In-Vitro Pollination

and
Fertilization
 INTRODUCTION
• Cultivation of plant tissue or other organs on
artificial media in a test tube or conical flask is
called in vitro technique.
• Under controlled conditions aseptic transfer of
pollen grains on stigma to produce hybrid
embryos among plants that can’t cross by
conventional method of plant breeding is called
as in vitro pollination and fertilization.
 HISTORY

• German Botanist Harberlandt (1902)

develops the concept of in vitro culture.
• This in vitro pollination technique was
developed at university of Delhi to
produce hybrid among species of
Pavaceraceae and Solanaceae. [Ref:
Maheshwari and kanta, 1964]
• Some of the barriers to fertilizations are
 Pre-fertilization/pre zygotic Barriers:
 Inability of pollen to germinate on foreign stigma.
 Failure of the pollen tube to reach the ovule due to
excessive length of the style or slow growth of the
pollen tube, which fails to reach the base of the
style before the ovary abscises.
 Bursting of the pollen tube in the style.

 Post Zygotic Barriers: Fertilization may occur

normally, but the hybrid embryo fails to attain
maturing due to embryo-endosperm
incompatibility, or poor development of endosperm.
It is called post zygotic barriers.
In vitro fertilization using isolated single gamete
A landmark technique developed recently in the field of
plant biotechnology has been the successful fusion of
male and female gametes isolated from higher plants in
vitro and subsequent regeneration of fusion product into
embryo and finally a plant.[Kraz and Lorz, 1993]. This
process is known as in vitro fertilization requires isolation
of male gametes sperms from germinating pollen tube
and of the female gamete (egg) from embryo sac.

Purpose of in vitro pollination

• Intergeneric hybridization
• Intraspecific hybridization
• Interspecific hybridization
• Intra-familiar crossing/
 Types of In Vitro Pollination

• Ovular pollination: Application of pollen to

excised ovule.
• Ovarian pollination: Application of pollen
to excised ovary.
• Placental pollination: Application of pollen
to ovules attached to the placenta.
• Stigmatic pollination: Application of
pollen to stigma.
 In vitro Pollination

Stigmatic pollination

Ovarian pollination

Placental pollination
 Technique
• Conditions required for successful IVF
 Ovaries large in size and contain many ovules are the
best experimental material for in vitro pollination.
 Isolation of ovules without damage as possible.
 Pollen which should be viable and able to germinate.
 There must be abundant growth of pollen tubes all over
the ovules and placenta in culture.
 1% CaCl2 solution that favour the growth of pollen tube.
• Other requirements:
Before start, the information on
 Time of anthesis
 Time of dehiscence
 Time of germination of pollen tubes into ovules.
 Viability of ovules.
 Disinfection of materials

• A reasonable disinfection of ovule and pollen is the

principle requirement for in vitro pollination.
• Flower buds are emasculated before anthesis and
bagged in order to prevent pollination. The buds
are brought to the laboratory for aseptic culture.
The whole pistil are sterilized by 70% alcohol
surface sterilized with a suitable agent and finally
washed with distil water.
• To collect pollen under aseptic condition, anthers
removed from the flower are kept in sterile petri
plates containing a filter paper until their
dehiscence. The pollen is then aseptically
deposited on the cultured ovules, placental or
stigma depending on the nature of the experiment.
 Culture of ovules, ovary and stigma

1. Ovules:
• The growth of pollen tube attached to bare
ovules is inhibited by the presence of water on
the surface of the ovules.
• This film of water should be dried with filter
paper and later the dried ovules covered by the
pollen grain.
• In Nicotiana tabacum, Allium cepa, Gynandropsis
gynandra seeds are raised from ovules which
contain globular or older embryo.
• 6 days after in vitro pollination ovules contain a
single celled zygote which requires more complex
growth condition.
• For the development of subsequent embryonic
stages, ovules which have been self-pollinated
are usually kept on the placenta until seed
formation while cross pollinated ovules regain
placenta only during the initial 6-8 days of
culture.
• Afterwards, they can be transferred to fresh
medium without placenta.
• Use: Ovule culture has proved to be very useful
technique for raising inter specific hybrids
within genus Gossypium herbacium and
Trifalium.
2. Ovary:

• The technique of ovary culture was developed by

Nitsch in 1951 by the ovaries of Cucumis and
Lycopersicum excised from pollinated flower in vitro
to develop into mature fruits
• The addition of vitamin B to the medium resulted in
the development of fruits of normal size with viable
seeds
• Further enrichment of medium with IAA or coconut
milk induced even larger fruits than the fruits
formed in in vivo conditions.
• Successful culture excised ovaries from a number of
species such as Linaria macroccana, Hyoscymus
niger on a medium containing mineral salts and
sucrose.
• Floret envelops: The floret envelops play an
important role in the development of the fruit
and the embryo of monocots. Ovary excised
soon after pollination only when the floret
envelop remain intact. E.g. Triticum aestivum
and Triticum spelta.
• Hull factor: This requirement of the floret
envelops associating with excised monocot
ovules in vitro is known as ‘hull factor’.
• In the elongation of barely embryo cells can take
place but cell division doesn’t occur.
• Use of ovary culture: Several interspecific and
intergeneric hybrids can be produced between
sexually incompatible parents in the family
3. Stigma
• The entire placenta or part of it bearing the
ovules is used in placenta pollination.
• To perform in vitro stigmatic pollination the
excised pistils are carefully surface sterilized
without wetting the stigma with the sterilant
solution.
• Sometimes the entire pistil in which the
placental bearing ovules have been exposed
are cultured to study the effect of placental
and stigmatic pollination in the same pistil.
• In stigmatic pollination presence of perianth
is important factor in dicot.
 Factor affecting seed set
 Physiological state of the explant
• 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.
• Pollen germination on the stigma and growth of the pollen
tube influences the synthesis of proteins which may
sometimes inhibit the entry of the pollen tube into the
ovary.
• To improve the chances of success of in vitro pollination
the level of incompatibility should be reduced.
• 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.
 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.
• Source of reduced N2 as a complete amino acid mixture is
require for optimal kernel development and growth.
• Kinetin promotes the initial growth of the embryo. IAA or
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.
Storage condition
• Usually the first step of this process occurs at
room temperature and without special lighting.
[Zenk teller-1980]
• The ovary cultures are maintained at 22-260C
and other suitable conditions favouring
embryogenesis.
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 culture.
• Brassica oleracea ovules in 1% solution of CaCl2
with 10% gelatin and then pollinated with pollen
 Application of in vitro pollination
• In plant breeding programs, the technique of in vitro
pollination has potential application in different areas-
• 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.
• 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: Another application of in vitro
pollination reported, is the production of haploids of Mimulus
luteus CV. Tigrinus grandiflorus by pollinating its exposed
ovules with Torenia fournieri. The haploids of Mimulus luteus
developed parthenogenetically, which otherwise could not be
obtained through anther culture.
• Some other examples that produces hybrid
parthenogenitically through in vitro pollination
are Nicotiana tabacum, Hordeum vulgare,
Triticum aestivum.
• Production of stress-tolerant plant: Maize plants
tolerant to beat stress have been produced
through in vitro pollination at high temperature.
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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