, PeruPeruandand VenezuelaVenezuela..

•• ConsideredConsidered toto bebe oneone ofof thethe mostmost dangerousdangerous•• ConsideredConsidered toto bebe oneone ofof thethe mostmost dangerousdangerousdiseasesdiseases inin cassavacassava.. YieldYield lostlost upup toto ofof 9090 %%(Lozano(Lozano yy NoltNolt,, 19891989))..

•• TheThe rootsroots areare stuntedstunted andand notnot fillfill withwith starchstarch..

•• TheThe productionproduction ofof cassavacassava certifiedcertified materialmaterial isisthethe bestbest strategystrategy forfor thethe controlcontrol ofof thethe CFDCFD..

Partículas observadas en Partículas observadas en Partículas observadas en Partículas observadas en corte de tejido foliarcorte de tejido foliarcorte de tejido foliarcorte de tejido foliar

Partículas observadas durante Partículas observadas durante Partículas observadas durante Partículas observadas durante el proceso de purificaciónel proceso de purificaciónel proceso de purificaciónel proceso de purificación

According to experimental evidences accumulated for 30 years, it was demonstrated that the cassava Frogskin is associated (100 %) with a reovirus called the Frogskin

virus (Virology Unit, CIAT).

�L. A. Calvert, M. Cuervo, I. Lozano, N. Villareal, and J. Arroyave. Identification of three strains of a virus associated with cassava plants affected byfrogskin disease. Journal of Phytopathology. 156,647-653 (2008).�Historia, caracterización y manejo de la enfermedad viral más importante de la especie Manihot esculenta en su centro de origen. Seminariopúblico presentado por: Iván Lozano, Maritza Cuervo y Francisco Morales. Noviembre 26, 2008. CIAT.

WashWash

2) 2) Atingen additionAtingen addition 1) 1) Addition of primary Addition of primary antibodyantibody 11

cDNAcDNA SyntesisSyntesis

dsds--RNARNA extractionextraction

CASSAVA X VIRUSCASSAVA X VIRUSCASSAVA COMMON MOSAIC VIRUSCASSAVA COMMON MOSAIC VIRUS

DASDAS--ELISAELISA

FROGSKIN VIRUSFROGSKIN VIRUS

GraftingGrafting TestTest

Molecular Method Molecular Method RTRT--PCRPCR

FLOW CHART CASSAVA INDEXATION (Manithot esculenta)

WashWash

WashWash

1: graphs taken from: 1: graphs taken from: GoldsbyGoldsbyet al. Immunology. Freeman et al. Immunology. Freeman editorial; 5editorial; 5°° edition. 149 pp.edition. 149 pp.

3) 3) Addition of secondary antibody Addition of secondary antibody conjugated enzyme conjugated enzyme

4) 4) Addition of Addition of substrate to enzyme.substrate to enzyme.

R1067F110

RTRT--PCRPCR

PCR: 958 b

ElectrophoresisElectrophoresis

Data base GRUData base GRUPositive Positive NegativeNegative In vitro and Bonsai In vitro and Bonsai collection conservationcollection conservation

Thermotherapy and Thermotherapy and meristem culturemeristem culture

Establishment of plants in the greenhouse: Disease indexing

Grafting test for diagnose CFSV in cassava

72 weeks

TIME TO DIAGNOSTIC:

IMPROVEMENT IN THE METHODOLOGY FOR CFSV DETECTION

IMPROVEMENT IN THE METHODOLOGY FOR CFSV DETECTION

(1984-1998)

0.7 weeks

21 weeks(1999-2007)

(2007- )M. esculenta -FULLY CERTIFIEDFULLY CERTIFIED

Source: Cuervo et al 2009

Indexing

Negative

(GHL)

MICROPROPAGATIONMICROPROPAGATION

Conservation medium

(8S/ NP/12A) - (5 tubes)

3 Explants/Tubes

INTRODUCTION FOR INTRODUCTION FOR IN VITRO IN VITRO CONSERVATIONCONSERVATION

GROWTH ROOMGROWTH ROOM

2-3 WEEKS

CONSERVATION ROOMCONSERVATION ROOM

Tubes: 25x150 mm

Cap: foil paper

Seal: extensible tape

Lighting: 18.5 µmol.m-2.s-1

Photoperiod: 12 h light / 12 h dark

Light quality: fluorescent lamp,

Light day type

Relative humidity: 50-70%

Temperature 23-24oC Temperature: 27-28oC

**

** **

ContaminationLeaf senescenceNumber of green shootsNumber of viable nodesPresence or absence of rootsOccurrence of callusDead cultures

MONITORING MONITORING

EXITEXIT

MONTHLY MONTHLY

GROWTH ROOMGROWTH ROOM(2-3 WEEKS)

CONSERVATION ROOMCONSERVATION ROOM

SPECIFIC PROTOCOLS FOR MEDIUM TERM STORAGESPECIFIC PROTOCOLS FOR MEDIUM TERM STORAGE

EXITEXIT

REGISTRATIONREGISTRATION

SUBCULTURESUBCULTURE

ENTRANCE ENTRANCE

REGISTRATIONREGISTRATION

(2-3 WEEKS)

� Increased time of conservation� Safely duplicated

ARG 2 BRA 337 COL 2056 NGA 16 VEN 329A CM 2177-2

8S1 2

Ag31 2

Ag41 2

8S1 2

Ag31 2

Ag41 2

8S1 2

Ag31 2

Ag41 2

8S1 2

Ag31 2

Ag41 2

8S1 2

Ag31 2

Ag41 2

8S1 2

Ag31 2

Ag41 2

SLOW GROWTHSLOW GROWTH

CONTROLCONTROL SILVER NITRATESILVER NITRATE

Fuente: Mafla et al., 2004

�Genetic stability checked through- AFLPs

IN VITROIN VITRO

BANKBANKBONSAIBONSAI CRYOCONSERVATIONCRYOCONSERVATION DNADNA ININ VITRO VITRO

DUPLICATEDDUPLICATED

CONSERVATION STRATEGYCONSERVATION STRATEGY

CIATCIAT CIPCIP

6,592(in trust)

928 619 (9.4%)

188 (2.8%)

4,888 (74.1 %)

DISTRIBUTIONDISTRIBUTION

REQUESTREQUEST

Physical mail, electronic mail or through the website of the Program

of Genetic Resources(http://www.ciat.cgiar.org/urg)

Material defined - suggested

PURPOSE PURPOSE FOOD AND AGRICULTURE FOOD AND AGRICULTURE

PHYTOSANITARY RULESPHYTOSANITARY RULES

Import permit

ACCEPT THE STANDARD MATERIAL TRANSFER AGREEMENT (SMTA)ACCEPT THE STANDARD MATERIAL TRANSFER AGREEMENT (SMTA)

OPTION 1: OPTION 1: Physical signature on the acceptance document of the Standard Material Transfer Agreement.OPTION 2: Acceptance of the Standard Material Transfer Agreement through sealed shipments. OPTION 3: OPTION 3: Electronic acceptance of the Standard Material Transfer Agreement.

Import permit

Phytosanitary certificate

Phytosanitary Movilization Certificate

DISTRIBUTION SYSTEMSDISTRIBUTION SYSTEMS

No. Accessions designated FAO

No. Accessions distributed

Percentage of the collection

distributed

CASSAVA 6,592 6,110 92.7%

Distribution of cassava Distribution of cassava germplasmgermplasm from from CIAT CIAT genebankgenebank -- 19791979--20102010

CASSAVA 1979-2010 (No. Samples)

CIAT PROJECTS EXTERNAL INSTITUTIONS TOTAL

21,69964.6%

11,85735.4%

33,556100%

Source: CIAT - GRP, March 2011

Distribution of cassava germplasm from CIAT genebank in 2009 and 2010

(as samples to internal and external recipients)


The kind of purposes

Distribution of cassava germplasm from CIAT genebank 1979-2010

33,556 samples ( 6,110 accessions)69 countries


NARS

Universities

CIAT users

CIAT Users21,699 samples

65.0%

External Users11,857 samples

35.0%

Distribution of cassava germplasm from CIAT genebank 1979-2010

Universities

Others

Commercialcompanies

33,556 samples ( 6,110 accessions)69 countries


Search for genetic duplicates in the cassava collectionSearch for genetic duplicates in the cassava collection

Initiated on the collection from Colombia: 1,986 accessions10% redundancy or 202 accessions that can be merged

Annual saving of US$2,088

Source: Ocampo et al 2008

RESEARCH CRYOCONSERVATIONRESEARCH CRYOCONSERVATION

� Establishment of the encapsulation- dehydration technique

� 619 clones of the core evaluated

� 3 groups of response found

� 60 clones evaluated in the field High Intermediate Low

Source: Escobar et al 2009

82%Morphology

Passport 98%

Progress in documenting the cassava germplasm collection maintained at CIAT

100

(%)

806040200

82%

91%

14%

Morphology

Molecular

Evaluation ????

Isozyme


CASSAVA REGISTRYCASSAVA REGISTRY

www.cassavaregistry.com


WEB SITE WEB SITE –– INFORMATION AVAILABLE INFORMATION AVAILABLE

http://www.ciat.cgiar.org/urg

GENETIC RESOURCES PROGRAM TRAINING1988 - 2010

Thesis TrainingTotal 64 Total 233

Colombia 52 Colombia 157

OPEN HOUSE EVENTS (10)

2005 - 20101,367 Students from 22 Colombian Universities

Training - In vitro Lab 372008-2010

www. ciat.cgiar.org/urg

2011. aranzales rondon. use of tissue culture techniques techniques to the conservation and exchange...

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