Anther and Pollen Culture
One of the very popular methods for production of
haploids is through culturing anthers or microspores on artificial culture
medium. The basic principle of anther and pollen culture is the production of
haploid plants. It is based on the totipotency of microspores.
Anther culture was first carried out by Shimakura with
the aim of understanding the physiology of meiosis. Later Guha and Maheshwari
accidentally noticed the development of embryo-like structures inside the
cultured anthers fo Datura. Subsequently, Bourgin and Nitsch
obtained the first haploid plants from isolated anthers of Nicotiana.
Technique
Mature anthers are selected and excised from the
flower buds.
They are surface sterilized with ethyl alcohol or
chlorine water.
The anthers are planted aseptically on the nutrient
medium. Anthers can be grown on a basal medium supplemented with coconut milk
or kinetin. Of the various mineral salts iron plays an important role for the
induction of haploids. Addition of activated charcoal (0.5 – 2%) in to the medium stimulate
androgenesis in cultures.
The cultures are then maintained in alternating light
(12-18h) and dark periods (2-6 h) at 280 C
After 3-8 weeks they burst open exposing the
multicellular tissue.
The tissue divides repeatedly to form callus. From
this callus several embryoids are differentiated. Each embryoid grow into a
plantlet on isolation.
In some species the tissue gradually organise into a
globular embryo. This embryo grow into a single haploid plantlet.
The invitro development of haploid plants
originating from totipotent pollen grain is called Androgenesis.
Diploidization of Haploids:
The haploid plants obtained either from anther or
pollen culture grow normally up to flowering stage. But these plants cannot
produce viable gametes, due to absence of homologous chromosomes. Consequently,
the plants are sterile and there is no seed set. The haploids may be
perpetuated by duplicating the chromosome complement.
The haploids can be made homozygous diploids by
treating with 0.4% colchicine for 24-28 hours.
Importance of Haploids:
Among the different importance of haploids, their use
in crop improvement is considered to be the most significant and stands out as
the most important reason for emphasis on haploid research.
Haploids have also been successfully utilized for
barley, maize, sugarcane, oilseed
Homozygous diploid plants can be used as pure lines in
breeding programme.
Haploid plants are useful
in cytogenetic studies like (i) Production of aneuploids (ii) determination of
basic chromosome number, and (iii) determination of the nature of polyploidy.
Recessive mutations can be easily detected in
haploids, because they contain one set of chromosomes.
Haploid plants become a good source for biochemical
and for physiological studies, just like in microorganisms.
Homozygous diploids (Isogenic lines) are beneficial
when the plants are self-incompatable, e.g. Rye.
In genetic engineering, haploids can be successfully
used for gene transfer.
Ovary Culture
The in vitro culturing of
ovaries isolated from pollinated or un pollinated flowers is called ovary
culture.
The technique of ovary
culture was developed by Nitsch (1951). He successfully reared the ovaries of
Cucumis, Lycopersicon,Nicotiana etc. on synthetic medium.
The ovaries excised from
pollinated flowers developed into mature fruits containing viable seeds. But
these fruits were smaller in size when compared to naturally developed fruits.
Maheswari (1958) cultured
the ovaries of Iberis amara, excised from flowers one day after
pollination. At this stage the ovules contained a zygote and a few free
endosperm nuclei. On a simple nutrient medium, the embryo remained smaller in
size. When B-complex vitamins are added to the medium, normal sized fruits were
obtained. The fruits became even larger in size than the normal fruits, when
IAA is incorporated into the medium.
The ovaries from
unpollinated flowers usually fail to grow on a simple nutrient medium. The
medium should be supplemented with auxins to provide the pollination stimulus.
Seedless fruits of tomato were obtained by Nitsch (1951), when unpollinated
ovaries were cultured on a medium supplemented with 2,4 dichlorophenoxy acetic
acid (2,4 D) or 2-napnthoxy acetic acid (NDA).
Importance of ovary
culture:
Ovary culture is an useful
technique to investigate many fundamental and applied aspects. Some of them are
(a) In vitro culture of
ovary has revealed the role of floral leaves, especially calyx, on the
development of fruits.
(b) This technique is
useful to study the early stages in the development of embryo, seed and fruit.
This knowledge is of great value to horticulturists in improving the quality of
fruits.
(c) The effect of
phytohormones on parthenocarpic fruit development can be studied from the
culture of unpollinated pistil.
(d) Ovary culture is of
immense value in plant breeding. It has potential applications in at least
three different areas viz. (a) over coming self-incompatability (b) over coming
cross-incompatability and (c) haploid production.
OVULE CULTURE
Ovule culture is an
elegant experimental system by which ovules are aseptically isolated from the
ovary and are grown aseptically on chemically defined nutrient medium.
Ovule culture is useful
to study the behaviour of zygote (or) very young embryos which are difficult to
culture.
Ovule culture was carried
for the first time by White (1932) in Antirrhinum majus. However, the technique
of ovule culture was perfected by Maheswari (1958)
Importance of ovule
culture
1. The most important
achievement of ovule culture is the development of technique of test tube
pollination and fertilization. By this technique, it is possible to germinate
pollen in the same culture along with the excised ovule and to induce invitro fertilization.
It has been possible to
fertilize the ovules of Argemone, Papaver, Nicotiana and Datura.
EMBRYO CULTURE
Like any other plant
organ, sexually produced embryos (zygotic embryos) can be used as explant and
cultured aseptically in a test tube containing nutrient medium.
Zygotic embryo culture
was first carried out by Hanning (1904) in two crucifers, Cochleria and
Raphanus. He successfully raised transplantable seedlings from the embryos
cultured on a semi-solid medium containing mineral salts and sugar.
Subsequently, many workers obtained plants by culturing embryos especially
obtained from abortive seeds.
There are two types of
embryo culture.
(a) Culture of immature
embryos : This type of culture is mainly used to grow immature embryos
originating from unripe (or) hybrid seed which failed to germinate. Excising
such embryos is very difficult. A complex nutrient medium is required to raise
them to produce plants. The objective of this technique is to understand the
factors that are regulating the development of embryos under natural
conditions.
6) Culture of mature
embryos: Mature embryos are excised from ripe seeds and are cultured in
nutrient medium. These embryos require a simple nutrient medium containing
mineral salts, sugar and agar. This type of culture is precised mainly to avoid
dormancy of the seed before germination.
Dietrich (1924) studied
the physiological behaviour of immature embryo's under invitro conditions. He
noticed that excised immature embryos on
a nutrient medium tend to bypass certain developmental stages. These embryos
grew directly into weak seedlings. This phenomenon of seedling formation
without completing normal embryonic developinent is called precocious germination.
Importance of embryo
culture:
1. Embryo culture helps
to study the metabolic and biochemical aspects of dormancy and seed
germination.
2. Long periods of
dormancy in seeds delay breeding work, expecially in horticultural and crop
plants. Using embryo culture technique, the life cycle can be shortened in
these plants. For example, the life cycle of Iris was reduced from 2-3 years to
less than one year.
3. The most useful and
popular application of embryo culture is to raise rare hybrids by rescuing
embryos of incompatible crosses.
Such rare hybrids are successfully
produced in case of RICE (interspecific) and also intergeneric crosses between
Barley X wheat, Barley X Rye.
4. Germination of excised
embryos is the more reliable test for rapid testing of viability in seeds,
especially during the dormancy period.
5. Seeds and embryos of
several obligate parasites can be 8rown under aseptic conditions to study their
dependence on the host. Johri and Bajaj (1962) sucessfully cultured the young
embryo of CusCuta reflexa, a stem parasite in the absence of host tissue.
Propagation of rare
plants. It is achieved in Musa and Colocasia plants.
Embryo culture
experiments has also helped in finding out the nature of factors which are
involved in dormancy of seeds after ripening and also vernalization of seeds to
some extent.
2. Embryo culture experiments has also made it
clear that the physiological differences between different species are due to
genetic differences.
3. Embryo culture
technique has given very clear understanding about the environment inside the
seed.
4. Crosses between plants
of different ploidy within the same species have been found to be possible.
e.g. Iris and Zea.
5. Inter specific hybrids
have been obtained in many cases, e.g. Gossypium, Datura, Lycopersicum.
6. Some inter-generic
hybrids have been obtained e.g. Hordeum and Secale, between Datura and
Brugmansia, between Triticum and Elymus etc.
7. The culture of embryos
from normal seeds has been used to shorten breeding cycles. In roses also,
which requires a whole year to come into flowering, embryo culture has made it
possible to shorten the breeding cycle and produce two generations in a year.
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