kodiswaran kandasamy © 2006 kodiswaran kandasamy tissue culture unit forest biotechnology division...

Kodiswaran KandasamyTissue Culture UnitForest Biotechnology DivisionFRIMkodiswaran@frim.gov.my

PLANT PROPAGATION(AN OVERVIEW)

PLANT PROPAGATION

- conventional

- tissue culture

1. Cuttings (leaf, stem, root)

2. Layering (stem)

3. Marcotting

4. Grafting

5. Seed

CONVENTIONAL PROPAGATION

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

plant tissue culture

some basics

Description

Growing surface sterilised plant tissue in a nutrient medium under aseptic conditions in a controlled environment

Hormonal control of growth

AUXINLow

IBANAAIAA

CYTOKININHigh

BAPKinetin2iPZeatin

shoots

Effect of IAA and Kinetin on Proliferation and morphogenesis:

Auxin - 3 3 0.03Cytokinin 0.2 0.2 0.02 1

(mg/l)

MICROPROPAGATION(commercial)

• Laboratory design• Defined culture procedures• Scheduling & Stock keeping• Calculations• Human resource

Key factors

• 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

Micropropagation cycle

Laboratory

Greenhouse

• 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

• 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

• Production estimations• Input requirement & cost

• Raw material• Labour

• Production efficiency• Cost estimations

• Budget estimation• Business plan• Cash flow (P&L)

• Marketing• Delivery schedule

Calculations

• 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

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

work @ frim

at a brief

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

• 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

IntroductionIntroduction R&DR&D ServicesServices StaffStaff Products Products ContactsContacts

TISSUE CULTURE LABORATORY(MAKMAL KULTUR TISU)

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

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

Shorea leprosula

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

MATURE PODS IMMATURE PODS

• 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

for Somatic Embryogenesis for Micropropagation

Explant sourceShoot formationRegeneration

Culture initiation

Shoot multiplication

Callus initiationCallus proliferation

Neobalanocarpus heimii (Chengal)

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)

Tongkat Ali (somatic embryogenesis)

Mangrove species

Weaning Chamber

Patent Pending:(# PI 2005 2865)

(Tectona grandis)

Acacia

(Acacia hybrids)

Jelutong(Dyera costulata)

Sesenduk(Endospermum diadenum)

(Aquilaria malaccensis)

Sungkai(Peronema canescens)

MAIN ACTIVITY FLOW-CHART

SampleReceive

Reject sample(Sample again)

Order (received)

Record informatio

PROTOCOL DEVELOPMENT

Sterilisation Method

Culture Initiation

Multiplication

(culture)

Rooting (of

shoots)

Weaning

Delivered( to client)

Prepare for

production

Shoot multiplicatio

Rooting of

shoots

Sales(ex-lab)

Weaning(ex-

nursery)

other experiences

a quick recap

Somatic embryogenesis

Suspension cell culture

Artificial seed production

Anther & pollen culture

Protoplast culture

Protoplast fusion

Fungal interaction studies

Associated problems

THANK YOUTHANK YOU

Kodiswaran KandasamyTissue Culture UnitForest Biotechnology DivisionFRIMkodiswaran@frim.gov.my http://www.frim.gov.my/knowledge_centre

1. Disease tolerance

2. Climatic tolerance

3. Value added products

4. Higher/improved yield

5. Change characteristics

Cost & time

Kodiswaran

Kandasamy

Genetic modification

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

What it means?

Plant Production Processing & formulating

Marketing

Hydroponic farming for

clean & vigorous growth &

quick harvest

Processing plant

Freeze drying

Grinding Product formulation

MARKET

Pegaga grown via

Tissue Culture for

mass volume production

1. Health food supplement2. Natural medicine3. Drugs Cost & time

Extraction, Purification &

Standardisation

Pharmaceuticals/Nutraceuticals

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

1. Time: 15 years

2. Cost: Ca RM15 million

3. Screen: 10,000 compounds

4. Chance: 1 drug

Drugs from microbes

1. Polyploidization

2. Protoplast fusion

3. Haploid plants

4. Artificial seeds

Miscellaneous tools

kodiswaran kandasamy © 2006 kodiswaran kandasamy tissue culture unit forest biotechnology division...

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