Plant Tissue Culture

and Micropropagation
 Lecture Outline-Introduction to Tissue Culture

1. Significance of Plant Tissue Culture p. 690-694

1. Tissue Culture vs Micropropagation…….... Terms : explant, in vitro,

2. Advantages of Micropropagation…………. 1. Mass Production

2. Pathogen free plants
3. Germplasm preservation
4. Year-round production
5. Stock plant rejuvenation (juvenility)

3. Disadvantages of Micropropagation………. 1. Specialized equipment

2. High labor cost
3. Contamination risk
4. Risk of off-types arising
5. Difficult to determine optimum conditions

2. Principles of Micropropagation……….…. Terms : Totipotency, Meristematic cells, Parenchyma cells

3. Forms of Micropropagation….……..…… 1. Callus- adventitious shoots from callus

2. Microcuttings- no callus formed, shoots arise from buds

4. Stages of Micropropagation p.702-710…. 1. Establish explant- cut, sterilize, place on media

2. Shoot initiation – high [cytokinin]
3. Root formation- high [auxin]
4. Acclimatization- Transition to greenhouse environment
 Plant Tissue Culture
z In the US/ developed countries:
– > $15 billion/ yr
– >100 million plants/ yr
– Ornamental plant production
– Stable industry growth

z In developing countries:
– Rapid growth industry
– Production of food, fiber, forestry and medicine
– Future- ornamental exports?
 Micropropagation vs. Plant Tissue Culture

z Micropropagation-
–– The production of whole plants from small sections of a plant,
called an “explant”.
“explant”.
z
z Stem
Stem tip
tip
z
z Node
Node
z
z Meristem
Meristem
z
z Embryo
Embryo
z
z Seed.
Seed.
z Plant tissue culture-
–– Inclusive term for growth of cells and tissues in a sterile
microenvironment
z
z undifferentiated
undifferentiated plant
plant cells
cells
z
z plant
plant callus
callus
z
z plant
plant tissue
tissue
 Micropropagation

in vitro culture In vitro:

From Latin- “within the glass”
performing an experiment in a test tube

Explant
Any part of the plant (cells, tissue,
organs etc.) used to start in vitro cultures.
 Micropropagation
Advantages:
z Mass production of various plant cultivars
– Widespread availability and low cost of exotics
– 10 66 plants / year from a single explant
– Compare with scale of other asexual techniques
–– Example:
Example: Hosta
Hosta division
division

– Especially beneficial:
–– ↑↑ demand
demand oror value
value plants
plants
–– Plants
Plants are
are slow
slow or
or difficult
difficult to
to propagate
propagate
–– Endangered
Endangered species
species

•
 Micropropagation
Advantages:

z Production of pathogen-free plants

z
z Many plants can only maintain disease-free stock
plants by micropropagation.

z Germplasm preservation
z
z Germplasm: the DNA of a species.
z
z In the past: seeds
• limited shelf-life
• require considerable storage space
• don’t preserve uniform characteristic
(variablility)
 Micropropagation
Advantages:

z Year round nursery production.

z Stock plant rejuvenation.

z
z Adult phase plant material roots poorly
z
z Micropropagation induces phase reversal
z
z Plants produced by tissue culture are more juvenile and can be
used for rooting cuttings
 Micropropagation
Disadvantages:
z Specialized equipment required.
z
z Laminar
Laminar flow
flow hoods
hoods
z
z Autoclaves
Autoclaves
z
z Water
Water purification
purification systems
systems
z
z Glassware
Glassware etc.
etc. (TABLE
(TABLE 18-1)
18-1)

z High labor costs.

z
z Most limiting factor
 Micropropagation
Disadvantages:
z Contamination risks.
z
z Maintenance of aseptic (sterile) environment difficult.
z
z Rapid spread of contaminants = widespread losses.

z Risk of off-type individuals arising.

z
z Artificial environment induces mutations.

z Individual response to TC conditions varies.

z
z Trial and error to determine optimum media or
conditions.
 Principles of Micropropagation
z Why does micropropagation work?
“totipotency”
z
z the ability of a single cell to express the entire genetic
potential of the DNA of that cell. (reproduce a whole
plant from a single cell).

z
z Any tissue or cell type can be used for
micropropagation
• Meristematic
Meristematic Tissue
Tissue
• Parenchyma Tissue
• Adventitious growth
• Virtually any plant cell
 Principles of Micropropagation

Terms-
z Meristematic cells
– undifferentiated cells at shoot and root tips.
– greatest potential to produce cells that will become shoots or
roots.
z Parenchyma cells
– thin-walled cells that make up the bulk of most nonwoody
structures.
– can be induced to divide and differentiate.
 Forms of Micropropagation

z Two general forms of plantlet production:

– 1. Callus production
Callus is made first.
Shoots and roots arise from callus cells.
– 2. Microcuttings
No callus formed.
Explant has buds- microcuttings
Rooted in vitro or transferred to growing media to root.
 Forms of Micropropagation
1. Callus Culture:
z
z Explant cells dedifferentiate.
z
z Callus is a mass of undifferentiated plant cells.
z
z Balance of PGR causes cells to differentiate
(shoots
(shoots ->
-> roots->
roots-> plantlets)
plantlets)

Callus
shoots initiating
from callus
 Forms of Micropropagation
2. Microcuttings:
z
z The explant has buds (produce shoots directly-no callus).
z
z Shoots are separated from the explant and rooted.

root initiation

Cross section of a microshoot

 Micropropagation - The Basics

z Four stages:
I. Explant establishment (microshoot initiation)
II. Multiplication of microshoots
III. Rooting
IV. Acclimatization (hardening-off)
 Micropropagation - The Basics
z Stage I – Explant establishment/initiation
1. Select explant.
2. Disinfestation (surface-sterilization).
3. Place explant in media.
–– Provides:
Provides:
•• Nutrients
Nutrients
•• Vitamins
Vitamins
•• Hormones
Hormones

fungal contamination
 Micropropagation - The Basics
z Stage II – Multiplication of shoots
“Subcultured” or “transferred” to media with
↑[cytokinin] = shoot initiation

callus

CIM: callus inducing media – [auxin] = [cytokinin]

SIM: shoot inducing media – ↑ [cytokinin ]
RIM: root inducing media – ↑ [high auxin]
 Micropropagation - The Basics
[auxin] : [cytokinin] regulate organogenesis
“Organogenesis” – the formation of shoots or roots

[cytokinin] 0.3 0.3 0.3 0.3 0.3 mg/L

[auxin] 0 0.1 0.3 1.0 3.0 mg/L

 Micropropagation - The Basics
z Stage III– Formation of roots

Microshoots are transferred to ↑ [auxin] media to

initiate rooting.
 Micropropagation - The Basics

[auxin] 1 1 1 10

[cytokinin] 1 10 30 1
 Micropropagation - The Basics
z Stage IV– Acclimatization or Hardening off

100% humidity in the test tube

- no wax
- poor stomatal response

Hardening off :
- transplanting the plantlet into soilless media with mist
- placing a clear plastic cover over the plantlet
Micropropagation - Summary
Explant placed in growth
Explant harvested media containing nutrients
and plant hormones

New plantlets
develop

Plantlets transplanted into

soilless media and hardened off