This document discusses seed and embryo culture techniques. It begins by explaining that seed culture involves generating a complete plant from a seed explant in vitro. It is commonly used for orchid plants, which do not germinate well in vivo. The document then provides details on the methodology for seed culture, including surface sterilization and germination of seeds on filter paper or cotton swabs. It discusses factors that affect seed culture, such as hormones, nutrients, and abiotic conditions. Applications include increasing germination efficiency and eliminating viruses. The document also covers embryo culture techniques, including culturing immature, inviable, mature, and undifferentiated embryos. It discusses the embryo nurse endosperm technique and applications such as

1. Seed & Embryo
Culture

2. SEED CULTURE
 Seed culture is a technique in which a complete plant or
a seedling can be generated in vitro by taking the seed
as explant.
 This technique is primary used for orchid plants as the
seed of these plants do not germinate well in in vivo
conditions.
 Seed culture is an important technique when explants
are taken from in vitro-derived plants and in propagation
of orchids.
 It has been reported that orchids live in symbiotic
relationship with the fungus from the moment of
germination.
 Symbiosis is the association of two organisms to their
mutual advantage.

3. METHODOLOGY
The seeds are treated with 70% alcohol for about 2 minutes, washed
with sterile distilled water, treated with surface sterilizing agent for
specific period.
Once again rinsed with sterilized distilled water and kept for
germination by placing them on double layers of pre-sterilized filter
paper, placed in petri-dish moistened with sterilized distilled water
or placed on moistened cotton swab in petri-dish.
The seeds are germinated in dark at 25-28°C and small part of the
seedling is utilized for the initiation of callus.

4.  The research carried out by Knudson around 1922 showed that seeds of Cattleya, Laelia, Epidendrum,
and many other orchids were able to germinate asymbiotically in vitro.
 In nature germination of orchid seedlings is dependent on a symbiotic relationship with a fungus.
However, in vitro it is possible to be independent of the fungus by substituting its action with a nutrient
medium, and this is known as asymbiotic germination.
 Orchid seeds are propagated vegetatively as well as generatively. With vegetative propagation, the
progeny is identical to the parent plants.
 However, with generative propagation (by seed), identical progeny is rarely obtained. If seeds from a
cultivated orchid are used (mainly obtained from a cross and strongly heterozygous), the progeny will
be extremely heterogeneous and seldom identical to the starting material.
 In principle, cultivated orchids can only be propagated vegetatively. Orchid cloning in vivo is a very slow process. Thus seeds
can be germinated in vitro and meri-cloning (vegetative propagation by meristem culture) is then carried out on a large scale.
SEED CULTURE OF ORCHID PLANTS

5. Most of the orchids are sown in vitro because
 Orchid seeds are very small and contain very little or no food reserves.
 Their small size (1.0 to 2.0 mm long and 0.5-1.0 mm wide) makes it very likely that they can be lost if
sown in vivo, and the limited food reserves also make survival in vivo unlikely.
 The seed consists of a thickened testa, enclosing an embryo of about 100 cells. The embryo has a
round or spherical form.
 Most orchid seeds are not differentiated: there are no cotyledons, roots and no endosperm. The cells
of the embryo have a simple structure made and are poorly differentiated.
 Sowing in vitro makes it possible to germinate immature orchid embryos, thus shortening the
breeding cycle.
 Germination and development take place much quicker in vitro since there is a conditioned
environment and no competition with fungi or bacteria.
 Orchid seeds imbibe water via the testa and becomes swollen.

6.  After cell division has begun, the embryo cracks out of the seed coat.
 A protocorm-like structure is formed from the clump of cells and on this a shoot meristem can be
embryo distinguished.
 Protocorm has a morphological state that lies between an undifferentiated embryo and a shoot.
 Protocorms obtained by seed germination have many close similarities with those produced from
isolated shoot tips; the term protocorm like-bodies has been introduced culture is while cloning
orchids by meristem culture.
 The vegetative propagation of orchids follows culture of seeds, transformation of meristem into
protocorm-like bodies, the propagation of protocorms by cutting them into pieces and the
development of these protocorms to rooted shoots.

7. MONOPOLAR
SEEDLING
FORMATION
BIPOLAR
SEEDLING
FORMATION

8. SEED CULTURE OF PARASITIC PLANTS
 In seed culture of Orobanche aegyptica both monopolar and bipolar seedling formation occurs
depending upon the composition of the culture media (Usha '68) In a medium containing coconut
milk or yeast extract monopolar seedling formation occurs. If the medium is supplemented with IAA
or gibberellin or kinetin bipolar seedling is formed.
 In seed culture of some obligate root parasites such as Orobanche, Striga, the host stimulus can be
replaced by some chemicals. The seeds cultured on a medium supplemented with gibberellin,
cytokinin or strigol can produce seedlings in absence of host stimulus.
 In Orobanche crenata the radicular pole (i.e. the pole proximal to the micropyle) produces the
seedling.
 Some stem parasitic species of Loranthaceae (Scurrula pulverulenta) when cultured on White's
medium supplemented with 'casein hydrolysate can produce haustoria. Rao ('63) successfully
cultured seeds of some interspecific hybrids of Vanda and obtained seedlings either directly or
callus is formed from which seedling later differentiates.
 Seed cultures of parasitic angiosperms in absence of host usually do not produce haustoria. But
certain root-parasitic species of Scrophulariaceae on culture produce haustoria in absence of host
when some chemically undefined substance, such as aqueous extract of gum tragacanth is present
in the medium.

9. SEED CULTURE OF PLANTS WITH REDUCED
EMBRYOS
 Some plants produce unorganised embryos,
which are not differentiated into radicle, plumule
and cotyledons. This is observed in some
species of Orchidaceae and Orobanchaceae. In
these plants seedlings are formed by the
globose embryo directly.
 In Eranthis sp. belonging to
Ranunculaceae the seeds contains
unorganised embryos during
shedding. After the seeds have fallen to
the ground, these by intraseminal growth
form typical dicot embryos.

10. Cymbidium giganteum
Vigna subterranea
Calopogon tuberosus
Ruditapes decussatus
Vanilla planifolia

11. EFFECT OF ATMOSPHERE &
ITS COMPONENTS
EFFECT OF
SUGARS &
CARBOHYD-
RATES
EFFECT OF
VITAMINS
EFFECT OF
HORMONES
EFFECT OF
LIGHT &
TEMPERATU-
RE
EFFECT OF
COMPLEX
ADDITIVES
EFFECT OF pH
EFFECT OF
MOISTURE

12. APPLICATIONS
Increasing
efficiency of
germination
and germling
production in
seeds, difficult
to germinate in
vivo.
Precocious
germination by
application of
plant growth
regulators.
Induction of
multiple shoot
formation and
organogenesis
by application
of plant growth
regulators.
Elimination of
viruses as
seeds do not
carry viruses.

13. EMBRYO CULTURE
 Plant embryogenesis is the process that produces a
plant embryo from a fertilized ovule by asymmetric cell
division and the differentiation of undifferentiated cells
into tissues and organs.
 Embryogenesis occurs naturally as a result of sexual
fertilisation resulting in the formation of Zygotic
Embryos.
 It occurs during seed development, when the single-
celled zygote undergoes a programmed pattern of cell
division resulting in a mature embryo.
 Embryogenesis involves cell growth and division, cell
differentiation and programmed cellular death.
 Plant somatic cells can also be induced to
form embryos without fertilization during
invitro culture technique; such embryos are
Somatic Embryos.

14.  The embryos of different developmental stages
found within the female gametophyte through
sexual process can be isolated aseptically
without any damage/dissected embryo from the
bulk of maternal tissues of ovule, seed or
capsule.
 They are cultured in vitro under aseptic and
controlled physical conditions in glass vials/tubes
containing nutrient medium to grow directly into
plantlets.
 Embryo culture represents the earliest technique to obtain viable offspring following
interspecific and intergeneric hybridizations where routine fertilization failed to produce a
well-defined and full-term embryo.
 The successful regeneration of plants from the cultured zygotic embryos largely depends
upon the maturation stage of the developing zygotic embryos and the composition of the
nutrient medium.

15. The
young/immature
embryosare
removedfrom
developingseeds
andinoculatedon
asuitablemedium
toobtainplantlets
Theyoung
embryosin
culturegenerally
donotcomplete
theirnatural
courseof
developmentand
germinate
prematurely.
Sometimes,
embryosfrom
matureseedsmay
alsobeusedfor
embryoculture.
Sofar,ithasnot
beenpossibleto
cultureembryos
beforea certain
stage of
development.
However,insome
plantspecies,
evenveryyoung
embryosoffew
cellshavebeen
successfully
cultured.
Youngembryos
needto be
isolatedwiththeir
intactsuspensor
astheyprovide
gibberellinstothe
developing
embryo.

16. The following protocol for embryo culture is based on the method used for Capsella bursapastoris.
With modification, this basic protocol should be applicable to embryo culture in general.
 The capsules in the desired stages of development are surface sterilized for 5-10 minutes in 0.1%
HgCl2 in a laminar air flow.
 Wash repeatedly in sterile water.
 Further operations are carried out under a specifically design dissecting microscope at a
magnification of about 90x. The capsules are kept in a depression slide containing few drops of
liquid medium.
 The outer wall of capsule is removed by a cut in the region of the placenta, the halves are push
apart with forceps to expose the ovules.
 A small incision in the ovule followed by slight pressure with a blunt needle is enough to free the
embryos.
 The excised embryos are transferred by micropipette or small spoon headed spatula to standard
10cm petri dishes containing 25ml of solidified standard medium. Usually 6-8 embryos are cultured
in petri dish.

17.  The petri dishes are sealed with cello tape to prevent desiccation of the culture.
 The cultures are kept in culture room at 25+ -0.1C and given 16hrs illumination by cool white
fluorescent tube.
 Subcultures into fresh medium are made at approximately four weeks interval. In case of fresh seed or
dry and imbibed seeds, the schedule is slightly changed
 Seeds are cleaned by 5% Teepol for 10 mins and dipped in 70% ethyl alcohol for 60 sec, surface
sterilization in 0.1% HgCl2 is followed by washing in sterile water, then the seeds are decotylated using
a sharp scalpel and embryos are transferred to solid nutrient medium.

18. Knudson (1923)
- Invitro germination
of Orchid embryo
HISTORY
Hanning (1904)
- Embryo from
seeds of Radish
Dubard et.al (1913)
- Role of endosperm
Laibach (1929)
- Embryo culture to
obtain interspecific
cross of Linum sps..
Lee (1955)
- Cultured various
segments of Lupin
embryos
Van Overbeek (1942)
- Mature embryo in
simple while proembryo
in nutrient rich medium
Bajaj (1966)
- Culture embryos of
Dendrophthoe falcata
Monnier (1978) -
New culture method
Kruse (1974)
- Endosperm
transplant
De Lautour
(1980)
- Rescuing hybrids

19. FACTORS INVOLVED IN INVITRO
EMBRYOGENY
Mannitol at 120g/l is proved to be suitable for the
growth and development of heart-shaped embryos.
ABA checks precocious germination, and
promotes orderly embryo development and
maturation.
Light does not appear to be a critical factor
in several cases, in Barley, it suppresses
precocious germination.
Embryo cultures are kept at temperatures range
from 25-30°C; but in some species of Datura,
higher temperature may be more significant.
Capsella embryos show improved survival
when iron level of the MS medium is lowered to
two-thirds of the normal and CaCl2
concentration is doubled.
In addition, increased potassium and micronutrient
concentration promotes embryo growth without
affecting survival.
The best recommended medium should contain 180g/l
sucrose, high Ca+, no Fe2+, low salts and should be rich in
amino acids.

20. I. IMMATURE EMBRYO
CULTURE
II. INVIABLE EMBRYO
CULTURE
III. MATURE EMBRYO
CULTURE
IV. UNDIFFERENTIATED
CULTURE
V. ADVENTITIOUS EMBRYO
CULTURE
I. Proembryo taken from immature or
unripe fruits.
II. Raise hybrid plants by culture of abortive
embryos invitro.
III. Analyse various parameters of
embryonic growth.
IV. Culture the undifferentiated embryos of
Orchids.
V. Additional abortive embryos of Citrus can
be exploited in culture.

21. EMBRYO NURSE ENDOSPERM
TECHNIQUE
 Often very young embryos may prove to be difficult to culture directly on the medium. In such
cases, embryos maybe placed onto or implanted into developing endosperms that are in
culture; this technique is known as embryo nurse endosperm technique. For example,
Hordeum x Secale hybrid, interspecific hybrids in Trifolium etc..
 In case of very small embryos like Orchid seeds,
entire seed or ovule is cultured because the seed
contains morphologically undifferentiated, spherical
embryos having no endosperm. Orchid fruit has
many tinny seeds, so a large quantity of sample
can be cultivated easily after excision of surface-
sterilized fruits.
 In interspecific cross of Gossypium arboretum x
Gossypium hirsutum, flowers may drop much before
embryo as explants reach a certain size. In such cases,
whole ovaries or even flowers can be cultured for a
period of time, so that embryo become large enough to
be isolated.

22. APPLICATIONS
Degeneration of
embryos
Degeneration of
endosperm
Produce Disease -
resistant varieties of
Lycopersicon
Overcome seed
dormancy
Shortened breeding
cycles
Hybridisation in
Jute plants
Overcome natural
sterility of some
economic plants
Produce harmless
varieties of Melilotus
Conventional
hybridisation
 Obtain rare hybrids of some plants
 Determining the factors that regulate the growth of
the primordial organs

23. EMBRYO RESCUE
 Embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining
haploid plants as well as for shortening the breeding cycle.
 One of the classic examples of successful application of embryo rescue techniques in plant
nematology in the production of tomato cultivar, resistant to root knot nematode.
 Lycopersicon esculentum (Cultivated species) is highly susceptible to Meloidogyne sp. But wild
species Lycopersicon peruvianum has a high degree of resistance against these nematodes.
 A cross between the two, yielded a hybrid in which the endosperm did not develop and therefore,
the embryo was aborted at an early stage.
 The problem was solved by the application of embryo culture. This provided a mechanism to
transfer nematode resistance gene from L.peruvianum into cultivated species of tomato.

24. SIGNIFICANCE
The hybrids raised through embryo culture have been utilized for the following purposes:
Phylogenetic studies and genome analysis
To raise synthetic crops like Triticaleby producingamphiploidsfrom the
hybrids
Transferof useful agronomictrats fromwild genera and species to the
cultivated crops
Production of haploidsthrough distanthybridisationfollowed by elimination
of the chromosomes of one parent in the hybridembryo culture.

25. Recently a
successful protocol
has been developed
for the in vitro
propagation of
Khaya grandiflora by
excising embryos
from mature seeds.
Somatic embryogenesis and
plant regeneration has been
carried out in embryo cultures
of Jucara palm for rapid cloning
and improvement of selected
individuals.
Intra-varietal hybrids
of an economically
important energy
plant “Jatropha”
have been produced
successfully with the
specific objective of
mass multiplication.

26. REFERENCES
REFERENCE BOOKS:
 Slater, A.Scott, N.W. & Fowler, M.R,(2003). Plant Biotechnology, New York: Oxford University Press, Print.
 Shekhawat.M.S, Vikrant, (2015). Plant Biotechnology- invitro principles, techniques and applications,
Chennai: MJP publishers.
REFERENCE ARTICLES:
 M.M. Hossain,Madhu Sharma,Jaime A. Teixeira da Silva & Promila Pathak., (2010). Seed germination
and tissue culture of Cymbidium giganteum.Sci. Hortic. 123,479 - 487.
 Philip J. Kauth, Wagner A. Vendrame & Michael E. Kane., (2006). In vitro seed culture and seedling
development of Calopogon tuberosus. Plant Cell, Tissue and Organ Culture (2006) 85: 91-102, DOI
10.1007/s11240-005-9055-1.
 Dave I. Thompson, Trevor J. Edwards & Johannes van Staden., (2006). Evaluating asymbiotic seed
culture methods and establishing Disa (Orchidaceae) germinability in vitro: relationships, requirements
and first-time reports. Plant Growth Regul (2006) 49:269 - 284, DOI 10.1007/s10725-006-9137-z.
REFERENCE LINKS:
 https://www.researchgate.net/publication/261070877_Studies_on_in_vitro_seed_culture_in_vanilla
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621365/
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549959/
 https://www.intechopen.com/chapters/40180
 http://www.jstor.org/stable/4353735