This content explains the anatomy of plant callus, a mass of undifferentiated cells, and the crucial process of subculturing for its maintenance and further development into specialized tissues or whole plants.
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So last class we spoke about the callus
and we told that callus is obtained from
an expplant and expplant I told you it
can be any part or piece of the plant.
So when we provide
the exact nutrition
particularly a growth hormone and
environmental conditions
we can form calluses
and today we shall go a step ahead and
see what is the anatomy of the calluses.
Last week we saw that how we can
initiate last class we saw that how can
we initiate a callus
and today we shall look into the anatomy
of the callus. So we define callus as an undifferentiated
undifferentiated
okay unorganized
mass of parentus tissue they are
amorphous in the form. We told that the
texture of the callus and the
pigmentation of the callus. Look at
this. The color of the callus that is
the pigmentation of the callus or the
texture of the callus all depends on
your expplant. What type of expplant you
have chosen? What is the species of
expplant you have chosen? Based on that
we get this type of different colors and
And also remember we told that callus is
just not formed in the laboratory.
It can be formed by the nature also when
there is an infection from the
microorganism or by some insect or some
mechanical and physical harm caused to
the plant that time also callousing can occur.
occur. So
we shall look into the anatomy of the
callus. So we define a callus as a
homogeneous. See that what we mean to
tell is like we have told that it is an
undifferiated unorganized mass of parent
cells. But when you look at a callus it
is not homogeneous. It is not same.
Okay. So a homogeneous callus consisting
entirely of parenyatic cell is rarely
found. So we can't find the entire
callus truly being having a paranky
cells. There is some sort of
differentiation. Okay. Like I told you
trachal cells are present in the callus.
So that is the meaning of your first
sentence. They cannot be truly parenyatis.
parenyatis.
Now we let us define a word cyto
differentiation which is very important
for callus. So what do you mean by cyto
differentiation means? Cyto means you
know all of you know that it is a cell
and the cell has to differentiate. It
has to undergo morphogenesis into
different types. It has to undergo the
cell division and finally form a whole
plant. This is called as cytoiation.
The morphological development of
undifferentiated cell into more
specialized one is called as the cytoiation.
cytoiation.
So undifferentiated
whose here are undifferentiated it is
our callus.
Okay we told it is an undifferiated mass
of tissue. But what happens this
undifferentiated mass of tissue should undergo
undergo
certain changes so that it can grow into
a specialized cells.
So that is what is our goal or aim is
once we develop a callus in the
laboratory our duty is just not to
maintain the callus as it
okay sometimes you require the callus in
its natural form. So there are other
means but my main idea is like from the
callus if I want to develop a whole plant
plant
and so what should I do I should see
that word becomes important. So from
undifferiated mass of cell it should
undergo differentiation it should
undergo differentiation and it should
form a specialized
cells or organs.
So what happens is like we told that it
is having some amount of vascular
elements. These vascular elements formed
by the meristematic cells. It can be
formed from your meristismatic cells of
your root tip or stem aex and vascular
cambium. Look at this word meristmoids.
Okay. So these callus they'll be having
a small nest of dividing cells. Okay. If
this is my callus they'll be having a
nest of dividing cells somewhere. Okay.
And they form a vascular nodules. Okay.
So malus is having a vascular nodules
which we term it as a meristtomoid. What
is a meristtomoid? A small triangular
stomatal precursor cell that function
Okay. So what happens like from the stem
cell different organs are been formed.
Okay. your hands, legs or whatever your
anatomy of your body is formed.
Similarly in the plant also in the
callus what we call it as a meristemoids
which are nothing but the which
represents the stem cells in the meristem
meristem
and this is a precursor.
Okay, prepare means which is going to initiate
initiate
into the formation of the chute or root
or embryo
or any other part of the plant. So this
becomes very important. So if there is a
meristemmoid it is going to behave as a
stem cells and these stem cells are the
precursor cells.
Okay, their function is only temporary.
And what is their function? It is to
produce shootute epexes, root primordial,
primordial,
vascular cambium. These are the
functions of it. So what is happening
from an undifferiated mass of cell it is
going to develop into all this because
of this meristtomid or because of this
So always remember whenever you come
across this word primordia
primordia
okay it is the earliest stage of
development the starting stage of the
development is known as the primordia.
So this is called as a primordial. From
So how are you going to subculture a
callus? You cannot keep a callus in the
same media and you cannot grow it. You
have to subculture it. So another
important question what do you mean by subculturing?
subculturing?
So once the callus tissue is formed from
the expplant okay the callus is
transferred to the fresh media media
and this is known as subculturing. So
So
I have an expplant from this expplant
what did I develop? I developed a callus.
Okay, I developed a callus.
Now what you have to do this callus?
Okay, it is transferred into a fresh
media. Here it is. It is transferred
So how long you should transfer it to a
fresh media? It takes approximately 3 to
6 weeks.
So remember after the callus has been
grown from the expplant
it should it becomes necessary to
subculture the callus into the fresh media
media
while still it is in association with
the original tissue. Who is your
original tissue here? My original tissue
here is the expplant. So this is the
meaning of subculturing.
So you grow that expplant develop it
into a callus and you're growing that in
the nutrient media containing the growth regulators.
regulators.
After a period of 3 weeks or 6 weeks,
what you're doing is you're transferring
this callus into your fresh media. And
this fresh media know it will have all
constituents same as the previous media
only that it is newly prepared, freshly
prepared. Why are we doing this? Let us
see that.
So when you're growing your callus in
the same media that is your first media
in which you have put your callus what
happens is there is a depletion of the
essential nutrients. So when the callus
is growing in the media what happens it
uses up all the nutrients in your media.
So what happens the nutrients comes down
there is decrease in the nutrients. Why?
Because the callus has used up the
nutrients and also what happens we can
grow callus in the solidifying agents.
What are your solidifying agents? They
are your jelling agents. What is my
jelling agent? It can be agar. It can be
your agurus. It can be your gelatin.
Particularly we are going to use the
agar. So what is a jelling agent? Means
it is an solidifying agent. It just
solidifies your media and nothing else
function it has. It is just an inert
material. Okay. So what happens?
Desiccation of your jelling agent
happen. Desiccation means drying. Okay.
See if this is your co
conical flask.
Okay. If this is your conical flask and
this is my media and here is my callus.
What happens? You know it starts
desiccating. it becomes dry and it will
be only this much no more to the walls
your media is present.
Okay. So what happens that is why we go
for the fresh media. So growth on the
same medium for a extended period will
cause depletion of essential nutrients
and gradual desiccation of your jelling
agent. Therefore, it is necessary for me
to transfer my callus along with its
associated original tissue into the
fresh media. Apart from these two
problem, I have another problem. What is
that? The metabolite secreted by the
growing callus may accumulate to toxic
level in the media. So, we told that
naturally every plant will produce a
metabolite. This is a separate chapter.
you'll be studying in detail the
metabolites. Okay. So I told you I took
an example of apple and I told you like
um it turns brown in color. Why it turns
brown in color? Because there is polyphenolic
polyphenolic
acids which are present there. Phenolic
compounds are present there. Flavonoids
may be present there. Tarpen can be present.
present.
So these are the metabolites which are
secreted by your plant.
But what happens if they are over
secreted in that same media?
profusely. What is happening? The
metabolites are being produced by the
plant and you are not removing that
metabolites. So what happens? base
metabolites will accumulate into your
media and it reaches a level such a way
that it becomes toxic to your own
callus. That is why I go for a fresh
media. Now when I'm telling that I am I
will take my callus put it in the fresh
media so that it grows again well.
So you cannot do it as your wish.
There is a particular protocol or
procedure for transferring. Okay, of
course you do all aseptic methods. That
is right. But what is another important
point you have to remember is the
transfer fragment of callus must be of
It should have a definite size so that
it can again it can grow in the fresh
media. I cannot take a large catalyst
and put it into the media or take a
small piece of catalyst and put it into
the media.
Both can have a negative effect. Okay.
So the sufficient size becomes
important. So you can see here if I'm
transferring a small inoculum of my
callus into the media into the fresh media
media
my callus may not grow otherwise it'll
grow very slowly.
So which is not advisable. Therefore a
scientist named has treat recommended
that the inoculum inoculum here means
your callus be at least 5 to 10 mm in
diameter and weigh 20 to 100 mg. So your
callus should be at least 5 to 10 mm in
diameter and its weight should be
somewhere around 20 to 100 mg so that
you can transfer that callus from your
previous from this okay to your fresh
media. So this is what is known as
subculturing of the callus. So once the
callus tissue is formed from your
expplant the callus is transferred to
your fresh media
along with your original tissue. This
process is known as subculturing. Why
should I go for subculturing means?
Because my media
there will be depletion of the essential
nutrients because my callus is taking up
all the nutrients. Otherwise there can
be desiccation or drying of the jelling
agent or my agar. Otherwise the
metabolites produced by my callus will
accumulate in the media and it'll reach
to a toxic level that my callus will not
be able to grow. That is why I transfer
my callus into the fresh media. When I
am transferring my callus to the fresh
media I should take certain precaution.
I should follow all the aseptic
condition sterilized condition and the
most important is the size of my callus.
The size of my callus should be 10 mm in
diameter and the weight should be
somewhere around 20 to 100 mg
otherwise my callus will not be able to
So when are you going to perform your
subculturing? That is your next
question. I have grown by callus. Now
when should I do the subculturing? Throw
it in in a fresh media. Successive
subcultures are usually performed four
to 6 weeks with the culture maintained
in an agar media at 25° centigrade or above.
above.
So you do your subculturing
for every four to two 6 weeks and see to
that you maintain your callus in the
agar media and you provide your
temperature at least 25° centigrades or
above or usually 25° centigrade plus or
minus 2 and you see to that depending on
the species what type of photo period I
should give should I give 16 hours or
should I keep it in the light or should
I keep it in completely dark? That all
Now comes this word fryable callus.
Okay. So we are more interested the
scientists are particularly more
interested in a callus which is soft
which is fryable. What is it? We shall
look into it. A fryable callus can be
subdivided with a thin spatula or
scalpel. So whenever I want to do
further experiment with my callus,
forget about developing a new expplant.
Suppose say I want to produce a single
cell. You studied that experiment. Okay. wherein
wherein
we kept a callus above that we kept a
filter paper and we kept a small cell
and we saw that the small cell was able
to grow into a new callus.
Okay. So whenever we want to grow a
single cell
we usually use a fryable callus. Why we
use a fryable callus means because this
callus can fall apart easily and can be
used to generate suspension culture. So
you can disassociate easily. For
example, this is my callus. I can just
using a thin spatula or a scalpel. So it
Okay. So I can get small pieces of it. I
can get a small I can get a single cell
out of this fryable callus. It's easy
for me to disassociate it. So it can
fall apart easily. So how I'm going to
cut my callus by either using the
spatula or I can use the scarpel. Put it
in a sterile petry dish and then you can
use your scarpel to cut it into small pieces.
pieces.
Remember whenever you are using callus
for any type of future experiment you
and brown color or necrotic tissue must
be discarded. The problem of tissue
culture. You studied the brown color. We
saw that how the plant was expplant was
developing the brown color. The phenolic
compound your metabolites it was
releasing into the media and ultimately
killing the plant itself. So whenever it
is brown in color please discard it.
Whenever it is a necrotic tissue,
remember that the die tip example we
spoke about in problems in tissue
culture. Necrotic tissue means that is
they have lost their ability to grow.
They are dying. Okay. Such type of
necrotic tissue also must be discarded.
These are very important precautions we
should take it in the both plant and
animal tissue culture. When you don't
find that the tissue is healthy, when
you find that they contaminated, when
you find they are weak, please
immediately discarded because you cannot
proceed with the experiment because
these are very lengthy experiments and
finally you cannot tell that nothing has
grown. Okay. So these precautions must
and should be taken care of. So now
there are many methods okay wherein you
can develop an alternate methods for
long-term maintenance of the culture.
So if I want to maintain it for a year
more than a year what can I do? I can
freeze them. Okay there are techniques
like triop preservation freeze preservation.
preservation.
So I can store them for a long period of
time. So in this slide what we spoke was
we told that we can go for subculturing
for every four to six weeks and we
should maintain a proper temperature of
25° centigrade plus or minus2 and
particularly if I want to carry out
suspension culture next you will shall
speak that is a separate chapter
suspension culture
that is I'm growing single culture dex
or single cell then what is important is
it is important callus should be fryable
that is it should easily fall apart or
easily it should be disassociated
and I always should choose an healthy
plant for doing any type of transfer
experiment if I want a longtime storage
of my expplant or whatever tissue
culture plants or even callus I can
always go for freeze preservation
depending on what type of expplant
see this is the
a very good example of your uh callus
how they are growing it look at this
this is your expplant okay I'll simply
explain to you all this part it is quite
interesting freshly cut pieces from
surface sterilized plant or any organ
okay they are placed in the newut
nutrient media. This is your nutrient
media. It has agar. Okay. So here it is
the leaf plant we sorry the leaf which
we have used. So it is placed on the
nutrient media consisting of suitable
proportion of oxen cytoinance to
initiate culture so that it can develop
into a new plant.
So the expplants on such media exhibit
call ink. See I have cut the plant under
all aseptic condition. I have placed it
into my solidified media. I have added
my cytoinine and oxin. And then you see
my expplant turning into a callus. Okay.
Okay.
So you can see here from where the
callus has been developing. Clearly
observe this. This expplant on such a
media exhibit callusing at the cut ends
which gradually extends to the entire
surface of the tissue.
The callus is separated from the
expplant and transferred to the fresh
media of the same composition to enable
to build up the mass of tissue.
So repeated subculture on agar media
improves the fryability of the callus.
That is what we were speaking. This is a
small picture which shows that.
Okay. So I have cut the eggplant for
example here it is a leaf. I have
provided the nutrients. I'm growing it
in the agaria.
I have provided the growth regulators
oxen and cytokinine. And you can see at
the cut edges of my expplant it has
started to callus and the entire callus
can be formed and then I can subculture
it in the same constituent of the media.
Whatever the first constituent I have
used freshly prepared media also should
have the same constituents.
Okay. When I'm doing such type of
subculturing, I will get the priable
cultures which can be easily disassociated.
So this is an example of suspension
culture. We'll not go into it for now.
And this is simply showing an plant le
okay like how small plantlet can be used
as an expplant for growing into the callus.
So let us look into the
why are we doing the scalus culture?
What is its importance? We shall look
So the first one is it helps in the
production of secondary metabolites. So
I told you the secondary metabolites you
have primary and secondary metabolites.
Okay, do not worry about it now but just
remember that your plants produce
secondary metabolites which are
economically useful for mankind.
Okay, I told you the medicinal plants,
the flavvenoids, the turppins, um the
saponins, the alkoids, phenoids,
flavorons, flavoroid, whatever name them
Okay. So these are very important. They
are secreted by the plant and it is
important for us. So we will what we do
is we will try to extract all the
secondary metabolites. So it helps us in
the production of the secondary. When my
plant is producing secondary
metabolites, what will I do is I will
extract all the secondary metabolites
for my use for the use of mankind. For
that we use the cal. Second is it is
useful for synthesizing starting
compounds that are subsequently modified
to yield a desided product. That is I
have taken the expplant I have produced
a callus. Okay. from the callus then
what I can do whatever the decided if I
want I can develop a shootute I can
develop only the root or I can develop
an whole plant so by that callus I can
modify it to yield a desided product I
can genetically modify my callus I can
induce a gene into it a new gene which
was not present so I can get a decided
product out of it so that is the meaning
of your second
application starting compost that can be
subsequently modified to yield a desired
product. It is a starting material for
your vegetative propagation. That is if
you are not if the plant is not
undergoing any sexual, we do not want
the sexual propagation. We can always
produce a vegetative propagation by
using the callus culture. One one of the
methods for producing vegetative propagation.
propagation.
Callus is the starting material of your
suspension culture.
So what is the sub suspension culture
means? Just remember for time being it
is to obtain a single cell. Remember the
nerve cell experiment. Okay. To obtain a
single cell we use the scalance. Okay.
You have obtained the single cells. So
what are you going to do it? I can do
many things about it. When we come to
the chapter we will study. But one
another method by using suspension
culture I can study the morphology.
I can study the biochemical changes.
I can study the metabolic pathway.
How the cellular metabolites is
occurring in my plant. I can study all
this by using suspension culture.
So by using callus I can start off with
the suspension culture. Suspension
culture consist of the priable callus
that is single cell callus. By using the
single cell callus I can study the
morphology of my plant. I can study the
physiology of my plant. I can see what
are the biochemical reaction is
happening. How a root is growing. How a
shoot is developing. What are the
cellular metabolites are present in that
plant. This all I can study by using the
One more point see
students that whenever we are
multiplying a callus okay like when we
I told you that we should follow subculturing
subculturing
but sometime most of the time what it
can happen is your callus loses the morphogenesis
morphogenesis
particularly if your callus is too old
okay I told you this is very important
this part
should have be a healthy tissue.
So it can be multiplied indefinitely.
Your callus can keep on be multiplying.
You can maintain it in the callus form
throughout when you're doing
subculturing. But most of the time what
particularly if your callus is too old
or it depends on the type of species you
have collect you forming the callus.
Okay. So callus varies from species to
species that is from expplant to
expplant from which expplant you have taken.
taken.
If I taken from a expplant I cannot
expect even my bpl plant should produce
the same type of callus
and it is also healthy. No it cannot
happen. So species to species also it varies.
please remember this terminology for the examination.
examination.
What do you mean by organogenesis?
See a callous tissue is a good source
from which the whole plant can be
regenerated by changing the nutrient and
the hormonal constituents in the culture media.
media.
This kind of plant regeneration is known
as organogenesis or it is also known as embryogenesis
embryogenesis
where the cells will form an entirely
new plant.
So a callous tissue from this callous
tissue I'm regenerating a whole plant.
How? By providing the nutrient by
changing the hormonal constituents
and this process wherein your
undifferiated unorganized mass of your
callus when you're supplying it with proper
proper
growth hormones it can develop into an
organ is known as the organogenesis or
it is known as the embryogenesis. So
organogenesis means the development of
the adventicious organ of primordial.
Primordial I told you is a developmental
stage. A small triangular shaped cell
which can develop into a shoot root or
whatever. Okay. From undifferiated cell
mass okay this undifferiated means from
the callus
to a tissue by a process of
differentiation. So an undifferiated
mass of tissue which is able to
differentiate and differentiate and
develop into an organ is known as organogenesis.
Another terminology is calioenesis.
Calioenesis is the type of organogenesis
by which only adventicious shootute bud
initiation takes place. Only you're
developing the chute from where? from
the callus. How? By adding your nutrient cytokinine.
cytokinine.
What is risogenesis? Is a type of
organogenesis by which only advantageous
root formation takes place in the callus.
callus.
So you know what is organogenesis?
You know what is calogenesis? That is
shoot butt formation. When I form the
root formation means it is risoenesis.
What is meristimbo means? Meristtomids I
told it is a a triangular shaped nodules
which are present. Okay. These
meristtomid is a localized group of
meristematic cells or the stem cells
that arise in the callus tissue and may
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