n vitro

Pollination & Fertilization

Reproductive isolation is considered to play akey part in evolution.

Plants and animals have developed a range ofstrategies that minimizegene

flowbetweenspecies.

In plants, these strategies involve eitherpre-zygoticbarriers, such as differences in

floralstructure and pollen-stigma recognition(inhibition of pollen tube
growth),orpost-zygotic barriers(malformation of endospermand the inhibition of
germination)which are lesswell understood and affect seed developmentfrom
fertilization to seed set.
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Wide Hybridization

In most crop improvement programme oftenwide hybridizationis resorted to transfer

thegenes for abiotic and biotic stress fromaliengenera.

Involves interaverietal, inter-varietal, inter-genera and inter-family crosses to

transfer thetarget genes from the donors.

Eg. Wheat & Rye (Inter-genera)Rice: Wide crosses of interaverietalMost failure in

these crosses are due toi) self incompatibility,ii) cross incompatibility etc.
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In vitro
pollination & Fertilization

To overcome the barrier ofhindering the growth of thepollen grainon the stigma or
style, a part of the stigmaor style may becutand the pollen grain may be placedon
the cut surface of the ovary or transferred through ahole in the ovary wall called
intraovarian pollinationEg.
Papaver somniferu
,
P. rhoeas
,
Argemone mexicana

Another approach to overcome the barrier topollentube growthis direct pollination

ofcultured ovules(
in vitro
ovular pollination) or excised ovules along withplacenta (
in vitro
placental pollination)

(Developed at University of Delhi byMaheswari andKranta, 1954to overcome the

self incompatibility inPapaveraceae and Solanaceae)
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image
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Other techniques (
in vivo
) toovercome prezygotic barriers are:
a.Bud pollinationb.Stump pollinationc.Heat treatment of the styled.Irradiation
ande.Mixed pollination
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in vitro pollination

Development of seed through

in vitro
pollinationof exposed ovules is termed as Test tubefertilization

Seed formation following stigmatic pollination istermed as

in vitro
pollination

In vitro
fertilization (IVF)is a process wherebyreproductive structures are isolated
andintroduced to each other enablingfusion ofgametesto proceed under culture
conditions.

IVF has been accomplished by using isolatedmale and female gametes

ofmaize(Kranzet al.1991; Kranzand Lrz, 1993; Faure
et al
., 1994),wheat (Kovcs
et al
., 1995), and tobacco (Tianand Russell, 1997).
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In vitro
pollination: Methodology

Ovaries should containlarge number of ovules(Insolanaceae many members viz.,

Nicotiana tabacum, N. alata, N. rusticaand Petunia hybrida
),and in Papavaraceaeand cryophillaceae, theplacenta are covered with several
hundreds ofovules.

Isolation of ovules with out any damage aspossible in these spp. which
contributingmaximum to the success in the
in vitro
pollination.

The other requirement is thepollenwhich shouldbeviableand able togerminate.

There must beabundant growth of pollen tubesallover the ovules and placenta in
the culture.
Conditions required for successful IVF
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Other requirements:
Before to start, the information on1.Time of anthesis2.Time of dehiscence3.Time of
germination of pollen tubes intoovules4.Viability of ovules and fertilization insidethe
embryo sacs etc. are essential forsuccessful IVF.
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Disinfection of materials

The buds to be brought to the laboratory foraseptic culturejust before the anthers
are at thestage of dehiscence

Thewhole pistil(after removing petals andsepals) or theovariesalone are sterilized by

aquick rinse of70% alcohol. (Ovaries of plantsgrown in open air requires longer
period ofsterilization)

The ovary wall should be carefully peeled withscalpel, needle to expose the mass of
ovulesattached to the placenta.

To perform stigmatic pollination the excisedpistils are to be carefullysurface

sterilizedwithout wetting the stigma.
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Preparation of pollen & ovary for IVF

Anthers collected form the bud are kept in assterile Petri dish containing apre-
sterilized filterpaper until their dehiscence.

Generally the pollendeposited directlyon thecultured part of the pistil performs

better thanthat spread on the medium around the ovules.

In graminaceous family the ovaries are wellprotected by many layers ofhusksand

hencethesurface sterilization is not required.

In maize husks are severed after 2-4 days ofsilking with a scalpel.

Ovaries are removed and transferred to themedium in a Petri dish.

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image 2
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Isolation of dimorphic sperm cells of
Nicotiana tabacum
image 3
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image 4
sperm cell isolaiton scan image
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Pollination is done directly on the silks or theovules
in vitro
.

Twenty four hours after the pollination silksare clipped off and the Petri plates
sealed.

To avoid the contamination with bacteria abrief disinfection with95% alcohol(a

briefrinse of inner husks or 30 min. treatment with1%Famosept(Sladkyand Havel,
1976) may benecessary.
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 Culture of ovule & ovary after IVP
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The growth of the pollen tube on the barren ovule isaffected by the presence
ofmoisture on the surface ofthe ovule.

The ovules may be wiped with a filter paper and thencovered with pollen grains.

After 4-6 days the ovules contain single celled zygotewhich requires acomplex
growth condition.

In self pollinated species, the ovules with zygotes arekept along with placenta until
seed formation while incross pollinated species they require the placenta onlyin the
initial6-8 days.

Afterwards they can be transferred to a fresh mediumwithout placenta

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Ovary Culture after Pollination
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Nitsch(1951)developed the
in vitro
technique for theovary culture and successfully cultured the ovaries of
Cucumis
and
Lycopersicum .

Addition ofvitamin Bto the medium resulted in thedevelopment of normal fruits and
viable seeds

Enrichment of medium withIAA and coconut milkinduced larger fruits than the fruits
formed in
in vivo
condition (Kanta& Maheswari, 1963).

The floral envelops (lemma & palea) play an importantrole in the development of
fruit & embryo in monocots.

Ovaries excised soon after the fertilization in

Triticum aestivum
&
T. spelta
develop in the culture only whenthefloret envelops remain intact.

This requirement of floral envelop with the excisedovule in monocots for the fruit
development is knownasHull factor.
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Ovary Culture after Pollination contd
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