In vitro pollination and
fertilization
In vitro pollination
Pollination and fertilization under in vitro conditions hybrid embryos in plants that cannot cross by
conventional methods.
In nature, Intergeneric or Interspecific hybridization
occurs less frequently.
This is due to barriers hindering the growth of the
pollen tube on the stigma or style.
As early as 1926, Dahlgren reported fertilization and
seed development in Codonopsis ovata - in vitro
pollination after cutting off the style at its base.
In most crop improvement program often
wide hybridization is resorted to transfer the
genes for abiotic and biotic stress from alien
genera.
Most failure in these crosses are due to
self incompatibility, and cross
incompatibility etc.
Hybridization barriers
Pre-zygotic barriers
Differences in floral
structure and inhibition
of pollen tube growth.
Post-zygotic barriers
Malformation
of
endosperm
and
the
inhibition of germination
intra ovarian pollination
To overcome the barrier of hindering the growth of
the pollen grain on the stigma or style, a part of
the stigma or style may be cut and the pollen grain
may be placed on the cut surface of the ovary or
transferred through a hole in the ovary wall is
called intra ovarian pollination.
Eg. Papaver somniferum, Argemone mexicana
Another approach to overcome the barrier to
pollen tube growth is direct pollination of
cultured ovules or excised ovules together with
placenta.
Various other techniques developed to
overcome the prezygotic barriers to fertility
include:
a) Bud pollination, b) Sub pollination, c) Heat
treatment of style, d) Irradiation and e) Mixed
pollination.
Seed formation following stigmatic pollination
is termed
as in-vitro pollination.
Development of seed through
in-vitro
pollination of excised ovules is termed as Test
tube fertilization (Kanta et al. 1962).
Considering the fact that male gametes in
plants do not float freely and are delivered by
pollen tube,
a general terms in vitro pollination has been
used for ovular pollination, ovarian pollination,
placental pollination and stigmatic pollination
under in vitro conditions.
In vitro
pollination
In vitro fertilization (IVF)
is a process whereby reproductive structures
are isolated and introduced to each other
enabling fusion of gametes to proceed under
culture conditions.
Kranz et al. (1991) reported the first successful
in vitro fertilization experiments with an
angiosperm.
In vitro fertilization
In vitro fertilization involves three basic
microtechniques:
isolation, handling and selection of egg cell,
sperm cell and central cell
in vitro fusion of isolated sperm cell with egg
cell or central cell and
Culture of the in vitro formed zygotes and
primary endosperm cells (both single cells).
In-vitro fertilization
Conditions for successful IVF
Ovaries should contain large number of ovules
Isolation of ovules with out any damage
Pollen should be viable and able to germinate
There must be abundant growth of pollen tubes
all over the ovules and placenta in the culture
Essentialities
Time of anthesis
Time of dehiscence
Time of germination of pollen tubes into ovules
Viability of ovules
viability of pollen grains
receptivity of stigma to pollen grains
pollen germination and pollen tube growth
fertilization and embryo and endosperm
development
Anthers collected form the bud are kept
in sterile Petri dish containing a presterilized filter paper until their
dehiscence.
Generally the pollen deposited directly on
the cultured part of the pistil performs
better than that spread on the medium
around the ovules.
In graminaeceous family the ovaries are
well protected by many layers of husks
and hence the surface sterilization is not
required.
For Eg. In maize husks are severed after
2-4 days of silking with a scalpel.
Ovaries are removed and transferred to
the medium in a Petri dish
Pollination is done directly on the silks or
the ovules in vitro.
Twenty four hours after the pollination
silks are clipped off and the Petri plates
sealed.
To avoid the contamination with bacteria
a brief disinfection with 95% alcohol
Culture of ovule & ovary after IVP
The growth of the pollen tube on the barren
ovule is
affected by the presence of moisture on the
surface of
the ovule.
The ovules may be wiped with a filter paper
and then
covered with pollen grains.
After 4-6 days, the ovules contain single celled
zygote
which requires a complex growth condition.
Factors affecting the seed set
after IVP/IVF
Physiological status of the explant
Culture medium
Storage condition: dark & 25 C
Genotype
Applications
Overcoming self-incompatibility
Overcoming cross-incompatibility
Haploid
production
parthenogenesis
through
Production of stress-tolerant plants.
Basic research in the cellular and molecular
control of fertilization in higher plants.
In vitro fertilization also provides a route to new
and simpler transformation method, such as
micro-injection of DNA into the nucleus of an
