Kodiswaran Kandasamy © 2006
Kodiswaran KandasamyTissue Culture UnitForest Biotechnology [email protected]
PLANT PROPAGATION(AN OVERVIEW)
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PLANT PROPAGATION
- conventional
- tissue culture
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1. Cuttings (leaf, stem, root)
2. Layering (stem)
3. Marcotting
4. Grafting
5. Seed
CONVENTIONAL PROPAGATION
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1. Micropropagation
2. Somatic embryogenesis
3. Suspension cell Culture
4. Protoplast fusion
5. Micrografting
6. Polyploidisation
7. Haploid Cell Culture
8. Mutation Breeding
9. Artificial Seeds
TISSUE CULTURE
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Kodiswaran Kandasamy © 2006
plant tissue culture
some basics
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Description
Growing surface sterilised plant tissue in a nutrient medium under aseptic conditions in a controlled environment
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Hormonal control of growth
AUXINLow
High
IBANAAIAA
CYTOKININHigh
Low
BAPKinetin2iPZeatin
shoots
roots
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Effect of IAA and Kinetin on Proliferation and morphogenesis:
Auxin - 3 3 0.03Cytokinin 0.2 0.2 0.02 1
(mg/l)
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MICROPROPAGATION(commercial)
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• Laboratory design• Defined culture procedures• Scheduling & Stock keeping• Calculations• Human resource
Key factors
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• Appropriate size of each section• Media Prep & Washing• Autoclave & Media room• Transfer Room• Growth Rooms
• Work Flow (Layout) • From non-clean to clean
• Utilities & Specification• Clean water • Reliable power supply• Floor & Wall finish• Equipment layout
Laboratory design
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Micropropagation cycle
Laboratory
Greenhouse
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• Define Explant Type & Size• Define Developmental Stages• Define Media for Each Stage• Define Growth Room Conditions• Define Number of Explant/Vessel• Define Sub-Culture Interval (cycle)• A Complete Step-by-Step guide
Shoot-tipNodal explant
Plantlets ready fornext sub-culture
Culture procedures
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• Continuous production • Multiplication cycle - numbers• Rooting Stages - numbers• Ready for Sale (ex-lab) - numbers
• Allocation of resources• Equipment• Manpower• Raw material
• Stock keeping (database)• Production record & estimation• Plan production to meet targets (demand)• Tracking & able to trace stock
Scheduling & stock keeping
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• Production estimations• Input requirement & cost
• Raw material• Labour
• Production efficiency• Cost estimations
• Budget estimation• Business plan• Cash flow (P&L)
• Marketing• Delivery schedule
Calculations
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• Smooth running of production line• Skilled & semi-skilled• Strict job specification
• Production Manager• Production Supervisor• Operators• Support staff
• Monitor & evaluate performance• Healthy and safe workplace
Human resource
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Planning production
Y = No. of plantlets producedA = No. of shoots produced at each subcultureB = No. of initial explants availablen = No. of subculture passages
F1 = Proportion of usable shoots after Stage IIF2 = Proportion of usable shoots after Stage IIIF3 = Proportion of usable shoots after Stage IV
Y = An x B x F1 x F2 x F3
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work @ frim
at a brief
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Genetic resources
Forests are the main home of Earth's species. While covering only 6% of the planet's land surface, tropical forests contain at least 50%, possibly 70% and conceivably 90% of all species.
½ km2 of Malaysia's forests can feature as many tree and shrub species as the whole of the United States and Canada, while a single bush in Peruvian Amazonia has revealed as many ant species as in the British Isles.
Norman Myers
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• Growing more crops and trees per acre leaves more land for Nature
• We cannot choose between malnourished children & saving endangered wild species
• Without higher yields, peasant farmers will destroy the wildlands and species to keep their children from starving
• Sustainably higher yields of crops and trees are the only visible way to safe both
Dr. Norman Borlaug
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IntroductionIntroduction R&DR&D ServicesServices StaffStaff Products Products ContactsContacts
TISSUE CULTURE LABORATORY(MAKMAL KULTUR TISU)
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Facilities & InfrastructureWe are fully equipped to undertake a complete range of in vitro studies, i.e. micropropagation,
suspension cell culture, somatic embryogenesis, etc.
Media Preparation Room
Weaning Chamber
Transfer Room Growth Rooms Culture Racks
Holding AreaGrowing AreaMisting Area
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Research & DevelopmentDevelopment of suitable, economically viable mass propagation
protocol for various forest species.
Selected Parent Explant Material Culture Initiation Multiplication Weaned Plantlet
Somatic Embryos S.E. germination Shorea (Rita System)Embryogenic callus Cell suspension
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Shorea leprosula
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Wood density(AB6 x MB20)
Fiber Length(AB3 x MB22
Harvest (pods/seeds)
Transfer to laboratory
Surface sterilisation
In vitro germination
Confirmation (DNA/Isozyme)
MultiplicationTransplant
Hybrids
Non-hybrids
Discarded
Clonal Trial
Acacia hybrids
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MATURE PODS IMMATURE PODS
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• Crosses > 70,000• Seed Pods > 380• Seeds > 2,000• Seedlings (ca 70%) > 1,400• Selected clones = 400• Plantlets (for clonal trial) = 20,000
In vitro germination & plant production
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for Somatic Embryogenesis for Micropropagation
Explant sourceShoot formationRegeneration
Culture initiation
Shoot multiplication
Callus initiationCallus proliferation
Neobalanocarpus heimii (Chengal)
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Culture Establishment from Seed Explant
ex vitro germination
in vitro germination
Seed explantsSeed dissected
Germination (in vitro)
Seedling (in vitro)
Germination (ex vitro) Seeding (ex vitro)
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Tongkat Ali (somatic embryogenesis)
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Mangrove species
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Weaning Chamber
Patent Pending:(# PI 2005 2865)
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Teak
(Tectona grandis)
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Acacia
(Acacia hybrids)
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Jelutong(Dyera costulata)
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Sesenduk(Endospermum diadenum)
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Karas
(Aquilaria malaccensis)
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Sungkai(Peronema canescens)
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MAIN ACTIVITY FLOW-CHART
SampleReceive
d
Reject sample(Sample again)
Order (received)
Record informatio
n
PROTOCOL DEVELOPMENT
Sterilisation Method
R & D
Culture Initiation
Multiplication
(culture)
Rooting (of
shoots)
Weaning
No
No
No
No
No
Delivered( to client)
Prepare for
production
Shoot multiplicatio
n
Rooting of
shoots
Sales(ex-lab)
Weaning(ex-
nursery)
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other experiences
a quick recap
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Kodiswaran Kandasamy © 2006
Somatic embryogenesis
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Suspension cell culture
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Artificial seed production
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Anther & pollen culture
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Protoplast culture
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Protoplast fusion
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Fungal interaction studies
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Associated problems
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THANK YOUTHANK YOU
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Kodiswaran KandasamyTissue Culture UnitForest Biotechnology [email protected] http://www.frim.gov.my/knowledge_centre
Kodiswaran Kandasamy © 2006
1. Disease tolerance
2. Climatic tolerance
3. Value added products
4. Higher/improved yield
5. Change characteristics
Cost & time
Kodiswaran
Kandasamy
Genetic modification
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
1. Look for the gene (s) &
remove it
2. Clone it & place it in the
desired plant
3. Ensure it functions well & does
not disrupt other functions
4. Produce many copies of the
plant
What it means?
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Plant Production Processing & formulating
Marketing
Hydroponic farming for
clean & vigorous growth &
quick harvest
Processing plant
Freeze drying
Grinding Product formulation
MARKET
R&D
Pegaga grown via
Tissue Culture for
mass volume production
1. Health food supplement2. Natural medicine3. Drugs Cost & time
Extraction, Purification &
Standardisation
Pharmaceuticals/Nutraceuticals
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1. Plantibodies (diagnostics &
vaccines)
2. Therapeutic proteins (e.g.
Interferon, growth hormones)
3. High value enzymes for
industry
4. Plant defense (e.g. Virus
resistant plants)Cost & time
Green pharmacy
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1. Time: 15 years
2. Cost: Ca RM15 million
3. Screen: 10,000 compounds
4. Chance: 1 drug
Drugs from microbes
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1. Polyploidization
2. Protoplast fusion
3. Haploid plants
4. Artificial seeds
Miscellaneous tools