Plant tissue culture media are carefully formulated nutrient solutions providing essential inorganic and organic components, including vitamins, amino acids, and growth regulators, to support plant cell, tissue, or organ survival and growth in vitro. The specific composition of this media is crucial and varies depending on the plant species and the desired outcome, often requiring empirical determination by researchers.
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So how do we define media?
Media means it is the nutrient
containing various composition of
ingredients for the survival of your
plant, cell, tissue or organ when they
are cultured in in vitro. So we are
providing all the nutritional
requirements for that particular plant
so that it can grow under the invitro
condition. In vitro means as I told you
in the laboratory.
But you see that all plants will not
grow at the same time. Okay. Like I mean
or the same type of media what you are
providing. Some of them will grow in
solid media. Some prefer liquid media.
So there is lot of variations in which
the plant grows. Some tissues grow in
simple media containing only inorganic
salts and carbon. But then some other
tissues require a complex media. So it
is up to us or up to the researchers to
find out what type of media is required
for my plant media in the sense what
type of nutrition I have to provide.
Should I provide a solid media for it to
grow or should I provide it a semi-olid
media or should I provide liquid media
or should I provide a complex media to
them or simple media. So this should be
done by the researchers who are doing
the plant tissue culture by a trial and
error experiment.
So the nutritional composition promoting
optimal growth
of tissue under laboratory condition
vary with the respect to the particular
species. With particular species the
your nutritional components vary when
you're growing them in the laboratory.
Therefore your media composition should
be formulated
considering the requirement of that
particular species and we have to
provide it with the media to that plant.
So what are the meds
which is required? For example, like
when we were talking about the history
of plant tissue culture, we came across
a word called wites media.
So this wes media is particularly I told
you it is used for
growing your root culture. Similarly I
told MS media. What is the function of
MS media? Your MS media I told you it is
a hallmark media even today it is uso.
So it induces organogenesis and
regeneration of plant. Organogenesis
means the formation of organ because we
are using simple cells simple tissues
and sometimes even the organs and we
want or your callus you're using we
wanted to develop all this organs
develop the cell division it should
undergo so probably we use a MS media.
Similarly for example we have this B5
media. Okay. So this is another example
like B5 media which is particularly used
for suspension culture. So there are
many types of medias which are
available. Okay. Of which MS media is
important for you all from their point
So now we shall look into the functions
of the media. What all is required for
the preparation of the media or the
ingredients what you require? First we
require the inorganic nutrients. Okay,
your inorganic nutrients can be macro
and micronutrients. We shall explain
this all in the next slides. We have to
provide water if it is essential.
Minerals is required. We should provide
vitamins and amino acids. The most
important we have to provide growth regulators.
regulators.
We saw that habberlant has missed the
growth regulator to add growth regulator
in his differentiated cells and we saw
that it not divide. So growth regulators
means it is the hormones growth
promoting hormones for the plant that is
required and sometime gases exchange is
also required that is when you're
preparing this suspension culture we
shall focus on the gaseous exchange
there and you should also have a
provision to remove the plant metabolic
waste that is plant also do produce some
waste when they growing in vitro under
laboratory condition you have to remove
that waste if If you're not removing
that waste, it can kill your culture.
Okay? So that should be removed. Now
each of this we will study taking into
So according to this international
association of plant physiology
okay what they have told is
for a macro nutrient
the unit is mill mole per liter
that is you should use mill per liter of
whatever substance whatever ingredients
you're using for macronutrients
and for organic nutrients
And for uh sorry for micronutrients and
hormones and vitamins you calculate
under micro mole per liter. So macro
means you know it is more. So that is
what it is mill per liter and
micronutrients hormones and vitamins are
required in small amount that is why we
weigh it in under micro mole per liter.
We do the calculation based on that.
So the elements required in the concentration
concentration
if the if it is a concentration more
than 0.5 m per liter then it is called
as macronutrient
and if it is less okay then 0.05 05 mm
mole per liter it is a micro nutrient
otherwise you can convert this into this
mill mole per liter you can convert it
into micro mole also
so this is the concentration required or
the unit in which whatever you're
weighing is represented okay for example
you weigh it in grams per liter mig per liter
liter
but here we use this term minology mill
Now first coming to the inorganic
nutrients. So what did I tell your
inorganic nutrient? It consists of macro
and micro nutrients. So macronutrients
means immediately what crashes to your
mind? It is required in larger quantity.
So what are my uh inorganic nutrients are?
are?
It can be your nitrogen
okay or it can be your phosphorus,
potassium, calcium, magnesium and
sulfur. These forms a macro nutrient.
Now all this are essential to your plant.
plant.
Suppose say
uh like uh see if if my plant require
nitrogen I cannot add the nitrogen gas
into my plant.
Okay, I should use and substitute for
it. For example, I can use ammonium
nitrate. Okay, I'll write it in. I can
use ammonium nitrate
or even I can use uh what potassium
uh dihydrogen phosphate.
Okay. So these all I can use it. So what
is happening? I'm providing a mixture of
salt to my plant. So when I'm adding
ammonia nitrate and I put it into water,
what will happen? It gets disassociated.
There is disassociation and ionization.
So what happens is for your plant it'll
become or for your culture it become
easy to observe. So when it
disassociates into ammonia and nitrogen
what happens? It can easily get the
nitrogen from here. It can get easily
your potassium from here. So like this
we use a um chemicals or mixture of
chemicals from which your plant or your
So why is this nitrogen
very essential? See nitrogen what it
does? It influences your plant growth
rate. It is a part of your DNA, part of
your protein chlorophyll. Okay? It is
present in all the amino acids and
hormones. Similarly, you see your
phosphorus. It is present abundantly in
your meristematics tissue.
Meismatic tissues are what? They are the
fastest growing tissue and also the
phosphorus is essential for the
photosynthesis and respiration. When we
come to potassium, you can see that
potassium is essential for the cell
division of our culture.
Also, it is required for a meristematic
tissue. And you can see that it helps in
many of the pathways like your protein,
carbohydrate and chlorophylls.
Similarly, you can see your calcium.
Okay, it is involved in the cell wall
and root and leaf formation. And also it
is involved in other functions like
transllocation of your sugars, amino
acid and oxylo acidic acid. Magnesium is
another macronutrient which is required
for the plant. It is again involved in
the photosynthetic
and the respiration system and also it
is involved in the transllocation of the
phosphate and starches wherever it is
required. And then lastly the sulfur.
You know that the sulfur is present in
the amino acids. It is a part of amino
acids and
enzymes and also it helps in the
formation of nodules. So these are okay
your calcium, magnesium, sulfur,
phosphorus, potassium, nitrogen. These
are all macro nutrient which comes under
inorganic compound which are must and
should you should provide to the plant
for their survival. because you have
seen that these are required for various
metabolic activity and to perform
So the most important of macronutrients
like when we say we tell it is nitrogen.
Okay. So nitrogen you can usually add it
in the concentration of 2 to 20 m per liter.
liter.
Otherwise I can provide nitrate or I can
provide ammonia. But the problem you can
see I have put an asteric mark here.
Okay, this ammonia has a problem because
it is poisonous.
In case of ammonia only you're using
ammonia there is need to add one or more
triricaroxilic acid citrate or malate so
that any delitrious effect caused to
caused by this ammonia
okay can be neutralized.
So when nitrate and am ammonia ions are
present together in the culture media
you see that this is used up
hopefully you understood once again I
will tell whenever in a media you have
nitrate and ammonia present usually your
cultures they take up the nitrate
and not much of the ammonia but suppose
say you are adding something ammonia salt
salt
you shouldn't didn't use ammonia salt
alone. Why? Because your ammonia when
you're using it is alone, it is
poisonous to the plant. That is why to
neutralize its poison okay we use
certain like this triricaroxilic acid
or citrate or suinate or malate is
added. So
and other what all macronutrients we are
using no you can use it at a
Now coming to your micronutrients.
So micro means you all know that it is
in small quantity. Okay, these inorganic
elements are required in small quantity
but essential for the plant cell and
tissue growth.
Though they are required in trace
amount, they are very essential for your
plant growth. And these include your
what? Iron, manganese, zinc, boron,
copper, moly blade. All this can be used
as your micronutrient. And look at the
concentration what you're using. It is
quite less when compared to your macro nutrient.
So of all the micronutrient you see that
the iron seems to be more critical
more important.
Why? Because it is in the chilated form
of iron. We don't use simply iron as
such. We use it in the form of chilated
form like it is in the combination with
sodium EDTA or only EDTA.
Why? Means
this iron what happens is um when you're
using we can't use iron directly. We are
using it in the form of iron tartrate
and citrate.
This iron uh citrate or tartrate when
you're using when you're dissolving in
the media it starts to precipitate.
Okay. Once it starts to precipitate what
happens if it in the media see if it
starts to precipitate what happens they
are not soluble. So your plant cannot
consume that because it has already
precipitated. So that is why usually we
use this EDTA
okay iron with EDTA. So EDTA is what it
acts as a chillator. So what it does
what is the function of the chillators?
So it has the ability to form complex
bound with your metal. See here this is
my later that is your EDTA. Now when you
have added along with iron what happens?
They form a complex. Okay. Once they
form a complex with your metal ion so
that your metal ions will remain in the
solution despite of precipitating agent.
So your plants can utilize them. Once
again I will tell you all if I'm going
to use iron tartrate or iron citrate
these iron will start to precipitate and
they will not be available for your
culture to grow because they have
precipitated. Therefore we will use
certain killators like EDPA which forms
a chemical bond with your iron. when
such type of bond is formed. Okay, here
you see sodium salt of EDTA. They act as
a sequence or they act as a killer. That
is what happens is even though there are
certain precipitating salt in your
media. Okay, your iron will not
precipitate. So it is easily available
for your plant as a nutrient. They can
take up as a nutrient.
So it is easy for the plant to utilize
them instead of using this precipitate.
Again the other chemical what we use is
the magnes. It is involved in the
regulation of enzymes, growth hormones
and even it helps in the photosynthesis
[Music]
So this is the importance of your iron.
iron.
So these are other micronutrients as
earlier I mentioned and you can use it
Next coming to the carbon and energy source.
source.
So the most preferred carbon source for
the plant is sucrose.
Okay. Sucrose is very
easily available for the plant to
utilize. After sucrose it is the glucose
which supports equally the plant growth.
While fructose is less efficient. Okay.
Sucrose is the main carbon source what
is used. Equally even your glucose can
be used and fructose can be used but it
is not as efficient as sucrose and glucose.
glucose.
So one important point what some of the
scientists have noticed is
um when you're autoclaning sucrose okay
when we that is when you're sterilizing
this uh sucrose what happens is it is
converted into glucose and fructose. So
when I'm autoclaving this sucrose
whenever I autoclave this sucrose what
has they have observed is it the sucrose
gets converted into glucose
glucose okay
okay
and fructose
so when I'm adding such type of
autoclave sucrose to the media it has
been observed that the cultures
immediately take up the glucose first
indicating that glucose can be easily
absorbed by the culture plant than your fructose.
fructose.
This is an important point what they
have noted. So the first glucose is used
and then they utilize the fructose. When
all the glucose are finished in their
medium only then they use the fructose.
Other carbohydrates whatever we have
like lactose, galactose, maltos okay
these are not used why because they are
usually they have provided inferior
results they have not given us any
beneficial results that is therefore we
are not going to use these type of carbohydrates
carbohydrates
so what is this from where I'm getting
my carbon and energy how we humans get
our energy. First source is your
carbohydrates, right? Similarly for your
plant also the important source of
carbon and energy is coming from the
carbohydrate. And which are the
carbohydrate? It is the sucrose and
glucose and then fructose. Okay. To
lesser amount whereas the others what we
mentioned your maltos, your lactose or
galactose, refinos these do not provide
any good results.
And another important when we are
speaking about the carbon and energy we
know that all our plants are photosynthetic
photosynthetic
that is in the field but what about in
my laboratory
the cells tissues
okay they do not have chlorophyll so
they are not auto tropes that is they
cannot synthesize their own food.
Sometimes you will see some green color
growing in your plant that the plant
which is growing in your laboratory. So
you may think that okay that green color
is what chlorophyll and it has a ability
to undergo photosynthesis by itself. No
that is not the case. Your tissue
culture plants are not auto troes. Okay.
So that you have to provide carbon and
energy to them. Only after the plantlet
starts developing after it has gone cell
division when it has started
started the cell division and when it's
starting to
produce different tissues or organs only
then it'll try to become autorophic
otherwise they are not autorophic.
Therefore, it is up to us to provide all
Now, we have spoken about our inorganic
components. Then, we spoke about the
carbon and energy sources.
Okay. Then
we'll be speaking about the organic
supplement. So, organic supplement the
first point is the vitamins.
In vitamins what happens is your plant
synthesizes vitamins endogenously that
is within itself it can by itself it can
produce the vitamins endogenously and
these are used as a catalysis for
various metabolic functions.
Similarly in our body how this vitamins
we consume it acts as a co-enzyme and
for the pro continuation of metabolic
processes. Similarly, the plants also
produces vitamins endogenously for
various metabolic process. But when
plant cells and tissues are grown in
vitro, whenever you are growing them in vitro,
vitro, okay,
when you're growing them under vitro condition,
condition,
you must provide the vitamins.
Though it is required in trace amount
also vitamins are required for us also
in trace amount. But just imagine if we
are deprived from vitamins what happens?
We so many disease can happen to us like
if vitamin A is not present, blindness
is okay. Thyine is not present very very
so though it is required in trace amount
it is very essential for the development
of the plant particularly the plant
which is produced which is grown in vitro.
vitro.
So when plant cells and tissues are
grown in vitro, some essential vitamins
are synthesized, they do synthesize but
only in suboptimal quantity. Okay, they
do not produce it up to the maximum
level. They produce it only to the
suboptimal quantity. So hence it is
necessary to supplement the media with
the required
vitamins and amino acids also. So we
supply the thymine nicotenic acid
pyodoxil or calcium pentathinate all
this myioinino as we have mentioned
here. So we provided. So once again let
us come back. We have told that vitamins
are produced endogenously but in the
plant in vitro also they start producing
that is when we are growing in
laboratory condition also they produce
vitamins but it is very in suboptimal
condition which is not sufficient to
proceed with their metabolic function.
So therefore we provide the vitamins for
the plant. And as I mentioned here,
these are some of the vitamins which we
supplement to the plant and we can see
that these are essential for all their
See essential has a co-enzyme for citric
acid cycle
and here you can see that it is part of
cell membrane.
So these are all required for the plant
group. So coming to amino acids okay
amino acids also are produced by the
plant but even these are not in
sufficient amount they not being produced.
produced.
So your cultured tissue are normally
capable of synthesizing amino acid
necessary for various metabolic
processes. But in spite of this the
addition of amino acid to the media
becomes important. Why? Because we have
to stimulate the cell growth
particularly your protolast culture and
for establishing cell lines
for that and all amino acid is required.
Whatever is synthesized by the culture
is not sufficient. So we have to add the
amino acids.
So you can see here the most common
source of organic nitrogen used in the
So which are the common ones we use? We
use cassine, hydroxilate. We can use
glutamine, asperen and adinine.
So one important very important point
what you have to notice is when these
amino acids are added alone. Okay, when
you add individual amino acid to the
media, they can be inhibitory to the
cell. They do not allow the culture to
grow when you are adding single amino
acid. This has been noticed. Therefore,
it is always essential for you all to
take precaution. Instead of adding
single amino acid, you add mixture of
amino acid to your culture plant
culture. Okay, this is your media and if
this is your culture growing, you add a
mixture of amino acids into it so that
it can grow. Otherwise it is proven to
show that it has the inhibitory effect.
Tyrrosin has been used to stimulate
morphogenesis that is the different
phases of the cell cultures.
So also it is noticed that you cannot
use tyrosin alone. Okay. It should be
used in agar media. Only when it is used
in the agar media it becomes active. See
some of the reasons why this happens it
is still not known.
But what happens is when we are adding
this individually we see that your
plants do not grow properly. That is why
we add it in the form of the mixture.
So supplementation of culture media with
adinine sulfate can stimulate cell
growth and greatly enhance the shootute
formation. So whenever we are using this
adinine sulfate okay see adinine in the
form of we are using it in the form of
adinine sulfate it is very good for the
development of the formation of the
shoot. So here the most important point
in amino acid what you have to consider
is the plants require if you remember
even um white when he did his
experiments he used east extract and
later on what did he do he shifted to B
complex vitamins and he saw that if you
use these thymine and all it is not
necessary for us to use east so he
showed us the importance of
vit vitam ments or or these amino acids.
Okay. So remember you should not add
single amino acid. Instead of that you
add the mixture of amino acid so you get
Now lastly
so these all are I've used now inorganic organic
organic
uh we have used vitamins amino acid and
now we'll see with some of the natural
organic supplement okay this also I told
you when the history of tissue culture
certain scientists started using coconut
milk and they found that its property
was very much different from the oxen
oxen is of what plant hormone
Okay. So we can use this coconut milk,
cassin hydroate,
protein hydroate, tomato juice, ease,
many of this are being used.
So what they have seen is that
like even you can use your orange
juices, tomato juices. So in tissue
culture the success and achievement with
the use of coconut milk and protein cine
hydroate has been significant. So when
you're using coconut milk and cassin
hydroilate it has shown that these have
positive effect on your culture
whichever you're growing that is your
tissue culture.
Similarly, potato extract has been found
to be suitable media for anthther culture.
culture.
I'm not mentioned potato here. So, even
this potato cultures.
So, most of you have used this potato
dextrose media. Okay. This potato
culture media is also used for the
So generally the use of this natural
extract is avoided
though you see that they have beneficial
effect like coconut milk or hydrolace or
potato. We saw that they have the
positive effect on the culture. Usually
these should be avoided in this tissue
culture. Why means? Because we don't
know the quality and quantity of this
growth promoting. Okay. Because we are
all measuring and adding but here we
don't know the quality of it from where
it is derived. We don't know what is the
quantity of content what are present in
the coconut milk or your fish emulsion.
So probably because of these reason it
is particularly avoided and therefore
now what the scientists are doing is
like instead of using tomato juice for
example it is replaced with asperine
your amino acid. Okay. Now we see that
yeast and malt extract it has been
replaced by aspergin. So instead of me
using tomato juice or yeast extract or
malt extract I can weigh this aspergin
because I know the quality of it. I know
the quantity of it. I can use it instead
of using these natural complexes. But
still we see that some of some labs
still using this coconut milk and
hydroxilate and potato cultures.
cultures.
Okay, here it is. Potato extract for anthoculture.
Lastly, we shall talk about the charcoal
activated charcoal. You can see in the
box, I've written a paradox. Okay, why?
We shall see that. See the addition of
activated charcoal to your culture media
is reported to stimulate the growth and
differentiation in some plant. Okay,
it's when you add this activated
charcoal. Okay, you can see it's
positive effect that is it is
stimulating the growth and
differentiation of your orchid plant or
carrot or tomato. But the same activated
charcoal when it is added to soy bean or
tobacco it has shown negative effect.
That is why it is a paradox. Either it
can activate or it act it can act as an
inhibitor. Now why this is happening? So
why charcoal is doing this? Because
this activated charcoal okay here for
this plant wherein it is acting as an
activator. What it is doing is it is
trying to remove the all the waste from
that culture. So what it is doing when
the waste products waste metabolites
from this is being removed it is
actually enhancing their growth.
That is why whereas while here it is
acting as an inhibitor means when we are
adding activated charcoal here it is
showing negative effect. What it is
doing is it is taking off the hormones
the phyto hormones which are essential
for the growth. Okay, that is what the
scientists have observed. So it can have
beneficial effect it can have this
negative non-beneficial effect also when
it is having its benefit effect we come
we see that it is trying to remove the
impurities in the culture. On the other
hand here what it is doing it is not
removing the waste from the plant
culture but instead of this it's
removing the hormones itself from the
culture media. So suppose say this is
your culture media and you're growing
your culture here. Okay. And I have
added this is your soya bean plant or
tobacco when I'm adding the activated
charcoal. All your nutrients are present
here. What it is doing? It is absorbing
of your nutrients itself. Then where is
the chance for the plant to grow? it
will not grow. Okay. So, it is having
its merits and demerits. But
particularly remember activated charcoal
is used for detoxifying the waste from
the plant.
So whenever you are using this activated
charcoal first you have to see that it
is acid washed. It should be neutralized
because we are using acid. It should be
neutralized with a base and then if it
is necessary only then you use your charcoal.
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