In vitro pollination technique

1. In-Vitro Pollination
and
Fertilization
Presented by:
Yashika
Shoolini University, Solan

2. 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.

3. 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]

4. Barriers during pollination and fertilization of in
vitro technique
• Pollination and fertilization under in vitro
condition offer an opportunity for producing
hybrid embryos among plants that can’t be
crossed by conventional method of plant
breeding.
• In hybridization programs, transferring viable
pollen from one parent to another does not
always lead to seed setting.

5. • 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.

6. Overcome the Barriers:
• From time to time various techniques have been developed to
overcome the pre zygotic Barriers to fertility. Some of these are:
 Bud pollination
 Stub pollination
 Heat treatment of the style
 Mixed pollination
 Irradication
• The possibility of effecting fertilization by introducing pollen grains
directly into the ovary (intra-ovarian pollination) is yet another
approach to bypass the pre-fertilization barriers.
• A still more promising and proven technique developed by
Maheshwari and his students to overcome the pre-zygotic barriers to
fertility is what they have described as ‘test tube fertilization’.
• Post fertilization barrier may overcome by the culture of ovaries by
ovule culture after pollination

7. Why in vitro pollination is needed?
• For the production of homozygous plant.
• For the conservation of extinct plant species.
• Hybrid production
• Reducing the breeding cycle.
• Overcome the dormant period.
• Production of hybrid species by distant
hybridization.
• Production of haploid plant.
• Conservation of germplasm.

8.  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 grainer tube and of the female gamete
(egg) from embryo sac.
Purpose of in vitro pollination
• Intergeneric hybridization
• Intraspecific hybridization
• Interspecific hybridization
• Intra-familiar crossing/

9. 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.

10. In vitro Pollination
Stigmatic pollination
Ovarian pollination
Placental pollination

11. 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.

12. 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.

13. 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.

14. • 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.

16. 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.

17. • 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
Cruciferae with the aid of ovary culture.

18. 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.

19. 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.

20.  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.

21. 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
transferred to Nitsch’s medium until seed set.

22. 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.

23. • 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.

24. THANK YOU