ANTHER CULTURE AND

SOMATIC HYBRIDIZATION

Akshay ChittoraMPUA&T Udaipur

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.

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.

HISTORYW. 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.

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.

PLOIDY LEVEL AND CHROMOSOME DOUBLINGThe 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 cellCounting of chromosome numberNumber of nucleoli- haploids contain one nucleolus while diploids contain

two nucleoliFlow cytometric analysis- nuclear DNA content reflects the ploidy status

The haploid plants are diplodized to produce homozygous plants by colchicine treatment (0.5%)

FACTORS AFFECTING ANTHER CULTUREGenotype 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- EthrelCulture medium- Sucrose, chelated iron, glutamine, activated charcoal, auxin,

cytokinin etc. in media are beneficial

IMPORTANCE OF ANTHER CULTURE1) 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

LIMITATIONS1. High level of management and expertise required2. Tissue or callus comprises a chimera of diploid, tetraploid and haploid

cells3. There is little chance of isolating a haploid from a mixture of various

ploidy levels since higher ploidy levels are easily outgrown4. Formation of albinos especially with cereals and effect the loss of

plants due to albinism5. 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

SOMATIC HYBRIDIZATION

• 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

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

Procedure

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

Mechanical Method

Plant Tissue

Collection of protoplasts

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife Release of protoplasm

Enzymatic MethodLeaf sterilization, removal of epidermis

Plasmolysed cells

Plasmolysed cells

Pectinase +cellulase Pectinase

Protoplasm released Release of isolated cells

cellulase

Protoplasm released

Isolated Protoplasts

• 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

Protoplast Fusion(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion 2. Induced Fusion

Intraspecific Intergeneric ElectrofusionMechanical Fusion

Chemofusion

Spontaneous FusionProtoplast fuse spontaneously during isolation process mainly due to physical contact. It is observed when protoplasts are isolated from callus cultures.Intraspecific produce homokaryonesIntergeneric have no importance

Induced Fusion• Chemofusion- fusion induced by chemicalsTypes of fusogensPEG, 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

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

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

Morphology

Cytoplasmic markers

Isozyme analysis

Molecular techniques

Genetic characterization

HYBRID VERIFICATION AND CHARACTERIZATION

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 CELLSHybrid cells are cultured on suitable medium provided with the

appropriate culture conditions.

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

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.

CYBRIDSCybrids 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.

ADVANTAGES OF SOMATIC HYBRIDIZATION1. 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 characters8. Study of cytoplasmic genes

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)