Plant Tissue Culture: Somatic Embryogenesis Direct and Indirect Somatic Embryogenesis

1. Plant Tissue Culture:
Somatic Embryogenesis
Dr. Divya Sharma
Assistant Professor
A Biodiction (A Unit of Dr. Divya Sharma)

2. Somatic Embryogenesis
 Ordinary plant tissue ----- Somatic embryo are formed from somatic plant
cells
(Those cells are not involved in the development of embryos)
 No endosperm or seed coat is formed around a somatic embryo
 Applications of this process include:
o Clonal propagation of genetically uniform in plant material
o Elimination of viruses
o Provision of source tissue for genetic transformation; generation of whole
plants from single cells ------- Protoplasts
o Development of Synthetic seed technology

3.  Callus ------ Cells derived from competent source tissue are cultured to form an
undifferentiated mass of cells
 Plant growth regulators in the tissue culture medium can be manipulated to induce
callus formation and subsequently changed to induce embryos to form from the
 Ratio of different plant growth regulators to induce callus or embryo formation
varies with the type of plant
A process where an embryo is derived from a single somatic cell or group of somatic cells.
Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo
formation
Embryos formed by somatic embryogenesis are called Embryoids

4. Features of Embryo
 Bipolar axis embryo
 Has cotyledons
 Shoot meristem at one end of the axis
 Root meristem at the other end of the
axis
 With the hypocotyls in the middle
Properties of embryo

5. Types of Embryo
 Kohlenbach (1978) has proposed the following classification of embryo:
1. Zygotic embryos: Formed by Fertilized egg or the zygote
2. Non-Zygotic embryos: Formed by cells other than the zygote
 Somatic embryo - Formed either in-vitro or in-vivo by the sporophytic cells
(except zygote)
 Parthenocarpic embryo - Formed by unfertilized egg
 Androgenic embryo - Formed by the microspore pollen grain (male
gametophyte)

6. Embryogenesis
 The process of development of plant embryos, being either a sexual or asexual
reproductive process that forms new plants are called Plant Embryogenesis.
 Embryogenesis may occur naturally in the plant as a result of sexual fertilization,
and these embryos are called Zygotic embryos and develop into seeds, which
can germinate and give rise to seedlings. Plant cells can also be induced to form
embryos in plant tissue culture; these embryos are called Somatic Embryos (SEs).
 The process was discovered for the first time in Daucas carota L. (carrot) by
Steward (1958) and Reinert (1959).

7. Stages of Embryogenesis
Zygote ----- Globular Embryo ----- Heart Shaped ---- Mature Embryo

8. Somatic Embryogenesis
 When embryos regenerate from somatic cells or tissues, that are haploid, diploid
etc; termed as a Somatic Embryogenesis.
 Somatic Embryogenesis: A process by which the somatic cells or tissues develops
into differentiated embryos.
 Somatic embryogenesis was first induced in:
Suspension culture (Stewart et al., 1958) and in Callus culture (Reinert, 1959) of
carrot.
In addition to the members of Umbelliferae and Solanaceae, a range of
dicotyledonous families have produced somatic embryos.
 Somatic embryogenesis most frequently in tissue culture and as an alternative
Organogenesis for regeneration of whole plant.

9. Steps of Somatic Embryogenesis
Formation of plant via somatic embryogenesis process

10. Factors associated in Somatic
Embryogenesis
 Media components
 50% normal concentration of media components and in some cases lacking
Phytohormones
 Light, temperature etc.
 Light intensities, particular wavelength and photoperiods, moderate and
stimulate the formation and development of embryoids
 Exudates
 Extracts (Tomato, Banana, Yeast/ Coconut water)

11. Others Factors Affecting Somatic
Embryogenesis
a. Explant
 Explant as source material to induce SEs are very diverse. There are very
responsive plants such as carrot in which any part of the plant can be use to
induce embryogenic cultures.
 There are some very recalcitrant plants such as Cereals and Legumes which
explant varies even within the genetically identical species.
 Various types of explants used like:
Immature zygotic embryos, Inflorescence, Cell suspension cultures, Petioles,
Protoplasts, Leaves, Stems and Roots

12. b. Plant Growth Regulators
 Auxins
 2,4-D has been the best synthetic Auxin used for inducing somatic embryos
 Continuous supply of Auxin causes embryonic cells to divide (Proliferation
without the appearance of embryos
 Witherell, 1971 have suggested that continuous supply of Auxin induces
endogenous ethylene production which suppresses embryo development
 Embryonic cells after treatment with Auxin must be transferred to Auxin free
medium that constitute the embryo development medium

13.  Cytokinins
 Cytokinin produces globular embryo from initial embryos
 Zeatin is promotive when applied to embryogenic cells after days 3-4 transfer
the proliferation medium to nutrient medium whereas BAP and Kinetin have
inhibitory effect on embryogenesis.
 High rate of cytokinins than Auxin induces shoot formation and reverse ratio
rooting
 Gibberellins
 Inhibits Somatic embryos
 Abscisic Acid
 Promote embryo maturation and prevent precocious germination and secondary
embryogenesis

14.  Nitrogen Source
 Reduced from of nitrogen is the sole source of embryo formation
 Polyamines
 Involved in-vitro and in-vivo somatic embryos
 Involved in cell growth, proliferation and aging
 Interact with negatively charged molecules DNA, RNA and Proteins
 Three polyamines (Putrescine, Spermidine, Spermine) Putrescine showed the
drastic increase in somatic embryos
 Genotype
 Genotype effect on somatic embryogenesis occurs as for regeneration via shoot
bud differentiation
 Genotypic variations could be due to endogenous levels of hormones

15.  Nitrogen Source
 Exposure of explant to mild electric current of 0.02V DC for 20 h promoted
embryogenesis in Alfalfa and Tobacco (Rathore and Goldsworthy, 1985)
 The electric stimulus seems to promote the differentiation of organized shoot/
embryo by affecting cell polarity through changes in organization of microtubes
and induction of asymmetric first division

16. Stages of Embryogenesis
Induction  Globular stage: Embryo is small and
round (multicellular)
 Heart stage (Bilateral symmetry):
Shape changes to a heart shape
with more cotyledon development
 Torpedo-shaped stage: Consists of
initial cells for the shoot/root
meristem
 Mature stage: Embryo becomes
cylindrical
Development & Maturation
Globular Shaped
Heart stage Shaped
Torpedo Shaped
Germination & Conversion

17. Stage 1: Induction
 Auxin required for induction
 Proembryogenic masses are formed
 2,4-D mostly used
 NAA, Dicamba are also used
Requirement of exogenous
Auxin for induction of somatic
embryogenesis depends on
nature of explants used with
relative concentration of Auxin

18. Stage 2: Development
 Auxin must be removed for embryo
development
 Continuous use of Auxin inhibits
embryogenesis
 Stages are similar to those of zygotic
embryogenesis:
o Globular
o Heart
o Torpedo
o Cotyledonary
o Germination (conversion)
After reinitiation of cell division
and a period of cell proliferation
in presence of Auxin
embryogenesis cells are
released into Auxin free
medium. These cells are in the
clusters of cytoplasmic cells are
called Proembryonic Mass of
Cells (PEMs)

19. Stage 3: Maturation
 Require complete maturation with apical
meristem, radicle and cotyledons
 Often obtain repetitive embryony
 Storage protein production necessary
 Often require ABA for complete maturation
 ABA promotes for normal embryo
morphology
 ABA prevent Precocious germination
(Dure et al., 1981, Triggering expression of
genes which normally express during drying
down stage of seeds)
Quality of somatic
embryogenesis with regards to
their germinability or
conversion into plants is very
poor due to the apparently
normal looking somatic
embryogenesis are actually
incomplete in their
development
Unlike seed embryos, somatic
embryogenesis do not go
through the final phase of
embryogenesis, called Embryo
maturation

20. Morphology of Stages of Somatic Embryo
Morphological stages of somatic embryo development if Alfalfa (Medicago sativa L.)

21. Types of Somatic Embryogenesis
Two route to somatic embryogenesis (Sharp et al., 1980)
 Direct Embryogenesis
The embryos initiate directly from
explants in the absence of callus
formation.
Explant  Meristemoid  Primordium
 Indirect Embryogenesis
Callus from explants takes place from
which embryos are developed.
Explant  Callus  Meristemoid 
Primordium

22. Types of Embryogenic cells
Pre-Embryogenic
Determined Cells (PEDCs)
 Cells are committed to embryonic
development and need only to be
released, such cells are found in
embryonic tissues.
 The explants capable of direct
embryogenesis seem to carry
competent or PEDCs
 These cells are committed to
embryo development and need only
to be released
Induced Embryogenic
determined cells (IEDCs)
 In majority of cases embryogenesis
is through indirect method.
 Specific growth regulator
concentrations and/or cultural
conditions are required for initiation
of callus and then redetermination of
these cells into the embryogenic
pattern of development

23. Direct Somatic Embryogenesis
 In direct somatic embryogenesis, the
embryoids are formed directly from a
cell or small group of cells without the
production of an intervening callus
(common among reproductive tissues).
It is rare in comparison with indirect
somatic embryogenesis
Eg: Coffea Arabica, Alfalfa, Daucus carota,
Ranunculus scleratus, Linum
usitatissimum,Brassica napus, Arachis
hypogea etc.
Leaves, scutellum, hypocotyl, nucellus and
embryo-sac etc are used as explants

24. Indirect Somatic Embryogenesis
 In indirect somatic embryogenesis,
callus is first produced from the
explants.
 Embryoids can then be produced from
the callus tissue or form cell suspension
produced from the callus.
Eg.: Secondary phloem of carrot, leaf
tissues of Coffee, Petunia, Asparagus etc.
In majority of cases embryogenesis is
through indirect method.
Indirect shoot formation from callus of tobacco

25. Steps of Indirect Embryogenesis
Explants
Culture of explants in the medium
Callus formation
Embryo
Maturation
Germination (Bipolar structure)
Complete plant

26. Steps involved in Indirect Somatic Embryogenesis

27. Systematic representation of Direct and Indirect
Somatic Embryogenesis

28. General rules for the induction of somatic
embryo
 A high Auxin concentration is often required for embryo induction. For further,
development of the embryo, Auxin conc., should be lowered completely
eliminated from the medium.
 Gibberellins and ethylene usually inhibit embryogenesis
 Callus from juvenile plant are more efficient for embryogenesis
 Reduced nitrogen in the form of ammonium ions can be an important factor in
embryogenesis
 Light generally promotes embryogenesis
 High temperature usually favorable for somatic embryogenesis. Coconut milk
often promotes embryogenesis
 Abscisic acid, exerts a number of striking effects on the somatic embryos on
suspension culture.
 The physiological state of the plant from which the explants is taken is extremely
important, as is the season during which the material is removed.

29. Differentiate Between Zygotic and
Somatic Embryo
Variables Somatic Embryo (SE) Zygotic Embryo (ZE)
Formed by Sporophytic cells Fertilized egg or zygote
Covered by No covering Seed coat
Result Only from embryo Seed
Nature of plantlets weak Healthy
Nutrient contents Less storage, specific reserves
Seed storage proteins, carbohydrates are
the characteristic features
Embryogenesis and
Pluricotyledony
Present in lack of dormant phase Not present
Vascular connections No Yes
Alike Mother plant Not like mother plant
Distinct suspensor
Absent
(If present which may not be functional
as in seed embryos)
Present
(well developed)
Propagation rate High Comparatively low

30. Variables Somatic Embryo (SE) Zygotic Embryo (ZE)

31. Various Stages of Somatic and Zygotic Embryo in
Cultures

32. Advantages of
Somatic
Embryogenesis
 Higher propagation rate
 Suitable for suspension culture
 Plantlets from single genetically modified
single cells
 Somaclonal variation
 Germplasm preservation
 Labor savings
 Elimination of diseases and viruses
 Artificial or synthetic seed production:
Synthetic seeds not commercially viable yet:
o No endosperm – limited shelf life
o No protective seed coat that can withstand dry conditions

33. Schematic representation showing Artificial or
Synthetic Seed Production
Cultured cells Single cell origin
Somatic Embryo
Encapsulation
Somatic Seeds
Germination

34. Diagrammatic representation of Haploid
production of through Somatic Embryogenesis
Haploid plantlets from anther culture may obtain

35. Disadvantages of Somatic Embryogenesis
 Response tissue specific (explants)
 Confined to few species
 Inability to generate large numbers of normal, free living plantlets
 Barriers to high frequency plantlets production may occur at any of a number of
points between induction and the production of a plantlets capable of surviving
transfer to ex-vitro condition
 May also include, low frequency embryo production, production of malformed
embryos, incomplete embryo maturation, unbreakable embryo dormancy or low
plantlet vigor.
 May create unwanted genetic variation (Somaclonal variation)

36. Role of Somatic Embryogenesis in Cereals
 Due to change of emphases from medium manipulation to explant and genotype
selection, several species of Cereals have been regenerated which were once
regarded recalcitrant
 High frequency of somatic embryos were obtained in Indian bread wheat cultivar
HD967 by using mature embryos and increasing concentration of Agar gel in the
medium (Gill et al., 2015)
 Use of somaclonal variation in somatic embryogenesis has broaden the genetic
variation in crop plants
 Several lines of disease resistant Wheat, Rice, Barley have been isolated from
somaclones (Jain et al., 1998)
 By using a Cephalosporin antibiotic, Cefotaxime several varieties of rice have been
developed by PAU, Ludhiana, 2009

37. Thankyou
A Biodiction (A Unit of Dr. Divya Sharma)