ANTHER CULTURE POLLEN CULTURE.

Angiosperm (tlowering plants) bears tlowers (2n) which are designated as reproductive organs.
Flower stamen: pollen sac and anthers
Stigma: (mega sporangium)
Pollen sac and anthers produces a large number of micro spores or pollen grains i.e. they are the first cells of the
maie gametophyte.
Mega sporangium contains a single diploid mega spore mother cell (n) produces linear tetrad of a haploid
megaspore ie. the female gametophyte of which only one of this is functional & gives rise to embryo sac and
others degenerate.
So in nature. these haploid discrete units are designed to cary only single complete set of chromosomes
from each parent to the progeny through fertilization and formation of baby plants through z gotic embryo(n
nder the in vitro conditions. the production of haploid plantlets can be achieved using mature pollen grains of
many angiospermns.
This was first demonstrated by two Indian scientists: Guha and S.C. Maheshwari
(1962) of Delhi University
They were able to regenerate haploid plants (gametic "n number) from pollen of Darura inmxia by
culturing intact anthers.
The basic of pollen and anther culture is that on an appropriate medium. the pollen microspores of some
plant species can regenerate instead of pollen grains. This change from a normal gamatophytie patterm of
development into a vegetativ e(sporophyte) patern appears to be initiated in an early phase ofthe cell cycle. when
transeription ofgenes for sporophy tic are activ ated .Consequently. callus or pro -embryos are formed.

EACTORSAFFECTING THE ANTHER CULTURE:

. Phy siological status of clonal plant: The plants grown under controlled climatic conditions (e.g.
photoperiod. light intensity and tem and mineral nutrition) gives better result plus anthers taken from voung plants
and at the beginning of the following time may provide superiority.
2. Developmental stage of anthers: for this we should have knowleige about the following time and
appropriate size of tlower bud at which correct stage of pollen development will be taking place. In most species.
anther is best cultures when the pollen is unicellular and uninucleate or inert entering tirst
pollen mitosis. eg
tomato when bud is between 3mm to ómm depending growth condition and genotype of the plant.
on

3. Pre-treatment of tlower buds and intlorescence: the following type of pretreatments ar generally tollowed
to get better response in anther pollen to get better response in anther polen culture.
. Low temp pretreatment in the range of 40 to 10 c for 2.5 days betore excision of culture of the anthers
b. High temp shock in range of 30-5" c for 1-4days.
Effect of anther wall: the anther wall acts as a barrier
by which direct etfeets of exogenous media
components on pollen is reduced to various degrees. Anther walls being diploid, acts rapidly on the maiia and
degeneration these anther walls releases some inhibitory compounds and growth of these microspores. However it
is also reported that the anther wall releases another r substance that
help mierospore division. These problem can
be reduced by using a high concentration of sugar in the semi solid media as well as
following culture of anthers
5. Genotype: in some species the response is very easy good but in some very poor. Ex: on corps like rice.
wheat, and solanum -it is good.
6. Culture media: Nitsch and Nitsch (1969) is known as a specific anther cultur madia. But M.S moadia
(1962) as well as the Ns medium of Chu et al (1978) and their moditication have reeeis od a wide acceptance
7. Culture condition:
a. Incubation temp 25-28 "C.
b. Photoperiod: - 16-18 hours light.
C. Float culture: Sunderland et al (1977) showed that tloat culture technique i.e. tloating anther on the surfaee
of the liquid media, showed better response in release
of embryonic
microspores
APPLICATIONS OFANTHER CULTURE very useful for the
mainly developed for obtaining haploid plants, haploid plants
are
1. Anther culture is
establishment of isogenic lines for genetic and plant breeding research.
2. Anther cultures are used to develop homozygous diploid within a short period of the time as compared to the

conventional method by colchicines induced chromosome doubling.

3. The anthers from FI hybrid when cultured, it results in the production of sterile, haploid plants & can produce
seced. The chromosome of these haploid are then doubled to obtain homozygous
doubled haploid (disomic)
plants. Which do not segregate and can be used for the producing the inbreed lines. The same in conventional
method required 6-7 generations to get inbreed lines.
Pollen Culture: Isolated pollen grains, when cultured in vitro, give rise to haploid embryos or callus, this approach
is called "pollen culture". Anther culture gives higher yield in terms of number of plants recovered compared to
the pollen culture (where it is difficult to get repeatable results). Hence anther culture is more commonly followed.

Procedure For The Pollen Culture: Collect about 50 anthers and place them in 20ml medium, squeeze with the
glass rod and filter the solution through a nylon mesh or suitable pore size (25-100um) and centrifuge at 500-
800rpm for 5 min. The pollen pellet is collected, washed twice and suspended at the final density of 10-10°
pollen/ml.

Float Culture: In this method, the excised anthers are floated on a shallow liquid medium in Petri dishes; the
anthers dehisce in a few days releasing their pollen grains into the medium. These anthers continue to shed pollens
that their serial subculture yield pollen samples in different stages of androgenesis.

Pollen culture offers many advantages over anther culture, mainly due to the elimination of the anther wall.
. Studies on differentiation and development are easier and more precise.
2. No callus formation can occur from wall tissue.
3. Produces from different pollen grains, ordinarily do not get mixed up

OVULE AND OVARY CULTURE

In angiosperms the female gametophytes enclosing the egg (female gametes) is deep seated in the ovarian
cavity, well protected by the ovular tissue. The pollen grains are normally fall on to the stigma and there is no
devise for them to reach the egg. To effect fertilization the pollen grains germinate on the stigma by putting forth
a tube(pollen tube) which grows through the stigma, and style ,reaches the ovule and discharges two sperm in the
vicinity of the egg, forming a zygote, while the other fuses with the polar nuclei forming the primary endosperm
nucleus.

During the above process, some barriers are seen Viz.

. Pre-fertilization barriers- such as
a. Inability of pollen to germinate on foreign stigma.
b. Failure of pollen tube to reach the ovary due to the excessive length of the style or slow growth of

the pollen tube.

C. Bursting of the pollen tube in the style.
2. fertilization barriers
PostFertilization may occur normally but the hybrid embryos fail to obtain maturity due to embryo-

endosperm incompatibility or poor development of the endosperm.

To overcome the above problems the ovule or ovary cultures are followed with in-vitro pollination

techniques.
 ROCEDURE /PROTOCOL: -MANNUAL
APPLICATONS:
Ovule culture has
1. To
some
important
applications like
study the in-vitro response of zygote and
2. For
raising hybrid which normal fail due to very young embryos.
excision very tedious or the abortion of the embryos at a rather early stage when its
3. The loss of
impossible.
the hybrid embryos due to the premature abscission of fruits,
prevented by thee ovule culture. as crosses in cotton could be

Gynogenesis:
It is due to in-vitrodevelopment of a haploid plant from| Androgenesis:
It is in-vitro
| fertilized
a
female gametophyte. Ex: ovule development of haploid plant from the
- culture. mature pollen grains, Ex: -

Pollen culture.
EMBRYORESCUE: The term embryo rescue is used for the
case the objective is embryo culture, ovule or ovary culture, since in each
to rescue the embryo.
During
the distance
hybridization, often the embryo aborts at an early stage of
mature seed can be obtained. This
problem can be if
development, so that no
overcome, young hybrid embryos are excised and cultured on
a
synthetic medium. These cultured embryos can
When embryos cannot be
directly develop into young seedlings.
easily excised, whole ovules can be cultured and
too small to be
removed, whole ovaries may be cultured. These similarly when ovule is also
Ovary culture are as follows. techniques of embryo culture, ovule culture &

The embryo is considered EMBRYO CULTURE

of the fundamental
one
evolutionary organs of the
In vitro culture of
embryo first initiated by Hanning (1904) & now it is a angiospermic
was
routine
sporophyte.
Embryo culture can be defined as: in vitro development of isolated operation for the plants.
mature or immature
seed in a suitable culture medium to obtain zygotic embryos from
It is more useful in
seedlings.
the plants, when
. Seeds lose their abitity Viabihl
2. In seed which take
due to many reasons.
longtime for germination.
3. In seed which do not
4. To rescue the
germinate at all.
hybrid embryo obtaining by crossing distant species (which otherwise aborts
development) at different stage of
The most
imp aspect in the embryo culture is that, the culture
media used should sustain the
development of embryo. Because in a less than optimum growth &
into undifferentiated callus or medium, the immature embryo may fail to survive &turn
germinated prematurely to give rise to weak
phases of embryo development have been seedlings.
ith respect to nutrition two
Autotrophic Phase: This starts at the late heart shaped stage. The recognize by Raghavan (1996)
-

is concerned. embryo is fairly as far independent as nutrition

Heterotrophic Phase: - Which may last up to the globular stage. The
endosperm for its nutrition. embryo is dependent and draws upon the
So, isolated mature embryo can
germinate and grow on a
simple inorganic medium supplemented with an
source. energy
Whereas younger embryos require an elaborate medium for their successful culture
Asif et al 2001). (Fregene et al 1998 &
 EMBRYOCULTURE PROCEDURE:
The technique of E.C involves two imp steps-Removal or excision of the embryo & Taking care of
nutritional requirement.
EXCISION OF THE EMBRYO: This depends on the type of plant material used and the stage of the
embryogenesis at which the embryos are excised
The embryo progresses through successive stages namely,
a) Undifferentiated pro-embryonic stage
b) Globular stage
c) Differentiated stages at which the embryo shift from the heterotrophic to
The selection of an appropriate embryonal stage requires a clear concept of zygotic embryogenesis of that
particular species as well as knowledge of when and why the embryo aborts in vivo
Once the embryo stage at which it has to be excised is decided .then the entire seed/fruits are sterilized and
the embryos removed aseptically under a stereomicroscope (if such are very small) entire operation is carried out
in front of a laminar air flow cabinet.
NUTRITION OF THE MEDIUM: proper balance in the macro, micro nutrients plus carbon source.
Supplementary the medium with the organic nitrogen like amino acids or casein hydrolysate can enhance the
growth of excised embryos sucrose has proved to be the best carbon source through other sugars have also been
used. The photo hormones like IAA, NAA, 2-4 D ga3ect have a profound influence on the growth of the cultured
embryos. Several other substance like vitamins meso -inositol, meet extract, yeast extract etc induce growth of
embryos when added as a supplement to the basal medium. Coconut milk is most common additive for embryos
culture.
PRINCIPAL APPLICATIONS OFEMBRYO CULTURE:
1. Embryo rescue: Main /principal application of embryo culture technique are in the area of overcoming

embryo absorption in non-viable hybrids. Ex: In rice, brassica, helianthus.

Agronomically useful hybrid between closely related species often cannot be obtained because of sexual
incompatibility. In such cases the embryo absorbs at varies stages of development.
Thus, the embryo culture technique is more useful in rescue& growing of such embryos to maturity and in
obtained hybrid plants.
It is also followed in triploids which are obtained by the crossing diploid tetraploids. Ex:-cit
Followed in some ornamental plants ex: Iris, Lolium, and Geranium etc.
Seed dormancy: - In plants, it may be caused due to chemical inhibitors or mechanical resistance offered by
2.
the seed coat, under seed cases, remove embryo and culture under in-vitro conditions to overeome seed
dormancy.
in
3. Somatic embryogenesis: -

Embryo culture provides a better and direct somatic embryogenesis system

some plants species.
maturation of the embryo and mutations in the seed coat. For
4. Seed sterility: -
It may be due to incomplete
this is the only viable altemative for seedling development.

ENDOSPERM CULTURE:
This endosperm forms the
.In angiosperms gives rise to the embryo and endosperm.
double fertilization
substance.
immediate environment of the embryo and it is rich in nutrition and growth
one male gamete with two polar
nuclei-
2. It is a genetically unique triploid tissue that arises from the fusion of
an event of double fertilization. (Where as in gymnosperms,
the endosperm is a haploid tissue).
About 81% of the angiospermic families are known to possess triploid endosperms in their developing
3.
seeds.
 Ihe presence of endosperm in the seed up to the germination stage designates them as "endospermous
seed" Ex: cereals, coconut, coffee, rhododendron.
While the seed have endosperm at the beginning but are consumed during maturity are called non-
endospermous seed. Ex: Legumes, cucurbits.
In-vitro eulture of the endosperm tissue began in the 1930s with Zea maize but it did not give good

response, later in1975 Nakano et al, cultured immature endosperms of rice and achieved
successfully
organogenesis
Lakshmi Sita et al (1980) reported successful regeneration of sandalwood from endosperm culture.
7.
endosperms of
8 Thus during last threc decades many have reported successful culture of immature & mature
different taxa mainly belongings to Euphorbiaccac, Loranthaceae and Scentalaceae.

CULTURAL PROTOCOL
Isolation of the uninjured callus endosperm is a prerequisite for endosperm culture. Selection of particular

endosperm is important factor and it may vary from species to species.

ge of responding a very
Ex: 4-7 days in rice
7-10 days in lolium
7-11 /12 days in maize,wheat, barley
cytokinin as growth regulator and tomato juice, yeast
extract
MIEDIA:- Any basal medium (B.M) with auxin and
substances are some
casein hydrolyses, coconut milk (liquid endosperm), young corn juice, as undefined
general requirements for endosperm culture.
APPLICATION;
the and it is not in agricultural plants, but it is
Seed sterility is the prime character of triploid plants
where seed loss is having some commercial values.
preformed some tropical fruits,
The regeneration of triploid plants from the endosperm of citrus by Wa and Chang (1978) was landmark
achievement, but they failed to transfer the plants to the field. 1 111 on in I990 Gmitter et al successfully cultured
the triploid hybrid plants of citrus from endosperm tissues.

TEST TUBE FERTlLIZATION OR IN-VITROFERTILIZATION:

To overcome some of the pre fertilization barriers, test tube fertilization/ in-vitro fertilization can be carried out.
This involves collection of pollen at the appropriate state of development and using them for in-vitro fertilization.
The pollination may be carried out on the stigma, placenta or ovules of an excised ovary cultured on an artificial
medium (under aseptie conditions)

The choice between the organs used for the pollination is made through a detailed study of crossing barriers. For
instance, if lack of stigma receptivity of slow pollen tube growth is the erossing barriers pollen cannot be carried
out on the stigma. But instead placenta or ovules will have to be used for pollination.

This bisexual flowers need to be emasculated and bagged before ovaries are excised for in-vitro operations. After
fertilization, the ovule or ovaries are transferred to a fresh medium for seed development.
In contrast to the above situation in plants (i.e. where ovules or ovary is used for in-vitro pollination).The in vitro
fertilization in human and other animals always involves fusion of an isolated egg with a sperm in test tube.
However till early 1990s plant gametes could not be isolated and fused in the test tube, only in 1993 Erhard Kranz
and Horst Lorz from Hamberg (Germany) reported ,in corn, the successful fusion of an egg cell and a sperm to
form a zygote in a test tube. The fusion was facilitated by a short pulse of electricity and was shotgun "weeding"