Biotechnology and

Germplasm
Resources

NOOR ZUHAIRAH BINTI SAMSUDDIN

 AT THE END OF THE LECTURE
STUDENTS ARE ABLE TO:
• Define the term biotechnology and
germplasm resources
• Clonal propagation by tissue culture
• Somatic cell hybridization
• Plant genetic engineering
• Germplasm resources and conservation
• Plant Genetic Resources utilization
What is Germplasm?
 GERMPLASM RESOURCES
• Germplasm is living tissue from which new
plants can be grown.
• It can be a seed or another plant part – a leaf,
a piece of stem, pollen or even just a few cells
that can be turned into a whole plant.
• Germplasm contains the information for a
species' genetic makeup, a valuable natural
resource of plant diversity.
 GERMPLASM
• It can be a seed or another plant part – a leaf,
a piece of stem, pollen or even just a few cells
that can be turned into a whole plant.
Ex situ Germplasm conservation
 Types of germplasm
1. Land races
• Land races are nothing but primitive cultivar which were selected
and cultivated by the farmers for many generations.
• Land races were not deliberately bred like modem cultivars. They
evolved under subsistence agriculture.
• Land races have high level of genetic diversity which provides them
high degree of resistance to biotic and a biotic stresses. Biotic stress
refers to hazards of diseases and insects, whereas a biotic stress
means, drought, salinity, cold, frost, etc.
• Land races have broad genetic base which again provides them
wider adaptability and protection from epidemic of diseases and
insects.
• May be used as starting material in mass selection or pure line
breeding project
 Types of germplasm
2. Commercial cultivars.
• There are two forms of this material – current cultivars and retired
or obsolete cultivars.
• These are products of formal plant breeding for specific objectives.
• Expected to have superior gene combinations, be adapted to a
growing area, and have a generally good performance.
• The obsolete cultivars were taken out of commercial production
because they may have suffered a set-back (e.g., susceptible to
disease) or higher performing cultivars were developed to replace
them.
• If desirable parents are found in commercial cultivars, the breeder
has a head start on breeding since most of the gene combinations
would already be desirable and adapted to the production
environment.
Commercial cultivars (MR219 2001) (MR263, MR253 2010)
 Types of germplasm
3. Breeding materials.
• On going or more established breeding programs
maintain variability from previous projects.
• For example, a breeder may release one genotype as a
commercial cultivar after yield tests.
• Many of the genotypes that made it to the final stage
or have unique traits will be retained as breeding
materials to be considered in future projects.
• Similarly, genotypes with unique combinations may be
retained.
 Types of germplasm
4. Genetic stock
• This consists of products of specialized genetic
manipulations by researchers
• (e.g., by using mutagenesis to generate
various chromosomal and genomic mutants).
 Types of germplasm
5. Plant introduction
• The plant breeder may import new, unadapted
genotypes from outside the production region.
• Usually from another country (called plant
introductions).
• These new materials may be evaluated and
adapted to new production regions as new
cultivars, or used as parents for crossing in
breeding projects
 BIOTECHNOLOGY?
• use of living systems and organisms to
develop or make products, or
• "any technological application that uses
biological systems, living organisms, or
derivatives thereof, to make or modify
products or processes for specific use" (UN
Convention on Biological Diversity, Art. 2).
Clonal propagation by tissue culture
Clonal propagation by tissue culture
Clonal propagation by tissue culture
 Plant tissue culture
• procedures used to maintain and grow
• plant tissue (callus, cells, protoplats) or organs
(roots, stems, embryos)
• under controlled environment (aseptic
conditions) and nutritive growth medium
• which lead to cell multiplication or
regeneration of organs or whole plants.

AGR232 N.S. ZUHAIRAH 2017 18

Clonal propagation by tissue culture
SOMATIC CELL HYBRIDIZATION
Development of hybrid plants through
the fusion of somatic protoplasts of two
different plant species/varieties is called
somatic hybridization
Somatic hybridization
 Somatic hybridization technique

1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells

5. Regeneration of hybrid plants

 Isolation of Protoplast
(Separation of protoplasts from plant tissue)

1. Mechanical Method 2. Enzymatic Method

 1. Mechanical Method

Cells Plasmolysis
Plant Tissue

Microscope Observation of cells

Release of protoplasm
Cutting cell wall with knife

Collection of protoplasm
 1. Mechanical Method

• Used for vacuolated cells like onion bulb scale,

radish and beet root tissues
• Low yield of protoplast
• Laborious and tedious process
• Low protoplast viability
 Enzymatic Method

Leaf sterlization, removal of

epidermis

Plasmolysed Plasmolysed
cells cells

Pectinase +cellulase Pectinase

Release of Protoplasm
Protoplasm released released
isolated cells
cellulase

Isolated
Protoplasm
 Enzymatic Method

• Used for variety of tissues and organs including

leaves, petioles, fruits, roots, coleoptiles,
hypocotyls, stem, shoot apices, embryo
microspores
• Mesophyll tissue - most suitable source
• High yield of protoplast
• Easy to perform
• More protoplast viability
 Protoplast Fusion
(Fusion of protoplasts of
two different genomes)

1. Spontaneous Fusion 2. Induced Fusion

Chemofusion Mechanical
Intraspecific Intergeneric Electrofusion
Fusion
 Spontaneous Fusion
• Protoplast fuse spontaneously during isolation
process mainly due to physical contact

• Intraspecific produce homokaryones

• Intergeneric have no importance
 Induced Fusion

• Chemofusion- fusion induced by chemicals

• Types of fusogens
• PEG
• NaNo3
• Ca 2+ ions
• Polyvinyl alcohal
 Induced Fusion

• Mechanical Fusion- Physical fusion of protoplasts

under microscope by using micromanipulator and
perfusion micropipette
• Electrofusion- Fusion induced by electrical
stimulation
• Pearl chain of protoplasts is formed by low strength electric field
(10kv m-1)
• Fusion of protoplasts of pearl chain is induced by the application
of high strength electric field (100kv m-1) for few microseco
Identification and Selection of somatic hybrid cells
• Hybrid identification- Based on difference between
the parental cells and hybrid cell with respect to
• Pigmentation
• Cytoplasmic markers
• Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC
(Rhodamine isothiocyanate) are used for labelling of hybrid cells
• Presence of chloroplast
• Nuclear staining
• Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-
orcein stain
 Hybrid Selection
(Several markers are used )

1. Phytotoxins
2. Specific amino acid
3. Auxin autotrophy
4. Antibiotics
5. Auxotrophic and metabolic mutants
6. Chromosomal analysis
7. Herbicides
 Culture of the hybrid cells

Hybrid cells are cultured on suitable

medium provided with the appropriate
culture conditions.
 Regeneration of hybrid plants
• Plants are induced to regenerate from hybrid
calli
• These hybrid plants must be at least partially
fertile, in addition to having some useful
property, to be of any use in breeding
schemes.
 Advantages of somatic hybridization
• Production of novel interspecific and intergenic
hybrid
– Pomato (Hybrid of potato and tomato)
• Production of fertile diploids and polypoids from
sexually sterile haploids, triploids and aneuploids
• Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other
quality characters
 Advantages of somatic hybridization
• Production of heterozygous lines in the single
species which cannot be propagated by
vegetative means
• Studies on the fate of plasma genes
• Production of unique hybrids of nucleus and
cytoplasm
 Limitations of Somatic hybridization
1. Poor regeneration of hybrid plants
2. Non-viability of fused products
3. Not successful in all plants.
4. Production of unfavorable hybrids
5. Lack of an efficient method for selection of
hybrids
6. No confirmation of expression of particular
trait in somatic hybrids
PLANT GENETIC ENGINEERING