2. • TOPIC:
• TRIPLOID PRODUCTION (ENDOSPERM
CULTURE)- DIFFERENTIATION AND FACTORS
AFFECTING THEM- APPLICATIONS
• SOMATIC EMBRYOGENESIS- FACTORS
AFFECTING, STAGES OF SOMATIC
EMBRYOGENESIS, LARGE SCALE PRODUCTION,
SYNTHETIC SEEDS CONCEPT AND ITS PRO’S
AND CON’S
PRESENTED BY
ZUBY GOHAR ANSARI
TAM/14-26
3. ENDOSPERM CULTURE (HISTORY AND DEF.)
• Double fertilization occurs in majority of
angiosperms which results in two fusion
products i.e. Zygote (fusion product of egg and
one of the male gamete) and triploid endosperm
(fusion product of polar nuclei and second male
gamete).
• The endosperm lack many organogenic potential
and vascular differentiation.
• Endosperm is unique tissue, firstly in its function
of supplying nutrition to developing embryo and
secondly in being triploid.
4. • Attempts to grow endosperm tissue in cultures
began in 1930’s and now mature and immature
endosperm of various taxa of angiosperm have
been grown.
• Lampe and Mills (1933) : They were first to
report the proliferation of immature endosperm
tissue of maize grown on medium containing
extract of potato.
• Nakano et al. (1975): Successfully cultured
immature endosperm of rice and achieved
organogenesis.
5. • Rangaswami and Rao (1963): Successfully
cultured mature endosperm of santalum
album (chandan) up to callus.
• Endosperm culture: It is the in vitro
development of isolated mature or immature
endosperm from seed at proper stage on a
suitable culture medium to obtain triploid
plantlet.
6.
7. TYPES OF ENDOSPERM CULTURE
• 1. Mature endosperm culture: It is the in vitro
development of isolated mature endosperm
from ripen endospermic seed on suitable
culture medium to obtain triploid plantlet is
called mature endosperm culture.
• 2. Immature endosperm culture: It is the in
vitro development of isolated immature
endosperm isolated at precise stage from
immature seed, cultured on suitable culture
medium to obtain triploid plantlet is called
immature endosperm culture.
8. STEPS IN ENDOSPERM CULTURE
• It consist of mainly 3 steps:
• 1. The immature or mature seeds are dissected
under aseptic conditions and endosperms
along with embryos are excised.
• 2. Excised endosperms are cultured on a
suitable medium and embryos are removed
after initial stage.
• 3. Callus followed by embryogenesis or shoot
bud differentiation.
• 4. Complete plant formation.
9.
10. PROCEDURE OF ENDOSPERM CULTURE
• 1. Explant source: In most of the cereals, mature
endosperm does not respond to cultural conditions. It is
therefore, excised at proper period of development.
Normally the endosperm of cereals undergo certain
changes 12 days after pollination making this able to
respond. Proper stage for maize is 8-11 days after
pollination, 4-7 days after pollination for rice, and 8 days
after pollination for wheat.
• In some families association of embryo tissue in initial
stages seems essential for proliferation of mature
endosperm. In such cases, entire seeds are used as
explant. Immature seeds provide explant for non-
endospermic seeds.
11. • 2. Inoculation of explant: For in vitro culture of
mature endosperm, seeds having massive endosperm
are decoated, surface sterilized with suitable
disinfectant and after 2-3 washings with sterile distilled
water, planted on the nutrient medium. For in vitro
culture of an immature endosperm, the entire seed or
kernel is surface sterilized and the endosperm tissue is
excised under aseptic conditions. In cereals, top of
kernel or immature ovaries (micropyle end) are cut off
with a sterile knife and exposed endosperm squeezed
out and placed on the callus induction nutrient
medium.
• The basal white and MS medium is generally used to
induce callus from an immature endosperm.
12.
13. • The basal medium is supplemented with
tomato juice, yeast extract, grape juice.
Sucrose (2-4%) is used as a source of
carbohydrates. In some cases, addition of 2,4-
D or IAA, cytokinin necessary.
• 3. Incubation/ maintenance of culture: To
induce callus, the endosperm cultures are
maintained darkness or diffused light.
Differentiation take place when calli are
transferred to bright light (2000 lux – 4000
lux) and 25⁰C temperature
14. • 4. Shoot bud differentiation or
embryogenesis: Plantlet formation from
endosperm callus maturation follow organogenic or
embryogenic mode of development. Embryo
differentiation occurs when the proliferated tissue is
transferred from callusing to basal medium with or
without gibberellins. So culturing carried out up to
complete development of plantlet.
• 5. plantlet developed are hardened in green house
by transferring in vermiculture media and
maintaining proper humidity.
15. FACTORS AFFECTING ENDOSPERM CULTURE
• 1. Explant stage: proper stage may vary from
cellular (immature) to mature endosperm
depending upon species. (4-7 DAP in rice, 8
DAP in wheat and 12 DAP in maize and fully
matured in santalum album).
• 2. Nutrient medium: Low amount of reduced
nitrogen is required for proliferation.
Undefined source like tomato juice, yeast
extract, etc.
16. • 3. Physical factors: The pH 7.0 seems to be
effective for fresh weight increase. Maximum
growth of endosperm occurs between 24-
27⁰C temperature and 12-16 hours
photoperiod with diffuse day light supported
callusing as well as regeneration.
• 4. Embryo factors: association of embryo
tissue in the initial stages seems essential for
inducing proliferation of mature endosperm
tissue in some families. Immature seeds of
non endospermic seeds exhibit no
dependence on embryo factor.
17. APPLICATIONS OF ENDOSPERM CULTURE
• Techniques of endosperm culture has enabled
the production of triploid plants. Triploid
plants are self sterile and usually seedless. The
trait increases edibility of fruits and is
desirable in plants such as apple, banana,
grape, watermelon and mango which are
commercially important.
• In timber and fuel yielding plants, triploids
show better performance over their relative
diploids or tetraploids. Also there is no
problem of seed sterility as they can be
multiplied by vegetative means.
18. SOMATIC EMBRYOGENESIS
• Regeneration of embryos from somatic cells, tissues
or organs either denovo or directly in vitro conditions
is known as somatic embryogenesis.
• It is also known as non- zygotic/ non-sexual
embryogenesis.
• Various terms for non-zygotic embryos have been
reported such as,
• A. Adventive embryos: Somatic embryos arising
directly from other organs or embryos.
19. • B. Parthenogenic embryos: Formed by
unfertilized eggs.
• C. Androgenetic embryos: Formed by male
gametophyte.
• Occurence of asexual embryogenesis is
generally restricted to intraocular tissues.
20.
21. • Differences between somatic and
sexual embryos
SOMATIC EMBRYOS SEXUAL EMBRYOS
Embryos arises from single cell. Arises from multi cell.
Embryos have bipolar structure. It is monopolar structure.
Embryos has no vascular connections with
cultured explant.
Embryos has vascular connections with
cultured explant.
Induction of somatic embryogenesis
requires single hormonal signal.
Requires two hormonal signals.
22. • The initiation and development of embryos
from somatic tissues in plant culture was first
recognized by steward et al (1958) and Reinert
(1958- 59) in cultures of Daucus carota
(carrot).
• In addition to the development of somatic
embryos from sporophytic cells, embryos have
been obtained from generative cells, such as
the classic works done by Guha and
Maheswari (1964), with Datura innoxia
microspores.
23. TWO ROUTES TO SOMATIC EMBRYOGENESIS
• Sharp et al (1980) divided 2 routes to somatic
embryogenesis.
• 1. Direct embryogenesis: The embryo initiates
directly from the explant tissue in absence of
callus proliferation.
• This occurs through pre-embryonic determined
cells (PEDC). Such cells are found in embryonic
tissues.
• Eg: scutellum of cereals
24. • 2.Indirect embryogenesis: The embryos initiate
from the explant through callus proliferation.
• This occurs through Induced embryogenic
determined cells (IEDC).
• Eg: Secondary phloem of carrot, leaf tissue of coffee,
petunia, asparagus etc.
• For some species, any part of plant body serves as an
explant, for embryogenesis. But in some species only
certain regions of explant may respond in culture. Eg:
cereals
• Floral or reproductive tissue in general has proven to
be an excellent source of embryogenic material.
• The physiological states of the plant from which
explant is taken is also extremely important.
25. STAGES OF SOMATIC EMBRYOGENESIS
• Somatic embryogenesis encompasses
various stages such as
• 1. Callus initiation
• 2. Embryo development and maturation
• 3. Plantlet formation
26.
27. FACTORS AFFECTING SOMATIC
EMBRYOGENESIS
• 1. Genotype of explants: Explant genotype
has a marked influence on somatic embryo
regeneration and in many cases, it may
determine whether or not somatic embryo
regeneration will occur.
• Strong genotypic effect have been shown in
many species.
• Eg: Alfa-alfa , wheat, maize, rice and chick pea
etc.
28. • 2. Growth regulators:
• Auxins: Auxin alone or in combination with
cytokinin appear essential for the onset of growth
and the induction of embryogenesis of all the auxins,
2,4-D followed by NAA have proven to be extremely
useful.
• Effective concentration ranges are 0.5 – 27.6 μ M for
2,4-D and 0.5 – 10.7 μ M for NAA.
• Cytokinins: CKs have been used in the primary
medium invariably during embryogenesis of crop
plants.
• Effective concentrations for kinetin0.5 – 50.0μ M.
CKs are important in fastening somatic embryo
maturation and especially cotyledon development.
29. • ABA: It is added at inhibitory levels @0.1- 1 μM
promotes somatic embryo development and
maturation and at same time inhibits abnormal
proliferation and initiation of accessory embryos.
• 3. Nitrogen source: Form of nitrogen has marked
influenced on somatic embryogenesis. In carrot NH₄⁺
form has a promotive effect.
• Somatic embryos development occurs on a medium
containing NO₃⁻ as the sole nitrogen source.
• 4. Other factors: High K⁺ levels and low dissolved
O₂ levels prevents somatic embryo regeneration.
30. • In citrus medica, volatile compounds like
ethanol, inhibit somatic embryo regeneration.
• In soybean low sucrose conc. @ 5 to 10 g/l
promotes somatic embryogenesis than high
concentration.
• In Alfa-alfa, use of maltose as a carbon source
improved both somatic embryogenesis
induction and maturation as compared to
sucrose.
31. SYNTHETIC SEED CONCEPT
• Synthetic seeds are also known as artificial
seeds and also encapsulated embryos.
• The aim of somatic embryo encapsulation is to
produce an analog to true seeds, which is
based on the similarity of somatic embryo
with zygotic embryo with respect to their
morphology, physiology and biochemistry.
• Two type of synthetic seeds have been
developed namely,
• 1. Hydrated S.S.
• 2. Dessicated S.S.
32. • 1. Hydrated synthetic seeds: Renderbergh et al
(1986) developed hydrated artificial seeds by mixing
somatic embryos with an encapsulation matrix so
that embryos are well protected in that matrix and it
is rigid enough to allow for rough handling.
• Encapsulation matrix consists of hydrogels such as
agar, carrageenan, alginate or plant exudates of
arabica, karaya or seed gums of guar, tamarind or
microbial products like dextran, xantham gum and
divalent salts.
• Various divalent salts which can be used are CaCl₂,
Ca(OH)₂, ferrous chloride, cobaltous chloride etc.
• Sodium alginate + CaCl₂ = encapsulation of somatic
embryo.
33. • Procedure: Mixing the somatic embryos with
sodium alginate, followed by dropping into a solution
of CaCl₂, CaNO₃ to form calcium alginate beads.
• Calcium alginate capsules tend to stick together and
are difficult to handle because they lose water
rapidly and dry down to a hard pellet within a few
hours of exposure to the atmosphere.
• These problems can be offset by coating capsules
with Elvax 4260 (Ethylene vinyl acetate acrylic acid
tetrapolymer)
• Sodium alginate + CaCl₂= Calcium alginate
34. • 2. Dessicated synthetic seeds: Kim and Janic
(1989)applied synthetic seed coats to clumps
of carrot somatic embryo to develop
dessicated artificial seeds.
• Mixing of equal volume of embryo suspension
and 5% of polyethylene oxide (a water soluble
resin), which subsequently dried to form
polyembryonic dessicated seeds.
• The survival of encapsulated embryos was
further achieved by embryo hardening
treatments with 12% sucrose or 10⁻⁶ M ABA,
followed by chilling at high inoculum density.
35. • Potential uses of synthetic seeds:
• Produce large number of identical embryos .
• Determines the roles of endosperm in embryo
development and germination.
• Propagation with low cost, high volume
capabilities of seed propagation.
• Produced within short time and in any season,
at any time.
• Dormancy period can be reduced there by
shortening the life cycle of a plant.
• Useful in preserving germplasm.
36. • Provides knowledge to understand the
developmental, anatomical characteristics of
endosperm and seed coat formation.
• Synthetic seeds production has been
successfully obtained in Zea mays, Opium,
Daucus carota, Lactuca sativa, Madicago,
Brassica species, Gossypium, Santalum species
etc.
37. • Disadvantages with artificial seeds:
• 1. Facilities required are costly.
• 2. Special skills are required.
• 3. Errors in maintenance.
• 4. Specific kinds of genetic and epigenetic
modifications can develop.
• Problems in production of synthetic seeds:
• 1. Artificial seeds that are stable for several months
requires the procedures for making embryos
quiscent.
38. • 2. Synthetic seeds need to be protected
against dessication.
• 3. Recovery of plants is often very low due to
incomplete embryo formation or difficulties in
creating artificial endosperm.
• 4. Embryo must be protected against
microbes.
