Different aspects of anther culture and somatic hybridization in plants

1. ANTHER CULTURE
AND
SOMATIC HYBRIDIZATION

2. ANTHER CULTURE: Anther culture is a technique by which the developing
anthers at a precise and critical stage are excised aseptically from unopened
flower bud and are cultured on a nutrient medium where the microspores
within the cultured anther develop into callus tissue or embryoids that give
rise to haploid plantlets either though organogenesis or embryogenesis.
POLLEN CULTURE: Pollen or microspore culture is an in vitro technique by
which the pollen grains preferably at the uninucleated stage ,are squeezed
out aseptically from the intact anther and then cultured on nutrient medium
where the microspores, without producing male gametes , develop into
haploid embryoids or callus tissue that give rise to haploid plantlets by
embryogenesis or organogenesis.

3. PRINCIPLE OF ANTHER AND POLLEN CULTURE
• The production of haploid plants exploiting the totipotency of
microspore.
• In this process the normal development and function of the pollen
cell to become a male gamete is stopped and is diverted forcibly to a
new metabolic pathway for vegetative cell division.

4. HISTORY
W. TULECKE (1953)
First observed that mature pollen grains of Ginkgo biloba (a gymnosperm) can
be induced to prolifrate in culture to form haploid callus.
S.GUHA AND S.C. MAHESWARI (1964)
First reported the direct development of embryos from microspores of
Datura innoxia by the culture of excised anther.
J.P. BOURGIN AND J.P. NITSCH (1967)
Obtained complete haploid plantlets from anther culture of Nicotiana
tabacum.

5. ANDROGENESIS
• Androgenesis is the in vitro development of haploid plants originating from
totipotent pollen grains through a series of cell division and differentiation.
• It is of two types.
1) Direct androgeneis:-
The microspores behaves like a zygote and undergoes chance to form
embryoid which ultimately give rise to a plantlet.
2) Indirect androgenesis (Organogenic pathway):-
The microspores divide repeatedly to form a callus tissue which
differentiates into haploid plantlets.

8. PLOIDY LEVEL AND CHROMOSOME DOUBLING
The ploidy level of plants derived from anther or microspore culture is highly variable
due to endomitosis or fusion of various nuclei. Moreover, haploid tissues are quite
susceptible to change in ploidy level during cell proliferation and growth in vitro. So
the cultured plants must be analysed for ploidy status for obtaining homozygous lines.
Some approaches to determine ploidy level are:
Counting of plastids in stomata- e.g. in potato monohaploids have 5-8, dihaploids
have 10-15 and tetrahaploids have 18-24 chloroplasts per guard cell
Counting of chromosome number
Number of nucleoli- haploids contain one nucleolus while diploids contain two
nucleoli
Flow cytometric analysis- nuclear DNA content reflects the ploidy status
The haploid plants are diplodized to produce homozygous plants by
colchicine treatment (0.5%)

9. FACTORS AFFECTING ANTHER CULTURE
Genotype of donor plant
Anther wall factor
Stage of pollen
Physiological status of donor plant
Pre-treatment of anthers
• Cold treatment- 3 to 5⁰C for 2 days in tobacco
• Hot treatment- 30⁰C for 24 hours or 40⁰C for 1 hour (Brassica)
• Chemical treatment- Ethrel
Culture medium- Sucrose, chelated iron, glutamine, activated charcoal, auxin,
cytokinin etc. in media are beneficial

10. IMPORTANCE OF ANTHER CULTURE
1) Study of genetic recombination in higher plants.
2) Study of mode of differentiation from single cell to whole organism.
3) Study of factor controlling pollen embryogenesis of higher plants.
4) Anther culture are use to obtain the alkaloid Example :- Homozygous recombination
Hyoscyamus niger having higher alkaloid content is obtain by anther culture.
5) Formation of double haploids that are homozygous and fertile. Double haploids are used in:
 Development of pure homozygous lines
 Hybrid development
 Mutation study
 Transfer of disease and insect resistance
 Haploid are use in molecular biology and genetic engineering. Example:- Haploid tissue of
Arabidopsis and Lycopersicon have been used for the transfer and expression of three genes from
Escherchia coli
 Genome mapping

11. LIMITATIONS
1. High level of management and expertise required
2. Tissue or callus comprises a chimera of diploid, tetraploid and
haploid cells
3. There is little chance of isolating a haploid from a mixture of various
ploidy levels since higher ploidy levels are easily outgrown
4. Formation of albinos especially with cereals and effect the loss of
plants due to albinism
5. Callus in a medium supplemented with growth regulators is usually
detrimental for haploid production.
6. The doubling of haploid does not always result in production of
homozygote

12. • Development of hybrid plants through the fusion of somatic
protoplasts of two different plant species/varieties is called
somatic hybridization
• This is a non conventional genetic procedure involving fusion
between isolated protoplasts under in vitro condition and
subsequent development of their product (heterokaryon) to
a hybrid plant

13. 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

14. Procedure

15. Isolation of Protoplast
(Separartion of protoplasts from plant tissue)
1. Mechanical Method 2. Enzymatic Method
• First used by Klecker, 1892
• Laborious method
• Restricted to certain tissues which have
large vacuolated cells
• Yield of protoplast is low
• Viability of protoplast is low
• First used by Cocking, 1960
• Easy
• Good yield
• Most frequently used

16. Mechanical Method
Plant Tissue
Collection of protoplasts
Cells Plasmolysis
Microscope Observation of cells
Cutting cell wall with knife
Release of protoplasm

17. Enzymatic Method
Leaf sterilization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase Pectinase
Protoplasm released
Release of
isolated cells
cellulase
Protoplasm
released
Isolated
Protoplasts

18. • The most convenient and suitable source of protoplasts is mesophyll
tissue from fully expanded leaves of young plants or new shoots
• The most frequently used pectinase is macerozyme (macerase) derived
from Rhizopus fungus. Driselase enzyme has both cellulolytic and
pecteolytic activities.
• Enzymatic isolation is performed under the pH of 4.7-6.0 and at the
temperature of 25-30⁰C
• Osmoticum like mannitol, sucrose etc. are required to prevent the
protoplasts from bursting

19. Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous
Fusion
2. Induced Fusion
Intraspecific Intergeneric Electrofusion
Mechanical
Fusion
Chemofusion

20. Spontaneous Fusion
Protoplast fuse spontaneously during isolation process mainly due to physical contact. It is
observed when protoplasts are isolated from callus cultures.
Intraspecific produce homokaryones
Intergeneric have no importance
Induced Fusion
• Chemofusion- fusion induced by chemicals
Types of fusogens
PEG, NaNO3, Ca 2+ ions, Polyvinyl alcohol etc.
• Mechanical Fusion- Physical fusion of protoplasts under microscope by using
micromanipulator and perfusion micropipette
• Electrofusion- Fusion induced by electrical stimulation
Treatment with PEG in the presence of/or by high pH/Ca ions is reported to be
the most effective in enhancing the fusion frequency and survivability of protoplasts

21. IDENTIFICATION AND SELECTION OF SOMATIC HYBRID CELLS
• Selection of somatic hybrids by culturing them on such a medium on which
only somatic hybrids can grow (hybrid complementation)
• Mechanical isolation by visual means and knowledge of identification of
somatic hybrids (e.g. pigmentation)
• Morphology of the plant after regeneration
• Compound selection system

22. Fusion
Strain A (Albino-virescent)
vir +
II. Chlorophyll deficiency complementation method
vir +
Strain B (albino sub lethal)
+ sl
+ sl
+ sl
vir +
Green Colony
Most frequently used method
Albino Albino

23. Morphology
Cytoplasmic markers
Isozyme analysis
Molecular techniques
Genetic characterization
HYBRID VERIFICATION AND CHARACTERIZATION

24. 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.
Caipira sweet orange + Rangpur lime
CULTURE OF THE HYBRID CELLS
Hybrid cells are cultured on suitable medium provided with the
appropriate culture conditions.

25.  May be complete in two to several days
 Although protoplast in culture generally start regenerating a cell
wall within a few hours after isolation.
 Protoplast lose their characteristic spherical shape once the wall
formation is complete.
 Regeneration of cell wall can be demonstrated using Calcalfluor
White ST fluoresecent stain or Tinapol solution
CELL WALL REGENERATION

26. Symmetric hybrids: These contain the somatic chromosome of both
the parental species. These are very significant as they show all the
properties exhibited by parent species.
Asymmetric hybrids: These are those hybrids which preserve the
genetic material of one parent organism. The chromosome content of
other parent species is lost.

27. CYBRIDS
Cybrids or cytoplasmic hybrids are cells or plants containing nucleus of one species but
cytoplasm from both the parental species.
They are produced in variable frequencies in normal protoplast fusion due to:
• Fusion of normal protoplast of one species with an enucleate protoplast or a
protoplast having inactivated nucleus
• Elimination of nucleus of one species from a normal heterokaryon
• Gradual elimination of chromosome of one species from a hybrid cell during
mitotic divisions
Irradiating (with X or gamma rays) the protoplasts of one species
can inactivate their nuclei. Enucleated protoplasts can be obtained by high
speed centrifugation.

28. ADVANTAGES OF SOMATIC HYBRIDIZATION
1. Symmetric hybrids can be produced between species, which cannot be hybridized sexually. These
hybrids can be readily used in breeding programmes for transfer of useful genes to crops or may be
useful as new species.
2. Hybrids can be produced even between such strains, which are completely sterile, e.g., monoploids.
3. Cytoplasm transfers can be affected in one year, while backcrossing may take 15-16 years. Even
where backcrossing is not applicable, cytoplasm transfers can be made using this approach.
4. Mitochondria of one species can be combined with chloroplasts of another species. This may be
very important in some cases, and is not achievable by sexual means even between easily crossable
species.
5. Production of novel interspecific and intergenic hybrid e.g. Pomato (Hybrid of potato and tomato)
6. Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids.
7. Transfer gene for disease resistance, abiotic stress resistance, cytoplasmic male sterility, herbicide
resistance and many other quality characters
8. Study of cytoplasmic genes

29. LIMITATIONS OF SOMATIC HYBRIDIZATION
• Poor regeneration of hybrid plants
• Non-viability of fused products
• Not successful in all plant species
• Production of unfavorable hybrids
• Lack of an efficient method for selection of hybrids
• No confirmation of expression of particular trait in somatic hybrids
• The end product is often unbalanced (sterile, misformed or unstable)