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1. Ovule and Seed culture
Technique and
Applications
Prerna jain
11615509
1628
MSC botany (H)
A06

2. Contents
Ovule culture
 Principle
 Protocol
 Historical perspective of ovule culture
Applications and advantages with
examples
 In vitro pollination and fertilisation
 Ovule culture in hybridization
 Production of haploid callus

3. Contents
 Ovule culture of parasitic plants
 Ovule culture of orchids plants
 Induction of poly-embryony by ovule culture
 Virus irradiation through ovule culture
Seed culture
 Protocol

4. Contents
Applications and advantages of
seed culture
 Seed culture of plants with reduced embryos
 Seed culture of parasitic plants
 Seed culture of orchid plants
Examples of seed culture
 In –vitro technologies - a challenge

5.  Ovule culture is an elegant experimental
system by which ovules are aseptically
isolated from the ovary and are grown
aseptically on chemically defined nutrient
medium under controlled conditions.

6.  An ovule is a mega sporangium covered by integument.
Ovules are attached with placenta inside the ovary by
means of its funiculus. An ovule contains a megaspore or
an egg cell. After fertilization, a single cell zygote is
formed which ultimately leads to form a mature embryo
possessing shoot and root primordia.
 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 organised
stages to a mature embryo. Alternatively, it may be
possible to germinate pollen in the same culture as the
excised ovule and to induce in vitro fertilisation and sub-
sequently embryo production.

7.  (1) 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.
 (2) Remove sepals, petals, androecium etc. from
the ovaries containing either fertilized or unfertilized
ovules.
 (3) Soak the ovaries in 6% NaOCl solution.
 (4) Rinse the ovaries 3-4 times with sterile distilled
water.

8.  (5) Using sterile techniques, ovules are gently
prodded with the help of spoon shaped sptula
by breaking the funicles at its junction with
placental tissue.
 (6) The spatula with ovules is gently lowered
into the sterile solid or liquid medium as the
culture vial is slanted about 45°.
 (7) Damaged or undersized ovules are rejected
when possible, during transfer.
 (8) Incubate the ovule culture in either dark or
light (16 hrs. 3,000 lux) at 25°.C

10.  First attempt to isolate ovules and culture
them under aseptic conditions was made by
white (1932) in Antirrhinum majus. But
perfected by Maheshwari (1958).
 Maheshwari (1958) and Lal (1961) raise viable
seeds of Papaver somniferum starting with
ovules excised 6 days after pollination when
they contained zygote or 2-celled proembryo
and a few endosperm nuclei.

11.  Paddubnaya -arnoldi (1959,1960) grew ovules
from pollinated ovaries of several orchids on
simply 10% sucrose solution.
 Nakajima (1969) cultured ovules at the
zygote or 2-celled proembryo stage in the
medium supplemented with cucumber and
watermelon juices.

12.  Joshi and Johri (1972) made extensive studies
on the nutritional and hormonal factors
concerned in ovule and fibre development
after the unsuccessful attempts at the culture
of Gossypium hirsutum due to interference of
environmental conditions.
 Pundir (1967) successfully produced hybrids
by using a cross between Gossypium
arboreum and G. hirsutum.

13.  Ramming (1971) reported the various in vitro
conditions for culture of ovules of immature
embryo of peach.
 Ramming discovered when medium
supplemented with activated charcoal produced
larger embryos.
 Doi et al, demonstrated that the unfertilized
ovule culture techniques of gentians is a
powerful tool for obtaining haploids and double
haploids because of its reproducible and reliable
nature and application to a wide range of
genotypes.

14. Image of ovule culture

15. Applications and
advantages of
ovule culture

16. TEST TUBE POLLINATION AND FERTILIZATION
 An important achievement of research on
ovule culture has been the development
of the technique of test tube pollination
and fertilization.
 In this technique, the ovules are planted
on a suitable culture medium which
supports growth of ovules as well as
germination of pollen grain.
 Pollen grains are dusted around the
ovules. The pollen germinate and affect
fertilization.

17. EXAMPLES
 Normal viable seeds develop within a few days
example Argemone mexicana , Nicotiana
developed by P. Maheshwari in 1962.
 Using the same method, it has been possible to
fertilize the ovules of Melandrium album with
pollen grains from other species of
caryophyllaceae and subsequently even with
pollen of Datura stramonium. Employing ovule
culture technique, the incompatibility barrier in
Petunia axillaris has been overcome.

19.  In-vitro pollination via ovule culture has been
used to circumvent various problems
associated with hybridization such as failure
of pollen germination on stigmas, insufficient
growth of pollen tube, or precocious
abscission of flowers and self incompatibility
or sterility.

20. Application of Ovule Culture in
Hybridization
ovule culture has been successfully employed to obtain hybrid
seedlings.
 It has been observed that in several inter specific crosses; the
hybrid embryo of Abelmoschus fails to develop beyond the heart
or torpedo-shaped embryo.
 By ovule culture, viable hybrids have been obtained in three out
of five interspecific crosses attempted, namely, A esculentus x A
ficuneus Aesculentus x A moschatus and A tuberculatus x A
moschatus.
 Similarly, a true hybrid between Brassica chinensis and B.
pekinensis has been obtained by culturing the fertilized ovule in
vitro.
 A hybrid between Loluim perenne and Festuca rubra has also
been obtained successfully by means of ovule culture .
 Pundir successfully produced hybrids by using a cross between
Gossypium hirsutum and G.arboreum.

21. 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.
 Also, ovule culture of cotton offers an
unique method for the studies on the
effect of phytohormones on fibre and

22. Production of Haploid Callus through
Ovule Culture
• Uchimiya et al. (1971) attempted culturing
unfertilized ovules of Solanum melongena
and obtained vigorous callus formation on a
medium supplemented with IAA and kinetin. A
cytological assay revealed it to be haploid in
nature. So it is an important attempt to obtain a
haploid cell line or plant from an alternative
source rather than anther or pollen culture.

23. Advantage
• For haploid production , one can go for anther
culture but anther cultures of some plant such as
Mimulus luteus did not form haploids. So it
is an important attempt to obtain a haploid cell
line or plant from an alternative source rather
than anther or pollen culture.

24.  It is generally believed that in obligate root
parasites such as Striga or Orobanke the
formation of seedlings is dependent on some
stimulus from the host root.
 Studies on ovule culture of Orobanche
aegyptica and Cistanche tubulosa have
demonstrated that the formation of shoots in
vitro can be induced in any absence of any
stimulus from the host.

25.  In ovule cultures of these parasites, it is
possible to substitute the host stimulus with
some known chemical like medium enriched
with cytokinin , GA, scopoletin or strigol.
Thus one can able to study hormonal
requirement or nutritional requirement of
ovules of parasitic plants.

26. OVULE CULTURE OF ORCHID
PLANTS
 Poddubnaya-Arnoldi (1959, ’60) successfully
grew the fertilized ovule of orchids in vitro.
They grew the fertilized ovule of Calanthe
veitchn, Cypripedium insigne, Dendrobium
and Phalaenopsis schilleriana.

27. Phaleonopsis-Orchid flower
resembling bee

28. ADVANTAGE
 In nature, the seeds of orchid germinate only
in association with a proper fungus. As a
result numerous seeds are lost due to
unavailability of proper fungus. Secondly ,
the seed capsule of many orchid takes a long
time to mature. thirdly ,minute size of seeds.
To overcome such problems, several
attempts have been made to culture the
fertilized ovule of orchid in vitro.(asymbiotic
germination).

29.  Ovule culture induces polyembryony in
various parts of the plant.
 It has been observed that the nucellus of
mono-embryonic ovule of citrus can be
induced to form adventive embryos in
culture.

30.  In horticultural practices, the artificial
induction of polyembryo holds a great
potential. Therefore, such achievement is
very significant.
 Polyembryony associated with polyploids
helps them to counteract the disadvantage of
being sexually sterile and thus help them in
their perpetuation generation after
generation.

31. Virus Irradiation through
Ovule Culture
 In the varieties of Citrus which are
impossible to free of virus by other
means, the ovule culture has proved
decisively advantageous to make
them virus free.

32. • Thus , due to all these advantages
and applications, ovule culture is a
boon for the plant breeders in
obtaining seedlings from crosses
which are normally unsuccessful
because of abortive embryos.

33.  Germination of seeds in vitro conditions
involves the proper inoculation of seed in the
medium. It is better to inoculate the seeds
without seed coats as it reduce the
germination time and increases the
germination potential.

34.  Surface sterilize the seeds with teepol and
then 0.1% Hgcl2 for about 5 min.
 Wash 3-4 times thoroughly with deionized
water and then inoculate the seeds in basal
MS media under laminar air flow.

35. APPLICATIONS AND
ADVANTAGES OF
SEED CULTURE

36.  Plants such as Eranthis (ranunculaceae)
characterized by the presence of embryos
that lack differentiation into various
embryonic organs, namely radicle, plumule
and cotyledons.
 Thus in such a case the culture of seeds to
raise sterile seedlings is the best method.

37. Eranthis -Plant with reduced embryo

38. Seed culture of parasitic
plants
 In obligate root parasites such as
Orobanche and Striga , the seeds
germinate close to host roots ,and it is
only after the parasite establishes
contact with the host –roots that it
develops shoot .
 Thus ,through seed cultures of these
parasites it is possible to substitute the
host stimulus with some known
chemicals.

39. Example of seed culture of
parasitic plants
 The seeds of Orobanche germinate in
vitro in the absence of a natural host if
the nutrient medium is enriched with a
cytokinin ,GA, scopoletin or strigol
 The seeds of Scurrula pulverulenta a
stem parasite can also germinate on
plain white’s basal medium also
seedlings grow better when casein
hydrolysate is added to the medium.

40. Seed culture of orchid
plants
 There are several plant species in which
seed sizes are minute and do not germinate
well. orchid is one of them.
 Thus , to meet requirements and to conserve
natural resources in-vitro seed germination
has been utilized to produce large quantities
of uniform seedlings.
 In vitro seed culturing of such plants is
dependent on the growth medium
supplemented and not required fungus/orchid
symbiotic relationship dependent germination
and thus known as asymbiotic germination.

41. Seed culture of orchids plants

42. Seed culture is widely used for large, rapid
propagation and large-scale seedling
production. For producing large-scale
seedlings of Capparis spinosa sterilized
the seeds with 0.1% HgCl2 (12 min) and
utilized optimum MS medium
supplemented with activated carbon.
Several examples of seed cultures are
given in following table-

43. Examples of seed culture
Plant description method Effective media
Cephalanthera falcata Asymbiotic
germination
Kano medium and ND
medium
Malaxis khasiana Rapid in-vitro
propagation
Ms medium 2 %
sucrose + casein
hydrolysate +BAP
Dendrobium
hookerianum
Asymbiotic seed
germination
MS medium
Coelogyne nervosa Asymbiotic seed
germination
Ms medium + 30%
coconut water
Cymbidium mastersii Mass propagation Ms medium + BAP
+NAA

44. In –vitro technology –a challenge
• In-vitro culture technologies are still a challenge
because of the slow growth of plantlets, low
multiplication rate, poor rooting, and
somaclonal variation. In this regard,
micropropagation through protocorm-like
bodies obtained from germinating embryos and
somatic tissues is an important strategy in
obtaining genetically stable plants and the
improvement of quality.