BY:

DR. JAMILAH SYAFAWATI BINTI YAACOB, PhD

(Tel: 03-79674090, Fax: 03-79674178)

INSTITUTE OF BIOLOGICAL SCIENCES,

FACULTY OF SCIENCE,
UNIVERSITY OF MALAYA,
50603 KUALA LUMPUR,
MALAYSIA.
INTRODUCTION TO PLANT GENETIC
ENGINEERING

(CONCEPT OF TISSUE CULTURE)

Fundamentals of PTC

1. Concepts and principle

2. Culture Requirements
3. Plant regeneration
4. Plant regeneration pathway
5. Stages in PTC
 Definition: The culture of explants / cells /
organs in synthetic formulation (media) and
placed under controlled environment to
produce whole plants.
Or can be simplified as cloning of plants.
Based on the concept of totipotency (the
ability of a single cell to divide and produce
all of the differentiated cells in an organism,
and example totipotent cells are spores and
zygotes).
 Cloning of plants:
(1) Explant or excised plant
parts (from intact
plants)
(2) Media formulation
(3) Plant hormones to
promote growth
(4) Controlled environment
(growth parameters)
(5) Clean / free from
microorganism
 Regeneration of CLONES:
individual plants with
similar genetic make-up
with mother plant.

But in some instances,

somaclonal variants may
arise (PROS vs. CONS?)
 Pants are unable to seek out environmental conditions optimal
for their growth and development but instead must complete
their life cycles in the environment in which they are growing.
However, plants are remarkably plastic, such that a single
genotype is able to give rise to a wide range of phenotypes.
Developmental plasticity has profound implications for plant
evolution and ecology and can make important contributions
to improving yield stability in agriculture.
Highly adaptable to environmental conditions
Plasticity is an important concept in Plant tissue culture:
Ability to initiate cell division from almost any tissue
Regenerate lost organ
Undergo different developmental pathways in response to
particular stimuli
 Many somatic plant cells, including some fully
differentiated types (e.g. leaf mesophyll),
provided they contain intact nuclear, plastid and
mitochondrial genomes, have the capacity to
regenerate into whole plants.
All plant cells can, when given the right stimuli
express the total genetic potential of the parent
plant.
Maintenance of the genetic potential is called
totipotency.
 Every plant cell has the genetic blue-print of the
plant, thus the ability to form a complete plant.
Theoretically, a single cell is expected to be able
to regenerate into a complete plant.

Video: Totipotency
 Explants separated or excised from mother plant
are usually wounded.
In response to wounding, the surrounding cells
divide to cover the wound.
In nature division stops once the wound is sealed.

Under the influence of plant

growth regulators, cells form
structures such as roots, shoots,
embryos or callus /
undifferentiated cells.
 In vitro cultures require both chemical and physical needs:
(1) Culture vessels
(2) Growth media
(3) External environment (pH, temperature, gaseous
environment, light duration & quality, osmotic pressure,
etc.

Media formulations:
(1) Essential elements (nutrients) or mineral ions:
macroelements, microelements & iron source
(2) Organic supplements: vitamins & amino acids
(3) Fixed carbon
 Large amounts for growth and development
Nitrogen, phosphorus, potassium,
magnesium, calcium and sulphur
At least 0.1% of dry weight
 Trace amounts
Manganese, iodine, copper, cobalt, boron,
molybdenum, iron and zinc
Nickel and aluminium sometimes present
 Essential vitamins
Thiamine and myoinositol
Asid amino
Glycine
Casein (cheap source)
 Sucrose cheap, stabile, readily assimilated
Other carbohydrates
Glucose, maltose, galactose, sorbitol
Sometimes superior to sucrose
 May be used to solidify culture media (if do
not want to use liquid media)
Agar (quality and purity varies)
Other forms: agarose, fitagel, gelrite, etc.
 Critical in determining developmental pathway
Plant hormones and their synthetic analogues
(Naturally occurring hormone vs. synthetic PGR)
 Auxins
Cytokinins
Gibberelins
Abscisic acids
Ethylene
Brassinoids
Steroids
 Promotes callus growth, cell division, cell
enlargement, adventitious buds, and lateral
rooting.
Endogenous auxins : Natural
Indole-3-acetic (IAA).
Exogenous auxins : Synthetic.
- 2,4-Dichlorophenoxyacetic acid (2,4-D)
- Indole-3-Butyric acid (IBA)
- -Naphthaleneacetic acid (NAA)
- 4-Chlorophenoxyacetic acid (CPA).
 Regulate growth, morphogenesis and stimulate cell
division
Endogenous cytokinins : occur naturally
zeatin
6-,-dimethylallylaminopurine (2iP).
Exogenous cytokinins : synthetic
6-furfurylaminopurine (kinetin)
6-benzylaminopurine (BA or BAP).
Purine derivatives and Phenylureas as a substitute
 A family of over 70 related compounds, all
forms of Gibberellic acid.
Regulating cell enlargement and elongation,
etiolation of stems, help break bud and
seed dormancy
Produced in young leaves.
Agronomically for plant height and fruit set
Endogenous: Gibberellic acid (GA3)
Many isomers
 Only one natural compound.
Promotes leaf abscission and seed
dormancy.
Plays a dominant role in closing stomata in
response to water stress.
Has an important role in embryogenesis in
preparing embryos for desiccation.
Inhibits cell division
Promote somatic embryogenesis
 Ethylene is present in the tissues of ripening fruits,
nodes of stems, senescent leaves and flowers
In the form of gas
Ethylene leads to release of dormancy state
It stimulates shoot and root growth along with
differentiation
Regulates leaf and fruit abscission
Regulates flower induction in Bromiliad
The femaleness of dioecious flowers is stimulated
Flower opening is stimulated
Flower and leaf senescence stimulation
Fruit ripening is stimulated by ethylene
 Species and cultivar dependent
Widely used concept
Ratio of auxin to cytokinin
Auxin: Stimulates Root Development
Cytokinin: Stimulates Shoot Development
Ratio of these two hormones can determine plant
development:
Auxin Cytokinin = Root Development
Cytokinin Auxin = Shoot Development
Auxin = Cytokinin = Callus Development

High ratio value promotes root formation

Low ratio value promotes shoot formation
 Explants (Many features affect efficiency)
Callus
Cell suspension cultures
Protoplasts
Root cultures
Shoot tip and meristem cultures
Embryo cultures
Microspore cultures
Explants

Callus
Cell suspension

Root cultures
 Shoot tip cultures

1.Shoot tip in medium 2.Shoot callus 3.Growth of stem

 Protoplast cultures

A. Freshly isolated E. Callus

protoplast F. Plantlets from the callus
B. First mitotic division
C. Developed division
D. Microcolonies
Microspore/anther cultures
 Organogenesis
Production of organs directly from
explants or callus cultures
Somatic embryogenesis
Development into whole plants
analogous to zygotic embryos
Direct or indirect
Somatic cells
 Organs regenerated from explants and
callus
3 methods
Adventitious shoots from explants &
callus
Axillary buds from explants
Depends on auxin and cytokinin ratio
 Direct regeneration of banana
planting materials from suckers
 Multiple shoots

Explant

Plantlets
 Organogenesis through callus
intermediary

~
 Direct SE
Embryo formed directly from a cell or a
group of cells
Common explants are reproductive tissues
(nucellus, styles or pollen)
Indirect SE
Callus produced from explants
Embryos produced from callus
 Various stages
Initiation, maturation, germination
Distinct morphology: proembryos,
torpedo, heart (absent for monocots)
followed by cotyledons
Predetermined structures

 Explant

Plantlets

Multiple
Callus shoots
Indirect SE via callus formation
 Response to media
Explants giving
response to the media
Shoot formation
Root formation
Callus formation
 Explants must be
able to multiply
callus, roots or
shoots upon
subculture
Subculture- transfer
to fresh media after
applying wounding
 Ability to be
autotrophic when
free of
media/carbon
source
Shoot elongation
Root formation
 Acclimatization
period
Shoots in culture (in
vitro) grown in high
humidity, low light
intensity, supplied
with sugars
Conditions in natural
environment (in
vivo)- contrast to in
vitro