1.

An ideal PTC laboratory consists of 2 major rooms and a little room. One major room is for
general lab work like preparation of media and all. The other huge room is for holding
societies under controlled light, temperature and moistness. The little room is a storage crll
for aseptic autoclaved glasswares.
Washing Area:
It contains a huge sink with the facility of running hot and cold water, brushes of different
sizes, cleanser and a can of single refined water for a last flush of the washed glass
merchandise.
Hot Air Oven:
It is generally a normal oven that’s is used to heat sterilize most of the glasswares and
equipments. It is also equipped with a tray like structure that can be used to contain all the
other glass wares to be sterilized, all at a once in a stove.
Fridge:
Here all the thermos- labile synthetics like nutrients, chemicals, amino acids, casein-
hydrolysate, yeast extricate, coconut milk, and so on, are stored. Stock arrangements of salts
are kept in a same way for further use.
Gauging Balance:
Three sorts of gauging balances viz. container balance; substance equilibrium and electric
equilibrium are needed for gauging synthetic compounds, sugars, agar-agar and others.
pH Meter:It is fundamental for the estimation and adjustment of pH of the supplement
medium
Vacuum Pump:
It is needed for separating fluid media, sugar arrangement and so forth through channel
contraption us-ing air attractions.
Autoclave:
It is vital for disinfection of nu-trient media, glass products, instruments, and so on
Research center for Aseptic Inoculation:
This room ought to be with no window or ventilator to make it sans dust. The room ought to
be furnished with swinging doors. The entryways ought to have a programmed entryway
closer. In-side floor ought to be fitted with an elastic mat to work with cleaning. For going
into the room, shoes ought to be left outside.
For aseptic work, a huge wooden chamber (Ca, 4′ x 4′ x 7′) is made for transient work. Upper
portion of the side dividers of the chamber is made of huge glass sheets. The chamber ought
to likewise have swinging doors furnished with an entryway closer.
Applications:
• Micro propagation utilizing meristem and shoot culture to create enormous quantities
of indistinguishable people.
• Screening projects of cells, instead of plants for profitable characters .
• Enormous scope development of plant cells in fluid culture as a wellspring of optional
items .
• Crossing indirectly related species by protoplast combination and recovery of the
novel half breed .
• Creation of dihaploid plants from haploid societies to accomplish homozygous lines
all the more quickly in rearing projects.
• As a tissue for change, trailed by either momentary testing of hereditary develops or
recovery of transgenic plants.
• Expulsion of infections by proliferation from meristematic tissues.
2. Role of nitrogen
• Nitrogen is a very useful macronutrient as it is a constituent of some of the important
biological compounds like amino acids, proteins, certain hormones and chlorophyll.
Therefore it can help in the nourishment of the plantelets.
• It affects the tissue growth in an indirect way by influencing the ph of the medium.
• Morphogenic response of the plant tissues can be influenced by using different forms
of nitrogen like NH4+ and NH3+ ions. For example deficiency of NO3- can lead to
the formation of anthocyanin, a flavonoid that acts as an antioxidant.
Role of Calcium

• Calcium is a macronutrient that forms a very useful compound called calcium pectate
that is present in the cell walls and is a very useful component of the cell wall.
• Calcium helps in the inhibition of cell extension by promoting callose deposition.
• Calcium forms an ion exchange channel in the stomatal cells, which makes these pore
like cells even more active. They remain open in the presence of Ca2+ ions.
• As calcium is not very mobile in plants, so it may result in the calcium deficiency due
to non uniform concentration of the calcium. i.e. why the leaf tips start dying under
such conditions. That is why the higher concentration of calcium is very helpful in
establishing the uniform growth of the plant.
Role of magnesium
• Magnesium is a component of chlorophyll and a co factor for many enzymatic
reactions.
• Magnesium uptake is not usually limited, except at low pH.
3. Differentiate between solid and suspension media? Suppose the standard media
concentration is 5 gm of salt in 0.5 litres of solution. How will you make 0.05 litre (final
volume) of the media without standard concentration being altered?

Difference between solid and suspension media

Suspension media
• It is a liquid media without any gelling agent means there is no gelling agent in it that
can solidify it, so it remains in a liquid state.
• Mostly suspension cultures are made in it.
• Shoot proliferation is directly proportional to media concentration.
• Chlorophyll dominance is inversely proportional to media concentrations, means the
explant can be fully green even at lower concentration of liquid media but as the
concentration of media increases the chlorophyll pigment starts decreasing.

Solid media
• It is a media containing gelling agent like solidifying agar or agar agar, that’s why it is
known as a solid media.
• Shoot proliferation is inversely proportional to media concentration means, the
proliferation can still happen at minimum concentrations also.
• Chlorophyll dominance is directly proportional to media concentration, higher
concentration of media gives more green coloured plantelets.
4. Briefly explain the role of Tween-20 and Mercuric chloride in surface sterilisation of
seeds? What practice would lead to enhanced efficacy of Mercuric chloride?

Role of Tween 20
• For the surface sterilization of an explant, viscous yellow colored Tween 20 detergent
is used.
• It helps in removing chemical agents like herbicides, pesticides and fertilizers etc.
• It used in PTC because it can form a very stable oil and water emulsions, therefore it
acts as a good emulsifying agent.
• It also increases the surface area and removes the chemical contaminants.

Role of Mercury Chloride (HgCl2)

• 0.1% of HgCl2 is used as a disinfectant to remove the biological contaminants,

therefore it can be used as an antimicrobial solution.
• It is prepared in autoclaved distilled water because if it is prepared in the normal
distilled water then it may react with the metals present in it, then it is finally
sterilized by using syringe filters.
• 0.1% concentration is used because it is highly toxic in nature and may kill an
explant. Therefore higher concentrations are avoided

How to enhance the sterilizing efficiency of HgCl2

• Sterilization procedures may be enhanced by placing the material in a 70% ethyl

alcohol solution prior to treatment with another disinfectant solution then rinsing with
0.1% HgCl2for 1.0-1.5 min is done to achieve complete sterilization.
• The use of a two-step sterilization procedure has proven beneficial with certain
species.
• Using a wetting agent, such as Tween 20 or 80 the disinfectants to reduce surface
tension and allow better surface contact.

5. Seed dormancy is a natural phenomenon in seeds in which the seeds prevent themselves
from germinate even in the presence of favourable conditions like soil humidity, temperature,
nutrients etc. This is one of the stage in the natural seed germination process, which occurs
deliberately because seeds require a certain amount of time to rest before undergoing the
process of germination and this time differs from seed to seed.

Significance of seed coats in maintain seed dormancy

 • Seed coats are naturally hard in nature that prevents the entry of all the favourable
components like oxygen, water and other nutrients, make them torpid or dormant for a
while.
• On the other hand, while performing in vitro propogation of seeds, the seed coats are
first of all soaked on muslin cloth in autoclaved water for around 20 mins so that the
seeds can soften, often resulting in the removal of hard seed coat layer. As a result,
the dormant or torpid stage is now disrupted and the seed can now proliferate in a
lesser time.

Role of phenolic compounds in seed coats

• Phenolics are the aromatic benzene ring structures having more than one hydroxyl
groups.
• Phenolics are antioxidant, anticarcinogenic, anti-thrombotic, anti-ulcer, anti-
artherogenic, anti-allergenic, anti-inflammatory, antioxidant, immunemodulating,
anti-microbial, cardioprotective and analgesic agents.
• Phenolics are also responsible for hardening of seeds, therefore for dormancy also.
• For ex. Epicatechin, a phenolic compound, in the seed coat of wild soybean is
responsible for its hardness.