PRODUCTION OF NEW CULTIVARS
Consider
about the production of new cultivars there are three major parts,
- Somatic Hybridization
- Protoplast Fusion
- Somaclonal Variation
Somatic Hybridization
Production of hybrid plants
through the fusion of protoplasts of two different plant species/varieties is
called somatic hybridization, and such hybrids are known as somatic hybrids.
The technique of somatic hybridization involves the
following four steps:
(1) Isolation of protoplasts.
(2) Fusion of the protoplasts of desired species/varieties.
(3) Selection of somatic hybrid cells.
(4) Culture of the hybrid cells and regeneration of hybrid plants from them.
(1) Isolation of protoplasts
Isolation of protoplasts is
readily achieved by treating cells/tissues with a suitable mixture of cell wall
degrading enzymes. Usually, a mixture of pectinase or macerozyme and cellulase is appropriate for most plant materials.
Hemicellulase may be
necessary for some tissues. Generally, crude commercial preparations of enzymes
are used. The pH of enzyme solution is adjusted between 4.7 and 6.0 and the
temperature is kept at 25-30°C.
Usually, 50-100 m mol 1-1
CaCl2 is added to the osmoticum as it improves plasma membrane
stability. The cells and tissues are incubated in the enzyme mixture for few to
several (generally, 16-18) hours; naked protoplasts devoid of cell wall are
gradually released in the enzyme mixture.
Protoplasts have been
isolated from virtually all plant parts, but leaf mesophyll is the most
preferred tissue, for this purpose. In general, fully expanded leaves are
surface sterilized,
their lower epidermis is peeled off with a
pair of forceps and the peeled areas are cut into small pieces with a scalpel
and suspended in the enzyme mixture. When epidermis cannot be peeled, leaf may
be cut into pieces and treated with the enzyme mixture; vacuum infiltration may
be used to facilitate the entry of enzymes into the tissues.
After the period of
incubation, protoplasts are washed with a suitable washing medium in order to
remove the enzymes and the debris. The protoplasts may be cultured on a
suitable medium in a variety of ways:
·
Bergmann's plating technique (in agar
medium)
·
In a thin layer of liquid medium.
·
In small microdrops of 50-100 µl.
Protoplasts readily regenerate cell wall
(within 2-4 days) and undergo mitosis to form macroscopic colonies, which can
be induced to regenerate whole plants. The conditions for isolation and culture
of protoplasts and regeneration of complete plants have been standardized for a
large number of plant species, but cereals still present some problems.
Generally,
MS and B5 media, and their modifications are used for protoplast culture. The
media are supplemented with a suitable osmoticum and, almost always, with an
auxin and a cytokinin, their types and concentrations depending mainly on the
plant species. After 7-10 days of culture, protoplasts regenerate cell wall and
the osmolarity of medium is gradually reduced to that of normal medium.
The
macroscopic colonies are transferred onto normal tissue culture media.
Protoplasts are very sensitive to light; therefore, they are cultured in
diffuse light or dark for the first 4-7 days.
(2) Protoplast
Fusion
A number of strategies have
been used to induce fusion between protoplasts of different strains/species; of
these the following three have been relatively more successful. Protoplasts of
desired strains/species are mixed in almost equal proportion; generally they
are mixed while still suspended in the enzyme mixture.
The protoplast mixture is
then subjected to a high pH and high Ca2+ concentration at 37°C for
about 30 min (high pH high Ca2+ treatment). This technique is quite
suitable for some species, while for some others it may be toxic.
Polyethylene glycol (PEG)
induced protoplast fusion is the most commonly used as it induces reproducible
high frequency fusion accompanied with low toxicity to most cell types. The
protoplast mixture is treated with 28-50% PEG for 15-30 min, followed by
gradual washing of the protoplasts to remove PEG; protoplast fusion occurs
during the washing.
The washing medium may be
alkaline (pH 9-10) and contain a high Ca2+ ion concentration this
approach is a combination of PEG and high pH-high Ca2+ treatments,
and is usually more effective than either treatment alone. PEG is negatively
charged and may bind to cations like Ca2+, which, in turn, may bind
to the negatively charged molecules present in plasma lemma; they can also bind
to cationic molecules of plasma membrane.
During the washing process,
PEG molecules may pull out the plasma lemma components bound to them. This
would disturb plasma lemma organisation and may lead to the fusion of
protoplasts located close to each other.
The above fusion techniques
are nonselective in that they induce fusion between any two or more
protoplasts. A more selective and less drastic approach is the electrofusion
technique, which utilizes low voltage electric current pulses to align the
protoplasts in a single row like a
pearl-chain. The aligned protoplasts can be moved, with a micromanipulator, and
pairs of protoplasts may be isolated in individual microelectrofusion chambers.
The pairs of protoplasts
can be fused by a very brief (few microseconds) pulse of high voltage. Alternatively,
the protoplasts may be subjected to mass electrofusion; in such a case the
population of protoplasts is subjected to high voltage after they are brought
close to each other by the low voltage current.
The high voltage creates
transient disturbances in the organisation of plasma lemma, which leads to the
fusion of neighbouring protoplasts. The entire operation is carried out
manually in specially designed equipment, called electroporator. Many workers
feel that this fusion technique is more desirable than the others for a number
of important reasons.
(3) Selection
of Hybrid Cells
The protoplast suspension
recovered after a treatment with a fusion inducing agent (fusogen) consists of
the following cell types:
·
unfused protoplasts of the two
species/strains,
·
products of fusion between two or more
protoplasts of the same species (homokaryons),
·
'hybrid' protoplasts produced by fusion
between one (or more) protoplast(s) of each of the two species
(heterokaryotis).
In somatic hybridization
experiments, only the heterokaryotis or hybrid protoplasts, particularly those
resulting from fusion between one protoplast of each of the two species, are of
interest. However, they form only a small proportion of the population (usually
0.5-10%).
Therefore, an effective
Strategy has to be employed for their identification and isolation. This step
is called the selection of hybrid cells, and is the most critical, and is still
an active area of investigation.
A number of strategies have been used for the
selection of hybrid protoplasts.
(i) Some
visual markers, e.g., pigmentation, of the parental protoplasts may be used for
the identification of hybrid cells under a microscope; these are then
mechanically isolated and cultured. For example, the protoplasts of one species
may be green and vacuolated (from mesophyll cells), while those of the other
may be nonvacuolated and nongreen (from cell cultures). Where such features are
not available, the protoplasts of two parental species may be separately
labelled with different fluorescent agents.
This approach is time consuming, and requires considerable skill and effort. Several workers have attempted to devise systems, which specifically select for hybrid cells.
This approach is time consuming, and requires considerable skill and effort. Several workers have attempted to devise systems, which specifically select for hybrid cells.
(ii) These systems,
exploit some properties (usually, deficiencies) of the parental species, which
are not expressed in the hybrid cells due to complementarily between their
genetic systems. These properties may be sensitivity to culture medium
constituents, antimetabolites, temperature, etc. inability to produce an
essential biochemical (auxotrophic mutants), etc. These properties may be
naturally present in the parental species or may be artificially induced
through mutagenesis/genetic engineering.
For example, protoplasts of
Petunia hybrida form calli on the MS medium, while those of P. parodii produce
only small cell colonies. Further, inhibits cell division of P. hybrida protoplasts
but it has no effect on those of P. parodii.
Thus protoplasts of both
these Petunia species fail to produce macroscopic colonies (calli) on MS medium
supplemented with µg ml-l actinomycin D. However their hybrid cells (P. hybrida
+ P. parodii; somatic hybrids are denoted by a + sign divide normally on this
medium to produce macroscopic colonies. This selection strategy exploits those
natural properties of the two parental species, which show complementation in
the hybrid cells.
These strategies are simple,
highly effective and the least demanding. Recently, genetic engineering has
been used to transfer resistance to an antibiotic/herbicide in one fusion
parent and that to another one into the other parent; the hybrid cells are
selected using a medium containing both the concerned antibiotics/herbicides.
A more general and widely applicable strategy is,
(iii) To culture the
entire protoplast population without applying any selection for the hybrid
cells. All the types of protoplasts form calli; the hybrid calli are later
identified on the basis of callus morphology, chromosome constitution, protein
and enzyme banding patterns, etc.
(4) Regeneration
of Hybrid Plants
Once hybrid calli are
obtained, plants are induced to regenerate from them since this is a:
prerequisite for their exploitation in crop improvement.
Further, the hybrid plants
must be at least partially fertile, in addition to having some useful property,
to be of any use in breeding schemes. The culture techniques have been refined
to a state where plant regeneration has been obtained in a number of somatic
hybrids.
Somaclonal Variation
Shoot tips or axillary buds are used for
direct propagation on culture medium without intervening callus phase. It has
been shown in recent years, that regeneration from callus, leaf explants or
plant protoplasts leads to the generation of considerable variation, described
as ‘somaclonal variation’. This variation includes aneuploids,
sterile plants and morphological variants, sometimes involving traits of
economic importance in case of crop plants. This variation received increased
attention and excitement during 1980s in view of its potential in crop
improvement programmes. The usefulness of this variability in crop improvement
programmes, was first demonstrated through the recovery of disease resistant
plants in potato (resistance against late blight and early blight) and
sugarcane.
In plants regenerated from
callus, not only variability involving both nuclear and organellar DNA is
observed, but variability even in chromosome number is observed in long-time
cell cultures. This is exhibited in callus and in plants regenerated from the
callus.
It may be necessary to
remember that the above variation may be transient (epigenetic) or genetic;
only the latter is transmitted to the next generation and is thus important for
crop improvement of a sexually propagated crop. In several crops R0,
R1 and R2 progenies were subjected to genetic analyses
and 3:1 segregation was observed, leading to the isolation of true breeding
variants.
Although the details of the
genetic basis of somaclonal variation in most crops are still largely unknown,
variation in structure and number of chromosomes has been suggested to be one
possible basis. Polyploidy, aneuploidy, translocations, inversions and
deletions have been reported in several cases. Meiotic crossing over involving
symmetric and asymmetric recombination could also be responsible for a part of
the variation observed in the regenerated plants. Methylation at the DNA level
in tissue culture has also been considered as a possible reason for ‘somaclonal
variation’.
Somaclonal variation has
actually been used in plant breeding programmes. A number of plant species,
where useful somaclonal variation.
Mechanism of Somaclonal
Variations
1.
Genetic (Heritable Variations)
Genetic varations occucrs due to
•
point
mutations (e.g., Adh mutants in wheat)
•
cytoplasmic
(maternal inheritance)
•
gene
amplification (e.g., incr. gene copy no.)
•
activation
of transposable element
•
cytogenetic
(changes to genome structure)
aneuploidy – gain or loss of 1 or more chromosomes
polyploidy – gain or loss of an entire genome
translocation – arms of chromosomes switched
inversion – piece of chromosome inverted
2.
Epigenetic (Non-heritable Variations)
Variations
generated during tissue culture
Caused
by temporary phenotypic changes
Usually
undesirable in a breeding program, not always undesirable in propagation.
Steps involved in induction and
selection of Somaclonal Variations
Callus Tissue
Organogenesis Somaclonal Variants
Regenerated plants Hardening and Selfing
Physiological Causes,
Genetic Causes and Biochemical
Causes affect to Somaclonal Variations
Ø Physiological
Cause
Exposure of culture to plant growth
regulators.
Culture conditions
Ø Genetic Cause
1. Change in
chromosome number
§
Euploidy:
Changes chromosome Sets
§
Aneuploidy:
Changes in parts of chromosome Sets
§
Polyploidy:
Organisms with more than two chromosome sets
§
Monoploidy:
Organism with one chromasomes set
2. Change in
chromosome structure
§
Deletion
§
Inversion
§
Duplication
§
Translocation
3.
Gene Mutation
§
Tansition
§
Transversion
§
Insertion
§
Deletion
4. Plasmagene Mutation
5. Transposable element
activation
6. DNA sequence
o
Change
in DNA
§
Detection
of altered fragment size by using Restriction enzyme
o
Change
in Protein
§
Loss
or gain in protein band
§
Alteration
in level of specific protein
o
Methylation
of DNA
§
Methylation
inactivates transcription process.
Ø Biochemical
Cause
•
Lack
of photosynthetic ability due to
alteration in carbon metabolism
•
Biosynthesis
of starch via carotenoid pathway
•
Nitrogen
metabolism
•
Antibiotic
resistance.
Detection
and Isolation of Somaclonal Variants
Identification of possible somaclonal variants at anearly
stage of development is considered to be very useful for quality control in
plant tissue culture, transgenic plant production and in the introduction of
variants.
1.
Analysis
of morphological characters
•
Qualitative
characters: Plant height, maturity date, flowering date and leaf size
•
Quantitative
characters: yield of flower, seeds and wax contents in different plant parts
2.
Variant
detection by cytological Studies
•
Staining
of meristematic tissues like root tip, leaf tip with feulgen and acetocarmine
provide the number and morphology of chromosomes.
3.
Variant
detection by DNA contents
•
Cytophotometer
detection of feulgen stained nuclei can be used to measure the DNA contents
4. Variant detection by gel
electrophoresis
Change in concentration of enzymes,
proteins and hemical products like pigments,
amino acids and alkaloid can be detected by their electrophoretic pattern
5.
Detection of disease resistance variant
Pathogen or toxin responsible
for disease resistance can be used as selection agent
during culture.
6.
Detection of herbicide resistance
variant
Plantlets generated by the addition
of herbicide to the cell culture system can be used as herbicide resistance plant.
7.
Detection of environmental stress
tolerant variant
Selection of high salt tolerant
cell lines in tobacco
Selection of water-logging and
drought resistance cell lines in tomato
Selection of temperature stress
tolerant in cell lines in pear.
Selection of mineral toxicities
tolerant in sorghum plant (mainly for aluminium toxicity)
