BASICS & PROCEDURES OF PLANT TISSUE CULTURE TECHNIQUES

                                              

                                                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.
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•          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.
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•           Here, the salts with higher solubility dissolves first, generally the plants can takes the                 nutrients, ranges from PH 5.6-5.8.
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•                   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.
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•                 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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