2011. aranzales rondon. use of tissue culture techniques techniques to the conservation and exchange...
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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
e.aranzales@cgiar.org
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%)
Species Genotypes(No.)
Species Genotypes(No.)
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
CONSERVATION ALTERNATIVES FOR CASSAVA CONSERVATION ALTERNATIVES FOR CASSAVA
BONSAIBONSAI
CONSERVATION ALTERNATIVES FOR CASSAVA CONSERVATION ALTERNATIVES FOR CASSAVA
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.
CONSERVATION ALTERNATIVES FOR CASSAVA CONSERVATION ALTERNATIVES FOR CASSAVA
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
CONSERVATION ALTERNATIVES FOR CASSAVA CONSERVATION ALTERNATIVES FOR CASSAVA
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
DISTRIBUTION SYSTEMSDISTRIBUTION SYSTEMS
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)
Source: CIAT - GRP, March 2011
The kind of purposes
Distribution of cassava germplasm from CIAT genebank 1979-2010
33,556 samples ( 6,110 accessions)69 countries
Source: CIAT - GRP, March 2011
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
Source: CIAT - GRP, March 2011
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
Source: CIAT - GRP, March 2011
CASSAVA REGISTRYCASSAVA REGISTRY
www.cassavaregistry.com
Source: CIAT - GRP, March 2011
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
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