By,Pillai Aswathy viswanathPG 2 BotanySt. Thomas college kozhencherry

The dominant life form of higher plants is the free-living sporophyte.

The sporophyte is the resultant of fertilization of male and female gametes and contains a set of chromosomes from each parent which genomic constitution is 2n.

Cells of the gametophytes carry half the sporophytic set off of chromosomes [n]

In diploid plants, which contain two sets [2n] of chromosomes

Haploid plants are defined as sporophytes having only a single set of chromosomes (n;gametophytic number of chromosome).

The ability to produce haploid plants is a tremendous benefit in genetic, plant breeding, plant physiology and embryology studies.

Study of genetic recombination in higher plants.

Haploids are use for mutation study Heritability studies are simplified, due

to haploid plant having only one set of chromosome hence recessive mutation are easily identified.

Several strategies and methods have been worked for the production of haploid plants.

Two methods: Androgenic methods Gynogenic methods

Androgenic Methods: haploid production of

plants through anther or microspore culture has been referred to as androgenesis

Gynogenic methods: haploid production of

plants from ovary or ovule culture has been referred to as gynogenesis

It is the formation of sporophyte from the male gametophyte on artificial medium

It is most commonly found in family solanaceae and poaceae

In androgenesis immature pollen grains are induced to follow the sporophytic mode of development by various physical and chemical stimuli.

There are two methods for in vitro production of androgenic haploids

They are : Anther culture isolated Pollen [microspore] culture.

Anther Culture

The male reproductive part of a flower is called the stamen.

It is composed of a long tube called a filament and has a pollen-producing structure on the end. This oval-shaped structure is called the anther.

it produces the male gametophyte known as pollen.

Pollen: A fertilizing powder discharged from flowers anther

Anther culture is the process of using anthers to culture haploid plantlets.

The technique was discovered in 1964 by Guha and Maheshwari.

This technique can be used in over 200 species, including tomato, rice, tobacco, barley,datura ,brassica ,etc.

The success of androgenesis dependent on the variety used, the growth condition of the plants, and the quality of the donor material.

The natural flowering conditions are normally the best environment for donor plants to produce anthers to be used in successful regeneration experiments.

Age of the donor plants should be noted

Usually anther from the flower buds will give better response during androgenesis

Once the donor material containing the microspores has been selected , it requires specific pretreatment conditions

The pretreatment can be applied at different levels of explants , such as intact flowers (e.g. for barley complete spikes), isolated anthers, or isolated microspores.

With regard to different explants, the type, levels, and duration of pretreatments are different, and the regeneration efficiencies vary as well.

It is widely supposed that pretreatment plays a key role for anther callus induction.

The main pretreatments applied to anther culture are cold treatment, hot treatment , and so on.

Cold treatment: In general, cold treatment between 3-6 degree C for 3-15 days gives good response.

As a result of cold treatment , weak or non-viable anther and microspores are killed also it will retards aging of the anther wall

It will showed pronounced activity in multiplication of embryonic cells.

Hot treatment: Floral buds or the entire plant in some species when subjected to 30 degree C for 24h or 40 degree for 1h stimulates embryogenesis.

Also cause dissolution of microtubules and dislodging of the spindle which causes abnormal division of the microspore nucleus

Before culture, surface sterilization of flower buds was carried out in the Laminar Air Flow Cabinet.

Young flower buds with immature anthers are surface sterilized and rinsed with sterile water

The calyx from the flower buds will be removed by flamed forceps

The corolla is slit open and stamens are removed and placed on the sterile petri dish

Each anther is gently separated from the filament

Care should be taken to avoid injury to anthers since it may induce callus formation from anther walls.

The intact uninjured anthers are inoculated horizontally on nutrient media

Media: The medium requirement may vary

with the species For most of the species, the

commonly used media for anther culture include MS (Murashige and Skoog, 1962) medium.

The constituents of the basal medium and combinations of growth regulators are also an important factor in eliciting successful androgenesis

Anther culture media is often solidified using agar.

Agar may contain compounds inhibitory to the androgenic process in some species

The use of liquid medium has been advocated by some researchers as a way to avoid the potentially inhibitory substances in gelling agents.

Anthers may be placed on the surface of the medium

Alternatively, microspores may be isolated and cultured directly in liquid medium.

In androgenesis most of the species required complete nutrient medium [mineral salts, vitamins and sucrose] with growth regulators.

For many species (2–3%) sucrose is added to the media it will induced the pollens for androgenesis

Several reports are available in which either one or more hormone has been found necessary for an androgenic response.

Addition of activated charcoal to agar medium is advocated, since charcoal is thought to absorb inhibitory compounds present in trace elements in the culture medium

For a few species, such as tobacco, it is not necessary to add plant growth regulators to the anther culture media.

Most species, however, require a low concentration of some form of auxin in the media.

Cytokinin is sometimes used in combination with auxin, is necessary for pollen embryogenesis and pollen callusing

The addition of other substances in the medium such as glutamine, casein, proline, biotin, inositol, coconut water, silver nitrate and polyvinylpyrrolidone

The addition of glutamine and glutathione to the culture medium also enhances the embryogenic response.

After inoculation haploid plants develop from anther culture either directly or indirectly through a callus phase.

Direct androgenesis Indirect androgenesis

Direct androgenesis: It is also called pollen derived

embryogenesis Here pollen grains directly acts as a

zygote and passes through various embryogenic stages similar to zygotic embryogenesis.

When the pollen grains has reached globular stage of embryo, the wall of the pollen is broken and embryo is released

The released embryo develop cotyledons, which ultimately give rise to plantlets

Eg: Datura , Brassica campestris

Indirect androgenesis:- In indirect androgenesis

the pollen grains, instead of normal embryogenesis , divide erratically to develop callus

Callus tissue which is finally redifferentiates and forms haploid plantlets

Eg: rice , wheat, tomato

Depending on the composition of the medium, development pollen may leads to the formation of embryoids or a mass of parenchymatous callus

Based on the few initial divisions in the pollen grains or responds of pollen grains,4 pathways have been identified in in vitro androgenesis

The uninucleate pollen grains may divide symmetrically to yield two equal daughter cells, both of which undergo further divisions it will contribute to sporophyte development

Vegetative and generative cells are not distinctly formed in this pathway.

Example: Datura innoxia

In this case ,the uninucleate pollen divides unequally which will result in the formation of Vegetative and generative cells

The sporophyte or plantlet arises through further division in the vegetative cell while the generative cell does not divide.

Examples: Nicotiana tabacum , Hordeum vulgare , Triticum aestivum

The uninucleate pollen undergoes a normal division but pollen embryos are predominantly formed from generative cell alone. the generative cell either does not divide at all or does so only to limited extent

The vegetative cell does not divide.

Examples: Hyoscyamus niger

In some species, the uninucleate pollen grains divide unequally, producing generative and vegetative cell but both these cells divide repeatedly to contribute to the development of sporophyte

Examples: Datura metal

Of the above early pattern of divisions , the responsive pollen grains ultimately become multicellular and burst open to release cellular mass having an irregular shape

This tissue gradually becomes globular embryo and undergoes normal embryonic differentiation

Four to five weeks after inoculation of anthers, the calli attained convenient size.

Then they were removed aseptically from the petridish on a sterilized glass plate inside the laminar airflow cabinet and were placed again on freshly prepared sterilized medium containing appropriate hormonal supplements for plant regeneration from the callus.

Sub culture was done in the MS media The sub cultured calli continued to

proliferate and differentiated into shoots.

After shoot initiation, more light intensity was used for shoot elongation.

The plants with well developed shoots and roots are then transferred to pots

By this method ,the pollen grain give rise to haploid plant

The plants may originate not only from the pollen grains , but also from various parts of the anther

During anther culture there is always the possibility that somatic cells of the anther that are diploid will also respond to the culture condition and so produce unwanted diploid calli or plantlets.

Sometimes the development of microspores inside the anther may be interrupted due to growth inhibiting substances leaking out of the anther wall in contact with nutrient medium.

Anthers often fail to grow in vitro or the initial growth is followed by abortion of the embryos

Isolated microspore or pollen grains culture can be over come this

In 1953,Tulecke was able to obtain the callus from isolated pollen cultures of gymnosperms

This raised the possibility of obtaining haploids plants from isolated microspores or pollen culture

Isolated Pollen [Microspore] Culture

For microspore culture , anther are collected from sterilized flower buds and kept in a small beaker containing basal media

The microspore are then squeezed out of the anthers by pressing them against the side of beaker with a glass rod

Anther tissue debris is removed by filtering the suspension through a nylon sieve

This pollen suspension is then centrifuged

The supernatant containing fine debris is discarded and the pellet of pollen is resuspended in fresh media and washed at least twice

the final suspension is then pipetted in to small petri dishes

To ensure good aeration, the layer of liquid in the dish should be as thin as possible

Each dish is then sealed with parafilm to avoid dehydration and is incubated at 28 degree C in darkness for the first 14 days of culture

After 14 days ,the culture are transferred to suitable media

Their the microspores forms embryos or calluses which may later differentiate to form whole haploid plants

Genotype Of Donor Plant The genotype of the donor plant plays

a major role in determining the success or failure of an Androgenesis.

Some genotypes of a given species may show androgenesis, while some others may not.

The genetics of androgenic response has been analysed in several crops like wheat, barley, maize, etc.

The natural flowering conditions (light intensity, day-length, temperature, humidity, etc.) are normally the best environment for donor plants to produce anthers to be used in successful regeneration experiments.

Any infection or stress to the donor plants will lead to less success or complete failure for induction of androgenesis and further regeneration.

Age of the donor plants affect the embryogenic potential of the cultured anther

Usually anther from the flower buds give better response during androgenesis

Anther derived from the older inflorescences show decline in the frequency of haploid production due to reduced pollen viability

Anther wall factor Sometimes the development of

microspores inside the anther may be interrupted due to growth inhibiting substances leaking out of the anther wall in contact with nutrient medium.

During anther culture there is always the possibility that somatic cells of the anther that are diploid will also respond to the culture condition and so produce unwanted diploid calli or plantlets.

Culture medium The culture medium also play a vital

role , since the requirements will vary with the genotype and age of the anther as well as the condition under the donor plant are grown

The medium should contain the correct amount and proportion of inorganic nutrients

Growth regulators In cereals , auxin , particularly 2-4 D

promotes the induction of pollen callus

Kinetin or cytokinins are essential for induction of pollen embryos in solanaceae

The concentration of sucrose plays an important role in induction of pollen haploid plants

Activated charcoal is added to the culture medium

It helps in 2 ways:- The removal of inhibitors from the

agar medium The adsorption of 5-

hydroxymethylfurfural,a product of sucrose dehydration during autoclaving , assumed to be an inhibitor of growth in anther culture

Physical factors Temperature and light are two

physical factors which plays an important role in the culture of anthers

Frequency of haploid formation and growth of plantlets are generally better in light

A temperature in the range of 23-28 C is usually suitable for divisions in pollen grains

But it has been observed that chilling of anthers before inoculation , increases the number of pollen embryoids

On the other hand , high – temperature treatment of anther culture of brassica napus before transferring them to normal temperature resulted in more embryoid production

Other factors Certain organic supplements added

to culture medium often enhance the growth of anther culture

Some of these include: The hydrolyzed products of proteins

such as casein (found in milk),nucleic acids

Coconut milk obtained from tender coconuts is often added to tissue culture media

It contains a complex mixture of nucleic acids , sugar , growth hormones and some vitamins.

In addition, amino acids like glutamine, proline, serine, etc. enhance the frequency of responsive anthers

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