1. Zygotic embryo culture involves the aseptic isolation and growth of sexually produced embryos in vitro with the goal of obtaining viable plants. 2. There are two main types of embryo culture: culture of immature embryos from unripe or hybrid seeds that fail to germinate, and culture of mature embryos excised from ripe seeds to avoid seed inhibition. 3. Successful zygotic embryo culture depends on several factors, including the composition of the culture medium, excision technique, plant material used, and growth conditions like temperature, light and pH levels. The goal is to support the embryo's development from heterotrophic to autotrophic phases.

1. WELCOME TO THE
PRESENTATION
ON
Zygotic Embryo Culture

2. Introduction
Group Members:
Maisa Faria 1236
Mohoshina Hossain 1234
Abida Sultana 1245
MD. Shariful Islam 1276
Najia Sultana 2077
Shahariar Hossain 2228
MD. Iftekhar Hossain Dipto 2319
MD. Ariful Islam Sagar 2340
Arman-UI Hoque 2344
Hasan Ali 2554

3. Zygotic Embryo Culture

4. Introduction
Zygotic embryo is formed following double
fertilization of the ovule, forming the plant and the
endosperm to gather go into the seed.
Zygotic embryo culture is the aseptic isolation and
growth of sexually produce embryo in vitro with the
objectives of obtaining viable plant.

5. History
• Embryo culture in the field was provided by
Laibachi.
• In 1904,Hanning published a paper describing
the first systematic attempt to culture isolated
mature embryos of angiosperm.
• Laibachi(1925,1929) cultured excised embryo
from seeds of an interspecific cross Linum
perenne cross L.austrainum and succeded in
raising hybrid plant.

6. Types of embryo culture
According to Pierik (1989) there are
in principle 2 types of embryo
culture-
• Culture of immature embryo:
This type of embryo culture in
mainly used to grow immature
embryos originating from unripe or
hybrids seeds which tail to
germinate.
The chance of success in this type
of culture depend largely on the
development stage of the excised
embryo.
• Culture of mature embryo:
Mature embryo are excised from
ripe seeds and cultured mainly to
avoid inhibition in the seed for
germination.
Fig: Types of embryo

7. Important aspect for technique:
Two most important aspect of zygotic embryo is
1. Composition of the culture medium
2. Excision of the embryo
Technique: The choice of plant material may
become important when the objective is to
introduce the technique to beginner.

8. Plant Material:
• Embryo which can be easily dissected out should
select.
• Mature embryo of seed legumes and crucifers
possessing a large seeds are good starting material.
• At the same stage of development large number of
genetically uniform embryos can be obtained.
Surface Disinfection: Disinfection of embryo surface
is unnecessary unless a systematic infection is
present.
Instead mature seeds entire ovules or fruits are
surface sterilized and embryo removed aseptically
from the surrounding tissue.

9. Sterilization:
• Entire capsules are surface sterilized and seeds
removal under aseptic conditions.
• There are then spread in a single layer on the surface
of an agar medium using a sterile needle.
• Sterilization is carried out by immersing the material in
hypo chloride.
• Containing commercial bleach (5-10% clorox,0.45%
Caocl2 or NaOCl) for 10-5 minute or ethanol (70-75) for
5 minute.
• A small amount(0.01-0.1)% of a surfactant (Tween 20,
Tween 80, Teepol or Mannoxol) added to the
disinfection solution to increase the tissue wettability.

10. Excision of embryo: For the in vitro culture of embryos
generally it is necessary to free them from their surrounding
tissues.
• In case of dicot, the procedure of excision embryo is given
below-
• Keep disinfected capsules in a few drops of the sterile
culture medium.
• Remove outer walls by an incision in the region of the
placenta and pull two halves apart with forceps to expose
ovules.
• Detach a single ovule from the placenta and place in the
depression (cavity) of a new sterilized slide containing a drop
of medium.

11. • Split the ovule
longitudinally using a
shape mounted blade.
• Carefully tease apart the
ovule tissue in order to
free the entire embryo
along with the attached
suspensor.
• To excise older embryo
make side lacking the
embryo apply pressure
with a blust needle to set
the embryo free.

12. Fig:Isolationofembryo(monocote)
In case of monocot the
procedure of isolation
embryo is given below-
• Rinse the disinfected
caryopsis with sterile
water.
• Place it in a sterile
Petridis with the rachilla
underneath.
• Remove fruit wall and
seed coat.
• Carefully isolate the entire
embryo with plumule,
scutellum and radical.

13. Embryo-nurse endosperm transplant:
The endosperm
transplant technique for
culturing young
(immature) embryos is
explained in figure.
Williams and De Lautour
inserted an excised hybrid
embryo into a normally
developing ovule of ones
of the parents or a third
species and cultured the
nurse endosperm with the
transplanted embryo on
the nutrient medium.

14. Nutritional Requirement: The nutritional requirement
of an embryo during its development in vivo constitute
2 phases-
• Heterotrophic phase- An early phase where in the
embryo in dependent and draws upon the endosperm
and material tissue.
• Autotrophic phase- A later phase in which the
embryo in metabolically required for synthesizing
substances required fairly independent for nutrition.

15. Media constituents for in
vitro of young or
immature embryos also
differ from those od
mature embryo.
Monnier (1976)
developed a unique
method which allow
complete development
of younger embryo
(early globular stage) up
to germination without
moving them from their
original position in
culture.

16. The Composition Of Media:

19. Mineral Salts :
• Higher levels of Potassium (by adding 350 mgl-1)
• Ca ( Double concentration of Cacl2 )
• Reduce level of NH4NO3 & FeEDTA
• Double concentration of MS micro nutrient
Carbohydrate :
• Sucrose is most commonly used in embryo culture
• Glucose & Sucrose maintain osmolarity of the culture medium which is
critical with respect to the age of embryo
• Mature embryo grow fairly well at 2% low concentration of sucrose but
younger embryo at higher level of carbohydrate
• Various concentration of sucrose used in embryo culture depend on the
species and a size / age of embryo

20. Nitrogen & Vitamin :
• NH4NO3 is significantly superior to KNO3
• NaNO3 & (NH4)2 HPO4
• The presence of NH4
+ in the medium has been found essential for
proper growth & differentiation of embryo
• Aspargine & Glutamin is the superior source of Nitrogen
Natural Plant Extract :
• Coconut Milk
• Water extract of dates
• Water extract of bananas
• Hydrolyzation of wheat gluten
• Tomato juice
• Alcohol diffuseness of young seeds of Irish, Lupinus, Datura, etc.

21. Growth Regulator :
• Auxin & Cytokine are not used in embryo culture because they
induce callus formation
• GA3, ABA is generally used as growth regulator
PH : 5 - 7.5
Incubation Condition :
• Temperature :
1. Embryo culture shows a favorable response at elevated
temperature (27-30 °c)
2. Incubation temperature to culture embryos of species ( Brassica
hybrids) occurring in cold region or season range from 17-22 °c
(see Hu & Wang 1986)
• Light : A type of recalcitrant secondary dormancy is induced
when these embryos are excised to 4000 lx or more for a 4
hour period during the initial 4 days of incubation.

22. Suspensor in embryo culture
• The suspensor is ephemeral structure found at the radicular
end of the pro-embryo and attained maximum development.
• Without it embryo culture is difficult
• Older embryo grow well with or without suspensor
• The presence of suspensor is critical particularly for the
survival of young embryo
• It may be substituted by the addition GA or ABA to the culture
medium
Precocious Germination
• According to Walbot (1978) embryo development is classified
into 5 stages

23. • Excised immature plant
embryo on a nutrient
medium tend to bypass
the stage of dormancy
and cease to undergo
linear embryogenic
development. The
embryo developed
instead into weak
seedlings. The
phenomenon of seedling
formation without
completing normal
embryogenic
development is called
precocious germination.

24. Morphogenesis in culture of seeds with partially differentiated
embryo :
• An unorganized embryo where seedlings arise without under going
further embryogenic differentiation
• The embryonal & Proximal to the micropyle is regarded as the
radicular pole & distal to the micropyle as the plumular pole
• In monopolar pattern only one of the pole is involved in the
development of seedlings
• Plumular pole enlarges to form a spherule like structure called “
Protocorm ” after turning green attain certain size differentiates roots
& shoots
• Sometimes radicular pole of the given rise to radicular cylinder whose
tip penetrate the root of host while the portion of the cylinder remain
outside the proliferates into an irregular mass of tissue called tubercle
from which shoot differentiate.

25. Morphogenesis in culture of seeds with partially differentiated embryo :
• Monopolar pattern of seedlings development can be modified by media
composition in by polar pattern
 TB medium
 CM or yeast extract
 IAA (0.1 mgl-1)
 Kinetin ( 0.5-10 mgl-1)
 GA3 (0.5-30 mgl-1)
 Strigol (0.01μgl-1)
• In bipolar pattern plumule pole differentiates shoot bud & ridiculer pole roots
• Glucose, mannose or raffinose favored bipolar germination
Organogenenic potential of embryo callus :
• An embryo callus is reported to possess a high regenerative capacity compared
to those derived from mature organs such as - leaf, stem & roots
• No differentiation occurred if the callus originate from a mature embryo
• Immature embryo are good explants for initiating callus capable of plant
regeneration. Example – Oats, Barley etc
• To obtain Callus with morphogenic potential excised immature embryo are placed
on an agar medium with the scutellum facing up in the presence of 2,4-D alone or
in combination with cytokinin

26. Factors Affection Zygotic Embryo Culture :
• Nutritional requirement : It is vary at different stage of
embryo
• Carbohydrate : Sucrose is most commonly used in embryo
culture
• Natural Plant extract : Coconut milk is an important factor
• Nitrogen & Vitamin : CH is a very important factor which has
it’s optimum level (50-500mgl)
• Growth regulator : 0.01mgl-1GA promotes embryogenesis of
young barley embryo without inducing precautious germination
• Physical factor :
• PH : 5-7.5
• Light : 4000 lux for 4 hour period

27. Application of Zygotic Embryo Culture :
• Obtaining rare hybrids : Crossing cultivated tomato
(Lycorpersicon esculentum) with wild tomato (L.
peruvianum) has been considered describable from the
point of vies of transferring pests & diseases resistance
from the later to the former. The cross does not succeed
dew to pre-fertilization barriers. In the reciprocal cross
however fertilization occurs but the cross fails because of
embryo absorption. But by the aid of embryo culture
hybrid plants can be produced from these two species.
The embryo callus culture approach has also yielded hybrids from
the crosses Lycopersicon esculentum × Solanum hycopersicoides
in which embryos capable of direct plant formation do not
developed.

28. Application of Zygotic Embryo Culture
• Haploid Plant Production: In the cross of Hordeum vulgare ×
H. bulbosum fertilization proceeds readily but the chromosome
of H. bulbosum are preferentially lost during the first few
divisions of embryogenesis. As a result the haploid embryos
show slow growth. This coupled with the disintegration of the
endosperm 2-5 days. After fertilization necessitates the culture
of the excised embryo to raise the haploid Hordeum vulgare
plants.
• Overcoming dormancy & shortening the breeding cycle :
Occasionally the breeding on horticultural plants such as –
deciduous plants is delayed dew to long dormancy period of
their seeds. By growing excised embryos in nutrient medium
this period may be reduced. For example - using embryo
culture (Randolph & Cox 1943) could shorten the lifecycle of
Irish from 2/3 years less than 1 year.

29. Application of Zygotic Embryo Culture
• Rapid seed viability test: The possibility of breaking seed
dormancy by embryo culture also allows the use of this technique
for rapid testing of the viability of a particular batch of seeds.
Germination of excised embryos is regarded as a more reliable &
more exact test than the commonly used staining method for seed
viability.
• Propagation of rare plant: As an abnormality some coconuts
developed soft fatty tissue in place of the liquid endosperm. Such
nuts are called “Makapum”. Being rare makapuros are very
expensive and served only special banquets in the Philippines.
Using the technique of embryo culture, De Gezman & Del Rosario
succeeded in raising plant from makapuro nuts
• Clonal micro propagation: Embryos have high potential for
regeneration & may be used for in vitro clonal propagation.

30. Thank you