Anther culture

Anther and Microscope Culture – One of the very popular methods for production of haploids is through
culturing anthers or microspores on artificial culture medium. Each PMC in the anther produces 4
microspore or pollen grains. Thus anther with microspore or isolated pollen grains can be cultured on
artificial medium to raise haploid plant. Haploid production through anther culture has been referred to
as androgenesis.

Pathways of Development:
The early division in responding pollen grains may occur in one of the following four ways

(1) The uninucleate pollen grain may divide symmetrically to yield two equal daughter cells both of
which undergo further divisions.

(2) In some other cases, the uninucleate pollen divides unequally (as it does in nature). The generative
cell degenerates callus/embryo originates due to successive division of the vegetative cell.

( 3) In few species the pollen embryos originate from the generative cell alone; the vegetative cell
either does not divide or divides only to a limited extent forming a suspensor like structure

(4) finally in some species, the uninucleate pollen grain divide unequally producing generative &
vegetative cells, but both these cells divides repeatedly to contribute to the developing embryo/callus.
The responsive pollen grains become multicellular & ultimately burst open to release the cell mass. The
cell mass may either assume the shape of a globular embryo & undergo the developmental stages of
embryogeny or it may develop into a callus depending on the plant species.
Procedure:
Young flower bud with immature anther at the appropriate stage of pollen development is surface
sterilized & rinsed with sterile water. The buds are dissected by removing the sepals & petals. The anthers
are carefully dissected from the flower buds & kept in a petridish. Each anther is gently separated from
the filament & the intact uninjured anthers are placed horizontally on nutrient medium. If the flowers are
very small a stereo microscope can be used for dissecting the anthers. Isolated healthy anthers are
cultured on agar medium or in a liquid medium. The anther burst open within 3-8 weeks of culture. After
they have attained a height of about 3-5 cm the individual plantlets or shoots emerging from the callus
are separated & transferred to a medium that would support further development. The rooted plants are
transferred to soil mix in the pots.
Fig: A figure showing haploid plant production by anther culture

Limitations
1. The application of this technique is not yet possible in many crops where proper protocol is not
yet developed.
2. High level of management & expertise are required to operate the tissue culture production of
haploid.
3. Mixture of different ploidy levels makes the task difficult.
4. Relatively high incidence of albinism in some types of anther & pollen culture is defected.
5. The lack of selection of traits during the derivation of haploid material.
6. In some cases, the doubling of a haploid does not always result in the production of a homozygote

Ovary Culture
Ovary culture is the in-vitro technique carried outside, i.e. on a
suitable nutrient medium to grow new haploid plants by taking an
unpollinated flower. The ovary culture process is
called gynogenesis because an ovary is a female reproductive part
called gynoecium.
Procedure
collect open flower ( pollinated or , unpollinated ) from a healthy
plant .

Wash them thoroughly with tap water

Dip into 5% teepal solution for 10 min.

Wash with Distilled water

Immers the flower in 5% Sodium hypochlorite solution for 5-7 min.

Wash 3 times with sterile distilled water.

In laminar air flow cabinet, transfer the flowers to a sterile Petri

dish.

Using a flamed forceps and a surgical scalpel, dissect out the

calyx, petals, anther filaments etc. of the flower to isolate the
ovary and ouvle.

Damaged or Undersized ovules are rejected when possible, during

transfer.

Incubate the ovule in culture medium ( liquid/ solid)

Kept the ovule culture in dank or light (16Hrs , 3000 less , 25 c

temp.)

Limitation it has mainly two limitation:

1 . It has been successful only in a relatively small number of
species.

2 . The frequency of responding ovaries (1 -5%) and the number of

plantlet per ovary (2 – 8 OR 1-2) quiet low.
Endosperm culture
In angiosperms the endosperm is the main nutritive tissue for the embryo. The endosperm is the
product of double fertilization during which out of the two male gametes, one fertilizes the egg to
form zygote and other fuses with secondary nuclei to form triploid endosperm.

Endosperm Culture. It is the in vitro development of isolated mature or immature endosperm from
seed at particular stage on a suitable culture. Medium to obtain triploid plantlet.

"TYPES OF ENDOSPERM CULTURE

• 1. Mature endosperm culture: It is the in vitro development of isolated mature endosperm from
ripen endospermic seed on suitable culture medium to obtain triploid plantlet is called mature
endosperm culture. • 2. Immature endosperm culture: It is the in vitro development of isolated
immature endosperm isolated at precise stage from immature seed, cultured on suitable culture
medium to obtain triploid plantlet is called immature endosperm culture."

STEPS IN ENDOSPERMCULTURE It consist of mainly 3 steps:

1. The immature or mature seeds are dissected under aseptic conditions and endosperms along with
embryos are excised.

2. Excised endosperms are cultured on a suitable medium and embryos are removed after initial
stage.

3. Callus followed by embryogenesis or shoot bud differentiation.

4. Complete plant formation.

"Procedure of endosperm culture

Explant source.

Inoculation of explant.

Maintenance of culture.

Shoot but differentiation or embryogenesis.

Plantlet development."

"Factors affecting endosperm culture

 Explant storage.

Nutrient medium.

Physical factors.

Embryo factors."

APPLICATIONS OF ENDOSPERM CULTURE

1. A novel method for triploid plant production - this technique has enabled the production of triploid
plants. Triploid plants are self sterile and usually seedless.

2.Increases yield of timber and fuel

3. Rescuing Embryos from Incompatible Crosses – In Interspecific and Intergeneric hybridization

programmes, incompatibility barriers often prevent normal seed development and production of
hybrids.

4. Overcoming Dormancy and Shortening Breeding Cycle

5.Overcoming Seed Sterility In early ripening fruit cultivars, seed do not germinate because their
embryos are still immature.

Embryo culture deals with the sterile isolation and in vitro growth of a mature or an immature
embryo with an ultimate objective of obtaining a viable plant. Conventionally, the term embryo
culture refers to the sexually produced zygotic embryo culture. There are two types of embryo culture
— mature embryo culture and immature embryo culture (embryo rescue).

Types of Embryo Culture:

1. Mature Embryo Culture:

Mature embryos are isolated from ripe seeds and cultured in vitro. Mature embryo cultures are
carried out in the following conditions

i. When the embryos remain dormant for long periods.

ii. Low survival of embryos in vivo.
iii. To avoid inhibition in the seed for germination.
iv. For converting sterile seeds to viable

2. Embryo Rescue:

Embryo rescue involves the culture of immature embryos to rescue them from unripe or hybrid seeds
which fail to germinate. This approach is very useful to avoid embryo abortion and produce a viable
plant.
Embryo-endosperm transplant:

The endosperm transplant technique used for culturing immature embryos

8.11.3. Culture Strategies

The young embryos are ordinarily excised and cultured directly on the medium. But often very young
embryos may prove difficult to culture directly on the medium. In such cases, embryos may be placed
onto or implanted into developing endosperms that are cultured in vitro; this technique is known as
embryo-nurse endosperm technique.
Applications of Embryo Culture:

1. Prevention of Embryo Abortion:

Incompati-bility barriers in interspecific and inter-generic hybridization programmes leading to embryo

abortion can be successfully overcome by embryo rescue.

2. Overcoming Seed Dormancy

Embryo culture is successfully applied to overcome seed dormancy, and to produce viable seedlings in
these plant species

3.Shortening of Breeding Cycle:

The embryos can be excised and cultured in vitro to develop into plants within a short period

4.Production of Haploids:

Embryo culture has been successfully used for the production of haploid (or monoploid) plants e.g.
barley.

5. Overcoming Seed Sterility

Seed sterility is mostly associated with incomplete embryo development which leads to the death of the
germinating embryo. Using embryo cultures, it is possible to raise seedlings from sterile seeds of early
ripening fruits e.g. apricot, plum

6.. Clonal Propagation:

Embryos are ideally suited for in vitro clonal propagation. This is due to the fact that embryos are
juvenile in nature with high regenerative potential. Further, it is possible to induce organogenesis and
somatic embryogenesis from embryonic tissues.

PLANT TISSUE CULTURE •

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs
under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is
widely used to produce clones of a plant in a method known as micropropagation .

Scope and Training of Plant TissueCulture

1 Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and various parts of the
plants (organs, embryos, tissues, single cells, protoplasts).

2 Plant tissue culture is one of the most rapidly growing areas of biotechnology because of its high
potential to develop improved crops and ornamental plants.
3 With the advances made in the tissue culture technology, it is now possible to regenerate species of
any plant in the laboratory.

4 To achieve the target of creating a new plant or a plant with desired characteristics, tissue culture is
often coupled with recombinant DNA technology.

5 The techniques of plant tissue culture have largely helped in the green revolution by improving the
crop yield and quality.

6 To study the various plant diseases and work out methods-for their elimination.

7 Single cell clones are useful for genetic, morphological and pathological studies.

Embryonic cell suspensions can be used for large scale clonal propagation.

8 Somatic embryos from cell suspensions can be stored for long term in germplasm banks.

9 In the production of variant clones with new characteristics, a phenomenon referred to as

somaclonal variations.

10 Production of haploids (with a single set of chromosomes) for improving crops.

11 Mutant cells can be selected from cultures and used for crop improvement.

12 Immature embryos can be cultured in vitro to produce hybrids, a process referred to as embryo
rescue.

History of Plant Tissue Culture:

1. G. Haberlandt, a German botanist, in 1902 cultured fully differentiated plant cells isolated from
different plants. This was the very first step for the beginning of plant cell and tissue culture

2.ith the identification of a variety of chemicals like cytokinin, auxin, other hormones, vitamins, etc.
and their role in affecting cell division and differentiation, the methods of plant tissue culture
developed in a proper manner.

3. The first plant from a mature plant cell was regenerated by Braun in 1959.

4. Shri S.C. Maheshwari and Sipra Guha made a remarkable contribution in the development of plant
tissue culture in India.

5.Gottleib Haberlandt was the first person to make attempts for plant tissue culture, i.e., he
developed the concept of in-vitro culture of plant cells and is aptly regarded as the father of tissue
culture.

Applications of Plant Tissue Cultures:

1 Plant tissue cultures are associated with a wide range of applications—the most important being the
production of pharmaceutical, medicinal and other industrially important compounds.
2 To study the respiration and metabolism of plants.

3 For the evaluation of organ functions in plants.

4 To study the various plant diseases and work out methods-for their elimination.

5 Single cell clones are useful for genetic, morphological and pathological studies.

6 Embryonic cell suspensions can be used for large scale clonal propagation.

7 Somatic embryos from cell suspensions can be stored for long term in germplasm banks.

8 In the production of variant clones with new characteristics, a phenomenon referred to a

somaclonal variations.

9 Production of haploids (with a single set of chromosomes) for improving crops.

10 Mutant cells can be selected from cultures and used for crop improvement.

11 Immature embryos can be cultured in vitro to produce hybrids, a process referred to as embryo
rescue.

Aseptic Condition
•The plant materials (tissues), equipments, culture media and the room should be free from
microorganisms.

•Usually dry heat, wet heat, ultrafiltration and chemicals are used for the sterilisation process.

•Surface sterilisation of plant materials such as seed, fruit, stem, leaf etc. by agents like •9-10%
calcium hypochlorite for 5-30 minutes •2% sodium hypochlorite solution for 5-30 minutes.

The materials need to be washed thoroughly in double-distilled water, after sterilising in these
solutions. •10-12% of hydrogen peroxide solution for 5-15 minutes. •1-2% bromine water, for 2-10
minutes •1% solution of chlorine water, mercuric chloride, silver nitrate or antibiotics etc. can also be
used.

•Absolute alcohol is used for hard tissues

•Dry heat method is used for sterilisation of equipments in hot air oven.

•Sterilisation of equipment with chromic acid-sulphuric acid mixture, hydrochloric acid, nitric acid
strong detergent solution, alcohol, incubator or autoclaves etc. are use for this purpose.

•Wet heat method is used for sterilisation of glassware, culture media in autoclave at 121°C and 15 lb
pressure for 15 minutes.

•Ultrafiltration is used for sterilisation of liquid media which are unstable at high temperature.

•Antibiotics are added to medium to prevent the growth of the microorganisms e.g. Potassium benzyl
penicillin, strptomycin sulphate, gentamycin etc.
•Chemicals like alcohol are used for sterilisation of working area and the instruments.

•Sterilisation of the environment is done by fumigation method, the inoculation chamber is generally
laminar airflow cabinet is widely used these days.

General Technique of Plant Tissue Culture:

General technique of plant cell, tissue and organ culture is almost the same with a little variation for
different plant materials. There are certain basic steps for the regeneration of a complete plant from
an explant cultured on the nutrient medium
 A. Selection and Sterilisation of Explant:
Suitable explant is selected and is then excised from the donor plant. Explant is then sterilized using
disinfectants.

(b) Preparation and Sterilisation of Culture Medium:

A suitable culture medium is prepared with special attention towards the objectives of culture and
type of explant to be cultured. Prepared culture medium is transferred into sterilized vessels and then
sterilized in autoclave.

© Inoculation:

Sterilized explant is inoculated (transferred) on the culture medium under aseptic conditions.

(d) Incubation:
Cultures are then incubated in the culture room where appropriate conditions of light, temperature
and humidity are provided for successful culturing.

€ Sub culturing:

Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets.

(f) Transfer of Plantlets:

After the hardening process (i.e., acclimatization of plantlet to the environment), the plantlets are
transferred to green house or in pots.

Micropropagation (Clonal propagation)

Micropropagation or Clonal propagation is a field dealing with the ability to regenerate plants
directly from explants or from a single individual by asexual reproduction, constitute a clone.

It is defined as True-to-type propagation of selected genotypes using in vitro culture techniques.

Vegetative method of propagating plant is termed as micropropagation or cloning tissue culture

or growing in vitro.

Methods of Micropropagation are as follows:

a) Micropropagation by proliferation of axillary buds

b) Adventitious shoot proliferation

c) Artificial seed.

D) Plant regeneration by organogenesis from callus and cell suspension cultures.

E) Plant regeneration from callus and cell suspension culture by somatic embryogenesis.
F) Direct (adventitious) somatic embryogenesis (Non-zygotic embryogenesis)

Advantages of this method

is rapid multiplication of superior clones,

maintenance of genetic uniformity,

high yielding crops of the desirable characters in a short period of time,

multiplication of sexually derived sterile hybrids and improvement of plant by developing virus-free,

insect-resistant,

disease –resistant, herbicide-resistant plant.

Example: Fennel is genetically heterozygous and produces wide variation in oil yield and
composition. Also various plants like Garlic, Brahmi, Vinca, Eucalyptus, Gymnema, Liquorice etc.
are propagation by this technique.

Technique of Micro propagation:

Stage 0:This is the initial step in micro- propagation, and involves the selection and growth of stock
plants for about 3 months under controlled conditions.

Stage I:In this stage, the initiation and establishment of culture in a suitable medium is achieved.
Selection of appropriate explants is important. The most commonly used explants are organs, shoot
tips and axillary buds. The chosen explant is surface sterilized and washed before use.

Stage II:It is in this stage, the major activity of micro propagation occurs in a defined culture
medium. Stage II mainly involves multiplication of shoots or rapid embryo formation from the explant.

Stage III:This stage involves the transfer of shoots to a medium for rapid development into shoots.
Sometimes, the shoots are directly planted in soil to develop roots. In vitro rooting of shoots is
preferred while simultaneously handling a large number of species.

Stage IV:This stage involves the establishment of plantlets in soil. This is done by transferring the
plantlets of stage III from the laboratory to the environment of greenhouse. For some plant species,
stage III is skipped, and un-rooted stage II shoots are planted in pots or in suitable compost mixture.