In vitro Pollination & Fertilization

¾Reproductive isolation is considered to play a

key part in evolution.
¾Plants and animals have developed a range of
strategies that minimize gene flow between
species.
¾In plants, these strategies involve either pre-
zygotic barriers, such as differences in floral
structure and pollen-stigma recognition
(inhibition of pollen tube growth), or post-
zygotic barriers (malformation of endosperm
and the inhibition of germination) which are less
well understood and affect seed development
from fertilization to seed set.
 Wide Hybridization
¾ In most crop improvement programme often
wide hybridization is resorted to transfer the
genes for abiotic and biotic stress from alien
genera.
¾ Involves interaverietal, inter-varietal, inter-
genera and inter-family crosses to transfer the
target genes from the donors.
¾ Eg. Wheat & Rye (Inter-genera)
Rice: Wide crosses of interaverietal
Most failure in these crosses are due to
i) self incompatibility,
ii) cross incompatibility etc.
 In vitro pollination & Fertilization
¾ 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 called “intraovarian pollination”
Eg. Papaver somniferu, P. rhoeas,
Argemone mexicana
¾ Another approach to overcome the barrier to pollen
tube growth is direct pollination of cultured ovules (in
vitro ovular pollination) or excised ovules along with
placenta (in vitro placental pollination)
¾ (Developed at University of Delhi by Maheswari and
Kranta, 1954 to overcome the self incompatibility in
Papaveraceae and Solanaceae)
 In vitro pollination

Stigmatic pollination

Ovarian pollination

Placental pollination

Diagrammatic representation of the in vitro pollination

 Other techniques (in vivo) to
overcome prezygotic barriers are:
a. Bud pollination
b. Stump pollination
c. Heat treatment of the style
d. Irradiation and
e. Mixed pollination
 In vitro pollination
¾Development of seed through in vitro pollination
of exposed ovules is termed as “Test tube
fertilization”
¾Seed formation following stigmatic pollination is
termed as “ in vitro pollination”
¾In 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.
¾IVF has been accomplished by using isolated
male and female gametes of maize (Kranz et al.
1991; Kranz and Lörz, 1993; Faure et al., 1994),
wheat (Koväcs et al., 1995), and tobacco (Tian
and Russell, 1997).
 In vitro pollination: Methodology
Conditions required for successful IVF
¾ Ovaries should contain large number of ovules (In
solanaceae many members viz., Nicotiana
tabacum, N. alata, N. rustica and Petunia hybrida),
and in Papavaraceae and cryophillaceae, the
placenta are covered with several hundreds of
ovules.
¾ Isolation of ovules with out any damage as
possible in these spp. which contributing
maximum to the success in the in vitro pollination.
¾ The other requirement is the pollen which should
be viable and able to germinate.
¾ There must be abundant growth of pollen tubes all
over the ovules and placenta in the culture.
Other requirements:
Before to start, the information on

1. Time of anthesis
2. Time of dehiscence
3. Time of germination of pollen tubes into
ovules
4. Viability of ovules and fertilization inside
the embryo sacs etc. are essential for
successful IVF.
 Disinfection of materials
¾The buds to be brought to the laboratory for
aseptic culture just before the anthers are at the
stage of dehiscence
¾The whole pistil (after removing petals and
sepals) or the ovaries alone are sterilized by a
quick rinse of 70% alcohol. (Ovaries of plants
grown in open air requires longer period of
sterilization)
¾The ovary wall should be carefully peeled with
scalpel, needle to expose the mass of ovules
attached to the placenta.
¾To perform stigmatic pollination the excised
pistils are to be carefully surface sterilized
without wetting the stigma.
 Preparation of pollen & ovary for IVF
¾Anthers collected form the bud are kept in as
sterile Petri dish containing a pre-sterilized filter
paper until their dehiscence.
¾Generally the pollen deposited directly on the
cultured part of the pistil performs better than
that spread on the medium around the ovules.
¾In graminaceous family the ovaries are well
protected by many layers of husks and hence
the surface sterilization is not required.
¾In maize husks are severed after 2-4 days of
silking with a scalpel.
¾Ovaries are removed and transferred to the
medium in a Petri dish.
Isolation of dimorphic sperm cells of
Nicotiana tabacum

a: Population of larger sperm cells, representing the Sua.

b: Population of smaller sperm cells representing the Svn.
(Yang et al., 2005)
 In vitro pollination

¾Pollination is done directly on the silks or the

ovules in vitro.
¾Twenty four hours after the pollination silks
are clipped off and the Petri plates sealed.
¾To avoid the contamination with bacteria a
brief disinfection with 95% alcohol (a brief
rinse of inner husks or 30 min. treatment with
1% Famosept (Sladky and Havel, 1976) may be
necessary.
 Culture of ovule & ovary after IVP
¾ The growth of the pollen tube on the barren ovule is
affected by the presence of moisture on the surface of
the ovule.
¾ The ovules may be wiped with a filter paper and then
covered with pollen grains.
¾ After 4-6 days the ovules contain single celled zygote
which requires a complex growth condition.
¾ In self pollinated species, the ovules with zygotes are
kept along with placenta until seed formation while in
cross pollinated species they require the placenta only
in the initial 6-8 days.
¾ Afterwards they can be transferred to a fresh medium
without placenta
¾ Ovule culture is proved useful in raising interspecific
hybrids in Gossypium, Helianthus and Trifolium
 Ovary Culture after Pollination
¾ Nitsch (1951) developed the in vitro technique for the
ovary culture and successfully cultured the ovaries of
Cucumis and Lycopersicum.
¾ Addition of vitamin B to the medium resulted in the
development of normal fruits and viable seeds
¾ Enrichment of medium with IAA and coconut milk
induced larger fruits than the fruits formed in in vivo
condition (Kanta & Maheswari, 1963).
¾ The floral envelops (lemma & palea) play an important
role in the development of fruit & embryo in monocots.
¾ Ovaries excised soon after the fertilization in Triticum
aestivum & T. spelta develop in the culture only when
the floret envelops remain intact.
¾ This requirement of floral envelop with the excised
ovule in monocots for the fruit development is known
as “Hull factor”.
 Ovary Culture after Pollination contd…

¾ In the absence of the hull factor, DNA

synthesis and cell elongation in barley embryo
cells takes place but cell division does not
occur.
¾ Similarly the association of perianth in dicots
has been found necessary for the development
of fruits.
¾ Several interspecific and intergeneric hybrids
were produced between sexually incompatible
parents in the family, Cruciferae with the aid of
ovary culture (Batra et al., 1970).
Factors affecting the seed set after IVP/IVF
¾1. Physiological status of the explant
¾2. Culture medium
¾3. Storage condition
¾4. Genotype

Physiological status of the explant

¾ The physiological state of the pistil at the time
of excising the ovules or ovary highly influence
the seed set after the IVP.
¾ Wetting the surface of the stigma (in stigmatic
pollination) may leads to poor germination or
bursting of the pollen tube followed by poor
seed set.
 Physiological status of the explant contd….
¾Pollen germination & growth of pollen tube through
the style may influence the synthesis of some protein
which may prevent the entry of pollen tube into the
ovule.
¾Hence it is necessary to find which part of the pistil,
the barrier exists.
¾To increase the success rate, it is necessary to excise
the part which synthesis the inhibitory protein and to
directly pollinating the remaining portion of the pistil
under in vitro condition
¾The time of excising the ovules from pistil has a
definite influence on seed set after IVP.
¾Ovules excised 1-2 DAA showed higher seed set than
on the day of anthesis.
¾In maize spikes pollinated 3-4 days after silking yields
better results.
 2. Culture medium
¾ Nitsch’s mineral salt and White’s vitamins
were used along with 5% sucrose to culture
the ovules successfully by Maheswari, (1958).
(for the constituents refer Table 3.1 in pp. 118
of text book by Razdan).
¾ Addition of kinetin and CH (Casein
hydrolysate) was essential to promote the
initial growth of the embryo.
¾ Several orchid ovules grew successfully in a
simple 10% sucrose solution.
¾ But the ovules of Zephyranthes required
coconut milk or casamino acid in the Nitsch’s
medium.
 2. Culture medium contd……
¾ In Trifolium ripens, ovules (1-2 DAP) required the
supplementation of the medium with juice prepared from
young fruits of cucumber or water melon.
¾ GA3 (10 Mg l-1) in addition to fruit juice further improved
the seed development in the young ovules
¾ Steward & Hsu, (1978) developed a medium for raising
intraspecific & inter-specific hybrids from young ovules
¾ Presence of 10 μg l-1 IAA or kinetin improve the number
of seed per ovary.
¾ According to Genginbach (1985) the N source does not
affect the fertilization frequency in the excised ovaries of
maize, but the amino acids as the source of N is required
for the optimal kernel development & growth.
¾ Osmolarity of the medium also affects the development
of excised ovules.
¾ Normal sucrose 4-10%. But a ovule with zygote with few
endosperm nuclei- 6%; ovules just after fertilization - 8%
sucrose.
 3. Storage conditions
¾ No precise information on the effect of
temperature and light on the test tube
fertilization
¾ Usually the first step of the process occur at
room temperature without special lighting.
¾ Later the ovaries are transferred to 22-26°C
4. Genotype
¾Pollen grains of crucifer are difficult to
germinate and a modified technique is required
¾Dipping Brassica ovules in 1% CaCl2 before
pollination followed by transfer to Nitsch’s
medium is required to successful seed set.
 Application of in vitro pollination

Four different major areas are:

1. Overcoming self-incompatibility
2. Overcoming cross-incompatibility
3. Haploid production through parthenogenesis
4. Production of stress-tolerant plants.
5. The use of IVF for research into the cellular
and molecular control of fertilization in
higher plants and its application as a tool in
biotechnology
1. Overcoming self incompatibility thru IVP

¾ Petunia axillaris and P. hybrida are self

incompatible species.
¾ Barrier to the germination of pollen exists in
the ovary.
¾ This was overcome by IVP and seed set has
been reported.
¾ In Petunia the self incompatibility can also be
overcome by bud pollination
¾ Several interspecific, intergeneric and
interfamilial crosses were attempted through in
vitro ovular, placental pollination.
++ Abundant germination
3: embryo formed
Pollen grains of the
gymnosperm species
Ephedra distachya and Pinus
wallichiana germinated
abundantly on the in
vitro cultured placentae of
the angiosperm species

Germinating pollen
tubes

Twin Embryo 3 d after in

vitro pollination
Interspecific, intergeneric
and interfamilial crosses
attempted through IVP
 3. Haploid production through in vitro
pollination
¾ Haploid production through in vitro
pollination in Mumulus luticus when
pollinating its exposed ovules with Torena
founieri was reported.
¾ The haploid of M. luticus developed
parthenogenetically which otherwise could
not have been obtained through anther
culture.
¾ Such parthenogenetic development of
haploids also been reported in Hordeum
vulgare, Nicotiana tabacum & Triticum
aestivum
¾Haploid plantlet regeneration through
gynogenesis in Citrus clementina cv. Nules,
induced by in vitro pollination with pollen grains
of Oroblanco, a triploid cultivar of grapefruit.
¾It indicates that parthenogenesis induced in
vitro by triploid pollen can be an alternative
method to obtain haploids in monoembryonic
cultivars of Citrus.
¾Pollination and mature stage of pistils was
necessary for gynogenic embryo regeneration.
¾Fourteen haploid gynogenic embryos of Nules
clementine were obtained
 Stigma exudate and Oroblanco pollen grains

1 month old pollinated pistil

Germination of a haploid embryo

Gynogenic embryos breaking through the

Germination of a haploid embryo ovary
4 months old pollinated pistil
A haploid citrus plant obtained through in vitro pollination
 Haploid production through IVP in wheat
¾ To improve haploid plant production in durum
wheat the haplo-method involving intergeneric
crossing with maize followed by embryo rescue
was used.
¾ The wheat genotype was significant for ovary
development, embryo and plant formation,
whereas the maize genotype was significant
only for embryo formation
¾ The significant effect of the wheat genotype on
embryo formation was found.
¾ Effect of the rescue medium for embryos on
plant yields showed MS/2 and B4 gave
significantly higher percentages of plants
Haploid wheat embryo and its development
after a durum wheat x maize cross

a: Embryo 2 DAP at the time of its rescue in culture (bar = 1.5mm)

b: Germinated embryo after 2 week of culture (bar =1.5 mm)
c: Haploid plant at the four leaf stage after about 5 weeks in a culture tube
¾ Intergeneric hybridization between wheat
(Triticum aestivum L.) and a wild weedy species,
Imperata cylindrica (2n = 20) resulted in the
recovery of a high frequency of wheat haploids,
which were obtained through the elimination of
I. cylindrica chromosomes.
¾ Comparisons based on the efficiency of I.
cylindrica and maize (Zea mays) as pollen
sources indicated that Imperata-mediated
haploid production is equally efficient.
 Culturing condition
¾ The embryos obtained from wheat X I. cylindrica
crosses were excised under aseptic conditions
and cultured on a nutrient medium which
comprised the standard MS medium,
supplemented with 0.5 mg/l kinetin, 400 mg/l
glutamine, 20 mg/l each of L-arginine, L-cysteine
and L-leusine, 30 g/l sucrose and 8 g/l agar agar.
¾ The embryos obtained from crosses of wheat X I.
cylindrica possessed a haploid set of wheat
chromosomes because of elimination of I.
cylindrica chromosomes (as revealed by the
cytological study of the root tips of the haploids)
 4. Production of stress-tolerant plants
¾ Maize plants tolerant to heat can be
produced through in vitro pollination at high
temperature.
¾ Pollen grain at temperature 38ºC were able to
effect fertilization and the resulting maize
plants expressed the trait for heat tolerance.
¾ Additionally these plants exhibited increased
vigour and grain yield.
¾ IVP induced haploids and doubled haploids
are potential source for identifying plants
tolerant to many abiotic stresses combining
IVP & in vitro screening techniques.
 Other applications of IVF
¾Difficulties in isolating gametes of higher plants
have impeded our understanding of gamete
physiology, activation of development and early
embryogenesis in flowering plants.
¾However increasing number of tools now
available to manipulate male and female
gametes of higher plants provides numerous
opportunities for scientific and biotechnological
progress (Fig.).
¾Isolated gametes can be analyzed directly during
IVF with modern cellular and physiological
probes, while means of regulating sexual
reproductive development are being refined.
 (Zygotic) Embryo culture Technique
¾ In angiosperms, the embryo is the miniature
sporophyte resulting from the fertilized egg or
zygote and the endosperm is the main nutritive
tissue for the embryo.
¾ These are the products of double fertilization
during which out of the two male gametes, one
fertilizes the egg to form zygote and other fuses
with secondary nuclei to form triploid
endosperm.
¾ The culture of embryo and endosperm has been
practiced by plant breeders for over half a
century.
¾ Among the two techniques, the former has a
long list of success stories whereas the latter
has very narrow success
 Embryo culture
¾In seed bearing plants, embryos are easily
accessible.
¾They can be separated with relative ease from the
maternal tissues and cultured in vitro under aseptic
conditions in media of known chemical composition.
¾Hanning (1904) first reported his systematic attempt
to culture isolated embryo of Cochleria and
Raphanus (Cruciferae).
¾He successfully raised seedling from the cultured
embryo using semi-solid medium containing mineral
salts & sugar.
¾Later Laibach (1925 & 1929) cultured excised matured
embryos from the seeds of an interspecific cross,
Linum perenne X L. austrianum and succeeded in
raising the hybrid
 Types of Embryo culture
¾ According to Pierik (1989) there are two types
of embryo culture
¾ 1. Culture of immature embryo:
¾ Mainly used to grow immature embryos of
hybrids which fails to germinate which require a
complex medium.
¾ Success of this culture mainly depends upon
the developmental stage of the immature
embryo
¾ By culturing immature embryo, it was able to
develop bulkier seeds than in in vivo condition
as reported by Monnier (1980)
 Types of Embryo culture contd…

2. Culture of mature embryo

¾ Mature embryos are excised from the
seeds and cultured mainly to avoid
inhibition in the seed germination.
¾ This type of culture is relatively easier
as embryo require a simple nutrient
medium containing mineral salts, sugar
and agar.
 Embryo culture techniques
¾ 1. Disinfection
¾ Embryos develop normally inside the ovules
which in turn covered by ovaries
¾ Since they are already in a sterile environment
they do not require disinfection unless they are
injured or seriously infected.
¾ Entire ovules are just sterilized following the
standard methods of surface sterilization and
embryos are dissected out and transferred to
culture medium under aseptic conditions.
¾ In orchids, seeds are minute and with highly
reduced seed coat, the entire capsules are to be
sterilized and the seeds are removed under
aseptic condition.
 Surface sterilization of material
¾ By immersing the material in hypochlorite
containing commercial bleach (5-10% Chlorox,
0.45% sodium or calcium hypochlorite) for 5-10
min.
¾ Or ethanol (70%) for 5 min.
¾ A small amount of (0.01-0.1%) of a surfactant
(Tween-20, Tween-80,Teepol or Monnoxol) to
the disinfection solution increases the tissue
wettability.
¾ Magnetic stirring, ultrasonic vibrations or a
vacuum applied during soaking of the plant
material in the disinfectant solution will reduce
the possibility of trapping the air bubble on the
plant materials.
 Excision of embryos
¾ Excision operation should be performed under
aseptically in a laminar air flow hood.
¾ A stereomicroscope (90X) with a cool ray
fluorescent lamp is required for excising smaller
embryos.
¾ Common tools used are: Forceps, dissecting
needles, scalpels, razor blades and Pasteur
pipettes
¾ Mature embryos can be excised easily by
splitting open the seed.
¾ Excision of smaller embryos requires careful
dissection under a dissection microscope esp.
where the embryos are embedded in liquid
endosperm. (see Appendix 11.1 &11.2 in pp 145,
146 of text book by Razdan)
 Embryo-Endosperm transplant
¾ Abortion of embryos at early stage of
development is due to the non availability of
nutrients.
¾ Brink (1951) demonstrated the in vitro growth of
immature embryos (300-400μm long) of
Hordeum by surrounding them with embryos
excised from another seed of the same species.
¾ Kruse (1974) implanted immature embryos of
Hordeum X Secale on cultured Barley
endosperm and succeeded in getting hybrids
than the normal method of embryo culture.
¾ Generally endosperm older than the embryo by
5 days was more efficient as a nurse tissue for
the growing embryo
Endosperm Transplant Technique (Williams & De Lautour (1980)

Successfully
used in
developing
interspecific &
intergeneric
hybrids in
forage legumes
which do not
grow in normal
conditions

Hybrid embryo Normal endosperm

Culture Medium-Nutrition requirement
¾ The requirement of culture medium depends on the
types of embryo culture.
¾ They may be either post-germinal or pre-germinal.
¾ In the case of post-germinal embryo culture,
embryos are cultured only to speed up the process
after germination.
¾ This can be achieved with less complex medium or
even with sucrose or glucose solution.
¾ In pre-germinal embryo culture, immature embryos
are cultured to get plantlets, where the embryos
require a complex nutrient medium.
¾ Includes modifications in the composition of
mineral salts, organic nutrients and growth
regulators
 Nutrition requirement contd…..
¾ The composition of the culture medium has to be
formulated to suit the developmental phase of the
embryo.
¾ There are two phases in embryo development
(1) heterotrophic phase in which the embryo draws its
nutrients from the endosperm and the surrounding
maternal tissues and
(2) autotrophic phase in which the embryo is
metabolically capable of synthesizing substances
required for growth
¾ Embryos excised at near maturity stage are completely
autotrophic and grow on simple medium comprising
salts with a carbohydrate source.
¾ Immature embryo which are heterotrophic requires
complex medium including vitamins, plant extracts and
plant growth regulators.
¾ Monnier (1976) developed technique which
allows complete development of Capsella
embryo (early globular stage) upto germination
without moving them from the original position
in the culture plate as below:
 Mineral salts

¾ Inorganic nutrients of MS, B5 and White’s

media with certain degree of modification are
the most commonly used media for embryo
culture.
¾ Monnier (1976) modified the MS medium
composition for higher survival rate of cultured
immature embryo.
¾ Contained high level of potassium (adding 350
mg l-1 KCl) and calcium (double concentration
of CaCl2) and reduced levels (approx half) of
ammonium (NH4NO3) and FeEDTA and double
concentration of MS micronutrients.
Table contd…
 Carbohydrates
¾ Sucrose is the most commonly used source of
energy for embryo culture
¾ In maize, addition of maltose, lactose, raffinose, or
mannitol may be required.
¾ Superiority of Glucose over sucrose is reported in
the embryo growth of Carex lucida, several spp of
Rosaceae and Lilium hybrids.
¾ Glucose & sucrose besides nutrition, also maintain
osmolarity of the culture medium.
¾ Mature embryo grow fairly well at low sucrose, but
younger embryos require higher conc. of sucrose.
¾ Various concentrations of sucrose used for
different species band age of embryo is given
below:
Role of sucrose in the medium
 Nitrogen & Vitamins
¾ Embryos have enzyme system to reduce
nitrates to ammonium
¾ Ammonium nitrate is superior to sodium nitrate,
potassium nitrate and ammonium diphosphate
¾ Presence of ammonium is essential for the
growth & differentiation of embryos
¾ Hanning (1904) reported that asparagine
enhanced embryo growth
¾ Glutamine to be a superior source of N in
Capsell sp.
¾ CH (a amino acid complex) widely used as an
additive to embryo culture media.
¾ Optimum level of CH is 500 mg l-1.
 Natural Plant Extracts
¾ Coconut milk (CM) assumed to possess some
“embryo factor” which presumably make up
deficiencies of certain sugars, amino acids,
growth hormones and other metabolites of the
culture medium.
¾ CM stimulates the growth of young excised
embryos of sugar cane, barley, tomato, carrot,
interspecific hybrids of Vigna and fern spp.
¾ Water extracts of dates, bananas, hydrolysates
of wheat gluten and tomato juice were reported
to promote growth of embryos.
¾ Alcohol diffusates of young seeds of Datura
and Sechium had comparative effect of CM and
Lupinus has got twice the effect of CM.
 Growth Regulators
¾ Auxins & cytokinins are not generally used
since they induce callus formation.
¾ At very low level (0.01 mg l-1), GA promotes
embryogenesis of young barley embryos
(without inducing precocious germination)
¾ ABA (abscisic acid) also has similar effects on
barley and Phaseolus embryos

pH of the medium
¾ Embryos grow well in a medium with pH of 5-7.5
¾ Generally the medium pH is adjusted 0.5 above
the desired pH to compensate the uncontrollable
change during autoclaving process
 Incubation conditions

¾ Incubation temperature of 25 ±2°C normally

supports the growth of majority of embryos.
¾ Sometimes the incubation temperature may
vary with the genotypes of the same species
¾ Zennia, Phaseolus and cotton adopted to warm
temperature (27- 30°C) to culture embryos.
¾ Whereas in Brassica hybrids, rice, barley in
cold regions or seasons require temperature of
17-22°C (Hu & Wang, 1986).
 Light
¾It is advisable to incubate immature
embryos of barley, flax, Aegilops x
Hordeum hybrids and interspecific
hybrids of Allium in darkness before
transferring them to light for germination.
¾A recalcitrant secondary dormancy is
induced when these embryos are
exposed to 4000 lx for more than 4 hrs of
light during the initial four days.
¾This suggests that initial incubation of
embryos for 4 days is essential.
 Role of suspensor in embryo culture
¾ The suspensor is an ephemeral structure found at the
radicular end of the proembryo and attains maximum
development by the time the embryo reaches the
globular stage.
¾ Generally, it is difficult to excise the suspensor along
with the embryo because of its small size and delicate
structure.
¾ Older embryos (500 μm or more in length) appear to
grow well with or without a suspensor
¾ Observations showed that attachment of the suspensor
with young embryos of Phaseolus coccineus, or its
placement in close proximity (when detached) of an
embryo, on the culture medium strongly stimulated the
development of embryos to maturity compared to those
cultured without the suspensor.
¾ The requirement of the suspensor may be substituted
by the addition of GA or ABA to the culture medium
 Precocious Germination
¾ Plant physiologists and biochemists conceive
embryo development as a linear progression
from zygote formation to germination.
¾ According to Walbot (1978), embryo
development can be classified into five stages
(Table 11.6).
 Precocious Germination contd….
¾ Excised embryos on a nutrient medium tend to
bypass the stage of dormancy.
¾ Directly develop into a weak seedling.
¾ This phenomenon of seedling formation without
completing normal embryogenic development is
called precocious germination.
¾ CH promotes further embryogenic development
and delays germination of cultured immature
embryos of barley.
¾ High level of sucrose (12-18%) also inhibits the
germination of immature embryos.
¾ Other factors viz., reduced O2 tension, elevated
temperature, ABA also inhibits the precocious
germination
 Applications of embryo culture
1. Embryo rescue in wide crosses
¾In interspecific and intergeneric hybridization,
incompatibility barrier often prevents the normal
seed development and production of hybrids.
¾To overcome the above barriers for obtaining the
hybrids, the embryo culture technique is effective
utilized in which the nutritional relationship
between the embryo and endosperm is restored
by providing the artificial medium to induce and
complete growth of hybrids embryos and is called
as embryo rescuing.
¾Intergeneric hybrids have been obtained between
Hordeum and Secale; Hordeum and Hordelymus,
Triticum and Elymus; Triticum and Secale and
Tripsacum and Zea.
Wide hybridization thru’ embryo rescue
Wide crosses Purpose
Corchorus capsularis x Hybrids had fibres with quality of C.
C. Olitorius capsularis and strength of C. olitorius
The hybrids possessed winter hardiness
Hordeum vulgare x H.
and mildew resistance like H. bulbosum
Lycopersicon The hybrids possessed resistance to
esculentum x L. viruses, moulds and nematodes along
peruvianum with good fruit set like L. peruvianum
Hybrids resembling M. officinalis in
Melilotus officianalis x M.
agronomic characters and low coumarin
alba
content like M. alba
Nicotiana tabacum x N. To get plants with resistance to black
resophilia shank
Oryza sativa x O.
To transfer pest resistance
officinalis
Trifolium pratense x T. To impart perennial plant habit to red
sarosiense clover
 2. Shortening breeding cycle of plants
¾Useful in reducing the breeding cycle of new varieties
in cases where long dormancy causes extension of
breeding cycle.
¾Cultivated varieties of rose generally take about a
year to flower and two to three months for the
formation of fruits.
¾Seedlings produced from cultured embryos flower in
two to three months.
¾These flowers can serve as the male parent for further
crosses enabling the breeder to produce two
generations in one year or shortening the breeding
cycle to three or four months.
¾In weeping crap apple (Malus sps) the seeds cultured
in vitro produce seedling in four months, on the other
hand, seeds planted in the soil take about nine
months to germinate.
 3. Overcoming seed sterility
¾In early ripening fruit cultivars the seeds do not
germinate because their embryos are still immature.
¾Using the embryo culture method it is possible to
raise seedlings form the sterile seeds of stone fruits
(sweet cherry), peach, apricot and plum.
¾‘Makapuna” coconuts are realized for their soft fatty
endosperm in place of liquid endosperm.
¾Under normal conditions the seeds fail to germinate.
¾But De Guzman et al. (1985) obtained 85% success
in raising makapuna seedlings through embryo
culture.
¾Same techniques also used in Maranta, Colocassia,
Musa bulbisima and Pinus armandii X P. koriensis
 4. Monoploid production

Production of haploids in barley

¾H. vulgar X H. bulbosum where fertilization
occurs normally, but there after chromosome
elimination of H. bulbosum resulting in the
production haploid embryos carrying only barley
chromosomes which could be rescued by
embryo culture.
¾In other crosses viz., Agropyron tsukuishiens X
H. vulgare and other cereals haploids were
produced following this technique.
 5. Micropropagation
¾Because of their juvenile nature the embryos
have high potential for regeneration.
¾Complete plantlets in vitro from conifers were
achieved in long leaved pines (Pinus palustris)
through embryo culture.
¾Both organogenesis and embryogenesis have
been induced in major cereals and forage
grasses from embryonic tissues.
6. Endosperm culture
¾In 1947, LaRue, successfully cultured the corn
endosperm and obtained plantlets with root-shoot
axis and miniature leaves.
¾The triploidy can be exploited in the crops viz.
apple, banana, mulberry, sugar beet, tea and
watermelon where seeds are not of commercial
importance.
 Embryo culture and
back crossing in gene transfer
¾The embryo culture has been proved as a viable
technique for resynthesising some of the plant
hybrids
¾For example Brassica napus has been resynthesised
from the cross of B. campestris/B. oleracea using
embryo culture.
¾The recent approach is back crossing the
resynthesised B. napus (2n=38) to B. campestris
(2n=20), so that the genes from B. oleracea can be
transferred to B.campestris .
¾In 1988 Quazi made an attempt in this regard and
came out with successful results.
¾He got a line from the back crosses of (B. napus/B.
oleracea)/B. oleracea which is resistant to cabbage
aphid attack.
 Other biotechnological uses
¾The embryo culture technique can be effectively
engaged in seed testing of various tree species,
germinating seeds of obligate phanerogamic
parasites,
¾Studying the host-pathogen relationship in seed-
borne diseases and studying developmental
embryogenesis.
¾Genetic transformation using the desiccated zygotic
embryos which were generally perforated with larger
pores.
¾Toper et al. (1989) transformed some cereals by
imbibing mechanically isolated zygotic embryos in a
solution containing plasmids carrying chimeric
genes for successful transformation.
¾This approach is a potential tool in transforming
important horticultural crops in future.
 Mature embryo culture in Cassava

¾ Cassava fertility and seed viability are

frequently low, which can be a disadvantage in
a breeding programme.
¾ An embryo culture method in which embryonic
axes are excised from mature seeds and placed
on a culture medium containing 1.23 μl
indolebutyric acid (IBA) at 30°C under
continuous light.
¾ The number of plants recovered by embryo
culture was much greater than the number
recovered from conventional seed germination
procedures
¾ A rapid regeneration protocol for proembryos of
Phaseolus angustissimus as young as 1 day after
pollination (DAP) involving pod culture for 1 week
followed by embryo culture for 2 weeks and embryo
germination for 1 or 2 weeks is provided.
¾ Optimization of the media was conducted with pods
collected 3 DAP.
¾ The best pod culture medium was composed of basal
medium salts with vitamins, 1000 mg l-1 glutamine,
1000 mg l-1 casein hydrolysate 1.9 μM, 3% sucrose and
0.5% agar.
¾ Embryo culture medium consisted of basal medium
with 500 mg l-1 glutamine, 250 mg l-1 casein
hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-
agar
Plant regeneration via pod culture, embryo culture and embryo germination for pods of P. angustissimus
 Immature embryo culture in Mango
¾ For the first time Jie-ning Xiao et al. (2004), regenerated
plantlets were obtained from immature zygotic embryos
of mango (Mangifera indica L.) through direct somatic
embryogenesis.
¾ Pro-embryogenic mass (PEM)-like structures, which are
differentiated as clusters of globular structures, were
easily induced directly from the abaxial side of
cotyledons from immature fruits, 2.0–3.5 cm diameter
by a 2-wk culture period on a modified MS medium with
5mg (25 mM) indole-3-butyric acid (IBA).
¾ Conversion of somatic embryos into plantlets was
achieved after 4 wk of culture on the conversion
medium containing 5mg (23 mM) kinetin.
¾ Concluded that secondary somatic embryogenesis
could also be obtained directly from the hypocotyls of
mature primary somatic embryos cultured on the
conversion medium.
 Plantlet dev. Form imm. embryo

Immature embryo with seed

Proembryonic mass

Globular embryo

Mature somatic embryo

¾ An interspecific hybrid of the sexually incompatible
species G. hirsutum cv.Laxmi and G. arboreum cv.
Jyoti was obtained through in ovulo embryo culture.
¾ Eight to twelve-day-old ovules were excised and
cultured on BS medium supplemented with IAA (5 ×
10-6 to 7 × 10 -7M), Kinetin (5 X 10 -6 to 5 × 10- -8), GA (5
X 10 -7 to 5 × 10 -9), Ammonium chloride (5 to 15mM)
and Casein hydrolysate (50 to 200mg/l) added
individually and in various combinations along with
sucrose.
¾ No single medium was adequate to ensure complete
development of the fertilized ovules to plantlets, thus
necessitating a sequential five step transfer to
different media.
¾ Cytological studies confirmed the hybrid nature of the
plants
Ovules of 85 D old

Enlarged ovule after 35 D of

incubation

Triploid hybrid plant

2n = 39
¾Synthetic seed technology may be of value in breeding
programs and allow the propagation of many elite
genotype derived plants in a short time.
¾A range of artificial endosperm treatments of Cleopatra
tangerine zygotic embryos were evaluated for suitability
for encapsulation of somatic embryos.
¾An artificial endosperm was used to encapsulate
somatic and zygotic embryos.
¾After encapsulation, zygotic embryos germinated after
four days of culture while somatic embryos germinated
asynchronously after 20 days.
¾Somatic embryo-derived plantlets showed greater vigour
than zygotic embryo-derived plantlets.
¾Results showed that this artificial endosperm is
adequate for Cleopatra tangerine somatic embryo
germination and conversion into plants
Encapsulation of embryos with Calcium
arginate