ASSIGNMENT

HOW TO PREPARE STOCK

SOLUTIONS AND WORKING
SOLUTIONS IN PLANT TISSUE
CULTURE
SUBMITTED TO: - DR. SYED BILAL
HUSSAIN SUBMITTED BY: - BSBTM20
IGHNA JALEEL BSBTM21 MAHNOOR
KHAN
BSBTM22 IQRA ALI YAMEEN
BSBTM23 ALEENA AHMED SOMROO
5TH SEMESTER SESSION (2013-2017)

PLANT TISSUE CULTURE: - Plant tissue culture is a

collection of techniques used to maintain or grow plant
cells, tissues or organs under sterile conditions on a
nutrient culture medium of known composition. Plant
tissue culture is widely used to produce clones of a plant
in a method known as micro propagation
WHAT IS STOCK SOLUTION?
A Stock Solution is a concentrated solution that will be
diluted to some lower concentrated for actual use. Stock
solutions are used to save preparation time, conserve
materials, reduce storage space, and improve the
accuracy with which working lower concentration
solutions are prepared. The stock prepared can be stored
in the freezer and can be used for a long time
WHAT IS WORKING SOLUTION?
Working Solution is a chemical solution made for actual
use in the laboratory, usually made from diluting or
combining stock or standard solutions.
WHY PREPARING STOCK SOLUTIONS?
The amount of nutrients used are considerably very low.
Weighing out the salts for each time media being
prepared is a tedious job as the quantity required is very
small. Therefore, accuracy will not be established.
Preparing the stock solution requires vast quantity and it
is also time saving.
STOCK AND WORKING SOLUTIONS IN PLANT TISSUE
CULTURE
In plant tissue culture, many nutrients are required which
is essential for the growth of plants, these nutrients are
prepared and stored in the form of stock solutions at a
suitable temperature for medium preparations so that
when the nutrients are required they can be used easily.
Stock solutions are generally concentrated form of

solutions through which working solutions are prepared to

be used in laboratory for the plant tissue culture media. It
is a vital step in media preparations
A convenient method for preparation of culture media is
to make concentrated stock solutions which can be
immediately diluted to preferred concentration before
use. Solutions of macronutrients are better to be prepared
as stock solutions of 10 times the strength of the final
operative medium. Stock solutions can be stored in a
refrigerator at 2- 4oC. Micronutrients stock solutions are
made up at 100 times of the final concentration of the
working medium. The micronutrients stock solution can
also be stored in a refrigerator or a freezer until needed.
Iron stock solution should be 100 times concentrated than
the final working medium and stored in a refrigerator.
Vitamins are prepared as either 100 or 1000 times
concentrated stock solutions and stored in a freezer
(20oC) until used if it is desired to keep them for long
otherwise they can be stored in a refrigerator for 2-3
months and should be discarded thereafter. Stock
solutions of growth regulators are usually prepared at
100-1000 times the final desired concentration.
In plant tissue culture, MS media is commonly used MS
medium was found to be the most suitable medium used
for plant regeneration from tissues and calluses. MS
medium was invented by plant scientists Toshio Murashige
and Folke K. Skoog during Murashige's search for a new
plant growth regulator. Initially, the media was produced
based on mineral analysis of tobacco. Since the MS media
contains high amount of nitrogen fixing agents, it was
found out that the MS medium was equally compatible for
other plant species as well.
Nutrients required for plant tissue culture media are:Macronutrients

Macronutrients are normally required in milli molar (mM)

quantities in most plant media. The macronutrient
normally contains high amount of nitrogen fixing agents in
the form of ammonium (NH4+) and nitrate ions (NO3-)
ions. Apart from nitro genic substances, potassium,
magnesium, calcium, sulphur and phosphorus are also
added as macronutrient in the form of diluted salts. Also,
macronutrients serve as components for structural and
protoplasmic tissue.
Micronutrient
Micronutrients are needed in a very small amount. The low
requirement of micronutrients can be accounted for
participation of these elements in enzymatic reactions and
as constituents of growth hormones. The micronutrient is
the mixture of boron, cobalt, manganese, molybdenum,
copper and zinc. Excessive amount added to the medium
can cause suffocation and premature death of the
explants used.
Ferum
Ferum is required in metabolic functions such as nitrogen
fixation, photosynthesis and electron carrier during
respirations electron transfer process. It is usually
present in the form of FeSO4.7H2O and Na2EDTA. In some
cases, ferum is prepared together with micronutrient.
Ferum oxidises in the presence of sunlight. Furthermore,
high concentrations will cause precipitation to occur.
Therefore, preparing ferum and storing the stock solution
in dark environment can prolong the shelf life of ferum
stock solution.
Vitamin
Vitamin is essential as it is involved in carbohydrate
metabolism and the biosynthesis of some amino acids.
Normally thiamine is deemed as the most important
vitamin and it is introduced as thiamine hydrochloride.

Other vitamins like pyridoxine, nicotinic acid are added as

well. Certain plant species requires special requirements
of vitamin like biotin, riboflavin, folic acid and more.
Vitamins are associated with metabolic activity of the
plant. Therefore, to have good yield, sufficient amount of
vitamin should be added to the MS medium.
Plant growth regulators
Plant growth regulator (PGR), functions in initiating the
root and shoot development of explants and embryos.
They also stimulate cell division and expansion. Certain
parts of plants have plant growth regulators readily
available in the explants. In cases of PGR absence, PGR
supplemented through medium enables growth of the
explants. There are two major classes of PGRs; cytokinin
and auxin. Cytokinin (e.g .BAP) is used to generate shoots
while auxins (e.g. NAA) are used to induce roots.
Inconsiderably high concentrations, certain explants
prefer callus formation. Therefore, PGRs can be seen as
the growth inducers in plants
Carbon source
In plant cell culture media, besides the sucrose, frequently
used as carbon source at a concentration of 2-5%, other
carbohydrates are also used. These include lactose,
galactose, maltose and starch and they were reported to
be less effective than either sucrose or glucose, the
similarly more effective than fructose considering that
glucose is utilized by the cells in the beginning, followed
by fructose. It was frequently demonstrated that
autoclaved sucrose was better for growth than filter
sterilized sucrose. Autoclaving seems to hydrolyze sucrose
into more efficiently utilizable sugars such as fructose.
Sucrose was reported to act as morphogenetic trigger in
the formation of auxiliary buds and branching of
adventitious roots. It was found that supplements of sugar
cane molasses, banana extract and coconut water to basal

media can be a good alternative for reducing medium

costs. These substrates in addition to sugars, they are
sources of vitamins and inorganic ions required growth
The formula used for calculating the volume of stock
solution that we need to make our total volume of
solution.
C1V1=C2V2
Example:You are given a solution of amyloseazure at
2 mg/ml. You want your final concentration
to be 0.5 mg/ml in a 5 ml reaction. How much volume
of the stock amyloseazure solution will you use for th
at 5 ml reaction?
C1 = 2 mg/ml stock solution
V1 = volume of stock solution you need; this is your unkno
wn you solve for
C2 = 0.5 mg/ml
V2 = 5 ml
V1 = (0.5 mg/ml)*(5 ml) / 2 mg/ml
V1 = 1.25 ml
Each 5 ml reaction will contain 1.25 ml of 2 mg/ml amylose
azure solution + 3.75 ml of other liquids. If you started wit
h a stock solution of 5 mg/ml amyloseazure, the volume of
stock solution you would use would change, while the final
concentration and final volume would not. The final conce
ntration is the only concentration reported.