ANTHER AND POLLEN

CULTURE

INTRODUCTION:
Haploid plants have the gametophytic number of chromosomes, a
single set (n) of chromosomes in the sporophyte. These are of
great significance for plant improvement, useful in mutations and
for the production of homozygous plants. Conventional breeding
programmes are tedious and are not very efficient for the
production of homozygous lines or haploids. These methods are
not very successful in cross-pollinated crops for obtaining
homozygous plants. Use of anther and pollen culture for the
induction of haploids by Guha and Maheshwari in 1964 at Delhi
University opened new techniques for obtaining homozygous
plants in short duration and in large number for breeding
programmes.

The culture of immature anthers into embryos is called anther

culture. In young anthers, the cells are diploid. The diploid cells
undergo meosis to form haploid tetraspore. In the culture medium
the haploid tetraspores differentiated into haploid parenchyma
cells. Thus a mass of parenchyma cells is produced in the anther.
The cell mass becomes circular and forms a globular embryo and
then heart shapedembryo. Finally tarpedo-shaped embryoid
develops in the culture medium. The development of embryoid
from the anther looks like the development of zygotic embryo.

PROCEDURE:
Plant tissue culture techniques for obtaining haploid cultures is
sinple and efficient. Closed flower buds, which have anthers
containing uninucleated microspores, are most suitable for the
induction of androgenesis. Excised flower buds are sterilised like
other explants and cut open longitudinally with the help of scalpel
or can be opened with a pair of fine forceps and anthers are
transferred on the medium. Before transfer filaments should be
removed from the anthers. Anthers should not be damaged during
excision and transfer. Damaged anthers should be discarded, as
they often tend to produce callus from parts other than pollen.
Anthers can be grown on solid or in liquid medium. The cultures
are incubated at 24-27 degree celcius and under illumination of
about 2000 lux for 12-16 hours/day.

Depending up onthe plants species it takes about 1-8 weeks for

pollen plantlets to embryo from the anther s .plantlets of
appropriate size can be transferred directly in to pots after
necessary washing remove agar .all the care should be taken as
desirable for young micropropagation plants.

Haploid plants can be diploidized to produce homozygous plants

by two methods.

Treating the young plantlets for 24-48 hours with 0.5/ aqueous
colchicine solutinn while still attached to the anther. After washing
the are transplanted . mature plants are treated with colchicine
lanolin paste 0.4% to obtain diploid shoots. These leads ton
production of homozygous seeds by selfing diploid callus can be
obtained from such branches.

Haploid stem explants are cultured. Such cultures tend to

become diploid by endomitosis.Diploid homozygous plants are
obtained from such cultures.

CULTURE MEDIUM AND NUTRITIONAL

REQUIREMENT:
Basal media of White (1943), Murashige and Skoog (1962)m and
Nitsch (1969) used for culture of excised anthers. A combination
of plant growth regulators and suitable concentration of sugar
(2.4% ) are added to the media. Sometimes increased sucrose
level(6-12%) is benificial for androgenesis as in cs=ase of wheat,
barley and tomato. This seems to act as osmoticum rather than
nutrient because anthers produce small amount of tissues. The
nutritonal requirement of anther culture is simpler than that of
isolated pollen grains. Isolated pollen grains are highly specialised
cells and all the nutritional factors provided by anther wall are not
present for isolated pollen grains. Therefore, pollen cultures may
require additional nitrogen, particularly in the form of aminoacids.
L-glutamine is the most frequently used aminoacid for haploid
production from anther culture of potato, Brassica napus and
Triticum aestivum.

ANDROGENESIS:
There are several pathways of androgenesis which leads to the
formation of haplioids either directly by embyogenesis or indirectly
through callus formation. From breeding point of view direct
embryogenesis is preferred over indirect embryogenesis
involving callus formation which may produce variation. There are
several hundred plant species in which haploid callus are
completed platelets have been produced.

In nature, pollen mother cells(P.M.C) give rise to pollen tetrads

through meiotic division. These microspores are released in the
atmosphere. The first mitosis division in the microspores
produces a large vegetative cell and small generative cell. The
former remains quiescent while the latter divides to form two
male nuclei or sperms. In culture although androgenesis can be
induced in anthers in the terad stage at the binuclear pollen stage,
but microspores just before or at the time o first mitosis, are
mostb suitable at the induction of androgenesis. Androgenesis
may be produced by pollen grains through mitosis producing
egual or unequal nuclei. In all cases, they may give rise to
somatic embryos directly or may produce callus. Direct
androgenesis, behavior of microspores has zygote leading to
embryo formation, has been observed in Atropa belladonna,
Datura innoxia and Nicotiana tabacum. Indirect androgenesis is
the most common method (95%) of producing haploids. This type
of development may be due to disturbed polarity and /or complex
nutrient medium.

STABILITY OF HAPLOID CULTURES:

Like plant tissue culture of diploid plants, cultures of haploid origin
are difficult to maintain with genetic stability. Somoclonal variation
occurs durin long term culture as well as , cultures tend to
become haploid. There is continuous increase in proportion of
diplod cells in the haplod culture. Use of specific chemicals like
para-fluro phenylalanine has been tried to enhance haploid callus
growth and maintainance of haploid nature.
UTILIZATION OF HAPLOIDS IN
AGRICULTURE

AND TREE BREEDING:

Haploids are useful for two reasons:1) the presence of one set of
chromosomes facilitate the isolation of mutants, and 2)isogenic
diploids can be obtained by chromosome diploidization. Isogenic
lines are defined as individuals that possess the same genotype
irrespective of their homo or heterozygous nature.

HAPLOID PRODUCTION:
Production of haploids and their importance in plant breeding
have been established. Attension has foccused on how to
improve the frequency of hapliod formation and their best
utilization for the improvement of plants.

TRUE BREEDING LINES:

The usefullness of haoloids in obtaining true breeding diploid lines
by the appliation of chromosomes doubling techniques, finds its
practical utility in the production of new varieties of crops by
anther culture of monosomic tobacco plants. It has been possible
to isolate nulli haploids, and regeneration of nullisomic would
serve as a tool for studying linkage relationship.

VIRUS FREE PLANT:

Selection of disease resistant geranium plants fre from virus
infection has been obtainedb through anther culture. Haploid cells
lines have provided a basic for recovering defined biochemical
and temperature sensitive mutants as there is direct impression of
affected gene.

EXPERIMENTAL EMBRYOLOGY:
In plant breeding, haploids could be exploited for advantage in
studies concerned with experimental embryogenesis, plant
breeding and cytogenesis, because of the ease with hich
homozygous cultivars could be obtained in shorter times following
diplodization by colchicine. Several double haploid l;ines have
been selected for improved yield, early flowering and incresed
disease resistant.

MUTATION BREEDING:
To induce mutation young plantlets emerging from anthers are
subjected to gamme radiation 15500-3000 rads) and following
selection mutants arev easily selected. Similarly chemical
mutagens and x-rays can also be used. These are of great
economic importance in case ofb cereals.

VEGETATIVE PROPAGATING CROPS:

The vegetatively propogated crops , like potato, haploids are very
useful for breeding programmes. They are difficult to produce by
conventional breeding programme.

LONG DURATION CROPS:

In long duration crops, like coffee,haploids and homozygous
diploids are of great importance to find out resistant plants and
once resistant plant propogated, they show stable resitant as they
are homozygous for the character.

TREE BREEDING:
Long term haploid callus culture were first initiated from Taxus
brevifolia pollen in 1955 in the lab of professor C.D.La Rue.
Application of haplidy in the improvement of woody species is
being attempted but progress has been siow. Androgenic
haploids have been obtained in following genera-Albizzia,
Populus, Camellia.