Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976 Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc Whites or MS or N6 inorganic salt medium supplement with growth substances are used

1. Submitted by
Reshna K R
3 Semester Biotechnology
Culture of ovule and ovary
factor affecting seed set after in vitro
pollination , application

2. Culture of ovule and ovary
INTRODUCTION
 Invitro culture of unpollinated ovaries and ovules
represents an alternative for the production of
haploid plant
 First successful report on the induction of
gynogenic haploid was in barley by San Noeum
in 1976
 Haploid plants are obtained from ovary and ovule
culture of rice, wheat, maize, sunflower, tobacco,
poplar, mulberry etc
 Whites or MS or N6 inorganic salt medium
supplement with growth substances are used 2

3.  When liquid medium is employed the ovaries can
be placed on filter paper raft
 Sucrose – carbon source
 Maltose, lactose equally favorable
 Addition auxin – greater stimulation of growth
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4. Culture of ovule
 ovules are aseptically
isolated from the ovary and
are grown aseptically on
chemically defined nutrient
medium under controlled
conditions
 ovule is a mega
sporangium covered by
integument
 Ovules are attached with
placenta inside the ovary by
means of its funiculus
 ovule contains a
megaspore or an egg cell
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5.  Ovules can be isolated and cultured in nutrient
medium
 In vitro ovule culture helps to understand the
factors that regulate the development of a zygote
through organized stages to a mature embryo.
PROCEDURE
 Collect the open flower (unfertilized
ovules). If fertilized ovules are desired,
collect the open flower where the anthers
are dehisced and pollination has taken
place. To ensure the fertilization, collect the
flower after 48 hrs. of anther dehiscence.
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6.  Remove sepals, petals,
androecium etc. from the
ovaries containing either
fertilized or unfertilized
ovules.
 Soak the ovaries in 6%
NaOCl solution.
 Rinse the ovaries 3-4 times
with sterile distilled water.
 Using sterile techniques,
ovules are gently prodded
with the help of spoon
shaped spatula by breaking
the funicles at its junction
with placental tissue.
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7.  The spatula with ovules
is gently lowered into the
sterile solid or liquid
medium as the culture
vial is slanted about 45°.
 Damaged or undersized
ovules are rejected
when possible, during
transfer.
 Incubate the ovule
culture in either dark or
light (16 hrs. 3,000 lux)
at 25°.C
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8. Advantage
 In a normal process
these hybrids fail to
develop due to early
embryo abortion and
premature abscission
of fruits. Thus ovule
culture can be used
to rescue them.
 This technique omits
excision of the
embryo by culturing
the entire ovule. Thus
greatly facilitating the
time and effort
involved 8

9. Culture of ovary
 Ovary culture is a technique
of culture of ovaries isolated
either from pollinated or un-
pollinated flowers
 Developed by Nitsch in 1951
 Ovary is a ovule bearing
region of a pistil
 Medium containing mineral
salt and sucrose
 Vitamin B, IAA, coconut milk
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10.  For many species e.g. tomato, gherkin (Cucumis
anguria) excised ovaries grow in culture and form
the fruits that ripen and produce viable seeds
 provided the flowers have been fertilized two or
more days before excision
 Ovaries of un-pollinated flowers do not grow on
simple nutrient medium
 Use of some synthetic auxins such as 2, 4-D, 2,
4, 5- T (2, 4-5-trichlorophenoxyacetic acid), NOA
(2, Napthoxyacetic acid) in the nutrient medium
induces the development of ovaries of un-
pollinated flowers.
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11. Procedure
 Collect the pollinated or un-pollinated flowers from a
healthy plant.
 Wash them thoroughly with tap water, dip into 5% Teepol
solution for 10 minutes and again wash to remove the
trace of Teepol.
 Transfer the flowers to laminar air flow cabinet. Surface
sterilizes the flowers by immersing in 5% sodium
hypochlorite solution for 5-7 minutes. Wash them with
sterile distilled water.
 Transfer the flowers to a sterile petridish. Using a flamed
forceps and a surgical scalpel, dissect out the calyx,
petals, anther filaments etc. of the flower to isolate the
pistil. During isolation of pistils care should be taken to
ensure that the ovaries are not injured in any way.
Damaged pistil, should be discarded as they often form
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12.  Place the ovaries on agar solidified nutrient
medium.
 Incubate the cultures at 25°C in a 16 hrs, daylight
regime at about 2000 lux. The light is provided by
fluorescent lamp.
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13. Advantage
 useful to study the early development of embryo
development, fruit development, different aspects
of fruit physiology including respiration,
maturation and disease
 effect of phytohormones on parthenocarpic fruit
development can be studied from the culture of
un-pollinated pistil.
 successful in inducing polyembryony
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14. factors affecting seed set after in vitro
pollination
 Physiological state of explants
 The physiological state of the pistil at the time of
excising the ovules or ovary influences the seed
set after in vitro pollination.
 Wetting surface of the ovules or stigma may lead
to poor pollen germination or bursting of the
pollen tubes and poor seed set.
 To improve the chances of success of in vitro
pollination the level of incompatibility should be
reduced
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15.  The time of excising the ovules from pistil has a
definite influences on seed set after in vitro
pollination.
 Ovules excised 1-2 days after anthesis show a
higher seed set.
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16.  Culture medium
 The nutrient medium plays an important role in
supporting the normal development of ovary and
ovules in culture until seed formation.
 Nutrient medium on successful culture of ovules
include Nitsch’s mineral salts, white’s vitamins
and 5% sucrose.
 Several orchid ovules isolated from pollinated
ovaries can grow successful on simple 10%
sucrose solution and Zephyranthes require
coconut milk or casamino acids.
 Source of reduced N2 as a complete amino acid
mixture is require for optimal kernel development
and growth 16

17.  Kinetin promotes the initial growth of the embryo.
10mgL-1 IAA or 0.1mg kinetin improves the no. of
seeds per ovule.
 Nitsch’s medium is appropriate for in vitro culture
of pollinated ovules of most species.
 Osmolarity of the culture medium also affects the
development of excised ovule.
 Sucrose anywhere in the range 4-10%.
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18. Storage condition
 Usually the first step of this process occurs at
room temperature and without special lighting.
 The ovary cultures are maintained at 22-26 ºC
and other suitable conditions favouring
embryogenesis.
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19. Genotype
 The response of in vitro ovaries in relation to the
seed set depends on the species.
 Pollen grains of crucifers are difficult to germinate
in cultures and a modified technique is required to
obtain germinable seed.
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20. Application
 Overcoming self-incompatibility
 Petunia axilaris and Petunia hybrida are self-
incompatible species. Germination of pollen is
good on self- pollinated pistils but a barrier exists
in the zone of the ovary as a result, the pollen
tube cannot fertilize the ovule. The barrier of
these taxa can be overcome by in vitro
pollination.
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21.  Overcoming cross-incompatibility
 Successful culture of in vitro pollinated ovules
has raised the possibility of producing hybrids
which are unknown because of pre-fertilization
incompatibility barriers.
 Production of haploid plant
 The haploids of Mimulus luteus developed
parthenogenetically, which otherwise could not be
obtained through anther culture.
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22.  Production of stress-tolerant plant
 Maize plants tolerant to beat stress have been
produced through in vitro pollination. Additionally
these plants exhibited increased vigour and grain
yield.
 Development of young hybrid embryo
 Development of young hybrid embryos can be
achieved in extremely widely crosses through in
vitro pollination. The efficiency of this technique
needs much improvement.
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