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Kodiswaran Kandasamy © 2006
Kodiswaran KandasamyTissue Culture UnitForest Biotechnology DivisionFRIMkodiswaran@frim.gov.my
PLANT PROPAGATION(AN OVERVIEW)
Kodiswaran Kandasamy © 2006
PLANT PROPAGATION
- conventional
- tissue culture
Kodiswaran Kandasamy © 2006
1. Cuttings (leaf, stem, root)
2. Layering (stem)
3. Marcotting
4. Grafting
5. Seed
CONVENTIONAL PROPAGATION
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
plant tissue culture
some basics
Kodiswaran Kandasamy © 2006
Description
Growing surface sterilised plant tissue in a nutrient medium under aseptic conditions in a controlled environment
Kodiswaran Kandasamy © 2006
Hormonal control of growth
AUXINLow
High
IBANAAIAA
CYTOKININHigh
Low
BAPKinetin2iPZeatin
shoots
roots
Kodiswaran Kandasamy © 2006
Effect of IAA and Kinetin on Proliferation and morphogenesis:
Auxin - 3 3 0.03Cytokinin 0.2 0.2 0.02 1
(mg/l)
Kodiswaran Kandasamy © 2006
MICROPROPAGATION(commercial)
Kodiswaran Kandasamy © 2006
• Laboratory design• Defined culture procedures• Scheduling & Stock keeping• Calculations• Human resource
Key factors
Kodiswaran Kandasamy © 2006
• 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
Kodiswaran Kandasamy © 2006
Micropropagation cycle
Laboratory
Greenhouse
Kodiswaran Kandasamy © 2006
• 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
Kodiswaran Kandasamy © 2006
• 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
Kodiswaran Kandasamy © 2006
• Production estimations• Input requirement & cost
• Raw material• Labour
• Production efficiency• Cost estimations
• Budget estimation• Business plan• Cash flow (P&L)
• Marketing• Delivery schedule
Calculations
Kodiswaran Kandasamy © 2006
• 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
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
work @ frim
at a brief
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
• 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
Kodiswaran Kandasamy © 2006
IntroductionIntroduction R&DR&D ServicesServices StaffStaff Products Products ContactsContacts
TISSUE CULTURE LABORATORY(MAKMAL KULTUR TISU)
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
Shorea leprosula
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
MATURE PODS IMMATURE PODS
Kodiswaran Kandasamy © 2006
• 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
Kodiswaran Kandasamy © 2006
for Somatic Embryogenesis for Micropropagation
Explant sourceShoot formationRegeneration
Culture initiation
Shoot multiplication
Callus initiationCallus proliferation
Neobalanocarpus heimii (Chengal)
Kodiswaran Kandasamy © 2006
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)
Kodiswaran Kandasamy © 2006
Tongkat Ali (somatic embryogenesis)
Kodiswaran Kandasamy © 2006
Mangrove species
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
Weaning Chamber
Patent Pending:(# PI 2005 2865)
Kodiswaran Kandasamy © 2006
Teak
(Tectona grandis)
Kodiswaran Kandasamy © 2006
Acacia
(Acacia hybrids)
Kodiswaran Kandasamy © 2006
Jelutong(Dyera costulata)
Kodiswaran Kandasamy © 2006
Sesenduk(Endospermum diadenum)
Kodiswaran Kandasamy © 2006
Karas
(Aquilaria malaccensis)
Kodiswaran Kandasamy © 2006
Sungkai(Peronema canescens)
Kodiswaran Kandasamy © 2006
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)
Kodiswaran Kandasamy © 2006
other experiences
a quick recap
Kodiswaran Kandasamy © 2006
Kodiswaran Kandasamy © 2006
Somatic embryogenesis
Kodiswaran Kandasamy © 2006
Suspension cell culture
Kodiswaran Kandasamy © 2006
Artificial seed production
Kodiswaran Kandasamy © 2006
Anther & pollen culture
Kodiswaran Kandasamy © 2006
Protoplast culture
Kodiswaran Kandasamy © 2006
Protoplast fusion
Kodiswaran Kandasamy © 2006
Fungal interaction studies
Kodiswaran Kandasamy © 2006
Associated problems
Kodiswaran Kandasamy © 2006
THANK YOUTHANK YOU
Kodiswaran Kandasamy © 2006
Kodiswaran KandasamyTissue Culture UnitForest Biotechnology DivisionFRIMkodiswaran@frim.gov.my 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?
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
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
Kodiswaran Kandasamy © 2006
1. Time: 15 years
2. Cost: Ca RM15 million
3. Screen: 10,000 compounds
4. Chance: 1 drug
Drugs from microbes
Kodiswaran Kandasamy © 2006
1. Polyploidization
2. Protoplast fusion
3. Haploid plants
4. Artificial seeds
Miscellaneous tools
top related