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.

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 ANDROGENESIS –
The microspores behaves like a zygote and undergoes chance to form enbryoid which
ultimately give rise to a plantlet.
2) INDIRECT ANDROGENESIS:-
The microspores divide repeatedly to form a callus tissue which differentiates into haploid
plantlets.

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 forcely to a new metabolic pathway pathway for vegetative cell division .

FACTORS INFLUENCING ANTHER CULTLRE

1) Genotype of donor plants:- The genotype of the donor plant plays a significant role in
determining the frequency of pollen production. ¤ Example :- Horedum of each genotype differs
with respect to androgenic response in anther culture.

2) Anther wall factor:- The anther wall provide the nourishment in the development of isolated
pollen of a number of species. ¤ There are reports that glutamine alone or in combination with
serine and myoinositol could replace the anther wall factor for isolated cultures.

3) Culture Medium:-

The anther culture medium requirements vary with genotype and probably the age of the anther as
well as condition under which donor plants are grown. In corporation of activated charcol into the
medium has stimulated the induction of androgenesis. The iron in the medium plays a very
important role for the induction of haploids . Potato extracts ,coconut milk and growth regulators
like auxin and cytokininare used for anther and pollen culture.
4) Stage of microspores:-

In most of the cases anther are most productive when cultured at the uninucleate microspore stage.
Example ,barely, wheat , rice etc. Anther of some species give the best response if pollen is cultured
at first mitosis or later stage Example:-Datura ,tobacco.

5) Effect of temperature:-

Temperature enhance the induction frequency of microspore androgensis. ¨ The low temperature
treatment to anther or flower bud enhance the haploid formation. ¨ The low temperature effects the
number of factors such as dissolution of microtubules lowering of absicisic acid maintenance of
higher ratio of viable pollen capable of embryognesis.

6) PHYSIOLOGICAL STATUS OF DONAR PLANT:-

¨ Physiological status of donor plant such as water stress nitrogen requirement and age of donor
plant highly effect the pollen embryogenesis. ¨ Plants starved of nitrogen may give more responsive
anthers compared to those that are well fed with nitrogenous fertilizers.

DEVELOPMENT OF ANDROGENIC HAPLOIDS

Pathway -1:-

The microspores divide by an equal division and identical daughter cells contribute to the
saprophyte development. Vegetative and generative cells are not distinctly formed in this
pathway .Example:-Datura innoxia.

Pathway:II:-

The division of uninucleate microspores re un equal resulting in the formation of a vegetative and
generative cell. The saprophyte arise through further divisions in the vegetative cell while the
generative cell does not divide. Example:-Nicotina tabacum.

Pathway III:-

¨ The uninucleate Microspores undergoes a normal unequal division ¨ The pollen embryo are
formed from generative cell alone. Example ;- Hyoscyamus niger.

Pathway IV ;-

¨ The division of microspore is asymetrical. ¨ Both vegetative and generative cell divide further and
contribute to th dvelopment of the sporophyte.Example:- Atropa belladona.
METHOD OF ANTHER AND POLLEN CULTURE

ADVANTAGE OF POLLEN CULTURE OVER ANTHER CULTURE

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.

IMPORTANCE OF POLLEN AND ANTHER CULTURE

(1) Utility of anther and pollen culture for basic research:-

(a) cytogenetic studies.

(b)Study of genetic recombination in higher plants.

(c) Study of mode of differentiation from single cell to hole organism.

(d) Study of factor controlling pollen embryogenesis of higher plants.

(e) Formation of double haploid that are homozygous and fertile.

2) Anther and pollen culture are use for mutation study. Example :- Nitrate reductae mutants are
reported in Nicotiana tabacum.

3) Anther and pollen culture use for plant breeding and crop improvement.
4) Anther culture are use to obtain the alkaloid Example :- Homozygous recombination Hyoscyamus
niger having higher alkaloid content is obtain by anther culture.

5) Haploid are use in molecular biology and genetic engineering. Example:- Haploid tissue of
Arbidopsis and lycopersicon have been used for the transfer and expression of three genes from
Escherchia coli....

ORGAN CULTURE
As the name suggests, organ culture is in vitro generation of plants using plant
organs such as the leaf, node, internode, shoot, root, axillary bud, and seedling.

If you aim to avoid somaclonal variation in your experiments, organ culture is the
preferable technique. Some other applications of the technique include the
production of secondary metabolites; the study of the pattern of growth,
differentiation, and development of organisms; and biochemical and molecular
functions of an organ or tissue.

The best advantage of organ culture is the maintenance of organ structure and
function even after culturing. It preserves the originality of the plant and shows the
same characteristics as it would show in vivo.

The two main types of organ cultures include: Root culture and
Shoot culture.

1. ROOT CULTURE

Hairy root or root culture involves the infection of explant by gram-negative soil
bacterium Agrobacterium rhizogenes. The hairy root is induced by integrating
root-inducing (Ri) plasmid into the plant’s genome followed by infection with
Agrobacterium rhizogenes.

After 2-3 days, explants are transferred to a solid medium containing antibiotics
such as Cefatoxime or Ampicillin to kill redundant bacteria. The induced hairy root
will be observed between one week to over a month depending on the plant
species.
Hairy roots are genetically and biosynthetically stable and are perfect means to
produce secondary metabolites at the commercial level. The hairy root cultures do
not require any plant hormones and conditioning of the medium.

Apart from the production of secondary metabolites, hairy root cultures are used to
enhance the concentration of secondary compounds, produce those compounds that
are not found in non-transformed roots or regenerate whole plants.

2. SHOOT CULTURE

Shoot culture or shoot tip culture is the culture of the terminal portion of shoots
with primordia, developing shoot, and adjacent stem tissue on B5 or MS media.
The most popular application of shoot culture is virus elimination in plants. The
shoot tip is supposed to be virus-free because of the absence of cell differentiation
and vascular system, and intense metabolic activity.

Some other applications of shoot tip culture include international transportation of

plants and storage of genetic material of plants producing recalcitrant seeds.
The source of the material should be chosen properly. It’s better if the plants are
disease-free. Successful in vitro culture has been observed by using vascular
cambia, storage organs, pericycle of roots, endosperm, cotyledons, leaf mesophyll,
and provascular tissue. Also, the choice of explant depends on the purpose of
culturing.

Following are the steps for organ culture:

1. STERILIZATION OF SOURCE MATERIAL

The surface sterilization of the explant is an essential step. The explant collected
from the outside source may be infested with spores or other microbial cells. And,
if explants are not sterilized properly before culturing it will cause contamination
in all your cultures and lead to heavy culture loss. Also, if there are any damaged
or dead tissues, remove them completely before culturing the explant on the culture
media.

2. EXPLANT PREPARATION
Size and shape of the explant is a critical factor in callus and organ culture. If the
size of the explant is not optimum, it may lead to failure in callus induction and
culture loss as well. Culturists majorly prefer the use of large sizes of explant to
obtain viable cultures.

3. CULTURE MEDIUM

The type and composition of the culture medium are some of the essential factors
affecting callus and organ culture. The cultures are more affected by the level of
plant hormones—both endogenous and exogenous—in the medium. The callus or
organ cultures are generally grown on solid media but sometimes liquid media is
also preferred. They sometimes use of liquid media refers to the condition when a
plant is unable to grow in the solid media. It can be because of uneven distribution
of nutrients, improper exchange of gases, and accumulation of toxic waste products
that may develop between the callus and the medium.

4. SUBCULTURING

The culture should be transferred to fresh media after some time. Keeping cultures
on one media for a longer time leads to waste buildup, exhaustion of nutrients from
the media, and media dryness. So, it's recommended that cultures should be
subcultured every 4-6 weeks when incubated at 25°C.

What is Organogenesis and Somatic

Embryogenesis?
In plant tissue culture, organogenesis is the formation of organs from the cultured
explants (plant material such as roots, leaves and flowers).The organogenesis
process is where the plant organs, either shoots or roots, are developed.

Embryogenesis is the process of forming and developing embryos (Bhatia and Bera,
2015). Plant embryos are the area of the seed where the plant’s roots, stem and
leaves start their earliest formation.
The in vitro and in vivo plant embryogenic processes are very different from each
other. In nature, a mother plant is crossed with a parent plant to produce a seed; this
is sexual hybridization.

In tissue culture, sexual hybridation does not occur. Instead bipolar structures that
are embryo-like can regenerate from somatic cells (any plant cell other than a
gamete or germ cell) (Arnold et al., 2002). Because of the embryo origin in tissue
culture, this process is named somatic embryogenesis (SE).

Types of Organogenesis and somatic

embryogenesis
Indirect Organogenesis
The process in which plant organs are derived from a calli mass (tissue formation
occurring on a plant wound or at the site of a cut) in the explant is termed indirect
organogenesis.

Indirect organogenesis has been used to produce transgenic plants in two ways:

 A plant can be regenerated from a transformed callus.

 The initial explant is transformed, and the callus and shoots are developed from the
modified explants.

Typically, indirect organogenesis is more critical for transgenic plant production.

Direct Organogenesis
The production of direct buds or shoots from tissue with no intervening callus stage
is called direct organogenesis. Direct organogenesis has been used to propagate
plants with improved multiplication rates (high number of plant per explant) thus
reducing operational costs. It has also been used to produce more transgenic plants.
And, most importantly, it has been used for clonal propagation (genetic multiplication
of a cultivar without sexual reproduction) since it ensures the production of uniform
planting material without genetic variation.

Direct Somatic Embryogenesis

In direct somatic embryogenesis, embryos are formed directly from a cell or small
group of cells without an intervening callus production. However, direct somatic
embryogenesis is generally a rare event in tissue culture. The biological mechanism
of how direct SE occurs is not clear and it happens at random for some tested
plants.

Indirect Somatic Embryogenesis

Indirect somatic embryogenesis is a process where a callus is first produced from
the explant, and then embryos are formed from the callus tissue or a cell suspension
culture. This technique is commonly used in transgenic experiments.
Phases of organogenesis and somatic
embryogenesis
Organogenesis
Organogenesis can be split into three phases:

 In the first phase, cells in the explants acquire 'competence' which is defined as the
ability to respond to hormonal signals of organ induction. During this phase, the
process leading to organogenic competence is called dedifferentiation where
differentiated cells become undifferentiated.
 During the second phase, competent cells in cultured explants are destined and
determined for specific organ formation under the influence of the phytohormones
balance.
 Lastly, the third phase is where morphogenesis proceeds independently of the
exogenously supplied phytohormones. It means, plant organs take the shape of roots
or shoots when phytohormones are removed from the culture med
Somatic embryogenesis
SE can be split into four phases:
 The first phase is where embryogenic masses (callus retaining cells with
embryogenic competence) initiate from vegetative cells or tissues.
 In the second phase, embryogenic cell lines (a group of cells with an embryogenic
fate) are maintained and developed.
 The third phase involves somatic embryo formation (embryo undergoes globular,
heart shaped, torpedo and cotyledonary stages), and maturation (accumulation of
reserve substances).
 Somatic embryos are converted (germinated) into viable plantlets.
Advantages and disadvantages of
organogenesis and somatic embryogenesis
Organogenesis Somatic embryogenesis
Advantages Axillary shoots can be SE allows large-scale
formed directly from production of plants
preformed meristems at through the multiplication
nodes. of embryogenic cell lines.
The chances of the Simultaneous production
organized shoot meristem of roots and shoots are
undergoing mutation are present in the same
relatively low. system.
Many economically Cultures can be
manipulated so that
important plants have embryo formation and
been propagated using germination can be
multiple bud induction synchronized.
organogenesis.
Direct organogenesis has Somatic embryo
more chance to occur dormancy can be
favoring the clonal induced, enabling long-
propagation with high term storage.
fidelity.

Disadvantages There is a chance for The development of somatic

somaclonal variation embryos tends to be
through indirect nonsynchronous
organogenesis.
The organogenesis Over time, the proportion
process is not of cells that enter or
standardized for several complete embryogenesis
recalcitrant species. decreases so that
regeneration may become
impossible.
Direct embryogenesis is less
common.

Factors influencing somatic embryogenesis

There are five factors influencing somatic embryogenesis: explant, plant growth
regulators, minor components, culture conditions, and embryo maturation.

Explant
The type of explant and the age of the explant can have an impact on the success of
somatic embryogenesis. Young explants especially yield more somatic embryos than
older explants. Another challenge encountered is that, even when you’re choosing
different explant tissue (root, flower, shoots, etc.) from the same mother plant, the
embryogenic calli are produced at different frequencies. Here it is necessary to test
different concentrations of growth regulators for the induction of somatic embryos.
Furthermore, explants coming from different cultivars of the same species can
present varied responses to SE induction. Again, the evaluation of cultivar-specific
explants in similar culture conditions is required to determine those with positive
responsiveness to somatic embryo production.

Plant growth regulators

If you supplement your media with auxins, it can help promote callus proliferation
and inhibit differentiation. And if you remove or decrease auxins, this will help the
development of somatic embryos. Auxins are also responsible for establishing cell
polarity in the embryo (apical and basal axis). Something else we want to point out is
that, although cytokinins are also suitable candidates for induction of somatic
embryogenesis, fewer cytokinins are used to stimulate SE. In general, it has been
suggested that the cytokinin thidiazuron-TDZ is more effective than other cytokinins
for somatic embryogenesis.

Minor components
Amino acids like glutamine, proline, tryptophan, polyamines (putrescine, spermidine),
and brassinosteroids (e.g., 24-epibrassinolid) have been reported to enhance
somatic embryogenesis in some species. The potential of these compounds has
been attributed to their diverse role in cellular processes such as precursor
molecules for certain growth regulators and induce embryogenic pathways through
cell reprogramming processes.
Culture conditions
Light condition is a critical aspect when working with SE. For instance, white light
enhances growth but at the same time increases the production of phenolic
compounds and abscisic acid levels. These substances induce oxidative reactions
generating browning of the tissues. One way to mitigate these light-derived effects is
by using activated charcoal in the medium. For other plant cultures like larches,
darkness has proved to be as efficient for SE as with light (Lelu-Walter and Paques,
2009). Under this scenario, light conditions should be tested for SE, mainly for the
maturation stage.

Embryo maturation
Maturation is regarded as a crucial stage of embryogenesis. Maturation is a
preliminary stage for embryo development, which is essentially required for effective
germination. One critical factor in embryo maturation is water loss. Most of the tissue
culture treatments promoting the maturation of the embryo use osmoticums (osmotic
stress inducers) like sugars. For instance, apart from being an energy resource,
sucrose reduces the water potential of the culture medium, which ultimately leads to
water stress, thereby promoting embryo development during in vitro culture. In this
sense, desiccation treatments (by drying the embryos in a filter paper) are also
intended to favor embryo maturation.

Factors influencing the in vitro organogenesis

There are three primary factors influencing in vitro organogenesis. Among these
factors, organogenesis depends mainly on the balance of auxins and cytokinins and
the tissue's ability to respond to phytohormones during specific culture conditions.

Explant

A broad range of explants has been used to induce organogenesis. They include leaf
disks, petioles, stems, peduncles, stipules, roots, embryos, sepals, and protoplasts.
The optimal explant in organogenesis is usually adjusted for each plant species. The
next table shows some of the appropriate explants to induce organogenesis in
different plant species.

Plant growth regulators

The kind of plant growth regulators (PGRs) and the amount used for plant
regeneration varies considerably. In general, a combination of auxin and cytokinin is
necessary for successful regeneration. In recent years, the potent cytokinin TDZ has
been commonly used as a unique PGR in the medium favoring the shoot
regeneration. TDZ, a substituted phenyl urea (N-phenyl-1, 2, 3-thidiazol-5-ylurea),
exhibits strong cytokinin-like activity and has been shown to be an efficacious
regulator of in vitro morphogenesis of many dicot plant species (Ahmed et al. 2012).
Culture conditions

Light is essential in influencing organogenesis, mainly for shoot regeneration. In

exceptional cases, darkness favors in vitro organogenesis, as is the case of
strawberry shoots (Husaini et al., 2011).

Biotech applications of organogenesis and

somatic embryogenesis
Organogenesis has been used mainly for applications like:

 Plant multiplication: This application is ideal especially for clonal propagation. As

there is more chance to induce direct organogenesis, desirable traits can be
preserved from the mother plant. Transgenic plants produced from organs induced in
vitro also retain a great potential to be faithfully cloned if direct organogenesis is
promoted.
 Germplasm preservation: bud or shoots can be stored using gelling agents, to
preserve and delay the developmental process for the organs. The organs can be
later released from the gelling agents to continue the regeneration process.

SE has been used in applications such as:

 Cell selection: as the origin of the embryos can be unicellular, a cell line can be
derived from a single cell.
 Plant multiplication: SE is the preferred choice technique to propagate plants,
especially genetically transformed plants (by Agrobacterium and other transformation
protocols).
 Somatic hybrid regeneration: Through protoplast fusion, two genetic materials can be
joined to produce a plant with better traits. Here, SE is also used to regenerate plants
from these hybrid protoplasts.
 Regeneration of homozygous plants: plants can be regenerated from pollen or ovules
as explants. It produces homozygous plants (plants derived from germinal lines).
 Germplasm preservation: Somatic embryos can be stored as synthetic seed (using
gelling agents), to preserve the embryo and resembling the seed structure (embryo
plus nutritional substances).
 Virus elimination: As an embryo can be derived from non vascular tissues (usually
virus transport around there) and the origin can be unicellular, plants produced in
these conditions can be free from virus.