BSc (BT) Sem-V: BT-2 (Concepts of Plant Tissue Culture)

The concept of totipotency

Many somatic plant cells, including some fully differentiated types (e.g. leaf mesophyll),
provided they contain intact nuclear, plastid and mitochondrial genomes, have the capacity to
regenerate into whole plants. This phenomenon is totipotency is the inherent potential of living
nucleated somatic cell to develop into a complete organism. Totipotency is an amazing
developmental plasticity that sets plant cells apart from most of their animal counterparts. The
concept of totipotency was first given by Haberlandt and was first demonstrated by Steward and
Reinert in the 1950s. Totipotency conveys the idea that many mature plant cells are not
terminally differentiated but rather retain developmental plasticity. Except for certain types of
terminally differentiated cells (e.g., tracheary elements, sieve-tube cells, and highly lignified
cells such as mature fibers and sclereids) plant cells are capable under certain conditions to
dedifferentiate, re-enter the cell cycle, proliferate and regenerate tissues, organs and entire fertile
plants.

Often totipotency is revealed when cells or tissues are disturbed or removed from their normal
environment and, for example, placed onto artificial media in tissue culture. A differentiated
plant cell that is selectively expressing its genetic information can instead initiate expression of
the program required for generation of an entire new plant. Many plants have been regenerated
from single cells, but not all plant cells are totipotent; some are terminally differentiated, often
because of partial or complete genome loss. We can generalize by saying that most plants at
most stages of the life cycle have some populations of cells that are totipotent. Totipotency is of
course also a property of normal undifferentiated cells, for example in meristems.

The first step in expression of regenerative totipotency is for mature cells to re-enter the cell
cycle and resume cell division — a process known as dedifferentiation. This may lead directly to
organized development, such as occurs in the epidermal cells of immature hypocotyls of
Trifolium where somatic embryos develop (direct embryogenesis), or formation of shoots or
roots (direct organogenesis). Alternatively, there may be an intervening callus stage from which
organised structures can later be induced to develop — referred to as indirect organogenesis.

Expression of totipotency depends on competence, by which we mean the ability of cells to be

induced along a particular developmental pathway, and determination, in which cells become
irreversibly committed to a particular pathway. Convolvulus explants display an initial
competence to follow two possible developmental pathways — root or shoot formation. Later,
once induction of, say, shoots has begun, cells become determined and transfer to conditions that
normally induce root formation are now ineffective. However, formation of callus does not
necessarily guarantee subsequent organogenesis or that the direction of organogenesis can be
controlled. Commitment of root primordia in cereal callus cultures often seems to be irreversible
and a high proportion of cells become terminally differentiated root cap cells that secrete the
mucus normally associated with caps of intact roots. Calluses which do not lead to regeneration
are a common occurrence.

One intriguing question is whether expression of totipotency is a phenomenon of a single cell or

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normally results from the collective interaction of a cluster of cells. We might expect somatic
embryogenesis to be an ideal experimental system because normal zygotic embryogenesis
always starts from a single cell, the fertilised egg. Perhaps surprisingly, groups of hypocotyl cells
from very young embryos may contribute collectively to formation of an embryo ‘bud’; on the
other hand, single epi-dermal cells from more mature tissues can divide to produce an embryo.

The ability to undergo direct organogenesis may be linked to developmental age of explant
tissue, with cells progressively losing this potential as they mature. Fully mature cells, if they
retain any capacity for dedifferentiation, tend to exhibit totipotency via indirect organogenesis.
Loss of totipotency is probably due to genetic (physical changes to chromosomes, for example
loss of DNA, nucleotide substitution, endopolyploidy) or epigenetic (changes in gene expression
as a consequence of development, for example DNA methylation) blocks.

Differentiation

Most cells start their lives as small, non-vacuolate entities. Usually, after several rounds of cell
division and a phase of cell growth, they mature into one of the many plant cell types. This last
phase of development is known as cell differentiation.

Differentiation is often referred to as the sum of developmental processes whereby apparently

unspecialized cells acquire specific structure and function or as a process by which cells acquire
or possess a character or function different from that of the original cell type. At the gene
expression level differentiation refers to the acquisition or possession of a specific pattern of
gene expression (an interplay between transcribed and untranscribed genes), which is different
from that of the previous (often primordial) cell type that bring about specific form and function
of the cell.

In intact plants, simultaneous differentiation of several types is the norm, for example the
epidermis, stomata, mesophyll and vascular cells that, in combination, become a functional leaf.
Generation of organs is obviously an intricate process requiring precise control of positioning of
cell types and directions of growth. Complexity, however, makes the regulatory processes very
hard to study. Instead, much has been learned about differentiation from cells and tissues grown
in isolation, usually in axenic cultures. Many plant tissues and organs are suitable sources of
explants (meaning any small excised piece of tissue). An advantage of tissue culture is the ability
to define almost every component of the cells’ physical and chemical environment. Successful
tissue cultures can be grown almost indefinitely or can be manipulated to ensure regeneration of
large numbers of genetically identical intact plants or clones. Many of the processes exploited
are related to natural wound responses and vegetative propagation. In addition, specialised tissue
culture conditions allow living cells to survive in the absence of a cell wall, as naked protoplasts,
or whole plants to be generated with haploid instead of diploid genomes.

Types of differentiation

Differentiation implies development of organized structures, usually from undifferentiated tissue

but also from previously specialized cells that would not normally give rise to organized
multicellular growth (e.g. epidermal cells, pollen grains). In plant tissue culture, undifferentiated

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tissue is referred to as callus although a callus can contain meristematic nodules that may not be
obvious to the naked eye but which never develop further unless suitable conditions are supplied.

Development of organised structures can follow one of three pathways:

1. shoot regeneration, based on a unipolar structure with a shoot apical meristem

2. root regeneration, essentially a unipolar structure with a root apical meristem
3. somatic embryogenesis in which there is a bipolar structure.

Repeated division of immature post-meiotic pollen grains (microspores) leads to production of

haploid plants, and is another type of embryogenesis. The phenomenon was first discovered in
1966 by Guha and Maheshwari when studying meiosis in vitro in Datura innoxia anthers.
Sometimes differentiation takes place in the absence of cell division: in tissue culture systems,
such as Zinnia elegans, we commonly find xylem elements appearing among otherwise
undifferentiated cells. This form of xylogenesis represents the acquisition of a specific metabolic
competence that is quite different from that of the parental cell. Clearly, differentiation in vitro
can take several forms.

a. Indirect organogenesis: plant growth regulators and differentiation

An understanding of the mechanisms underlying regeneration of whole plants, or parts of plants,

from cells has come some way since the classic observations of Skoog and Miller that the
direction of differentiation could be influenced by the ratio of the exogenously supplied growth
regulators auxin and cytokinin. They observed in tobacco stem pith cultures that a high ratio of
auxin to cytokinin led to initiation of roots whereas a low ratio led to development of shoots.
Although there are many species for which this simple manipulation will not work, in general
auxins (e.g. IAA (indoleacetic acid), NAA (a-naphthaleneacetic acid) and IBA indolebutyric
acid)) will stimulate regeneration of roots, and cytokinins (e.g. BAP (6-benzylaminopurine) and
kinetin) will promote regeneration of shoots or embryos.

It is now obvious that the two groups of growth regulators play an important role in unlocking
totipotent expression. Dedifferentiation and callus formation occur naturally in response to
wounding. Indeed, wound responses involve auxin and cytokinins and seem to be the biological
trigger for plant regeneration from somatic cells. However, sustained callus growth in vitro
requires addition of one or more growth regulators. Prior to the chemical characterisation of IAA
in 1934, attempts to obtain long-term callus cultures failed. With very few exceptions, auxin is
essential for dedifferentiation and commonly 2,4-dichlorophenoxyacetic acid (2,4-D) is used to
promote callus; cytokinin often enhances this process. In tissues with a high endogenous level of
auxin, culture of explants on a medium containing cytokinin as the only growth regulator may
lead to development of shoots with very little callus. Not all living cells respond to auxin and this
is particularly true of mature cells of grasses. Without dedifferentiation, it is not possible to move
to the next stage of totipotent expression — plant regeneration. Until the late 1980s, grasses,
especially the economically important cereals, were regarded as recalcitrant in vitro. Since that
time, researchers have developed methods of regeneration for most of these species. This
includes work on sugar cane, sorghum, wheat and barley in several Australian laboratories.

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The first example of a single isolated cell dividing directly to produce an embryo was recorded
in 1970 in a suspension culture of Daucus carota. However, even in this carrot system, the
phenomenon is rare and normally somatic embryogenesis is indirect via an initial callus phase.

Protoplasts capable of undergoing cell division seldom give rise directly to organised structures
but ?rst synthesise a new cell wall, then produce a callus from which shoots or embryos can later
regenerate. Although auxin stimulates initial cell division in quiescent cells, continued presence
of auxin can inhibit organised out-growth. This is a typical example of the sequential functions
of a single hormone through a developmental progression. In practical terms, cultures are usually
transferred onto low or zero auxin media to permit or speed up shoot organogenesis. Sometimes
‘removal’ of auxin occurs when auxin in the medium is degraded either by the tissue itself or via
chemical reactions such as photooxidation. Cytokinins promote out-growth of shoots but are
normally kept at very low concentration when root regeneration is wanted.

b. Direct organogenesis: the role of growth regulators

Direct organogenesis bypasses the need for a callus phase. A good example is the formation of
somatic embryos. Most evidence suggests that direct embryogenesis proceeds from cells which
were already embryogenically competent while they were part of the original, differentiated
tissue. These preembryogenic cells appear only to require favourable conditions (such as
wounding or application of exogenous growth regulators) to allow release into cell division and
expression of embryogenesis. Such cells tend to be much more responsive than those involved in
indirect organogenesis and do not seem to require the same auxin ‘push’ to initiate division;
indeed, the cells may never have left the cell cycle and growth regulator application has some
more subtle role. In Trifolium repens hypocotyl epidermis, we see that BAP (a cytokinin)
promotes reorientation of the plane of cell division, leading to initiation of a promeristemoid. An
analogous response occurs in cotyledon explants of Abies amabilis where subepidermal cells
develop into shoots. In haploid embryos developed from Brassica napus anther cultures,
cytokinin actually suppresses secondary embryoid formation and instead promotes normal leafy
shoots. This suggests a role for cytokinin in switching between shoot development and
embryogenesis.

Dedifferentiation

Cellular dedifferentiation is a process in which differentiated somatic cells revert to meristematic

state and acquire the capacity of cell division. This process occurs in many multicellular
organisms after wounding. Plant callus cells, generated at the wound site, can continue to
proliferate or redifferentiate into roots, shoots, and even embryos in appropriate culture
conditions. The term dedifferentiation was initially coined to describe the reversal of cells from a
given differentiated state into a more primordial state (“an indifferent embryonic cell type”) as
deduced from changes in cell shape and morphology. Apparently, dedifferentiation and re-entry
into the cell cycle are two distinct processes and it is suggested that dedifferentiation represents a
transient phase conferring competence to switch fate and thus preceding not only re-entry to the
cell cycle but also re-differentiation/trans-differentiation and even a commitment for cell death
(See Figure 1).

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Figure 1. The characteristic features of cellular dedifferentiation. Somatic cells (such as
parenchyma, collenchyma, or mesophyll cells) can be reprogrammed following exposure to
various internal or external signals resulting in dedifferentiation and acquisition of a transient,
stem cell-like state. This transient state is accompanied by global chromatin decondensation – a
hallmark of stem cells. Depending on the type of stimulus, dedifferentiated cells can be induced
to trans-differentiate/re-differentiate, re-enter the cell cycle or undergo a programmed cell death.

Re-differentiation

The term re-differentiation is often understood as “a process by which a group of once

differentiated cells return to their original specialized form.” However, in plants, the term re-
differentiation is commonly used not in the sense of returning to a previous differentiated state
but rather to express the idea that differentiated plant cells do not lose their developmental
capacity and are capable of repeated cycles of differentiation (re-differentiation). Thus when
parenchyma cells are converted into tracheary elements (terminal differentiation) or into
meristematic cells, in both cases, cells are said to have undergone re-differentiation although
they have acquired completely different fates and certain cells may lose their capacity for
repeated cycles of differentiation (e.g., tracheary elements).

Tissue Competence

Competence means the ability of cells to be induced along a particular developmental pathway.
The ability to regenerate is widely exploited by multitude of organisms ranging from unicellular
bacteria to multicellular plants for their propagation and repair. But the levels of competence for
regeneration vary from species to species. A variety of living cells of a plant display regeneration
ability.This highly pliable nature of plant cells in-terms of regeneration can be attributed to their
high developmental plasticity. De novo organ initiation can be relatively easily achieved in
plants by proper hormonal regulations. Elevated levels of plant hormone auxin induces the
formation of proliferating mass of pluripotent cells called callus, which predominantly express
lateral root meristem markers and hence is having an identity similar to lateral root primordia.
Organ formation can be induced from the callus by modulating the ratio of hormones. An

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alternative for de novo organogenesis is by the forced expression of plant specific transcription
factors.

Acquisition of embryogenic competence largely relies on dedifferentiation, a process whereby

existing transcriptional and translational profiles are erased or altered in order to allow cells
to set a new develop- mental program. The activation of cell division is required to maintain
the dedifferentiated cell fate, as well as for embryo differentiation. This is not only true for
those embryogenic systems where embryogenic callus formation precedes somatic embryo
development (‘indirect somatic embryogenesis’), but also for those where somatic embryos
develop on primary explants without an intervening callus formation (‘direct embryogenesis’).
It has been reported that there are many cytological parameters serve as early indicators of cell
competence to undergo somatic embryogenesis and/or organogenesis. Water potential of the
culture medium declines before the onset of embryogenesis while at the same time the
intracellular osmolarity and cell surface increases in embryogenic cells. In addition, cellulose
accumulates in the walls of non-embryogenic cells while cell walls became thinner with onset of
embryogenesis, and diminishes further as embryos mature.

More than 50 years since Skoog and Miller (1957) described the hormonal control of organ
regeneration in plants, there is still little insight on the primary determinants of the capacity of
undifferentiated cells to regenerate shoots, and little is understood about the mechanisms through
which the auxin/cytokinin balance exerts its effects. It is generally accepted that organogenesis is
composed of three sequential phases: acquisition of ability to recognize hormonal or other
signals by cells that commit them to a particular developmental program, such signals induce a
process where competent cells alter their developmental fate, and finally differentiation to form
determinate organs, during which the inductive signals are no longer required. Although
experimental procedures for in vitro organogenesis vary among species, the first phase is
generally initiated by culturing on an auxin-rich callus-inducing medium (CIM), then explants
are cultured on a shoot-inducing medium (SIM) or root-inducing medium (RIM) that contains a
specific auxin/cytokinin ratio. The genetic determinism for the differences in regeneration
competence is still poorly understood though, and a number of genes have been identified that
positively influence the competence of plant cells for somatic and/or adventitious shoots
formation.