This content details the crucial steps and precautions in preparing plant tissue culture media, focusing on the proper dissolution of agar, pH adjustment, and the preparation methods using dry powders or stock solutions. It also covers the critical process of surface sterilizing explants and discusses common problems encountered in tissue culture, such as hyperhydricity, contamination, phenolic exudation, and somaclonal variation.
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Chapter in media composition
we ended up telling about the
solidifying agents or the jelling agents
and we saw why agar is important.
One more precautions what you have to
take is whenever you are using agar it
is necessary to ensure that the agar
powder is properly dissolved.
can see in this picture.
It should be properly dissolved so that
it is distributed evenly
or uniformly throughout the media so
that it can minimize the risk of post
sterilization contamination.
What we mean by post sterilization
contamination means after you have
sterilized the agar can result in
contamination because when you have put
the agar powder sometimes what happens
is the agar stays back on your sides of
your glass b okay
that is the dry powder remains above the
water level it is not completely
dissolved so this may impose a problem
later on. So the powder remains above
the level of the water resulting in post
sterilization contamination.
So the media containing agar should be
heated to dissolve the agar before autoclaving.
autoclaving.
So bring the media to boil without any
burning. Okay. So what we usually do is
we weigh the agar and then what we do we
add water and we keep it in the oven so
that there is completely the agar powder
is dissolved and we have to also see
that it doesn't get burnt up. Okay. Then
most of the culture media will require
the final sterilization in an autoclave
at 121° centigrade for 20 minutes. So
this precaution we have to keep it in
our mind that whenever we are dissolving
agar see to that agar is completely
dissolved in the water it doesn't have
any powder left on the conical flask and
then you dissolve it okay slightly you
boil it in your microwave so that the
agar powder is completely dissolved and
Next coming to the pH. So, pH plays a
very important role in PTC.
So, the plant and tissue culture
requires optimum pH for growth and
development in tissue culture.
So, while preparing a media, the pH can
be adjusted to the requirement of the
experiment. Whichever individual is
doing the experiment according to his
protocol he can adjust the pH but the
most optimum pH is the 5 to six and
always the pH is adjusted before the sterilization.
sterilization.
So the pH should not be high or low
because this can affect a media. How it
can affect a medium means like higher pH
See what happens is if suppose say your
pH is higher then what will happen
your media it can become too hard.
Okay. While if the pH is low it results
in unsatisfactory solidification of the
agar. So this both precautions we have
to take care of. It should not be too
high otherwise your media is going to
become too hard. If it is low then your
media is not going to solidify. So this
both can interfere with the optimum
culture growth. So the media the pH of
your media is usually 5 to six or if
your protocol if you are doing some
different type of experiment according
to that protocol you can go and adjust
the pH
and always remember that the pH should
be adjusted before the sterilization is
considered. This is another important
point which I would like to tell about
Now let us see how we are going to
prepare the media. Okay. See nowadays
the plant tissue culture media is most
commonly used are available in the
market as dry powder. You can just
purchase the media and according to your
protocol you can use it.
It's almost it's become very simple.
Okay. So the simplest method of
preparing the media is to dissolve these
powders all these dry powders containing
your organic or inorganic nutrients in
minimum amount of water.
That is in some quantity of distilled
water you'll dissolve. After the
contents have been dissolved thoroughly,
it is mixed with water, sugar and agar.
What type of agar? It should be an
melted agar. Then afterwards you can add
the organic supplements are added. Now
after we have done with this finally the
volume is made up to one liter and then
the pH
is adjusted and then go for
sterilization. This is the common method
we use. Okay. We take the
we purchase the media that is the dry powder
powder
and we try to dissolve the organic in
inorganic nutrients in some quantity of
water. We will try to mix it well and
then add sugar and melted agar and other
constituents and finally we'll make up
the water to 1,000 ml. Then we adjust
the pH and then go for sterilization in
So sometimes in experiments in which
changes in the quantity and quality of
media constituents becomes necessary
that time it is desirable to weigh and
dissolve each ingredient separately
before mixing them together
because in some experiment okay you need
a there there is a lot of changes what
we require in the quantity and the
quality of the media what is required
that time We can't put all the
ingredients together. Then what you can
do is dissolve each ingredient
separately before mixing them.
And then you can prepare the media. One
of the convenient method commonly used
in plant tissue culture is the
preparation of stock culture. Okay. When
the stock is there
then you can take some amount of the
stock and you can prepare the media.
Okay. Suppose say you have you cut the
vegetables and keep it in the
refrigerator so that you take the stock
from that for each day and you prepare.
Okay. So like that you can prepare a
So for to prepare a stock solution
what we do is we take the appropriate
quantities of the media. We actually
prepare four stock solutions. You can
see here major constituents minor salts
iron organic nutrients
except sucrossse we don't have. You can
see major concentration we are using up
to 20X minor salt 200 X means is the
concentration iron 200X
organic nutrients except sugar 200X. So
we are preparing all this in a separate
concentration and we are keeping it as a
stock solution. Then what about our
growth regulators?
For each growth regulator, a separate
stock solution is prepared by dissolving
it in the minimum quantity of
appropriate solvent. Why solvent? In
last class that most of our growth
regulators were not soluble in water. We
either use dilute alcohol or NaOH. Okay.
So that is why we use the solvent to
dissolve our growth regulators. And each
of these growth regulators also you can
prepare it separately and dissolve in
the respective solvent and you can keep it.
Then what you can do is you can adjust
all the ingredients with the distilled
water to a desided volume to make an
overall concentration. Overall
concentration you can make up to 1 mill
mole. Okay. This we have spoken already
1 m mole per liter or 10 m mole per
liter. These are stock solutions you
have prepared. So you can prepare this
and keep and how you are going to store
this. You have to store all these okay
in the plastic bottle or in the glass
bottles and whatever requires
refrigeration condition you have to keep
them in the refrigerator. But remember
students iron you have to store it in
the amber bottles that is the brown
bottle otherwise it will get oxidized
okay is called as the amber bottle that
is the brown bottle which we commonly
say. So
whenever I want to use the media I
already have the stock solution I can
dilute it from my stock solution and
prepare the media. But another
precaution what you have to take care is
shake well before use.
Okay? Because your media contents would
have been settled to the bottom.
Therefore, you have to shake them before
you use it for further experiment. And
also we told about natural organic
nutrients which are being used by a
scientist. Okay. And one such good
example is your coconut endosperm. So
whenever you are using coconut endosperm
also there should be lot of precaution
be taken because the this endosperm no
it should be boiled to deproinize
it because it'll be containing the
proteins which may interfere with our
experiment. Therefore you boil it so
that you can remove the protein from it
and then remember to store it at deep
freezer at -20° centigrade. This is how
I'm going to prepare my media. So either
you can use the D dry powder which are
available in the market. You purchase it
and you prepare it accordingly. You make
up the volume, adjust the pH and then
sterilize it. Otherwise some experiment
will require a particular quantity and
quality of your experiments. when you
want it like that you prepare dissolve
each of the ingredients separately
according to the protocol and then use
it otherwise as most of the lab what
they follow is the preparation that is
tissue culture lab I'm telling who are
particularly involved in that
experiments okay they what they do is
they prepare a stock solution of like
major minor iron organic nutrients
everything they prepare separately in a
particular concentration
And they store it in the plastic or
glass bottles and whenever they require
it they take the minimum amount of these
ingredients what is necessary and make
and suppose if you're using the organic
nutrients you have to take precautions
particularly I told you even in the
history of tissue culture most commonly
what we use is the coconut milk okay so
therefore the endosperm should be
deeproin organized before we use and
shake all your bottles well so that the
media is uniformly distributed before
the start of the preparation of the media.
Okay. Suppose say if you want to prepare
a new media.
So that time I told you the hallmark
media what we use usually is the MS
media and we know all the ingredients
now. Okay. Today for for past two three
classes you're studying only the
composition of the media. What all must
and should be present and should not be
present for the media. Something may
interfere with your media which in turn
will lead to your deterioration of your
culture that should be avoided. Suppose
say I want to prepare a new media. How
shall I prepare that new media?
Okay, that time what you do is you
divide all your components. Okay, into minerals,
minerals,
oxins, cytoinance and organic nutrients.
So I have divided all my ingredients
into four components which are my minerals,
minerals,
oxins, cytochenine and organic nutrients
and prepare this in three combination.
This is in four. Okay, my components are
divided into four. Next is you prepare
this inner combination of three that is
L HM. What L stands for is low. Okay, L
and M stands for medium, medium, sorry,
Okay.
So prepare these into three combination
of LHM low, high and medium. Suppose if
I'm using this in low quantity, I can
make this as in the high quantity. Other
two I can use it in medium quantity.
When I make such combination, I will get
totally 81 treatments. Okay,
Okay,
in this combination when I do I usually
end up in getting 81 treatments. Then
you perform your experiments and you see
that which combination is giving you the
best result
and that best result is denoted in the
four letter code. For example, what I
have mentioned here is LHM.
So what does L stands for? It stands for
low I told. So it is the first
constituent. What is that? My mineral.
My mineral is in low constituent. Second
one is is L. Again it stands for low.
What is the second combination here? It
is the oxen not combination and separate
ingredient. So it is oxen. So oxen is
also I've used low. Hi. What is the
third one? Cytokinine I've used in high
concentration. So yum medium that is the
fourth one that is the organic
nutrients. So low concentration of
minerals, low concentration of oxen,
high concentration of cytoinine and
medium concentration of organic nutrient
has given me the best result
for my particular experiment. So this
will become a standard for me to use
this in the next experiments whenever
I'm using the same experiment instead of
doing all the 81 experiments I have
already standardized with this.
Okay once again I shall tell you
whenever you are preparing new media you
divide your components
on one second
you divide your components into four
parts. Okay. Like minerals, oxins,
cytoinines, organic nutrients. Then you
prepare three combination of low, high
and medium of these
ingredients. So when you prepare
different combinations of this, we get
an 81 treatment. Then what you do is you
perform your experiments and finally
come to a standardization of procedure.
What for example I have written is L LHM
is my standardized procedure that is my
experiment has worked good in this. So
what is L means you know it is low. So
it is low concentration of mineral low
concentration of oxen high concentration
of cytochinine medium concentration of
organic nutrients as performed the best experiment.
experiment.
So I will consider this as my standard
protocol. When I want to do the same
experiment next time, it is not
necessary for me to do all the 81
experiments. It is not necessary. I can
do only this. So now you see that so
many combinations we are using, so many
ingredients and constituents, different
concentrations we are using, the
quantity, quality is varying. So the
entire plant tissue culture media is
ultimately is what it is complex.
So it always becomes an important point
to keep a reference sheet giving details
of the constituents and the sequency of
steps to be followed during the preparation.
preparation.
So what happen what we mean to tell is
because the entire tissue culture
constituents media what we are using is
complex you write it down in your
reference sheet what is the you give the
details of the constituents what you're
preparing what is the concentration you
have used what is the chemical you have
used okay how you have dissolved that
chemical it should be in a sequence of stepwise
stepwise
Okay. In a sequence manner you should
write it
and then prepare the media according to
the steps after you have weighed. Okay.
See for example I have to weigh some 20
chemicals. Okay. So 1 2 3 4. Write it in
sequency. Don't write it somewhere
randomly and keep it. Then each time
after you weighing and preparing sc it
out that doesn't lead to confusion
otherwise you'll end up in thinking that
know which step I am in. Okay. So this
is another precautions which you have to take.
take.
So that we come to the end of the media
constituents what are required for our
tissue culture which is an important
questions related to your plant tissue
culture. Now let us speak about the expplant.
expplant.
So here you can see in the petri dish we
have put some stock leaves okay some
ailary buds or lateral buds. So what do
you mean by an expplant? As earlier said
expplants are small pieces of your plant
part. Okay. It can be a cell or it can
be a tissue
like the different parts of your plant
such as the chute, leaves, stems,
flowers, roots, embryos
or it can be even your mature cells.
So they are cut or exised from the
plants aseptically
and it is used to initiate a culture in
the nutrient media. That is you are
going to grow these expplant in the
nutrient media
provided they can differentiate and into potencils
potencils
that is I have taken if this is my plant
for example this is my plant okay and
here these are all some flowers and all
what I'm going to do is using the aseptical
aseptical
method I'm going to cut this leaf or I'm
going will cut the flower or the stem
small pieces I can take. So whatever we
have mentioned here okay and then we use
all the aseptic techniques these are
called as expplant that small pieces of
plant parts what you're taking that is
called as an expplant using this we are
growing this expplant in the suitable media
okay and this expplant should have the
ability to
what mature and provide
Provided that they differentiate into
your purity potentials that is they
should undergo differentiation,
undergo cell division, undergo
redivision, they should have all this
capability. In one word we are saying it
is a toty potential. What is a toty
potential? The ability of your cells or
tissues or organs or expplants what you
have taken for your tissue culture
should have the ability to grow into a
fully developed plant under all provided
conditions. That is what is the meaning
So we have seen a laboratory
organization how medial is prepared, how
we wash and dry a glass bips
and instruments what we use in a tissue
culture what is all necessary we have
seen now the most important is the
expplant what I I we spoke about now. So
surface generalization of expplant is
important from the point of examination
and even in your practicals.
So how we are going to surface sterilize
our expplant. Now we have taken the
expplant I told you that aseptically you
remove your expplant. This expplant must
be surface sterilized to eliminate the
microbes present on the surface. So this
expplant will be carrying microbes. Can
you see the microbes in your naked eye?
No. Until it is something grown into
colonies, you can see otherwise it's not
possible to see. So there will be
microorganism present on your expplants.
There will be dust and dirt present on
your expplants. So I have to remove
these things first. Okay, that is called
as the surface sterilization of the
expplant. This is generally done by
treating with 1 to 2% solution of sodium
or calcium hypocchloride as I mentioned
earlier or with 0.1% solution of mercury
chloride. So I can use
1 to 2% of sodium hypocchlor sodium
hypocchloride or I can use even calcium hypocchloride.
hypocchloride.
So when you're using
um mercury chloride okay when you're
using mercury chloride I told you that
the an individual should take all
precautions because these are poisonous
so you have to wear your safety head
gear or gloves and mask because it
should not poison you okay so this is
how I go for the surface sterilization
so let us see the different
um steps how I go for the surface
sterilization. So I have excised my
plant I have to do the surface
sterilization of the plant. So first
when I get my plant here you can see I
have cut my plant. What will I do is I
wash it with the tap water that is the
running tap water. After that what I can
do is I can wash it with 5% of te what
It is a detergent.
So I have washed it with tap water. Then
I'm going to wash it with the detergent.
One example what I've given is the
teapole for 10 to 15 minutes. Again I'm
going to wash it with water. Okay? So
that I remove up all the detergent and
finally I'll wash it with distilled
water. So you should never ever forget
distilled water at all. Final wash is
always in distilled water. We should do
now this and all these steps. No, I can
perform it in the normal lab lab itself.
But subsequent steps I should perform it
in the laminina airflow. Remember
laminina airflow cabinet we have spoken
in detailing that is it'll be having a
UV light, air blower, illumination
light. You have to clean the swab the
floor with ethanol. All those steps you
have to perform in the lab and then you
have to bring this expplant into your
lab because if the lab itself is
contaminated what is the use of doing
the expplant sterilization. Okay. So
everything should be under the aseptic
Okay. The subsequent step I'll be doing
in the using the laminar airflow. So
here next what you do is dip your
expplant 70% of ethanol only for 60 seconds.
seconds.
Okay otherwise here I told you remember
students whatever solvent you are using
the time is also important otherwise
it'll it's definitely going to destroy
your microorganism simultaneously. It is
going to inactivate your bioactivities
of the plant also. It'll start
dehydrating even your plant. So we
should be careful. Our aim is to kill
the microorganism, remove the dirt and
dust from the plant but without causing
any harm to my expplant. So dip the
eggplant in 70% ethanol for 60 seconds
and then again you can transfer it as I
told earlier how you do surface
sterilization okay you can do it in 0.1%
of mercury chloride
okay for 10 to 15 minutes
then it should uniformly dip the plant material
material
okay what I mean to tell is suppose Say
this is my beaker and this is my
solvent. Okay. I have put my expplant here.
here.
Okay. Using a forceps I have put the
expplant. You see the expplant is
completely immersed and you un uniformly
you can shrill it. Okay. So that every
part of your expplant is exposed to the
detergent or your solvent what you're
using. Then you can decant the uh
expplant with that is all the solvent
you can decant it. Okay. So as I told
you the bottle is frequently stilled to
wash away whatever the solvent is there.
Again then you give a final wash with
your distilled water and then with the
autoclave distilled water to see that no
solvent is present there. whatever
detergents or chemical I have used to
treat my expplant even that should not
be otherwise I told you that it can have
an uh effect okay or a delous
consequency on your expplant. So this is
how I wash my expplant or the other word
sterilize my expplant that is the
correct word what we use and then we can
go for a
tissue culture experiment whatever we
want to perform we can do those
experiments. So hopefully you understood
what the expplant meant. Okay, here it
is. What all we use even the embryos all
those roots also we use it. So that part
of that piece of the plant is known as
the expplant. Then what I told you is
how you going to sterilize surface
sterilize your expplant. Okay. So you
can see that all the part of your plant
should be exposed to water. It should be
exposed to the solvent at a particular
time and then you wash it always with
distilled water. Do your performance all
in the laminina hair flow or hair hood
or the cabinet whatever is available to
you all. And then whatever experiments
you have to do you proceed with the
So now the
we will see into the problems
encountered in tissue culture. There are
many problems what the tissue culture
scientists faced. We will see some of
the problems what it is. First one is
the hyperhydricity.
Previously this hyper hydroity was known
as bitrification.
Okay. What do you mean by hyperhydricity
or in some textbook as it mentioned as
vitrification means it is a
physiological mal foration.
This is M. Okay. M A T I O N. This is a physiological
physiological
malf foration that results in excessive hydration.
hydration.
Okay. Lot of water when it is present.
Low ligignification
and your somatal functions are reduced.
Your mechanical strength of the tissue
is been reduced.
That is called as hyper hydricity.
Okay. The tissue becomes like water
soaked and transpulent as you can see
here. Okay, that is called what is
happening physiological malfformation is
resulting because of excess of water
which is leading to low lignon formation
and even the stomata of your plant is
not working properly and there is
reduced mechanical strength of the
tissue which you are generating. If
there is no mechanical strength what
happens? It just keeps falling off.
Okay. So if there is hyper um hydroity
how can I prevent it? So I can prevent
it by adding suitable concentration of
the agar
and also here what happens in
hyperhydricity is um the leaf okay see
here the leaf or the lamina of the leaf
all they look very transparent and transluent.
transluent.
So this is how you can easily identify
the hyperhydricity or the vitrification
of the tissue culture plant. This is one
of the problem of the tissue culture
plants you're facing because of high
hydration, low liignification,
So how can I prevent this hyper hydroity
is just like modification of your
hugger. you you slightly use higher
concentration of your jelling agent and
you can increase the carbohydrate levels
in the media also you can change the
light intensity
okay and reduce the humidity particularly
particularly
the agar
is the most important one by just by
changing the agar concentration okay by
modifying the agar concentration see
that you can prevent the hyper hydrocity
If it is not possible then you go for
increasing the carbohydrate levels or
changing the light and humidity. Okay.
Okay.
So it is caused by excessive water uptake.
uptake.
The next one is internal pathogens. We
have already spoken about this also.
What happens the surface when we are
doing surface sterilization of the plant
well and good but sometimes what
happened to those microorganism which
are interior in the tissue that becomes
an problem.
Okay that will act as a culprit to our
tissue culture. So when the tissue
culture here you can see the tissue
culture plant is growing along with that
you can see some funguses also are
growing some moles are growing. So what
this fellow moles will do is they'll
take up all the nutrients what is
required and they'll flourish in growth
without providing anything for your
plant and they are going to contaminate
your plant. This is the biggest problem
of the tissue culture that is the
contamination by the microorganism.
So this contamination can be your
viruses or bacteria even your fungal
pathogen. If any nematodes are present
also they can bring about such type of
um contamination.
So eliminating this contamination is a
problem. Okay. That is why starting
itself you should take all the
precautions to see that you avoid the
growth of microorganisms because after
doing all the experiments and you grow
growing your plant and then you see the
growth of the organism your entire
experiment is waste. how much time you
have invested on that, how much
chemicals you have used. So there is
economic loss, loss of manpower. Okay.
So bacteria are difficult to eliminate.
So what is the one choice is you can use
antibiotics. While using antibiotics you
should take lot of precaution because I
told you that antibiotics have a
like they are narrow range like they can
affect only one type of bacteria
okay and otherwise they can result in
even resistancy of the microorganisms.
So these all you have to take
precautions when you're using
antibiotics but I told you they are
systemic infection that is your plants
are always usually get infected very low
immunity they have so such type of
plants should must be treated with the
phenolic exution.
So this is another
problem with your tissue culture plants.
So here what happens phenolic exitution.
These phenolic exudence are frequently
encountered problem in the establishing
expplants. See here we are growing a
expplant. Okay. See it's growing and you
can already see something brown in color
and after few days you see there is no
expplant. There is only brown color
contamination which is present or
instead of contamination let we say that
it is a phenolic execution. Okay. And
particularly such type of phenolic
executions you can see only in
particularly in UDI perennials when you
want to grow such udy plants in the
laboratory it is usually
it faces this problem phenolic
execution. This phenolics execution are
part and parcel of the plant itself.
Okay. Because these are secondary
metabolit which are produced by your
plant. You know certain like u your very
good example is your apple. When you cut
the apple it turns brown when it is
exposed to the atmospheric oxygen. So
what has happened?
Okay the phenolic components in your
apple when it comes in contact with the
air it is getting oxidized and it is
turning brown and it will not give you
an appeal to eat that apple. Okay. So
these phenolic component oxidizes to
form a brown material in the media and
then it inhibits your shoot formation.
See here how it has inhibited my shoot
formation because of this brown color
phenolic. Now how can I overcome this
pholic exeution? How can I minimize it?
I can minimize it minimize it by using
antioxidants. You can use ascorbic acid
which is one of your good antioxidants.
Another one which we already spoke is
the activated charcoal. Remember when
you're using activated charcoal you have
to first clean the charcoal. Okay. Wash
your charcoal charcoal, dry it and then
you have to use it. Otherwise the other
method is the frequent transfer to the
new media. So when you are coming to
know that there is a phenolic exitution
or you already know whenever I'm using a
woody perennial this is what problem I'm
going to faces you subsequently transfer
to the fresh media
so that you can avoid this problem.
Okay. See when even in the apples like
when you want to bring it to when you to
your workplace when you cut it and bring
it you you if you apply that lemon juice
you see how fresh your apple is. What is
the lemon containing? It is containing
ascorbic acid the vitamin C which is
acting as an antioxidants that is
preventing your apple from turn avoiding
your apple from turning into brown.
Similarly here also we use antioxidants
like ascorbic acids or we can use
activated charcoal or we can frequently
transfer this plants into the tissue
culture new media.
Second is shoot tip necrosis. See here
the arrow marks what I have put that is
the shoot tip necrosis. Shoot tip
necrosis occur in the UI perennial
tissue cultures. When actively growing,
shoe tips develop what is known as the
die tip. Tip die back. Okay, that is you
can see that they just fall off back.
Even when you just plant a new plant,
sometimes you can see the tip dying off
very soon. Tip type back. Okay. In
tissue culture, why it is caused? Means
because there is lack of calcium in the
growing plant. So what I can do is
whenever I find such type of condition
this condition is usually caused by the
calcium deficiency in the media so that
I can see that I can improve the
concentration of the calcium so that I
prevent the chip dieback of the plants
of my tissue culture plants. I can avoid
it by increasing the concentration of
the calcium.
You have to know how what is the
concentration I I have to use. Simply I
cannot just go and add how much of a
calcium I want. No everything quantity
and quality is important in your tissue
culture. Next one another problem what
we see is the habituation.
Habituation is when a culture continues
to develop even in the absence of oxins
or cytoen.
For example, I have shown here. What is
this? You can see multiple shoots are
being formed. Okay. Shoot culture
habitated for cytokinine would continue
to produce a new shoot on a cytokinine
free media. That is what it tells means
the meaning of the sentence is
I have added cytoinine for the
development of shoot then I have removed
the cytokenine.
Now my media is completely free of
shoot. Simply then also your tissue
culture plant will be developing only
the shootute. This is another problem.
Though my media is not having cytoin,
it has become an habit for it to develop
into the shootute. That is known as the
habituation. Habitution is when the
culture continues to develop even in the
absence of your particular oxins or
cytokin whichever you have used. Okay.
Oxin is used for the root culture and
cycin is used for your shoot culture.
Even when they are not present, they
keep on growing. That is another
problem. One more is somoconal
variation. This is an individual chapter
in detail you'll be studying.
So somoconal variation occurs because of
genetic mutation.
Now you know what is genetic mutation
very well. You have studied in genetics.
It is caused by invitro condition or by
the chimeic separation. So why do we get
the somoconal variation? Soma means
somata. Okay. Clonal is the similarity
in propagation, vegetative propagation.
When we do they all appear same. Okay.
Somoconal variation occurs because of
genetic mutation that your culture is
undergoing in the invitro condition.
That is when you're growing your plant
in that laboratory because of the
genetic mutation it is leading to
somoconal variation or it can be due to
the chimeic separation. This chimer
means it is a plant or a part of a plant
that is a mixture of two or more
genetically different type of cells.
genetically different. Okay. Genetically
genetically
Okay. Different type of
cell that is known as your primer.
Primer means is a plant or plant part
that is a mixture of two or more
genetically different type of cells that
is called as chimeic separation. So in
when a growing in tissue culture that is
under laboratory condition there can be
a mutation happening which can lead to
somoconal variation and these are some
of the example. Here
you can see they're growing in the roset form.
form.
Change in the leaf size. Okay. Actual
leaf is different but they're growing in
a different form. Here you can see fasiation.
fasiation.
This type.
Okay. Like roset type formation. Suppose
your flower like u
very simple flower I'm drawing. If your
flower is growing like that sometime
what happens it'll be like this. The
center part will be like this and your
flower keeps growing like this.
So these are the some chromosomeal
rearrangements are happening in your
tissue culture plant which is resulting
in such type of combinations. But look
at this sentence. In some cases
variation can lead to new cultivors that
may have desirable ornamental characteristics.
characteristics.
Suppose you like you started liking this
plant than this. You will start to
propagate this one. Okay. Look at this
plant. It is having like I told you know
like two or more genetically different
cells. Some um part of the cell is light
green in color. Some the same plant is
dark green in color which looks
attractive. So I can use it as an
ornamental plant. So if it is a new
cultiv and it is having a desirable
all this okay
so the occurrence of somoconal but I
don't want somoconal variation means
avoid long-term culture that is don't
grow keep on growing the culture in
vitro use axillary shoot induction
system wherever possible
propagate primal by other clonal system.
If one clonal system is not working out
properly for you, use another system so
that you can avoid your somoconal
variation. We'll have a separate chapter
recalcitrance.
So what do we mean by this recalcitrance
means whenever you're growing a plant in
vitro? In vitro means kindly remember
that you are growing in the laboratory
using your glass v that is called as a
invitro. In vitro recalcitancy is the
inability of your plant cell or the
tissue or the organ whichever you have
taken as an expplant to respond to the
tissue culture manipulation. So you have
brought your cell your tissue or your
organ from the plant and that is acting
as an expplant and now you want to grow
it in your laboratory under in vitro in
aseptic condition. You want to do some manipulation
manipulation
but the plant is not accepting it.
Okay. It is failing to regenerate. It is
losing its ability of totency.
that is called as recalcitrance. So
recalcitrance can be defined as a
inability of your plant tissue culture
to respond to invitro manipulation.
It cannot withstand whatever
manipulation in vitro you're providing.
In the broadest term, tissue cultural
recansitance also concerns with time
related decline or loss of morphogenetic competency
competency
that is it is unable to grow through the
different stages of its growth
finally affecting its toy potency. So
these are some of the common problems we
are facing in tissue culture laboratory
recalcitrance. Somoclonal variation,
habituations, shoe tip necrosis,
phenolic exidation, internal pathogens
and hyper
hydricity or vitrification. The better
word we have to use is the hyper hydricity.
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