Description about phytohormones and their role in tissue culture, including descriptions about molecular basis of phytohormones with special focus on auxin and cytokinin and their role in calli development, organogenesis and somatic embryogenesis.

1. Role of Phytohormones in
Tissue Culture
Assignment Presentation for Plant Developmental Biology
(PP503) Course
By - Apoorva Ashu
Roll No. - 70022
MSc 1st Semester, Division of Plant Physiology, ICAR-NIASM
1

2. Introduction
2

3. Plant Development
Plant development is the process by
which structures originate and mature
as a plant grows. Plants produce new
tissues and structures throughout
their life from clusters of
undifferentiated cells called
meristems. These meristems then
give rise to specialized tissues in the
plant.
Stages of development of a plant - From zygote
to mature plant
3

4. Tissue Culture
It is a technique in which fragments
of tissue from an animal or plant are
transferred to an artificial
environment in which they can
continue to survive and function. The
cultured tissue may consist of a
single cell, a population of cells, or a
whole or part of an organ.
Example of tissue culture from plant cell
4

5. An overview of the steps involved in plant tissue culture
(http://ecoursesonline.iasri.res.in/mod/page/view.php?id=1655)
5

6. Different forms of tissue culture techniques
Plant Tissue Culture
Organogenesis/Micropropagation
Callogenesis
Genome Editing
Somatic Hybridization
Shoot Regenesis
Root Regenesis
Genetic
Transformation
6

7. Why the need of Tissue Culture?
7

8. Some advantages of plant tissue culture
1. Production of exact copies of plants that produce particularly good flowers
and fruits.
2. To quickly produce mature plants.
3. Production of multiples of plants in the absence of seeds.
4. The regeneration of whole plants from plant cells that have been genetically
modified.
8

9. 9

10. Few Important Terms
10

11. Signal Transduction
Signal Transduction is the cascade of
events that allow a signal, usually
from outside the cell to be interpreted
by the cell.
It usually results in a final biological
response, and often the response
can be measured.
11

12. Signals in Plants are of
Two Kinds
Along with Exogenous signals like
light and abiotic stress,
endogenous signals like plants
hormones are the major
developmental and physiological
signaling molecules in the plant
that coordinate the growth and
development of plants.
(Taken from Nature: Review of Cell Biology) 12

13. Phytohormones
Phytohormones are defined as
substances produced in some
tissues at certain developmental
stages of a plant, and are then
distributed by the vascular system,
often exerting functions at remote
tissues in very low concentrations.
They play important role in
germination, flowering, abscission,
dormancy, ripening and maturity
Action of Plant Hormones
13

14. Different kind of Phytohormones:
14

15. 1. Auxins - promotes cell growth and differentiation, especially on the tips of
plants. It controls plants' response to light.
2. Cytokinin - promotes cell division and lateral growth in plants. It helps in the
rapid division of seeds and fruits.
3. Gibberellins - helps in breaking dormancy in seeds and buds. It can be used in
the production of seedless fruits.
4. Abscisic acid - promotes dormancy in seeds and buds. It promotes wilting and
falling of leaves.
5. Ethylene - promotes fruit ripening.
Function of Plant Hormones in the Plant Development
15

16. Examples of different
phytohormones with
their representative
structures
(Progress in quantitative analysis of
plant hormones - accessed 16 May,
2023)
16

17. Phytohormones Can Work Together or Independently
Towards the Same Set of Function
17

18. How did We Establish the Importance of
Phytohormones in Plant Tissue Culture?
18

19. A Historical Breakthrough
Plant callus (plural calluses or
calli) is a growing mass of
unorganized plant parenchyma
cells.
It was a historical discovery that
callus formation in plants is
because of different endogenous
plant hormones.
Callus formation in vitro and in nature
(www.plantcell.org/cgi/doi/10.1105/tpc.113.116053)
19

20. Cultured cells grow in
response to
Phytohormones
In In-vitro conditions, cultured
cells are able to produce auxins,
cytokinins, and ethylene and are
able to establish a characteristic
endogenous hormonal system
Nicotiana tabacum parenchyma
cells in culture 20

21. Experiments were done to find out how to use these
phytohormones in tissue culture to grow explants
A major advancement
was the discovery of
liquid endosperms such
as coconut water to
culture media
dramatically increased
the amount and rate of
cell division.
Tobacco callus showing different growth in
Broad Spectrum Tests by de FOSSARD, R.A.,
MYINT, A. and LEE, E.C.M. (1974) 21

22. Studies on Tissue Culture
In tobacco it was shown that stem explants containing bits of vascular cylinder as
well as pith required only auxin as an additive to culture medium to achieve
significant callus growth.
22

23. Studies on Tissue Culture
● When tobacco pith explants without
vascular tissue were tested, they would
not grow well unless complex additives
such as yeast or malt extract, or coconut
water were supplied in addition to auxin.
● This work opened the way to the
discovery of kinetin, the first chemically
identified plant cell division factor. It soon
followed that kinetin in the presence of
IAA or a synthetic substitute could bring
about cell division in tobacco and a
number of other species in a completely
defined medium.
23

24. Studies on Tissue Culture
In the course of investigating the interaction of kinetin and auxin in cell division in
the tobacco pith system, it was learned that manipulating auxin:cytokinin ratios
could affect organogenesis.
24

25. Studies on Tissue Culture
When the level of auxin relative to that of cytokinin is high, roots form; when the
cytokinin relative to that of auxin is high, shoots form. When the ratios are about the
same, a callus mass is produced.
The device of adjusting auxin:cytokinin ratios in an attempt to induce shoots and roots
is now well established in tissue culture.
25

26. Genetic Basis of Phytohormones
26

27. It was found that there are numerous
examples of bacteria and fungi that
synthesize or metabolise phytohormones
27

28. Genes that direct synthesis of auxin and cytokinin are
located within the region of DNA transferred to the plant
during transformation (T-DNA).
Tumour Root Nodules 28

29. It was found that the more fully transformed the plant tissue, the more autotrophic
it was. In other words, crown gall tumors could be maintained on a simple medium
of mineral salts and a carbon source because they were synthesizing their own
growth substances.
29

30. Molecular Mechanism behind
action of Phytohormones in
Tissue Culture
Plant growth regulators and signaling are essential for defining cell fate in vitro in tissue culture.
Thus, uncovering the molecular mechanism that underlies between Phytohormones and cell
regeneration is fundamental to further improve agriculture and horticulture.
30

31. Callus
Formation
Some of the key regulators playing
a role in the spatial expression of
root meristem are WOX5
(WUSCHEL-RELATED
HOMEOBOX 5) and SHR (SHORT
ROOT). In other words, calli
formation in response to PGRs like
auxin rely on root initiation
pathways.
When explants are placed in an auxin-rich,
callus-inducing medium, the developed
calli resembles the root meristem
histologically.
31

32. Callus
Formation When a calli is formed in response
to wound, it promotes
transcriptional activation of genes
encoding the cytokinin biosynthesis
like IPT3 (ISOPENTENYL
TRANSFERASE 3), LOG1, LOG4,
and LOG5 (LONELY GUY 1/4/5)
32

33. De Novo Root
Formation
Polar auxin transport (auxin transport from cell-to-cell in a
directional way, from the root to the shoot and vice
versa) is critical for auxin-mediated promotion of de novo
root formation.
Key auxin transporters, such as
PIN1 (PIN-FORMED 1), PIN2, PIN3
and AUX1 (AUXIN RESISTANT 1),
have been identified by playing
roles in root regeneration from leaf
explants
33

34. Root Meristem
Regeneration
after Wounding
When the farthest part of the meristem is
lost to injury the quiescent center transfers
signals to surrounding cells to promote root
meristem restoration.
These two regulators promote
reconstruction of the meristem:
ERF115 (ETHYLENE RESPONSE
FACTOR 115)
PAT1 (PHYTOCHROME A SIGNAL
TRANSDUCTION 1).
34

35. Shoot
Formation
The mechanism for shoot regeneration relies more on
cytokinins.
The mechanism for shoot
regeneration relies more on
cytokinins.
The cytokinin receptor WOL
(WOODEN LEG) plays a major role
in shoot regeneration
35

36. Somatic
Embryogenesis
Zygotic embryos from Arabidopsis plant
species have been used to understand the
molecular basis for somatic embryogenesis.
These zygotic embryos cultured in auxin-rich
medium produce calli. Subsequently, after the
transfer to auxin-free medium, auxin response
maxima (point within cells where auxin content
is the highest) is established at external regions
within the callus via the gene PIN1, an auxin
transporter.
Genome-wide transcriptome analyses have
revealed important transcriptional regulators
that participate in embryogenesis like LEC1
and LEC2 (LEAFY COTYLEDON 1/2), AGL15
(AGAMOUS-LIKE 15) and BBM (BABY BOOM)
36

37. Conclusion
37

38. ● Humans have a long history of reliance on plants for a supply of food, shelter
and, medicine.
● in vitro plant tissue culture has given us an important alternative, as it assures
independence from geographical conditions by eliminating the need to rely on
conventional methods.
● Phytohormones play a key role in the developmental processes of the plants.
● They are essential in the determination of the fate of cell in vitro.
● Tissue culture techniques can help us grow and improve new plants by
making use of the phytohormones.
38

39. References
● Van Staden, J., C. W. Fennell, and N. J. Taylor. "Plant
stress in vitro: the role of phytohormones." V
International Symposium on In Vitro Culture and
Horticultural Breeding 725. 2004.
● Gaspar, Thomas, et al. "Plant hormones and plant
growth regulators in plant tissue culture." In vitro
Cellular & Developmental Biology-Plant 32 (1996):
272-289.
● Neumann, Karl-Hermann, et al. "Phytohormones and
growth regulators." Plant Cell and Tissue Culture–A
Tool in Biotechnology: Basics and Application (2020):
309-319.
● Phillips, Gregory C., and Martina Garda. "Plant tissue
culture media and practices: an overview." In Vitro
Cellular & Developmental Biology-Plant 55 (2019):
242-257.
● Klee, Harry, and Mark Estelle. "Molecular genetic
approaches to plant hormone biology." Annual review
of plant biology 42.1 (1991): 529-551.
● Tuskan, Gerald A., et al. "Defining the genetic components
of callus formation: A GWAS approach." PLoS One 13.8
(2018): e0202519
● Ikeuchi, Momoko, Keiko Sugimoto, and Akira Iwase. "Plant
callus: mechanisms of induction and repression." The plant
cell 25.9 (2013): 3159-3173.
● Neumann, Karl-Hermann, Ashwani Kumar, and Jafargholi
Imani. Plant cell and tissue culture: a tool in biotechnology.
Vol. 12. Berlin: Springer, 2009.
● Krikorian, Abraham D., Kevin Kelly, and David L. Smith.
"Hormones in tissue culture and micro-propagation." Plant
hormones and their role in plant growth and development
(1987): 593-613.
● De Fossard, R. A., Aung Myint, and EDWARD CM LEE. "A
broad spectrum tissue culture experiment with tobacco
(Nicotiana tabacum) pith tissue callus." Physiologia
Plantarum 31.2 (1974): 125-130.
● An overview of the molecular mechanisms of plant
regeneration by Adriana Gallego, PhD.
.
39

40. Thank You
40