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1. VEER KUNWAR SINGH COLLEGE OF AGRICULTURE
COURSE TITTLE:-MICRO-PROPAGATION TECHNOLOGIES
COURSE CODE:-AHT-311
SUBMITTED TO:- SUBMITTED BY:-
NANDITA MAM NAME:-VISHALKUMAR
DR.PAWAN SUKLA ROLLNO.-46/VKSCOA/2021-22

2. OVARY CULTURE
Culture of unfertilized ovaries to obtain haploid plants from egg
cell or other haploid cells of the embryo sac is called ovary
culture and this process is termed as gynogenesis.
San Noem first reported the gynogenesis in barley in 1976.
Subsequently, success has been obtained in several species
including wheat, rice, maize, tobacco, sugar beet, rubber etc.
About 0.2-6% of the cultured ovaries show gynogenesis and one
or two, rarely up to 8, plantlets originate from each ovary. The rate
of success varies considerably with species and is markedly
influenced by the genotype so that some cultivars do not respond
at all. E.g. In rice, japonica genotypes are far more responsive
than indica genotypes.

3. INTRODUCTION
 In most cases, the optimum stage for ovary culture is the nearly mature
embryo sac, but in rice ovaries at free nuclear embryo sac stage are the
most responsive. Generally, culture of whole flowers, ovary and ovules
attached to placenta respond better, but in Gerbera and Sunflower
isolated ovules show better response. Cold pretreatment (24-48 hr at 40C
in sunflower and 24 hr at 70C in rice) of the inflorescence before ovary
culture enhances gynogenesis.
 Growth regulators are crucial in gynogenesis and at higher levels they
may induce callusing of somatic tissues and even suppress gynogenesis.
Growth regulator and sucrose requirement seems to depend on species.
Ovaries are generally cultured in light, but at least in some species, e.g.
sunflower and rice, dark incubation favours gynogenesis and minimizes
somatic callusing; in rice light may lead to degeneration of gynogenic
proembryos.

4. Generally, gynogenesis has two or many stages and each stage has distinct
requirements. In rice, tow stages viz., induction and regeneration, are
recognized. During induction, ovaries are floated on a liquid medium having
low auxin and kept in dark, while for regeneration they are transferred on to
an agar medium with higher auxin concentration and incubated in light. As
in anther culture, gynogenesis may occur either via embryogenesis or
through plantlet regeneration from callus. In general, regeneration from a
callus phase appears to be easier than direct embryogenesis.

5. Ovary culture, also known as ovule culture or ovary rescue culture, is a technique used in plant
biotechnology and agriculture to rescue immature or unpollinated ovules from a plant's ovary and
culture them in a nutrient medium under controlled conditions. This technique is particularly useful in
plant breeding, where it allows for the production of seeds from crosses that would not occur
naturally.
Here's an overview of the process:
1.Collection of Ovaries: Ovaries are collected from plants shortly after flowering, typically when the
ovules are at an immature stage.
2.Surface Sterilization: The collected ovaries are surface sterilized to remove any contaminants,
such as bacteria or fungi, that could interfere with the culture process. This is usually done using a
combination of sterilizing agents like bleach or ethanol.
3.Isolation of Ovules: The ovaries are dissected under sterile conditions to isolate the ovules.
4.Culture Medium Preparation: A nutrient medium is prepared containing essential nutrients,
vitamins, plant growth regulators (such as auxins and cytokinins), and sugars. This medium provides
the necessary nutrients and hormones for the growth and development of the ovules.
5.Culture Initiation: The isolated ovules are placed onto the prepared culture medium in sterile
conditions. They are usually cultured in Petri dishes or test tubes.

6. 6.Inubation: The cultures are then incubated under controlled environmental conditions, typically
with controlled temperature, light, and humidity levels. This encourages the growth and development
of the ovules into embryos.
7.Embryo Development: Over time, the ovules develop into embryos within the culture medium.
This process may take several weeks depending on the plant species and specific conditions.
8.Plantlet Regeneration: Once embryos have developed, they can be transferred to another
medium that supports their further development into plantlets. This involves adjusting the nutrient
composition and growth regulators in the medium to promote shoot and root growth.
9.Acclimatization: Finally, the regenerated plantlets are acclimatized to ex vitro conditions (outside
of the laboratory) before being transferred to soil for further growth and development.
Ovary culture techniques have numerous applications in plant breeding, including the production of
hybrid seeds, the rescue of interspecific or intergeneric crosses, and the propagation of plants that
are difficult to propagate through traditional methods such as tissue culture. This technique has
contributed significantly to the development of new crop varieties and the preservation of genetic
diversity in plant species.

7. OVARY:
An ovary is a floral part, which typically represents the female reproductive part and also
called gynoecium. It is one of the components of carpel that we can see in the image given below. Carpel
includes a long style, terminal stigma and ovule within the ovary.
Style and Stigma: The ovary is the basal body from which a long and slender tube, i.e. style originates,
and a stigma is terminally present on the style.
Ovule: Within the ovary, an ovule (also called megasporangium) carries megaspore or egg-cell. The
integuments of an ovary encase the egg-cell. The egg cell goes through fertilization, after which a zygote
forms that later develops into a mature embryo.

8. Principle
The principle of gynogenesis is based on the regeneration principle, in which an ovary can regenerate into a fully
differentiated plant. In the ovary culture, the flowers are excised from the plant either in pollinated or non-
pollinated stage. Then, the ovary is removed from the flower’s pistil. The ovary possesses an ovule (a female
reproductive part of a flower), which can develop haploid plantlets by the growth on nutrient medium under
controlled conditions.
Method of Ovary Culture
Gynogenesis includes the following steps:
1.First, collect the open flowers from a healthy plant and keep it in a sterilized zip-
lock bag.
2.Wash the flowers with the distilled water.
3.Then, dip the flowers into a 5% Teepol solution for 10 minutes.
4.Rewash the flowers with the distilled water.
5.Bring the flowers to the laminar airflow chamber.
6.Then subject the flowers to surface-sterilization by immersing them in a 5%
sodium hypochlorite solution for 5-7 minutes. Again wash the flowers with the
distilled water.

9. 7. To a sterile Petri-plate, transfer the surface-sterilized flower, and by using a sharp
scalpel dissect the calyx, petal, and other filaments etc. to separate the ovary.
8.Then, the ovary can be cultured either through induction or regeneration process.
In induction, the ovaries float over the liquid medium. In contrast, the ovaries grow on
the solid nutrient medium in the regeneration process.
9.Incubate the cultures for 16 hours at 25 degrees C. For ovary regeneration, keep
the culture plates in a daylight regime by using a fluorescent lamp. Place the culture
tubes in the dark for the induction process.
10.After 2 weeks, haploid plantlets will grow either through embryogenesis or
through plant regeneration from callus.

11. Application of ovary culture
Ovary culture, also known as ovary tissue culture or ovarian tissue culture, is a technique used in
reproductive biology and assisted reproductive technology (ART) with various applications:
1.Research in reproductive biology: Ovary culture is used to study the development, maturation, and
functioning of ovarian tissues. It helps researchers understand folliculogenesis, oocyte maturation, and
hormone production in the ovary.
2.Fertility preservation: Ovary culture is increasingly being used as a method for fertility preservation,
especially in cases where cancer treatment or other medical interventions may damage or compromise
ovarian function. Ovarian tissue can be removed and cryopreserved, and later, it can be cultured to
obtain viable oocytes for in vitro maturation (IVM) and fertilization.
3.Improving in vitro fertilization (IVF) success rates: Ovary culture can contribute to improving the
success rates of IVF by providing a controlled environment for oocyte maturation. This is particularly
relevant in cases where conventional ovarian stimulation protocols are not suitable or effective for
patients.
4.Gene editing and manipulation: Ovary culture provides a platform for gene editing and
manipulation experiments aimed at studying gene function in ovarian cells, understanding genetic
causes of infertility, or developing gene therapy approaches for reproductive disorders.

12. 5.Animal breeding and conservation: In veterinary medicine and animal breeding programs, ovary
culture can be used for fertility preservation, assisted reproduction, and conservation of endangered
species. It allows researchers to manipulate reproductive processes and develop breeding strategies
for desired traits.
6.Drug testing and toxicity studies: Ovary culture can serve as a model system for testing the
effects of drugs, chemicals, or environmental toxins on ovarian function and fertility. This is valuable
for assessing potential risks to reproductive health and developing safer pharmaceuticals.
7.Regenerative medicine and tissue engineering: Ovary culture techniques are explored in
regenerative medicine and tissue engineering efforts aimed at developing artificial ovaries or ovarian
implants for restoring fertility in women with ovarian insufficiency or failure.
8.Understanding reproductive aging: Ovary culture can be used to investigate the mechanisms
underlying reproductive aging, including the decline in ovarian reserve and oocyte quality with
advancing age. Such studies may lead to insights into age-related infertility and potential
interventions to mitigate its effects.

13. Advantages
1.Gynogenetic haploids may be a valuable substitute for the production of
homozygous lines in cases where cytoplasmic male sterility prevents the use of
micropsores.
2.Reduction in the frequency of albino plants in some species especially cereals.
Limitations:
1. So far it has been successful only in less than two dozens species.
2. The frequency of responding ovaries (1-5%) and the number of plantslets/ovary
(1-2) I quite low. Therefore, anther culture is preferred over ovary culture. Only in
those cases where anther culture fails, e.g. sugarbeet and for male sterile lines,
ovary culture assumes significance.