Basic technique of plant tissue culture

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Following steps are used for plant tissue culture
1.In vetro lab:
       For in vitro culture a well equipped lab is require which must have a media room for nutrient medium preparation ,sterilization of glass,metal as well as others .Given facility are found on those lab are as follow .
I.Nutrient medium preparation ,sterilization and storage of supplies
ii.An artificial environment is require for the growth and development of culture plant .
iii.Observation and evaluation of culture as hoped.
Equipment for these culture
      Refrigerator,hot air ovan,Autoclave,Normal temperature balance,Normal chemical balance ,Microscope,PH meter,hot plate,gas stove/heater ,hot plate with regulator ,magnetic stirrer,water dissolution plant.
Apparatus required :
Test tube(20cc-2000cc),Measuring cylinder (5-1000ml),pipette(0.01-10ml),petri dish(4-8inches ),conical flask(50-3000ml),volumetric flask for storing chemicals50-500ml).
Others:
  Pointed forceps,blunt forceps,scalples,seissors,fine pointed needle,aluminium foils,medical cottons,filter papers,rubber bands,sprit lamp,sterilizing boxes and surgical blades.
2.Selection of nutrient medium :
      The nutrient medium provides nutrition and hormone as well as other biochemical requirement for the growth and multiplication culture plant.13 elements are necessary for the growth of plants which are divided into 2 groups ie Macronutrients and micronutrients.Macronutrients such as (C,H,O,Ca,Mg,K,P,S) and micro nutrient such as (Mn,Zn,Bo,Cu,Mo,Cl) among them these macro nutrient are used larger quantity in compare to micro nutrient .Some popular nutrient medium are
I.White'medium (WM)-1954
ii.Murasighe and skoog'(MS)-1962
iii.Nitech's medium (NM)-1969
iv.Nageta and Takebc's(NTM)-1972
3.Preparation of nutrient medium :
         Basically we prepare stock solution of nutrient medium maintaining PH 5.7to5.8
for one liter of nutrient medium we use
Macro nutrient -50ml
Micro nutrient -5ml
Iron source -5ml
succorose-30gm/lit
Plant hormone -depend upon 
basically low concentration of auxine and high concentration of cytokynine is used for shoot culture and vice-varsa for root culture .
4.Sterilization:
         Sterilization is prevention of contamination is one of the foremost requirement for in vitro technique .Basically it is divided into 3 groups .
A.physical sterilization
B.chemical sterilization
C.ultra filteration
A.physical sterilization:
I.by dry heat method :
      The glass/metals instruments used in culture were treated with ethyl alcohol as well as heated with hot air ovan about 160°C for few hours.
ii.by wet method 
        plastic,cotton and filterpaper are heated under pressure in autoclave for 20min .
iii.Light sterilization:
      A UV light is used in a room before a day of culture of plant and UV light is used in working table before one hour of culture.
B.chemical sterilization:
         Heat and rediation are not used for the sterilization of plant materials .Chemical are used to kill microbs.Sodium hypocjloride are used for sterilization of plant parts and 70% ethyl alcohol are used for cleaning our hands.
C.Ultra filteration:
        Heat sensitive hormone and vitamin are sterilized in this process .We used Milipone filter paper to sterilized such things .
5.Micropropagation:
   It deal with following 
I.establishment of culture 
ii.selection of explants
iii.Inoculation
iv.leveling of culture 

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