BASICS OF PLANT TISSUE CULTURE
Tissue culture is the
method of “in vitro” culture of plant or animal cells, tissue or organ grown on
nutrient medium under aseptic conditions usually in a glass container. It is sometimes referred to as ‘sterile culture’ or ‘in vitro’
culture.
By this technique living cells can be
maintained outside the body of the organism for considerable period. Tissue
culture involves both plant and animal cells, tissue culture produces clones,
in which all product cells have the same genotype.
A more
recent advance is the use of plant and animal tissue culture along with genetic
modification using viral and bacterial vectors and gene guns to create genetically engineered organisms.
INTRODUCTION:
Plant tissue culture is a method or technique
to isolate parts of plants (protoplasm, cells, tissues, and organs) and grow
them on artificial media in aseptic conditions in a controlled space so that
parts of these plants can grow and develop into complete plants.
It is a collection of techniques used to
maintain or grow plant cells, tissue or organ, under sterile condition on a
nutrient culture medium of known composition.
There are numerous methods to propagate plants
in tissue culture, but the one principle that is Constant (ie.) totipotency.
WHAT IS TOTIPOTENCY ?
The
property of a normal cells that they have the genetically potential or capacity
to give rise to a completely new individual plant species, referred as
totipotency.
This is because every cell
has the genetic potential of the parent plant, the basic steps starts with the
regeneration of whole plants from an explants or cell.
WHAT IS EX- PLANT ?
It is referred as that the any portion or a
piece of a plant material which can be used in a cultured medium to establish a tissue culture system or to regenerate a
new whole plant.
WHAT IS CALLUS ?
It is referred to as
undifferentiated mass of cells.
PROCEDURES OF PLANT TISSUE CULTURE :
Preparation of instruments and nutrients
culture medium
Sterilization of culture
medium
Preparation of Explant
Inoculation of Explant
Incubation for growth
Acclimatization of plantlets &
Hardening
Preparation of instruments and nutrient medium :
·
The area dedicated to media preparation should
comprise of sufficient storage space for the chemicals, culture glassware/vessels
etc.
·
And the lab procedures and rules should be
followed strictly to maintain aseptic conditions respectively.
·
The media room should be maintained with cleanliness,
removal of contaminated cultures, restricted entry.
Thus, a sterile area is required for
performing various aseptic manipulations during tissue culture. In other hand we can see about the preparation
of instruments, required for the plant tissue culture techniques.
APPARATUS REQUIRED :
·
Forceps
·
Scissors
·
Blade holders
·
Disposable and surgical blades
·
Petri plates with 2 filter papers
·
500 ml beaker
·
350 ml Erlenmeyer flask
·
500 ml test tubes
·
Disposable pipette tips
·
Pipettes and 100 ml bottle with screw cap
1.
Apparatus are trapped in newspaper or brown
paper to undergo autoclaving for an hour.
2.
Before the process of plant tissue before using culture,
it is required to sterilizes and clean the vessels respectively.
3.
Before using any glass wares for any culture
preparation, they are cleaned in 4 steps processes, as given below.
4.
Al glass wares & plastic wares should be
autoclaved for an hour.
5.
And, all wares were immersed in tub-full of
promic acid for 16 hours.
6.
Then it is washed or rinsed in water to remove
the promic acid.
7.
After this, all glass wares & plastic wares
are immersed and washed in detergent water with a hard nylon brushes.
8.
Then the glass wares & plastic wares are
immersed and cleaned in other tub-full of clean water, the detergent is rinsed
off.
9.
Finally the glass wares & plastic wares are
cleaned in the running tap water & kept in clean trays then place into the
oven with temperature of 60-80*c for drying process.
10.
Thus the dried glass wares are now ready for
use.
PREPARATION OF CULTURAL MEDIUM:
- Growing of desirable micro-organisms in
a nutrient medium is known as medium.
- Thus the next important method is preparation of
media, which is used in micro propagation techniques.
- Media, here used is composed of group of micro
and macro elements, vitamins, amino acids (glycine) and mostly sucrose, which
serves as a main source of carbon and carbohydrates for the plant.
- The amount of elements used are weighed
accurately as per the reported formulations and dissolved in de-ionized water
to avoid the precipitation of salts and it prevents the availability of salts
to the plants.
- Here, the salts with higher solubility dissolves
first, generally the plants can takes the nutrients, ranges from PH 5.6-5.8.
- After the medium at ranges the desired PH, it is
ready to be poured in 250ml of Erlenmeyer flask or 50ml test tubes as 100ml /
20ml.
- Semi-solid medium as to be prepared, so take
0.75 mg -0.8mg of Agar-Agar, this amount provide optimum solidification and
moisture content to the culture.
- 100ml of medium is carefully poured into the
each 250ml of Erlenmeyer flask and plucked with non- absorbent cotton plucks,
covered with muslin- cotton at the opening top of the flask, for easy holding
of the cotton plucks.
STERILIZATION OF CULTURAL MEDIUM:
·
Sterilization is a method of removal of unwanted
microbes from the desirable cultural medium. It can be done by following
methods,
v
Sterilization by moist heat
v
Sterilization by dry heat
v
Surface sterilization
STERILIZATION BY MOIST HEAT:
AUTO CLAVING:
Ø
Autoclave is a device used to sterilize the
instruments by means of moist heat such glass wares, cultured media with high-
pressure saturated steam.
Ø Autoclave the media for 20 minutes at high
temperature of 120*C and pressure 15 pascal.
PREPARATION OF EX-PLANTS :
Different kinds of explants such as leaves , petals, buds, ovaries , seeds, anthers and nodal segments can be used in tissue culture because , each & every cells of the cells of the plants are eligible to give rise to the new plants of individuals.
However it should be carefully to collect the plants as,
- Disease -free,
- Healthy & actively growing plants,
Immediately , after collections , the explants are placed in clean-water to avoid air-bubbles , microbes and contaminants from the cut - exposed plant parts.
And to avoid browning , due to the phenolic oxidation.
This explants are brought to the lab for the further sterilization processes.
First, this explants are cut into small pieces with the help of sterilized scissors, and then placed in a petri-plate containing a clean water.
The surface of the ex-plants are brushed clean with stable-hair brush to make it light-wet.
And all the small pieces of cut- explants were placed into a plastic - wares of containing the mild solution of fungicides as an antibiotic respectively.
Then, the pieces of the ex-plants are cleaned & rinsed well with a clean & de-ionized water.
Finally the ex-plant pieces are immersed in a clean de-ionized water and then taken to the inoculation chambers.
INOCULATION OF EX-PLANT :
While entering the inoculation chamber, it is neccessary to wear a clean cotton lab-coat.
Inoculation means transfer of plant specimen to the media under a septic conditions.
PREPARATION OF LAMINAR HOOD:
- Laminar hood is a special equipment for inoculation processes.
- Shud the shutter of the laminar-hood switch on the UV light for about 30 minutes.
- UV eyes can kill all microbes, it is highly dangerous to human eyes and skin.
- Before switching off the UV lights, the shutter should not be opened.
- After 30 minutes, switch off the UV lights and switch on the chamber lights with the shutters opened.
- And switch on the laminar - hood.
- While operating laminar-hood , it is neccessary to wear headmask and face masks.
- And use hand wash (detol) for hand in sterile conditions.
- The Ex-plants are washed with 70% of ethanol for few seconds and then treated with mild mercuric chloride solution for a few minutes.
- And keep the open mouth of the conical flask near to the spirit lamp to avoid the invasion of any other microbes.
- Keep on mind that,generally use a mild solution for longer duration, and use strong solution for short duration.
- After the sterilization process, the solution is decanted into the another beaker, then the Ex-plants are washed several times after and after to remove the mercuric chloride solution.
- Now, after cooling the forceps, before treated with 70% ethanol , used to transfer the ex-plant pieces from conical flask to the petri-plate.
- Before this the petri-dish is placed to sterilized above the spirit -lamp to avoid microbial invasions.
- Place the ex-plants over the filter paper placed over the petri-plate, were it absorbs the excess amount of water.
- The ex-plants which are exposed to the mercuric chloride are removed with blades.
- Each nodal-segments were carefully taken and transferred to the test tubes containing media.
- It is very important that forceps could not touches the rim of the test tube and mainly the media.
- After that the cap is put tightly over the test tube and sealed well..
- Finally, name of the plant, date of inoculation were labelled over the surface of the test tube.
- The culture are now ready to be inoculated.
- At optimum conditions, with 16 hours light alternating with 8 hour darkness, at 25-27 degree Celsius temperature and 40% relative humidity and it is monitered alternately.
- The time of culture grows starts from 12-15 days without any bacterial or fungal contaminants.
- And the corresponding cultures are allowed to grow respectively.
- And a period of time they arises like shoot developed in conditions.
- Once , the shoot is grown at the appreciable height(3-4 cm).
- Generally , auxins are used in culture medium .
- Once they are grown well in the lab conditions, they are ready for acclimatization .
ACCLIMATIZATION OF PLANTLETS &TRANSFER TO POTS :
- Now the tissue cultured plantlets are transferred to green house condition.
- Green houses normally has decreased relative humidity , intensity and aseptic conditions.
- And when the plantlets reaches 3-4 inches, transfer them into a new pots consists of garden sand,soil,well decomposed farm yard manure at the ratio of 1:1:1.
- After 45-60 days, these acclimatized plants can be shifted to the outer door conditions.
Thus , plant tissue culture plays a major role in production of transgenic and disease free resistance plants such as Bt Cotton, Amentotaxus assamica, Lotus corniculatus, Psilotum nudum . And also used in the used in the yielding of medicinal plants , also in the food industries,..
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