CHAPTER 4

Haploid, Embryo, and Protoplast Culture

Contents of the chapter
Introduction to haploid & Anther culture
Importance of haploid
Meristem culture
Protoplast isolation, culture, & fusion
Production of virus-free plants;
Application to forestry and horticulture
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Haploid Production
Spontaneous production of haploids usually occurs
through the process of parthenogenesis (embryo
development from an unfertilized egg)
Rarely, they reproduce the characters of the male parent
alone,

suggesting

their

origin

through

'ovule

androgenesis (Embryo development inside the ovule by

the activity of the male nucleus alone.)
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In 1953 Tulecke discovered that haploid tissue (i.e. tissue composed of

cells having half the chromosome number that is characteristic of a
species), could be produced by the culture of Ginkgo pollen

Until 1964 the artificial production of haploids was attempted through:

a.

Distant hybridization,

b.

Delayed pollination

c.

Application of irradiated pollen

d.

Hormone treatments and

e.

Temperature shocks.

None of these methods, however, proved dependable.

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 The technique of haploid production through anther culture

('anther androgenesis') has been extended successfully to
numerous plant species,
Including many economically important plants, such as cereals
and vegetable, oil and tree crops.
The basis of pollen and anther culture is that on an appropriate
medium the pollen microspores of some plant species can be
induced to give rise to vegetative cells, instead of pollen grains.

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 This change from a normal sexual gametophytic into a vegetative

(sporophytic) pattern, appears to be initiated in an early phase of
the cell cycle when transcription of genes concerned with
gametophytic development is blocked and genes concerned with
sporophytic development are activated.
The result is that in place of pollen with the capacity to produce
gametes and a pollen tube, microspores are produced capable of
forming haploid pro-embryos (somatic embryos formed directly
from the microspores), or callus tissue.
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 The formation of plants from pollen microspores in this

way is sometimes called androgenesis.

Haploid plants are more readily regenerated by culturing

microspores within anthers than by culturing isolated

pollen.
The presence of the anther wall provides a stimulus to

sporophytic development.
The nature of the stimulus is not known but it may be

nutritional and/or hormonal.

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 Actually, three experimental approaches are available to

produce haploid plants:
1. The anther culture method, or that of microspores
derived thereof
2. The embryogenesis of isolated unfertilized egg
cells, and
3. Production from hybrids of species from which the
set of chromosomes of one parent has been
eliminated during development
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 Basically, two methods are available, which are practiced with

many variations.
Anther culture
Isolated pollen culture
In the first method, which seems to be the more original,
whole anthers are placed on an agar medium, and
in the second the anthers are cultured in a liquid medium in
which the pollen is liberated by agitation
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Anther Culture
The experimental plants for anther and pollen culture should
ideally be grown under controlled conditions of temperature,
light and humidity, and anthers should be taken from young
plants.
Generally, with increasing age of the donor plants the
androgenic response declines and abnormalities appear.
These external markers can be used for selecting buds of
approximately the required stage.
However, the correlation is never absolute and, therefore, it is
always necessary to crush one of the anthers from each bud to
assess the exact stage of pollen development.
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 The selected buds are surface sterilized with a suitable

disinfectant
Anthers along with their filaments are excised under aseptic

conditions & placed on a sterilized petri-plate.

The anthers of the stamens are gently detached from their

filaments,

without

injuring

the

anthers,

and

placed

horizontally on the medium (anther culture).

In responsive anthers, the wall tissues gradually turn brown

and, depending on the species, after 3-8 weeks they burst

open due to the pressure exerted by the growing pollen callus
or pollen embryos

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 In responsive anthers, the wall tissues gradually turn brown and,

depending on the species, after 3-8 weeks they burst open due to
the pressure exerted by the growing pollen callus/ pollen embryos
The embryos may germinate on the original medium or require
transfer to another medium to form plantlets.
After they have attained a height of about 3-5 cm, the individual
plantlets or shoots are excised and transferred to a medium which
would support good development of the root system.
The rooted plants are transferred to sterilized potting-mix in small
pots or seed trays
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Factors Affecting and Stages of Androgenesis

1. Physiological status of the donor plants
2. Stage of pollen development
3. Anther wall factor(s)
4. Genotype
Based on the few initial divisions in the microspores
four modes of in vitro androgenesis have been
identified:
Pathway I, II, III and IV.
Read this!!!!!!!
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12

GYNOGENESIS
Development of plants from unfertilized cells of the female
gametophyte (embryo sac), in floret, ovary or ovule culture is an
attractive alternative to anther culture for haploid production.
Gynogenetic haploids mostly arise from unfertilized egg cell
(parthenogenesis).
The gynogenic plants may arise through direct embryogenesis, or
callusing followed by plant regeneration on another medium.
Young flowers, ovaries or ovules have been used as the explant to
produce gynogenic haploids.
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Characteristics of Haploid Plants

The most obvious morphological characteristics of haploid plants are:
A reduced height,
smaller leaves with a reduced diameter of the leaf lamina, and
an excessive number of small fruits
Seed formation, however, is absent, and
Since fruit size often depends on the development of seeds, the fruits
remain small
Most of the time they are infertile
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Diploidization to Raise Homozygous Diploids

Haploids may grow normally up to the flowering stage but in the absence
of homologous chromosomes meiosis is abnormal consequently, viable
gametes are not formed
To obtain fertile, homozygous diploids for analysing the progenies and the
breeding behaviour of the pollen plants the chromosome complement of
the haploids must be duplicated.
Spontaneous duplication of chromosomes in pollen-derived plants has been
observed but its frequency is fairly low.
This can be significantly enhanced by using artificial means.
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15

Cont
For Nicotiana tabacum a 0.4% solution of colchicine is recommended to
diploidize the pollen plants.
In practice, the young pollen-derived plants are immersed in a filter-sterilized
solution of colchicine for about 96 h and then transferred to a culture medium
to allow their further growth.
Besides bringing about chromosome duplication, colchicine treatment may also
result in chromosome and gene instabilities (Burk, 1970).
Therefore, the frequent occurrence of spontaneous duplication of chromosomes
in differentiated plant cells (cortex, pith) and callus cells in longterm cultures.
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Application and importance of Haploid

production

A prerequisite to use the heterosis effect reproducible in hybrid

breeding is the availability of homozygous parent lines.

The production of such inbred lines requires many back-crossings

of heterozygotic parent material.

Inbred lines with desired properties are also required for

outbreeding plant species.

A considerable reduction of the time required to produce such plant

material can be achieved by the use of haploids.

Haploid higher plants are infertile, and therefore before haploids

can be used in breeding programs, a diploidization is required

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Beside a reduction of time required to establish inbred lines, another benefit

of haploid plants is the possibility of bringing recessive genes to realization
hidden in the parent generation

To consider the gene dosage effect in judging the genomes of plants

derived by androgenesis, dihaploid plants are required.

Limitation of Haploid Production

1. Low yields
2. Conversion of pollen embryos into plants
3. Albinism in cereals
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EMBRYO CULTURE
The technique of embryo culture has been widely used to produce
hybrids which were otherwise not possible due to embryo abortion
The possibility of growing embryos outside the environment of
the ovule (ex-ovulo) provides an excellent opportunity to study
the nutrition of the embryos at various stages of development
Techniques of Embryo culture
From technical considerations the 2 most important aspects of embryo culture
are:
Excision of the embryo and
Composition of the culture medium
The composition of the medium and the preparation of the explant for aseptic
culture varies with the plant and the age of the embryo to be cultured
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Excision of Embryo
For the in vitro culture of embryos, generally it is necessary to
free them from their surrounding tissues .
The mature embryos can be isolted with relative ease by splitting
open the seed.
Seeds with a hard seed-coat are dissected after soaking them in
water .
Smaller embryos require careful dissection with the aid of a
stereoscopic microscope and quick transfer to the culture vial.

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20

Cont
With respect to its nutrition, two phases of embryo development
have been recognized:
The heterotrophic phase: during this early phase the embryo is
dependent and draws upon the endosperm and the surrounding
maternal tissues; and
The autotrophic phase: during this the embryo is metabolically
capable of synthesizing substances required for its growth from
the basic mineral salts and sugar and is, thus, fairly independent
for its nutrition.
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Protoplast Isolation Culture and fusion

In eukaryotes the transfer of genetic material from one individual to
another is conventionally achieved through sexual breeding, the
scope of which is extremely limited, particularly in animals.
Even in plants, where fairly distant species could be crossed, it has
not always been possible to obtain full hybrids between desired
individuals because of sexual incompatibility barriers
This is serious handicap in crop improvement programmes through
hybridization

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 Fusion of cells, whether in plants or animals, must occur

through the plasma membrane.
Unlike animals, in plants the plasma membrane is bound
by a rigid cellulosic wall, and the adjacent cells are
cemented together by a pectin-rich matrix.
Besides being able to fuse with each other, higher plant
protoplasts can also take up foreign DNA, through their
naked plasma membrane, under specific chemical and
physical treatments
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23

Essential ingredients of the technique of genetic modification

of plant cells through the protoplast system are:
a. isolation of protoplasts,
b. Culture of protoplasts to raise whole plants,
c. Cell fusion, and
d. Introduction of foreign genetic material into the
protoplasts.
The isolation of protoplasts from higher plants can be;
mechanical ( using fine knife), enzymatic (cellulase,
macerozyme and pectinase enzymes )
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Purification of protoplasts
After the material has been incubated in enzyme solution for an adequate period
the incubation vessel is gently swirled or the leaf pieces are gently squeezed to
release the protoplasts held in the original tissue
The digestion mixture at this stage would consist of
Subcellular debris, especially chloroplasts, vascular elements,
Undigested cells and broken protoplasts,
Besides intact and healthy protoplasts.

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25

 It is, therefore, necessary to remove these contaminants.

The large debris is removed by passing the digestion mixture
through a metal or nylon sieve (30-100 ttm pore size).
The filtrate is sedimented in a centrifuge tube at 100 x g for about
5 min, and the supernatant containing small debris is discarded.
The pellet is resuspended in the washing medium and washed
three times by repeated centrifugation at 50 x g for 3-5 min and
resuspension
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Viability of the protoplasts

Viability of the freshly isolated protoplasts can be checked by a number of

methods:

1.

Observation of cyclosis or cytoplasmic streaming as an indication of active

metabolism

2.

oxygen uptake measured by an oxygen electrode which indicates respiratory

metabolism

3.

Photosynthetic activity

4.

Exclusion of Evan's blue dye by intact membranes

5.

Staining with fluroescein diacetate.

The last method is most commonly used

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PROTOPLAST CULTURE
The culture methods and the culture requirements of isolated protoplasts are
often similar to those of single cells.
Protoplasts may be cultured in agar plates following the Bergmann's technique
of cell plating
An advantage in using semi-solid medium is that the protoplasts remain
stationary which makes it convenient to follow the development of specific
individuals

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Cont

However, liquid medium has been generally preferred for the following
reasons:

a.

Protoplasts of some species would not divide if plated in agarified medium

b.

The osmotic pressure of the medium can be effectively reduced after a few
days of culture,

c.

If the degenerating component of the protoplast population produces some

toxic substances which could kill the healthy cells it is possible to change
the medium

d.

The density of cells can be reduced or cells of special interest may be

isolated after culturing them for a few days at a high density
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Cell wall formation

Within 2-4 days in culture, protoplasts lose their characteristic spherical
shape and this has been taken as an indication of new wall
regeneration.
More reliable and direct demonstration of wall regeneration has been
through staining with Calcofluor White ST and from the use of a
variety of electron microscopic techniques
The regularity of cell wall regeneration and the lag period prior to the
onset of wall formation depends partly on:
the plant species and
the degree of differentiation of the cells used for protoplast isolation
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Somatic Hybridization and Cybridization

In culture, isolated protoplasts often perform better than single,
whole cells and should, therefore, serve as an excellent starting
material for cell cloning and development of mutant lines
Freshly isolated protoplasts have been employed in studies
related to cell wall synthesis, membrane properties & virus
infection
This technique of hybrid production through the fusion of body
cells, bypassing sex altogether, is called somatic hybridization.

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 Unlike sexual reproduction in which organelle genomes are generally

contributed by the maternal parent, somatic hybridization combines
cytoplasmic organelles from both parents.
In somatic hybrids recombination of mitochondrial genome occurs
frequently.
Fusion products with the nucleus of one parent and extra-nuclear
genome/s of the other parent are referred to as cybrid and
the process to obtain cells or plants with such genetic combination/s
is called cybridization.
Somatic cell fusion, thus, offers new ground to achieve novel genetic
changes in plants.
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32

PROTOPLAST FUSION

During enzymatic degradation of cell walls some of the adjacent

protoplasts fuse together forming homokaryons
also referred to as homokaryocytes, each with two to several
nuclei
This type of protoplast fusion, called 'spontaneous fusion
the

expansion

and

subsequent

coalescence

of

the

plasmodesmatal connections between the cells

The occurrence of multinucleate fusion bodies is more frequent
when protoplasts are prepared from actively dividing cultured
cells.
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 So far as somatic hybridization and cybridization are concerned spontaneous

fusion is of no value; these require the fusion of protoplasts of different origin.
To achieve induced fusion a suitable chemical agent (fusogen) or electric
stimulus is generally necessary
During the last decade fusion of protoplasts by electric stimulus (electrofusion)
has gained increasing popularity

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34

Chemical fusion
Since 1970 a variety of fusogens such asNaNO3, high pH and high Ca
2+ and PEG treatments & successfully used to produce somatic
hybrid/cybrid plants.
1. NaNOs treatment: Kuster demonstrated (1909) showed
hypotonic solution of NaNO3 induces the fusion of subprotoplasts within a plasmolysed epidermal cell
the technique suffers from a low frequency of heterokaryon
formation,
especially when highly vacuolated mesophyll protoplasts are
involved
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2.

High pH and high Ca 2+ treatment:In 1973 Keller and Melchers reported

mesophyll protoplasts of two lines of tobacco could be readily fused by
treating
highly alkaline (pH 10.5) solution of high Ca 2+ ions (50 mM
CaC12.2H20) at 37oC for about 30 min

Using this technique Melchers and Labib (1974) and Melchers (1977)
produced intraspecific and interspecific somatic hybrids

For somatic hybridization in petunias this method of protoplast fusion was

regarded as superior to the other two common chemical methods

However, for some protoplast systems such a high pH may be toxic

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3. Polyethylene glycol (PEG) treatment:

Since 1974 PEG has achieved widespread acceptance
as a fusogen of plant protoplasts because of:
The reproducible high frequency heterokaryon
formation
Comparatively low cytotoxicity to most cell types
The formation of a high proportion of binucleate
heterokaryons
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 PEG-induced fusion is non-specific

In addition to fusing soybean-maize and soybean-barley
PEG brings about effective fusion between
animal cells
animal cells with yeast protoplasts
animal cells with higher plant protoplasts
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Mechanism of fusion
Protoplast fusion consists of three main phases:
1. Agglutination, during which the plasma membrane of two or
more protoplasts are brought into close proximity
2. membrane fusion at small localized regions of close adhesion
resulting in the formation of cytoplasmic continuities or
bridges between protoplasts
3. rounding-off of the fused protoplast due to the expansion of
the cytoplasmic bridges forming spherical hetero- or
homokaryons
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Electrofusion
Chemical fusion of plant protoplasts has many disadvantages:
1. The fusogens are toxic to some cell systems
2. It produces random, multiple cell aggregates
3. The fusogen must be removed before culture
In contrast, electrofusion is rapid (usually complete within 15
min), simple, synchronous, and more easily controlled
The fusion systems generally consist of a DC pulse generator
and a sine wave generator connected in parallel to a fusion
chamber fitted with two electrodes
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Selection of Fusion Products

In somatic hybridization by electrofusion of protoplasts it may
not be difficult to follow the fate of the fusion products because:
the fusion frequency is very high and
possible to achieve one-to-one fusion of desired pairs of
protoplasts
However, a chemical fusion treatment results in a heterogeneous
mixture of
the parental type protoplasts, heterokaryons and
a variety of other nuclear-cytoplasmic combinations.
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 The heterokaryons which are the potential source of future

hybrids constitute very small (0.5-10%) proportion of the
mixture
Only a fraction of these heterokaryons show nuclear fusion
Moreover, being novel genetic combinations, several things
may happen following fusion treatment which further reduce
the number of potential hybrid cell lines to a very low level
It is, therefore, of key importance in somatic hybridization to
be able to select the hybrid cells or their products
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Selection methods
Morpho-physiological basis
The hybrid calli may exhibit heterosis and outgrow the parental
cell colonies
Selection of putative hybrids based on callus morphology has
been used in intra- and interspecific
Complementation
In this method complementation of genetic or metabolic
deficiencies of the two fusion partners are utilized to select the
hybrid component
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 When protoplasts of two parents, each carrying a non-allelic

genetic or metabolic defect are fused it reconstitutes a viable
hybrid cell of wild type
In which both defects are mutually

abolished by

complementation, and
The hybrid cells are able to grow on minimal medium nonpermissive to the growth of the parental cells
For such a complementation it is necessary that the defects
are recessive and expressed in cultures
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 Isolation of heterokaryons or hybrid cells

The most reliable and widely applicable selection
system is one which involves
isolation of the heterokaryons or hybrid cells and
their culture individually or at low density
This approach not only allows definitive picking up of
the hybrid components but also in purer forms.

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 Manual isolation of heterokaryons requires that the two parental

type protoplasts have distinct morphological markers and are

easily distinguishable.
Fluorescence-activated cell sorter (FACS) which is accurate and

exceptionally rapid is used for physical isolation of dual-labelled

heterokaryons
After fusing the protoplasts labelled with different fluorochromes

at different wavelengths, the mixture is fed to the FACS

In the machine the fluid stream carrying the protoplasts is passed

between focused laser light and photocells that detect the

fluorescence

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Verification of Hybridity
Most

of

the

selection

systems

have

their

characteristic

disadvantages which preclude their widespread use

Therefore, successful passage through a selection system should be

treated as first evidence for the hybridity of the selected materials.

Further proof must be added from other, independent markers to

finally prove or disprove the hybrid nature of the selected putative

hybrids.
This proof requires a demonstration of genetic contribution from

both parents
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1. Morphology
Intermediate expression of numerous vegetative and
floral characters,
such as stalk height and diameter, leaf shape, type of
trichomes formed, pigmentation, flower colour and
morphology,
have been screened for evaluation of presumed
somatic hybrids to demonstrate their hybridity
This criterion is, however, not very reliable
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2.

Cytological analysis
One of the primary features to characterize somatic

hybrids is the chromosome complement

Comparison of number and morphology of chromosomes

would reveal if the putative hybrid possesses the expected

chromosome complements from the two parents
This is relatively easy when the parental species exhibit

prominent differences in chromosome number and

morphology
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However, this approach will not be applicable to all species, if

the fusion involves closely related species or where the
chromosomes are very small

3.

Isozyme analysis
Isozymes are defined as multiple molecular forms of an
enzyme exhibiting similar or identical catalytic properties
If the two parents exhibit different band patterns for a
specific isozyme the putative hybrid can be easily verified.
The banding pattern displaying isozymes from both parents
are usually sufficient proof of hybridity
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4. DNA analysis
Recent developments of molecular biological techniques
have greatly expanded our analytical tools which can
serve to characterize somatic hybrids and cybrids.
Demonstration of the presence of DNA from both parents
provides the most direct proof of hybridity.
Restriction

fragment

length

polymorphism

(RFLP)

analysis of nuclear DNA has been widely used to verify

the hybrid and cybrid nature of the selected fusion
products

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Genetic Consequences of Protoplast Fusion

Fusion of protoplasts at the level of the plasmalemma is nonspecific, and there is no barrier to interspecific, intergeneric,
interfamily, or even interkingdom fusion of cells
Therefore, a range of wide crosses between sexually
incompatible parents to incorporate useful genes from wild
species into present day cultivars
Hybrid cells can give rise to hybrid plants with full nuclear
genomes from both the fusion partners
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52

More often, however, gradual elimination of chromosomes of

one of the partners occurs during successive cell cycles

The fate of the nuclear genome in the course of somatic

hybridization largely depends on three factors:
1. The number and type of parental cells participating in
fusion;
2. Genomic segregation during the first division of the fusion
product; and
3. Chromosome segregation and/or rearrangement during
colony formation and/or plant regeneration
PTC chapter 4

53

READING ASSIGNMENT
Production of virus-free plants; Application to
forestry and horticulture

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