FOODCROPS.VN. 2011. aranzales rondon. use of tissue culture techniques techniques to the conservation and exchange of cassava germplasm material of planting material

1. Use of Tissue Culture Techniques to theUse of Tissue Culture Techniques to the
Conservation and Exchange of CassavaConservation and Exchange of Cassava GermplasmGermplasm
Material / Health Certification of Planting MaterialMaterial / Health Certification of Planting Material
Ericson Aranzales Rondon
e.aranzales@cgiar.org
Graciela MaflaGraciela Mafla
Maritza Cuervo
GENETIC RESOURCES PROGRAMGENETIC 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

2. .
..
. .
.
CIAT
Cali
Colombia
.
..
. .
. .. ...

3. HoldingHolding GermplamGermplam in CIATin CIAT
Register into the International TreatyRegister into the International Treaty
Crops
Taxa
( No.)
Accessions
(No.)
Beans (Phaseolus) 36,407
Rank
(No.)
1 44
Country of
origin (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

4. - Manihot is a Neotropical genus, distributed
in its natural habitat from the southern United
States through Mesoamerica, South America,
Brazil, Perú, Bolivia, Paraguay and Argentina.
- Cassava is the fourth most important supplier
of food calories in the tropics.
CassavaCassava
MOCHICA-PERU CULTURE
AMANO MUSEUM-LIMA. 1300 B.C.
of food calories in the tropics.
-The principal economic product is starchy
roots, which are utilized in a wide range of end
uses, most notably including human food,
animal feed, and industrial products.

5. Landraces
Wild
5,301
883
INTERNATIONAL PUBLIC GOODSINTERNATIONAL PUBLIC GOODS
Status ofStatus 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

6. 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) 490Others, 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

7. Holdings of cassavaHoldings 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

8. 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 ofHoldings 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

9. SPECIES POTENTIAL BENEFITS
M.M. flabellifoliaflabellifolia PohlPohl Resistance to whitefly Aleurotrachelus socialis Bondar
M.M. alutaceaalutacea RogerRoger yy AppanAppan Resistance to mites
M.M. chlorostictachlorosticta StandleyStandley yy GolmanGolman Resistant to mosaic virus African and tolerant to
drought
M.M. carthaginensiscarthaginensis MuellerMueller vonvon ArqauArqau Tolerant to drought and the high activity to pyruvate
decarboxylase fosfoenol
Why conserve wild species?
M. filamentosaM. filamentosa PittierPittier High pontential as forages
M.M. glazioviiglaziovii MuellerMueller vonvon ArqauArqau Resistant to mosaic virus African
M.M. guaraniticaguaranitica ChodatChodat yy HasslerHassler Tolerance to low temperatures
M.M. longepetiolatalongepetiolata Roger yRoger y AppanAppan Lower length in plants
M.M. orbicularisorbicularis PohlPohl Tolerance to aluminum toxicity
M.M. peltatapeltata PohlPohl
M.M. TristisTristis MuellerMueller vonvon ArgauArgau
Tolerance to acid soils
High protein and starch content
Source: Chavez et al., 1987; Bellotti, 2008; Carabali, 2010

10. CONSERVATION ALTERNATIVES FOR CASSAVACONSERVATION ALTERNATIVES FOR CASSAVA
FIELDFIELD
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.

11. 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 frequent
Suitable for storage and distribution of species
CONSERVATION ALTERNATIVES FOR CASSAVACONSERVATION ALTERNATIVES FOR CASSAVA

12. BONSAIBONSAI
CONSERVATION ALTERNATIVES FOR CASSAVACONSERVATION 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 CASSAVACONSERVATION ALTERNATIVES FOR CASSAVA

13. 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 CASSAVACONSERVATION ALTERNATIVES FOR CASSAVA

15. Import permit issued byImport permit issued byImport permit issued byImport permit issued by
Colombian Agricultural InstituteColombian Agricultural Institute
(ICA)(ICA)
PhytosanitaryPhytosanitary CertificateCertificatePhytosanitaryPhytosanitary CertificateCertificate
Donor countryDonor country
PROCEDURES FOR INTRODUTION OF CASSAVA GERMPLASM
Virus eradicationVirus eradication
In vitroIn vitro conservationconservation
laboratorylaboratory
INDEXING (GHL)
Inspection of plants to determine ifInspection of plants to determine if
there is any plant health problem
(fungi and bacteria)

16. 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

18. PATHOGENS OF QUARANTINE IMPORTANCEPATHOGENS OF QUARANTINE IMPORTANCE
• Implementation of sanitationImplementation of sanitation
techniques:techniques:
Thermotherapy followed byThermotherapy followed by meristemsmeristems cultureculture
ThermotherapyThermotherapy in vitroin vitro
• Indexation of clonesIndexation of clones
GermplasmGermplasm Health LaboratoryHealth Laboratory-- GHLGHL
••Propagation of healthy materialPropagation of healthy material

19. PATHOGENS OF QUARANTINE SIGNIFICANCE TOPATHOGENS 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 ofof
SouthSouth AmericaAmerica (Brazil,(Brazil, Paraguay,Paraguay, México,México, NorthNorth CoastCoast
ofof 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. InNo 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 (LennonLennon
et. al., 1986)et. al., 1986)
CASSAVA X VIRUS (CSXV)CASSAVA X VIRUS (CSXV)

20. CASSAVA FROGSKIN DISEASE (CFD)CASSAVA FROGSKIN DISEASE (CFD)
•• ReportedReported inin 19711971 inin thethe DepartmentDepartment ofof thethe
CaucaCauca (Pineda(Pineda yy otrosotros,, 19831983))..
•• FoundFound inin thethe majoritymajority ofof thethe ColombianColombian
regionsregions andand hashas beenbeen spreadingspreading constantlyconstantly;; nownow
itit isis foundfound inin Brazil,Brazil, CostaCosta Rica,Rica, Panama,Panama, PeruPeru
andand VenezuelaVenezuela..
•• ConsideredConsidered toto bebe oneone ofof thethe mostmost dangerousdangerous•• ConsideredConsidered toto bebe oneone ofof thethe mostmost dangerousdangerous
diseasesdiseases 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 isis
thethe bestbest strategystrategy forfor thethe controlcontrol ofof thethe CFDCFD..

21. Partículas observadas enPartículas observadas enPartículas observadas enPartículas observadas en
corte de tejido foliarcorte de tejido foliarcorte de tejido foliarcorte de tejido foliar
Partículas observadas durantePartículas observadas durantePartículas observadas durantePartículas 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 by
frogskin 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. Seminario
público presentado por: Iván Lozano, Maritza Cuervo y Francisco Morales. Noviembre 26, 2008. CIAT.

22. WashWash
2)2) Atingen additionAtingen addition 1)1) Addition of primaryAddition 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 MethodMolecular Method RTRT--PCRPCR
FLOW CHART CASSAVA INDEXATION (Manithot esculenta)
WashWash
WashWash
1: graphs taken from:1: graphs taken from: GoldsbyGoldsby
et al. Immunology. Freemanet al. Immunology. Freeman
editorial; 5editorial; 5°° edition. 149 pp.edition. 149 pp.
3)3) Addition of secondary antibodyAddition of secondary antibody
conjugated enzymeconjugated enzyme
4)4) Addition ofAddition of
substrate to enzyme.substrate to enzyme.
R1067F110
RTRT--PCRPCR
PCR: 958 b
ElectrophoresisElectrophoresis
Data base GRUData base GRUPositivePositive NegativeNegative
In vitro and BonsaiIn vitro and Bonsai
collection conservationcollection conservation
Thermotherapy andThermotherapy and
meristem culturemeristem culture

23. Establishment of plants in the greenhouse: Disease indexing

24. Grafting test for diagnose CFSV in cassava

25. 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

27. Indexing
Negative
(GHL)
MICROPROPAGATIONMICROPROPAGATION
Conservation medium
(8S/ NP/12A) - (5 tubes)
3 Explants/Tubes
INTRODUCTION FORINTRODUCTION FOR IN VITROIN 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
**
** **

28. Contamination
Leaf senescence
Number of green shoots
Number of viable nodes
Presence or absence of
roots
Occurrence of callus
Dead cultures
MONITORINGMONITORING
EXITEXIT
MONTHLYMONTHLY
GROWTH ROOMGROWTH ROOM
(2-3 WEEKS)
CONSERVATION ROOMCONSERVATION ROOM
SPECIFIC PROTOCOLS FOR MEDIUM TERM STORAGESPECIFIC PROTOCOLS FOR MEDIUM TERM STORAGE
EXITEXIT
REGISTRATIONREGISTRATION
SUBCULTURESUBCULTURE
ENTRANCEENTRANCE
REGISTRATIONREGISTRATION
(2-3 WEEKS)

29. Increased time of
conservation
Safely duplicated
ARG 2 BRA 337 COL 2056 NGA 16 VEN 329A CM 2177-2
8S
1 2
Ag3
1 2
Ag4
1 2
8S
1 2
Ag3
1 2
Ag4
1 2
8S
1 2
Ag3
1 2
Ag4
1 2
8S
1 2
Ag3
1 2
Ag4
1 2
8S
1 2
Ag3
1 2
Ag4
1 2
8S
1 2
Ag3
1 2
Ag4
1 2
SLOW GROWTHSLOW GROWTH
CONTROLCONTROL SILVER NITRATESILVER NITRATE
Fuente: Mafla et al., 2004
Genetic stability checked through- AFLPs

30. IN VITROIN VITRO
BANKBANK
BONSAIBONSAI CRYOCONSERVATIONCRYOCONSERVATION DNADNA ININ VITROVITRO
DUPLICATEDDUPLICATED
CONSERVATION STRATEGYCONSERVATION STRATEGY
CIATCIAT CIPCIP
6,592
(in trust)
928 619
(9.4%)
188
(2.8%)
4,888 (74.1 %)

32. 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
PURPOSEPURPOSE FOOD AND AGRICULTUREFOOD 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

33. DISTRIBUTION SYSTEMSDISTRIBUTION SYSTEMS

34. DISTRIBUTION SYSTEMSDISTRIBUTION SYSTEMS

35. DISTRIBUTION SYSTEMSDISTRIBUTION SYSTEMS

36. No. Accessions
designated FAO
No. Accessions
distributed
Percentage of the
collection
distributed
CASSAVA 6,592 6,110 92.7%
Distribution of cassavaDistribution of cassava germplasmgermplasm fromfrom
CIATCIAT genebankgenebank -- 19791979--20102010
CASSAVA 1979-2010 (No. Samples)
CIAT PROJECTS EXTERNAL INSTITUTIONS TOTAL
21,699
64.6%
11,857
35.4%
33,556
100%
Source: CIAT - GRP, March 2011

37. Distribution of cassava germplasm from CIAT genebank
in 2009 and 2010
(as samples to internal and external recipients)
Source: CIAT - GRP, March 2011

38. 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

39. NARS
Universities
CIAT users
CIAT Users
21,699 samples
65.0%
External Users
11,857 samples
35.0%
Distribution of cassava germplasm from CIAT genebank
1979-2010
Universities
Others
Commercial
companies
33,556 samples ( 6,110 accessions)
69 countries
Source: CIAT - GRP, March 2011

40. Search for genetic duplicates in the cassava collectionSearch for genetic duplicates in the cassava collection
Initiated on the collection from Colombia: 1,986 accessions
10% redundancy or 202 accessions that can be merged
Annual saving of US$2,088
Source: Ocampo et al 2008

41. 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

42. 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

43. CASSAVA REGISTRYCASSAVA REGISTRY
www.cassavaregistry.com
Source: CIAT - GRP, March 2011

44. WEB SITEWEB SITE –– INFORMATION AVAILABLEINFORMATION AVAILABLE
http://www.ciat.cgiar.org/urg

45. GENETIC RESOURCES PROGRAM TRAINING
1988 - 2010
Thesis Training
Total 64 Total 233
Colombia 52 Colombia 157
OPEN HOUSE EVENTS (10)
2005 - 2010
1,367 Students from 22 Colombian Universities
Training - In vitro Lab 37
2008-2010

46. www. ciat.cgiar.org/urg