Morphogenesis, organogenesis, embryogenesis & other techniques

1. 1. Media preparation
2. Explant selection
3. Establishment of explant in media
4. Callus development
5. Plantlet development
6. Hardening or acclimatization
7. Open field planting
Lecture 8: Morphogenesis, organogenesis,
embryogenesis & other techniques
Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF
HORTICULTURAL CROPs

2. Introduction to Tissue Culture
⚫Tissue Culture (also known as Micropropagation or In vitro culture) is:
⚫The growing of plant cells, tissues, organs, seeds or other plant parts in
a sterile environment on a nutrient medium.

3. Steps of Micropropagation
⚫ Stage 0 – Selection & preparation of the mother plant
 sterilization of the plant tissue takes place
⚫ Stage I - Initiation of culture
 explant placed into growth media
⚫ Stage II - Multiplication
 explant transferred to shoot media; shoots can be constantly divided
⚫ Stage III - Rooting
 explant transferred to root media
⚫ Stage IV - Transfer to soil
 explant returned to soil; hardened off

7. Callus
Dedifferentiation
Callus growth &
cell division Redifferentiation
Plantlet
development

11. 3/27/2015 Deptt of Plant Biotechnology 11
EMBRYOGENESIS
:-
 Plant embryogenesis refers to the process of development
of plant embryos, being either a sexual or asexual
reproductive process that forms new plants.
 Embryogenesis may occur naturally in the plant as a result
of sexual fertilization, and those embryo 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 embryo are called somatic embryos.

12. Stages of Embryogenesis:-

13. 3/27/2015
Types of Embryogenesis:-
• Two types of embryogenesis:-
A) Zygotic embryogenesis
B) Somatic embryogenesis

14. Zygotic Embryogenesis:-
The zygotic embryo is formed following double
fertilization of the ovule, forming the plant
embryo and the endosperm which together go
into the seed, this process is known as zygotic
embryogenesis.
Seeds may also develop without fertilization
through pathways referred to as apomixis.

15. Somatic Embryogenesis:-
Somatic embryogenesis is a process by which
somatic cells or tissues develops into
differentiated embryos.
Embryos regenerate from somatic cells or
tissues ( haploid or diploid etc) it is termed as
Somatic Embryogenesis.

16.  Somatic embryogenesis was first induced in suspension culture
(Stewart et al, 1958) and in callus culture (Reinert, 1959) of carrot,
Umbelliferae and Solanaceae dicotyledonous families have
produced somatic embryos.
 SE occur most frequently in tissue culture as an alternative
organogenesis for regeneration of whole plant.
 In literature, somatic embryos are referred to by many names such
as embryo like structures, adventitious or vegetative embryos,
Embryoids; and the process is termed as adventitious , asexual or
somatic embryogenesis.

17. Stages of Somatic Embryogenesis:-

18. Stages of Somatic Embryogenesis:-
3/27/2015

19. Contd:-
 INDUCTION
 Development and
Maturation
 Globular
 Heart stage
 Torpedo
 Germination and
Conversion
• Globular stage: Embryo
is small and round
(multicellular).
• Heart stage (Bilateral
symmetry): Shape changes
to heart shape with more
cotyledon development.
• Torpedo shaped stage:
Consists of initial cells for
the shoot/root meristem.
• Mature stage: Embryo
becomes cylindrical.

20. Induction
Auxin required for induction
Pro embryonic masses are formed.
 2,4-D are mostly used.
 NAA, DICAMBA are also used.

21. Development
• Auxin must be removed for embryo development.
• Continuous use of Auxin inhibits embryogenesis.
• Stages are similar to those of Somatic embryogenesis:-
 Globular
 Heart
 Torpedo
 Cotyledonary
 Germination (Conversion)

22. Maturatio
n
Require complete maturation with apical meristem,
radicle and cotyledons.
Often obtained repetitive embryony.
Storage protein production necessary.
Often require ABA for complete maturation.
ABA often required for normal morphology.

23. Morphological Stages of Maize cv. Gaurav
3/27/2015 Deptt of Plant Biotechnology 23

24. Routes of Somatic Embryogenesis:-
Two routes to somatic embryogenesis
 Direct somatic embryogenesis
 The embryos initiate directly from explants in the
absence of callus formation. Embryos are formed
due to PEDCs cell.
 Indirect somatic embryogenesis
 Callus from explants takes place from which
embryos are developed. Embryos are formed due to
IEDCs cells.

25. Examples of Direct Somatic Embryogenesis:-
Figure :- Isolation of mature embryo from imbibed cereal grain. (a) A curved-tip scalpel
blade is inserted beneath the Coleoptilar region of the Mature embryo; (b) With a swift and
smooth scooping motion the mature embryo is dislodged from its attachment to the
scutellum; (c) Isolated mature embryo which will be inoculated with abaxial surface in
contact with culture medium. Ganeshan et al., 2006.

26. Contd:
- Mature embryos culture in the Murashige and Skoog, 1962
medium with supplements 1gm/l enzymatic casein
hydrolysate, 0.7 gm/l L-proline.
 4.5 µM of TDZ and 4.4 µM of BAP are best combination of
growth regulators in which Durum Wheat produces 35
number of shoots per explant and Mature embryos of CDC
Dancer oat produces 16 shoots per explant.
 Explants for direct embryogenesis include microspores,
ovules, scutellum, endosperm, embryos and seedlings.

27. Indirect Somatic Embryogenesis:-
In Indirect SE, callus is produced from explants.
Embryoids(suspensory cell to cotyledon) are
produced from callus tissue.
Explants are roots, shoots, leaf cells, anthers, seeds
etc.
Steps involved in Plant regeneration of Rice variety
through Indirect SE:-

28. a) Formation of callus b) Greening of callus c) Embryo at globular stage
d) Torpedo stage of embryo e) Cotyledonary stage and regeneration
of embryo f-g) Multiple shoot regeneration h) Complete plantlets i)
Hardening of plantlets. (Rice Variety:- Swarna)
Mondal et al., 2011
(a) (b) (c) (d)
(e) (f) (g) (h)
(i)

29. 3/27/2015
Factors affecting Somatic Embryogenesis:-
1) Genotype:-
 Genetically engineered / transgenic plant
does not regenerate through SE because due
to variation.
 Methylation occurs in the DNA during mitosis
then SE occurs. If Methylation occur in the
cytosine bases or H3 protein then SE get
stop.

30. 2) Explant:-
Totipotent somatic cell are used.
Immature inflorescence and Scutellar tissue of
immature seeds are used for the research. Ex:-
Triticum aestivum .
Epidermis, Procambial tissue are also produced
somatic embryo.

31. During Proembryonic
phase, 2,4-D generates
DNA Hyper methylation
so that cells in a highly
active mitotic stage.
High concentration of
auxin produces root in
somatic embryo.
2,4-D is one of the growth
regulator that produces
callus from cereals and
conc. of 2,4-D 0.1-10µM
3) Auxin:-
Polar transport of auxin
produces somatic
embryo.
Auxin concentration
will be more then
somatic embryogenesis
get stop. Ex:- Maize.
Auxin induces indirect
somatic embryogenesis
in monocots.

32. 4) Cytokinins:-
Cytokinin promote axial growth.
Cytokinin produces globular embryo from initial
embryo.
Cytokinin combination with auxin, induces somatic
embryogenesis and produce callus in cereals.
Cytokinin ratio more than auxin then it produces
Shoots.

33. 5) Gibberellic acid:-
 GA promote
elongations of embryo
axis, cell division.
It synthesized of
photosynthetic
pigments in developing
somatic embryo.
It improve
photosynthetic activity,
Extra storage reserves in
vitro germination.
Hypo cotyledon are used
as explant then GA inhibit
somatic embryogenesis.
Addition of Uniconazole,
Paclobutrazol inhibit
somatic embryogenesis.
GA higher in suspensory
embryo than the proper
embryo. So GA requires
early embryo
development.

34. 6) Abscisic acid (ABA):-
 ABA control tolerance and seed dormancy during
later stage of embryogenesis.
ABA induced somatic embryogenesis in high osmotic
stress and high temperature in auxin free medium.
Primary embryo contain more conc. of ABA than
secondary embryo.
Treatment of Fluridone inhibit ABA synthesis and
primary embryo does not produce secondary
embryo.

35. 7) Polyamines:-
 Spermidine, Spermine and
Putrescine are added as
growth regulators and
secondary messenger.
source for plants.
 Spermine act as a
 Polyamines serve as nitrogen
 It act as a free radical
scavengers by protecting
senescing membranes
against lipid per oxidation.
 In Maize, Putrescine are
most effective with varying
concentration of GA3.
antioxidant in a medium.
 It help in vegetative
growth, pollen
development, regulation of
DNA duplication,
transcription of genes, cell
division, development of
organs.

36. 8) Phytosulfokine
 It modulate the culture media.
 It promote somatic
embryogenesis by activating
cell division of embryogenic
cells, in presence auxin.
 Phytosulfokine increases the
cell through differentiation
process.
9) Phenolic compounds:-
 Phenolic compounds are inhibit
somatic embryogenesis.
 4hydroxy benzyl alcohol inhibits
the globular stages.
 Vanillyl benzyl ether are inhibit
the suspensor development.
 Recently identification of 4
[(phenyl methoxy) methyl]
phenol involves in seed
development stills unknown.

37. Differences between Zygotic and Somatic embryo:-
Zygotic embryo
 Fertilized egg or zygote.
 Contain seed coat.
 Produce seed.
 Plantlets are healthy.
 Not like to mother plant.
 Propagation is low.
Somatic embryo
 Sporophytic cells.
 Did not contain seed coat.
 Only form embryo.
 Plantlets are weaker
 Like to mother plant.
 Propagation is high.

38. Advantages and Disadvantages of Somatic
Embryogenesis:-
 Higher propagation rate.  Somaclonal variation.
 Suitable for Suspension
culture.
 Artificial seed production.
 Germplasm
conservation.
 Labour savings.

39. Disadvantages
Response tissue
specific (explants).
Low frequency embryo
production.
Incomplete embryo
production.
May create unwanted
genetic variation
(Somaclonal variation).
Inability to generate
large numbers of
normal, free living
plantlets.
Plantlets are weaker.

40. Somatic Embryo Germination Media:-
MS medium containing different concentrations of
BAP (0, 1, 2, 3, 4and 5 mg/l), in combination with
different concentrations of NAA (0, 0.5, 1.0, 1.5, 2.5
and 4.0 mgL-1) were used as treatments for the
germination of somatic embryos.
Media were kept in the incubation room 25±2°C
with 16 hrs of light provided by fluorescent bulbs
and a light intensity of 16.75 µmolm-²s-¹ for eight
weeks.
Calculate the Callus induction frequency(%) and
Regeneration frequency(%).

41. 3/27/2015 Deptt of Plant Biotechnology 41
Somatic embryogenesis is an efficient plant
regeneration system.
It is potentially useful tool for genetic transformation.
Cross linking between hormone and transcription
factors is likely to play an important part in SE.
But mechanism of plant embryogenesis is unclear
and comphrensive work in future it by studying the
interaction of various factors thereby entire picture of
regulatory mechanism of embryogenesis would be
transparent.

42. 3/27/2015 Deptt of Plant Biotechnology 42
Conclusion
 Indirect Somatic embryogenesis reduces the breeding
cycle.
 Indirect somatic embryogenesis are used in the crop
improvement.
 Indirect somatic embryogenesis are produce virus free
plants.
 Indirect somatic embryogenesis are better than the
Direct somatic embryogenesis.

43. Types of In Vitro Culture
 Culture of intact plants (seed and seedling culture)
 Embryo culture (immature embryo culture)
 Organ culture Callus culture
 Cell suspension culture
 Protoplast culture
 Somatic Embryogenesis
 Micropropagation
 Somaclonal variation

44. Micropropagation
⚫Embryogenesis
 Direct embryogenesis
 Indirect embryogenesis
⚫Organogenesis
 Organogenesis via callus formation
 Direct adventitious organ formation
⚫Microcutting
 Meristem and shoot tip culture
 Bud culture

45.  EXPLANT PREPARATION
EXPLANT : It is defined as a portion of plant body, which has been
taken from the plant to establish a culture
•Explant may be taken from any part of the plant like
root,stem,leaf,or meristematic tissue like cambium, floral parts like
anthers, stamens etc..
•Age of the explant.
• Homozygous plants are preferred.
45

46. we
r
46
leaf

47.  TYPES OF CULTURE
 Callus culture
 Suspension culture
 Root tip culture
 Leaf or leaf primordial culture
 Shoot tip culture
 Complete flower culture
 Anther & pollen culture
 Ovule & embryo culture
 Protoplast culture 31

48. Culture
type

49. Callus culture Suspension
culture
Pollen culture
Ovule culture Root tip culture Shoot tip culture
32
Protoplast culture Leaf primordial culture Flower culture

50. What is Callus development ?
⚫ A callus is a blob of tissue – (mostly undifferentiated cells)
⚫ A callus is naturally developed on a plant as a result of a
wound
⚫ This callus can be left to develop or can be further
divided

51. Callus Culture
⚫Equimolar amounts of auxin and cytokinin stimulate
cell division. Leads to a mass proliferation of an
unorganised mass of cells called a callus.
⚫Requirement for support ensures that scale-up is
limited.
⚫Callus Suspension Culture
⚫When callus pieces are agitated in a liquid medium,
they tend to break up.
⚫Suspensions are much easier to bulk up than callus
since there is no manual transfer or solid support.

52. Protoplast Isolation
⚫Created by degrading the cell wall using enzymes.
⚫Very fragile, can’t pipette.
⚫The membranes are made to fuse.
 osmotic shock, electrical current, virus
⚫Regenerate the hybrid fusion product.
⚫Contain genome from both organisms.
⚫Very, very difficult .

53. Use of enzymes results
in a high yield of
uniform protoplasts
after removal of cellular
debris Protoplasts can
originate from different
sources: greenhouse or
field material,
micropropagated
plants, calli,

54. Protoplast Fusion Techniques
⚫ Protoplast fuse spontaneously during isolation process
mainly due to physical contact.
⚫ Induced Fusion.
⚫ Chemofusion- fusion induced by chemicals.
⚫ Types of fusogens
⯍ PEG
⯍ NaNo3
⯍ Ca 2+ ions
⯍ Polyvinyl alcohal
⚫ Mechanical Fusion- Physical fusion of protoplasts under
microscope by using micromanipulator and perfusion
micropipette.

55. Somatic & Cybridization

56. Somatic & Cybridization

57. Uses for Protoplast Fusion
⚫Combine two complete genomes
 Another way to create allopolyploids
⚫Partial genome transfer
 Exchange single or few traits between species
 May or may not require ionizing radiation
⚫Genetic engineering
 Micro-injection, electroporation, Agrobacterium
⚫Transfer of organelles
 Unique to protoplast fusion
 The transfer of mitochondria and/or chloroplasts between species

58. Somaclonal Variation
 Variation found in somatic cells dividing mitotically in culture
 Ageneral phenomenon of all plant regeneration systems that involve a
callus phase
Some mechanisms:
 Karyotipic alteration
 Sequence variation
 Variation in DNA Methylation
Two general types of Somaclonal Variation:
 Heritable, genetic changes (alter the DNA)
 Stable, but non-heritable changes (alter gene expression, epigenetic)

59. Somaclonal Breeding Procedures
⚫Use plant cultures as starting material
 Idea is to target single cells in multi-cellular culture.
 Usually suspension culture, but callus culture can work (want as much
contact with selective agent as possible).
 Optional: apply physical or chemical mutagen.
⚫Apply selection pressure to culture.
 Target: very high kill rate, you want very few cells to survive, so long as
selection is effective.
⚫Regenerate whole plants from surviving cells.

60. Advantages of somatic hybridization
⚫Production of novel interspecific and intergenic hybrid
 Pomato (Hybrid of potato and tomato).
⚫Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other quality
characters.
⚫Production of heterozygous lines in the single species
which cannot be propagated by vegetative means.
⚫Production of unique hybrids of nucleus and cytoplasm.

61. Plant germplasm preservation
⚫ In situ : Conservation in ‘normal’ habitat
 rain forests, gardens, farms
⚫ Ex Situ :
 Field collection, Botanical gardens
 Seed collections
 In vitro collection: Extension of micropropagation techniques
⯍ Normal growth (short term storage)
⯍ Slow growth (medium term storage)
⯍ Cryopreservation (long term storage
⚫ DNA Banks

62. Cryopreservation
 Storage of living tissues at ultra-low temperatures (-196°C)
 Conservation of plant germplasm
⯍Vegetatively propagated species (root and tubers, ornamental,
fruit trees).
 Conservation of tissue with specific characteristics
⯍ Medicinal and alcohol producing cell lines
⯍ Genetically transformed tissues.
⯍ Transformation/Mutagenesis competent tissues (ECSs).
 Conservation of plant pathogens (fungi, nematodes)

63. Applications:
⚫ Study of Biochemical & Physiological activities.
⚫ The effect of various hormones.
⚫Production of Secondary Metabolites.
⚫To preserve the plant species which are on red-line.
⚫Improve crop yield with regard to molecular
breeding & Genetic Engineering.
⚫To make transgenic & cis-genic plants.

64. Commercial Applications of Clonal Propagation
⚫Clonal propagation has the potential for propagation of thousands of
plantlets from a single genetic stock.
⚫Examples:
 Orchids,
 Potato,
 Asparagus,
 Strawberry, And
 Various flowers or herbaceous ornamentals that set seed poorly.
⚫This may not be suitable for seeding field crops.

65. Problems in Tissue Culture
⚫ Application of protoplast technology requires efficient plant regeneration
system.
⚫ The lack of an efficient selection method for fused product is sometimes a
major problem.
⚫ The end-product after somatic hybridization is often unbalanced.
⚫ Regeneration products after somatic hybridization are often variable.
⚫ It is never certain that a particular characteristic will be expressed.
⚫ Genetic stability.
⚫ Sexual reproduction of somatic hybrids.

66. Conclusion
⚫PTC is the technique by which plant cells can be
grown in vitro sexually & asexually. By the help of this
we can study biochemical, physiological and
hormones activity.
⚫High yield, good quality of crops can be obtained.
⚫PTC , G.E. and Molecular breeding these techniques
are used to transfer the gene of same species or from
different species.

67. References
⚫Plant Tissue Culture, ELESIVISER Publishers
,Bhojwani & Rajdhan
⚫ H.S. Chawla
⚫M. S. Shekhawat
⚫Images from google search engine

68. Thank you 