Technical guidelines for the safe
movement and duplication of
Coconut (Cocos nucifera L.)
germplasm using embryo culture
transfer protocols
CA Cueto, VB Johnson, F Engelmann, A Kembu, JL Konan, M Kouassi Kan,
RL Rivera, V Vidhanaarachchi, R Bourdeix and SF Weise
RESEARCH PROGRAM ON Forests, Trees and Agroforestry
Technical guidelines for the
safe movement and duplication
of Coconut (Cocos nucifera L.)
germplasm using embryo culture
transfer protocols
CA Cueto1, VB Johnson2, R Bourdeix3, F Engelmann4, A Kembu5,
JL Konan6, M Kouassi Kan7, CM Oropeza Salín8 RL Rivera9,
V Vidhanaarachchi10 and SF Weise11
1 Philippines Coconut Authority, Albay Research Centre, Philippines 2 Bioversity International, Montpellier, France
3 COGENT, Montpellier, France; CIRAD Bios; Umr CEFE-CNRS, Montpellier, France 4 IRD, Montpellier, France
5 Cocoa and Coconut Institute of PNG, Madang, Papua New Guinea 6 Coconut Research Programme, CNRA, Abidjan, Côte d’Ivoire 7 Laboratoire de Biotechnologie, CNRA, Abidjan, Côte d’Ivoire
Centro de Investigación Científica de Yucatán, Mexico
9 Philippines Coconut Authority, Zamboanga Research Centre, Philippines 10 Coconut Research Institute, Sri Lanka
Acknowledgements
4
Contents
5
List of figures
7
Preface
9
Introduction
11
The coconut ... 11
Conservation of diversity ... 12
Limitations of field genebanks ... 14
Towards safe movement of coconut germplasm
15
A rationale for the guidelines ... 15
The technical guidelines
18
General guidelines and recommendations for coconut in vitro
transfer
19
The coconut embryo culture transfer protocol
20
Arrangements in donor and recipient countries ... 20
Selection and preparation of mother palms ... 21
Harvesting and processing of nuts ... 21
Labelling of palms, nuts, embryos and seedlings ... 23
Processing of endosperm plugs ... 24
Extraction of endosperm plugs ... 24
Disinfection of endosperm plugs ... 25
Storage of endosperm plugs ... 26
Preparation of materials for transit ... 27
Transit vials ... 27
Transit medium ... 28
Additional preparations: ... 29
Preparation of embryos ... 30
The embryo culture protocol (in the recipient country)
37
Inoculation and germination of the embryos ... 37
Seedling elongation and further growth and development ... 40
Acclimatization and ex vitro establishment ... 41
Other considerations
42
National or international exchange? ... 42
Special conditions ... 43
Others ... 43
Concluding comments
44
Traceability of the embryo cultured coconuts
45
Movement of seednuts
47
Coconut disease risks
49
Introduction ... 49
Lethal Yellowing Disease (LYD) ... 49
Symptoms ... 50
Diagnosis and detection ... 50
Cadang cadang ... 53
Symptoms ... 53
Diagnosis and detection ... 54
Weligama coconut leaf wilt disease and Kerala wilt ... 54
What is a Standard Material Transfer Agreement (SMTA)? ... 58
Indexing laboratories
61
Contributors
62
Contributing institutions (by alphabetical order) ... 62
Individual contributors ... 63
References
65
Annex A. Overall data summary for coconut embryo transfer 67
Figure 1. Stages in coconut embryo transfer ... 19
Figure 2. Over-mature, germinated coconut ... 22
Figure 3. Recognising 10-11 month old nuts ... 22
Figure 4. Nut labelling ... 23
Figure 5. De-husking ... 23
Figure 6. Nut splitting ... 24
Figure 7. endosperm plug extraction ... 25
Figure 8. Serrated cork-borer ... 25
Figure 9. Coconut endosperm-plug disinfection... 26
Figure 10. Packing and labelling endosperm plugs ... 27
Figure 11. Cryotubes in box for transit ... 27
Figure 12. Embryo excision and inoculation ... 32
Figure 14. Four month-old embryo cultured seedling ready for ex vitro establishment ... 34
Figure 13. Embryo culture and germination ... 35
Figure 15. Embryos three months after germination ... 38
Figure 16. Removed shoot cap ... 38
Figure 17. Filter sterilization ... 39
Figure 18. Embryo-cultured seedlings in pots. ... 42
Figure 19. Embryo-cultured coconut plantlets for transfer ... 44
Figure 20a. LYD devastated coconut palms, Ghana... 51
Figure 20b. Premature fruit drop, Ghana. ... 51
Figure 20c. Inflorescence necrosis caused by LYD, Cuba ... 51
Figure 20d. LYD leaf chlorosis, Mozambique. ... 52
Figure 21a. Coconut leaf symptoms of cadang cadang, Philippines. ... 54
Figure 21b. Coconut palms with cadang cadang, Philippines ... 54
Figure 23. Coconut embryo contamination levels ... 69 Figure 24. Coconut seedling establishment levels... 69 Figure 25. Germination levels of embryos without in vitro manipulation . 69 Figure 26.Germination levels of embryos with in vitro manipulation ... 69
Table 1. Documented data (as of April 2012) on national and international collections (with international genebanks in orange).
Countries & Genebanks # reg
ist er ed accession s # a ct ive accession s # u niq ue a ct ive po pu lat io ns& cu ltiva rs # Pass po rt Dat a # Char ac ter izat io n Dat a 1 Bangladesh Bari 40 16 16 699 932 2 Benin CRC Sémé Podji 4 0 0 116 20 3 Brazil EMBRAPA 29 23 23 1 286 1665
4 China Wenchang Coconut Research Inst. 17 17 3 366 312 5 Côte d'Ivoire CNRA Marc Delorme R. S. 149 124 61 5 782 163 10 6 Fiji Taveuni Coconut Centre 11 10 10 413 536
7 Ghana OPRI 16 15 13 206 71
8 India CPCRI 301 299 170 9 227 122 16
9 Indonesian Palm Research Institute 203 84 62 5 747 6 631 10 Jamaica Coconut Industry Board 60 47 28 923 522 11 Malaysia Dept. of Agric. Sabah 45 37 19 968 924 12 Malaysia MARDI Hilir Perak 44 44 44 1 213 2 621
13 Mexico CICY Yucatan 20 0 0 568 800
14 Pakistan 32 0 0 192 1148
15 Papua New Guinea Stewart Res. Centre 57 50 49 937 759 16 Philippine Coconut Authority 224 224 130 6 444 8 259 17 Solomon Islands Yandina Res. Centre 21 20 20 398 147 18 Sri Lanka Coconut Research Institute 157 154 127 6 322 950 10 19 Tanzania Nat. Coconut Dev. Programme 72 65 57 2 420 963 20 Thailand Chumphon Hort. Research Centre 52 51 49 1 480 760
21 Tonga Ministry of Agriculture 7 0 0 45 274
22 Vanuatu Saraoutou Research Centre 79 57 44 2 494 1 768 23 Vietnam Dong Go Experimental Centre 31 31 31 1 221 1 093
24 Western Samoa 9 6 6 84 36
Figure 1. Stages in coconut embryo transfer (CA Cueto, VB Johnson & R Bourdeix).
Selection and preparation of mother palms
Figure 2. Over-mature, germinated coconut (CA Cueto, PCA-ARC).
Labelling of palms, nuts, embryos and seedlings
Figure 4. Nut labelling
Figure 6. Nut splitting (RL Rivera, PCA-ZRC).
Processing of endosperm plugs
Extraction of endosperm plugs
Figure 7. endosperm plug extraction
(RL Rivera, PCA-ZRC) Figure 8. Serrated cork-borer (VB Johnson, Bioversity International)
Disinfection of endosperm plugs
Figure 9. Coconut endosperm-plug disinfection (RL Rivera, PCA-ZRC).
Figure 10. Packing and labelling endosperm plugs (RL Rivera, PCA-ZRC).
Preparation of materials for transit
Transit vials
Transit medium
Table 2. Concentration of stock solutions and volume of stock solution to prepare for 1x of 1 L Y3 medium.
Stock solution Volume for 1 L of Y3 medium (ml)
Macronutrients (10x) 100 Micronutrients (100x) 10 Vitamins (100x) 10 FeEDTA (100x) 10 Myo-inostiol (100x) 10
Additional preparations:
Preparation of embryos
Figure 12. Embryo excision and inoculation (PCA-ZRC).
PCA-ZRC Protocol for in vitro culture
(Laboratory operations)
Excision of the embryo from
the endosperm plug Disinfection of culture media receptacle opening
Note: If embryos and embryo cultured seedlings are coming from disease-risk
areas i.e. areas reported with coconut Cadang-cadang viroid and
Phytomonas-infected coconut palms, the FAO/IBPGR recommendation would be adopted
(FAO/IBPGR 1994).
Figure 14. Four month-old embryo cultured seedling ready for ex vitro establishment
Figure 13. Embryo culture and germination (PCA-ZRC.)
PCA-ZRC Protocol for in vitro culture
(Shelves/racks)
Embryos were singly inoculated onto “ketsup” bottle containing Y3 liquid media
Germinating embryo Developing embryo
It is lighted with 40 watts fluorescent tube with approximately 4000-5000 lux just above (6 inches) the bottles at 9 h photoperiod (15 h dark and 9 h light)
Mode of transfer
Table 3. Comparing transport by courier and hand-carrying.
Parameters Hand-carrying Via courier
Cost Entail additional expense of travelling specialist to collect embryos Entail cost of freight
Duration of transit
Depends on the distance of the originating laboratory to the receiving laboratory. Normally, a maximum of four days.
Depends on the courier service provider; could last up to two weeks.
Can take sometimes for the package to be released due to custom-related issues Handling during
transit Assurance of proper handling and storage conditions of the package
No assurance of the storage conditions provided by the courier service.
Inoculation and germination of the embryos
Figure 15. Embryos three months after germination
(CA Cueto, PCA-ARC).
Figure 16. Removed shoot cap (CA Cueto, PCA-ARC)
Notes:
Pech y Aké
et al. (2007) report that using Gibberellic acid (GA3) can benefit
coconut embryos in culture, favouring germination and conversion for plants
grown in a semi-solid medium.
For GA3-supplemented media, the GA3 should be filter-sterilized using
Millipore filtration:
Sterilize the filtration set-up, millipore membrane filters membranes
(0.45 and 0.20μm), and empty reagent bottles to contain the
filter-sterilized solution (Figure 17). In the absence of a filtration facility,
sterilize glass and millipore holders containing millipore membrane
filters. Disposable sterile syringes and Millipore filters are also
available.
Filter-sterilize the GA3 stock solution using sterile 0.45μm millipore
membrane filters.
Proceed with filter-sterilization using 0.20μm millipore membrane
filters.
Compute for the required volume of GA3 needed.
For BAP-supplemented medium, co-autoclave BAP with the culture medium.
Seedling elongation and further growth and development
Note: In laboratories which practice medium-replenishment, culture the embryos in
wide-mouthed bottles or ketchup bottles, whichever is available. With this method,
the embryos are not transferred to new culture vessels during subculture; instead,
the embryos and eventually, the growing seedlings are maintained in the same
culture vessel. The medium is replenished with the new medium either by decanting
or pumping out the old medium and pouring in the new medium after one to two
months. A suction pump may be used to remove the old and add the new media.
National or international exchange?
Figure 18. Embryo-cultured seedlings in pots (VB Johnson, Bioversity International).Special conditions
Figure 19. Embryo-cultured coconut plantlets for transfer (C Oropeza, CICY).
Introduction
Symptoms
Diagnosis and detection
Antibiotherapy in Florida as preventive treatments for non-food palms; curative treatments not possible; antibiotic treatments not allowed in several countries (M. Dollet 2012, Pers. comm.).
Figure 20a. LYD devastated coconut palms,
Ghana (M Dollet, CIRAD). Figure 20b. Premature fruit drop, Ghana (M Dollet, CIRAD).
Cadang cadang
Symptoms
Figure 21a. Coconut leaf symptoms of Cadang cadang: healthy palm (top) and palm with late stage of the disease showing non necrotic chlorotic spotting (J Randles, Waite Agricultural Research Institute, University of Adelaide).
Figure 21b. Area with high incidence of Cadang cadang disease, showing trees in early, mid and late stages (J Randles, Waite Agricultural Research Institute, University of Adelaide).
Weligama coconut leaf wilt disease and Kerala wilt
Box 1. Good practices at BPI Plant Quarantine Service in the Philippines
In transferring plant materials the Plant Quarantine Service requires completed forms for:
1. Exporting plant materials: Application for Inspection and Phytosanitary Inspection (BPI ‘Q’ FORM No. 10). Information required includes- Common
Name; Scientific Name; Quantity; Description & No. of Packages; Source of Plants/Plant Products; Name & Address of Consignee; Date and Place of Inspection Desired; Port Of Entry; Means of Conveyance; Flight No./Voyage No.; Departure Date; Import Permit No./Additional Declaration/Treatment (if any); (Signature of Applicant/Authorized Representative); stamp of the service (Name & Designation of Applicant/Authorized Representative).
2. Application for Accreditation- as an importer of plant materials, signed by
the public notary
3. Accreditation Validation of accreditation: Information required includes-
Company Name; Address of Present Office; Contact Numbers (Tel. & Fax No.); T.I.N. Number; Name of the Owner; Name of Representative/s; Commodity to be imported.
To be filled up by the PQ officer - Application for Accreditation (Notarized);
Company Profile; DTI Registration/ SEC Registration/CDA Certificate of Registration; Current Mayor’s Permit (2009); Valid Certificate/Contract of Lease of Storage Facilities; Sketch map of Storage Facilities; Special Power of Attorney of Representative/s; Current Bureau of Customs-Certification of Accreditation; Two (2) 2X2 ID Pictures of the Owner and the Representative/s; Notes:; PQ Officer signature.
4. Application for Plant Quarantine Clearance Plants/Plant Products (BPI
‘Q’ FORM No. 1). Information required includes- Common Name; Scientific Name; Quantity; Purpose of Importation; Place of Origin or Source of Plants/Plant Products; Name, Address & Country of Exporter; Port Of Entry; Means of Conveyance; Flight No./Voyage No.; Final Destination (Exact location and Sketch where plants are grown); Expected Date of Arrival; Signature of Applicant/Authorized Representative; stamp of the service (Name & Designation of Applicant/Authorized Representative).
Contributing institutions (by alphabetical order)
Institution Country Website Logo
Bioversity International Italy http://www.bioversityinternational.org/
the Central Plantation Crops Research Institute
(CPCRI) India http://www.cpcri.gov.in/ the Centro de
Investigación Científica
de Yucatán Mexico http://www.cicy.mx/ the Cocoa & Coconut
Institute (CCI) PNG the coconut genetic
resources network (COGENT)
France http://www.cogentnetwork.org/ the Coconut Research
Institute (CRI) Sri Lanka http://www.cri.gov.lk/ the Global Crop Diversity
Trust Germany http://www.croptrust.org/
L’Institut de recherche pour le développement
(IRD) - UMR DIADE
France http://www.diade-research.fr/ the Ministry of Coconut
Development and Janatha Estate Development
Sri Lanka http://www.cdjedmin.gov.lk/
the Philippine Coconut
Institution Country Website Logo
Station de recherche Marc Delorme, Centre national de recherche agronomique (CNRA)
Côte
d'Ivoire http://www.cnra.ci/descprog.php?id=3&prog=Cocotier&act=present
Arancon RN, Shantichandra WKN, Priyanthie Fernando LC, Perera C, Aucith
Dissanayake JD, Naynanayake A, Megahakummbura K, editors. 2011. Proceedings of the APCC/MCD&JED/CRI Consultative Meeting on Phytoplasmas/Wilt Diseases in Coconut, Coconut Research Institute, Luniwila, Sri Lanka, 15-17 June 2011. Batugal PA, Engelmann F, editors. 1998. Coconut embryo in vitro culture. Proceedings of
the first workshop on embryo culture, Banao, Guinobatan, Albay, Philippines, 27-31 Oct. 1997. IPGRI/APO, Serdang, Malaysia.
Bourdeix R, Johnson VB, Tuia VS, Weise S. 2012. Three declinations of the Polymotu Concept: “Inland ex situ”, “Ecotourism on Islands”, “Urban” and their possible applications in Brazil, Côte d’Ivoire, Indonesia, French Polynesia and Samoa (Paper presented at the 45th APCC COCOTECH Meeting, 2nd - 6th July 2012, Kochi, India). De Guzman, E, del Rosario DA. 1964. The growth and development of Cocos nucifera L
‘Makapuno’ embryo in vitro. Philipp. Agric. 48:82-94.
Dollet M, Quaicoe R, Pilet F. 2009. Review of coconut "Lethal Yellowing" type diseases. Diversity, variability and diagnosis. Oléagineux Corps gras Lipides. (16)2: 97-101. Eeuwens CJ. 1976. Mineral requirements for growth and callus initiation of tissue
explants excised from mature coconut palms (Cocos nucifera L.) and cultured in vitro. Physiol. Plant. 36:23-28.
Engelmann F, Batugal P, Oliver J, editors. 2002. Coconut embryo in vitro culture: part II. Proceedings of second international workshop on embryo culture, Mérida, Yucatán, Mexico, 14-17 March 2000.
Engelmann F, Malaurie B, Oulo N’Nan. 2011. In vitro culture of coconut zygotic embryos. pp. 63-74 In: T Thorpe, E Yeung, editors. Plant Embryo Culture: Methods and Protocols, Methods in Molecular Biology Series. Humana Press, Totowa, NJ. Frison EA, Putter CAJ, Diekman M editors. 1993. FAO/IBPGR Technical Guidelines for
the Safe Movement of Coconut Germplasm. Food and Agriculture Organization of the United Nations, Rome/International Board for Plant Genetic Resources, Rome, Italy. Available from the URL: (http://www.cogentnetwork.org/index.php/technical-bulletins) Ikin R, Batugal P, editors. 2004. Germplasm health management for COGENT’s multi-site
international coconut genebank. International Plant Genetic Resources Institute – Regional Office for Asia, the Pacific and Oceania (IPGRI-APO), Serdang, Selangor DE, Malaysia. Available from URL: http://www.cogentnetwork.org/index.php/manuals-and-handbooks.
IPGRI. 1995. Descriptors for Coconut (Cocos nucifera L.). International Plant Genetic Resources Institute, Rome, Italy.
Konan JL, Bourdeix R, George ML. 2008. Regeneration Guidelines: Coconut. In: E Dulloo E, I Thorman, MA Jorge, J Hansen, editors. Crop Specific Regeneration Guidelines (CD-ROM). CGIAR System-wide Genetic Resources Programme. Rome, Italy. 10pp. Orense OD, Rillo EP, Imperial LAI, Cueto CA, Lobos AA, Areza-Buena MBB. 2011. Rapid and cost-effective embryo culture technique for commercial production of Makapuno seedlings. CORD 27(1):20-41.
Pech y Aké AE, Maust B, Orozco-Segovia A, Oropeza C. 2007. The effect of gibberellic acid on the in vitro germination of coconut zygotic embryos and their conversion into plantlets. In Vitro Cell.Dev.Biol.—Plant (2007) 43:247–253.
Pech y Aké, AE, Souza, R, Maust, B, JM Santamaria, JM, Oropeza.C. 2004. Enhanced aerobic respiration improves in vitro coconut embryo germination and culture. In vitro Cell. Dev. Biol.—Plant 40(1):90–94.
Perera L, Meegahakumbura MK, Wijesekara HRT, Fernando WBS, Dickinson MJ. 2012. A phytoplasma is associated with the Weligama coconut leaf wilt disease in Sri Lanka. J. Plant Pathol. 94(1):2015-209.
Rillo EP, Cueto CA, Medes WR, Areza-Ubaldo MB. 2002. Development of an improved embryo culture protocol for coconut in the Philippines. In: F. Engelmann. P. Batugal and JT Oliver, editors. Coconut embryo in vitro culture: part II. Proceedings of 2nd international workshop on embryo culture, Mérida, Yucatán, Mexico, 14-17 March 2000. p. 41.
Rodriguez MJBR. 2011. The nature of the Cadang-Cadang disease of coconut in the Philippines and review of the R & D Programme: Strategies and accomplishments. In: RN Arancon, editor. Final proceedings report of the APCC/MCD and JED/CRI Consultative Meeting on Phytoplasma/Wilt diseases in Coconut. Coconut Research Institute, Lunuwila, Sri Lanka.
Santos GA, Batugal PA, Othman A, Baudouin L, Labouisse JP. 1996. Manual on Standardized Research Techniques in Coconut Breeding. IPGRI, Rome, Italy. Samosir YMS, Mashud N, Novarianto H, Vu Thi My Lien, Rillo E, Magdalita P,
Damasco O, Kembu A, Faure MG, Adkins SW. 2008. A New Embryo Culture Protocol for Coconut Germplasm Conservation and Elite-type Seedling Production. In: Final Report: Development of an embryo culture manual and an embryo transplantation technique for coconut. Australian Centre for International Agricultural Research, Canberra. pp. 14-30.
Shivashankar,S, Karun A and Sajini KK 1999 Embryo Culture of Coconut, , thr CPCRI Protocol, Indian J Hort, 56 (4) 348-353 , Kasaragod, India
Table A1. Overall data summary.
Element Partner Type Hand-carried Couriered
% embryo contamination PCAZRC WAT 43 98 MYD 83 40 CRISL WAT 10 55 MYD 15 23 CCIPNG WAT 78 13 MYD 43 73 Average WAT 45 55 MYD 47 45 % embryo germination (with in vitro manipulation)
PCAZRC WAT 73 90 MYD 50 83 CRISL WAT 54 3
MYD 55 60 CCIPNG WAT No data No data
MYD No data No data Average WAT 63 46
MYD 53 72
Table A1 (cont’d)
Element Partner Type Hand-carried Couriered
% embryo germination (without in vitro manipulation)
PCAZRC WAT 73 90 MYD 50 83 CRISL WAT 3 0
MYD 43 30 CCIPNG WAT No data No data
MYD No data No data Average WAT 38 45
MYD 46 56
% coconut seedlings established for field-planting
PCAZRC WAT 40 2.5 MYD 15 20 CRISL WAT 10 0
MYD 30 25 CCIPNG WAT No data No data
MYD No data No data Average WAT 25 1
Figure 23. Coconut embryo contamination
levels Figure 24. Coconut seedling establishment levels
Figure 25. Germination levels of embryos
Table B1. The composition of the Eeuwen’s (Y3, 1976) mineral and vitamin solutions.
COMPONENTS Amount
MACRO (mg/l) Calcium chloride di-hydrate CaCl2.2H2O 294
Magnesium sulphate heptahydrate MgSO4.7H2O 247
Potassium nitrate KNO3 2020
Potassium chloride KCl 1492 Sodium di-hydrogen phosphate di-hydrate NaH2PO4.2H2O 312
Ammonium chloride NH4Cl 535
MICRO 1x (mg/l) Manganese sulphate tetrahydrate MnSO4.4H2O 11.2
Zinc sulphate heptahydrate ZnSO4.7H2O 7.2
Boric acid H3BO3 3.1
Potassium iodide KI 8.3 Cupric sulphate pentahydrate CuSO4.5H2O 0.25
Sodium molybdate di-hydrate Na2MoO4.2H2O 0.24
Cobalt chloride hexahydrate CoCl.6H2O 0.24
Nickel chloride hexahydrate NiCl.6H2O 0.024
IRON 1x (mg/l) Ferrous sulphate heptahydrate FeSO4.7H2O 13.9
Ethylene dinitrilo tetra acetic acid disodium
Table B1 (cont’d)
COMPONENTS Amount
VITAMINS 1x (mg/l) Myo-inositol 100 Thiamine hydrochloride Thiamine HCl 0.5 Nicotinic acid 0.05 Pyridoxine hydrochloride Pyridoxine HCl 0.05 Calcium pantothenate) pantothenate Ca D- 0.05 Biotin 0.05
Table B2. The components and pH of the “hybrid medium” being used for coconut embryo culture (Rillo et al., 2002).
Particulars Medium Source Chemicals Weight
1x (mg/l) 10x (g/l)
Macro Eeuwens (Y3)
NH4Cl 535.00 5.35 KNO3 2020.00 20.20 MgSO4.7H2O 247.00 2.47 CaCl2.2H2O 294.00 2.94 KCl 1492.00 14.92 NaH2PO4.2H2O 312.00 3.12
Micro Eeuwens (Y3)
1x (mg/l) 100x (g/l) KI 8.30 0.830 H3BO3 3.10 0.310 MnSO4.4H2O 11.20 1.120 ZnSO4.7H2O 7.20 0.720 CuSO4.5H2O 0.250 0.0250 CoCl2.6H2O 0.240 0.0240 NaMoO4.H2O 0.240 0.0240 NiCl.6H2O 0.024 0.0024 EDTA UPLB 1x (mg/l) 100x (g/l) Fe2SO4.7H2O 41.70 4.170 Na2EDTA 55.80 5.580
Table B2 (cont’d)
Particulars Medium Source Chemicals Weight
1x (mg/l) 10x (g/l)
Vitamins UPLB + ARC
Pyridoxine HCl 0.05 0.005 Thiamine HCl 0.05 0.005 Nicotinic acid 0.05 0.005 Ca-D-pantothenate 0.05 0.005 Biotin 0.05 0.005 Folic acid 0.05 0.005 Glycine 1.00 0.1 Table grade sugar
60 g/l 60 g/l from culture initiation until seedlings have developed shoots and roots (until the 2nd to 3rd month) 45 g/l 45 g/l for maintenance prior to transplanting to the soil Activated charcoal (acid washed) 1 g/l Gelling agent 7-8 g/l State of the medium Liquid/ solid/ liquid
Culture initiation (So) = liquid +60g/l sugar 1st Subculture (S1) = solid + 60 g/l sugar
2nd Subculture (S2) = solid + 60 g/l sugar if not
germinated yet
= liquid +45 g/l sugar if
germinated
3rd Subculture (S3) = liquid +45 g/l sugar until the
seedlings are ready to be transplanted to the soil.
