CENTER FOR PLANT TISSUES CULTURE AND

BIOTECHNOLOGY

Summer training(7th may-7th June)

Report on some techniques of plant tissues culture,

phytochemicals, microbiology and bioinformatics

By: Subhalaxmi Swain

Expt: 1

Aim of the experiment:

To prepare the MS- B2 or the BAP (benzenyl Amino Purine) medium for the plant tissue culture.

Principle:
For tissue culture we need to first prepare the media which contain all the nutrient , vitamins ,
organic and inorganic salts and the growth factors also so that the plant can easily grow in our
laboratory. There are different type of medias are used in plant tissue culture like MS media, N6
media, B5 media and B2 media. Here we are making the MS- B2 media.

For preparation of MS-B2 media:

Components Stock(/lit) For 250ml

Stock A1 50ml/lit 12.5ml
StockA2 50ml/lit 12.5ml
Stock B 5ml/lit 1.25ml
Stock C 5ml/lit 1.25ml
Stock D 5ml/lit 1.25ml
Stock E 5ml/lit 1.25ml
Sucrose 30gm/lit 7.5gm
Hormone 2ml/lit o.5ml
Agar 4gm/lit 1gm

Requirements:

Chemical requirements:
 All the stock solutions (stock A to stock E)
 Autoclaved Distilled water
 Agar powder
 Sucrose
 Hormone (BAP)

Apparatus and other requirements:

  Beaker
 Measuring cylinder
 Test tube
 Aluminium foil
 Reagent bottle
 Amber bottle
 Spatula
 Cotton plug
 Weighing balance
 Ph meter
 Autoclave

Procedure:
1. Test tubes and beakers were taken and washed with the labeolin.
2. Then the test tubes and beakers were dried and cotton plugs were prepared for the test
tubes.
3. Then the MS- B2 media was prepared according to the chat and the ph of media was
maintained up to 5.7.
4. Then the prepared media was distributed equally among the test tubes and the test tubes
are plugged in with the help of cotton plug.
5. All the tubes were autoclave for 25mint.
6. Then all prepared media was placed in the cultured room for further use.

Fig: after the media was prepared and was stored for further use in the culture room.

Conclusion: The MS- B2 media is prepared and stored for further use.

Expt: 2
Aim of the experiment:
Initiation of Rauvolfia serpentina.

Requirements

Chemical requirements:
 Bavistin
 Hgcl2
 Autoclaved distilled water

Apparatus and other requirements:

 Explants( Rauvolfia)
 Laminar air flow
 Gas burner
 Culture tube
 Beakers
 Forceps
 Blade
 Petri dish
 Tissue papers
 70% ethanol
 Cotton

Procedure:
1. The explant was taken from the in vivo condition and soaked in 0.1% of Bavistin
solution for 10 to 15 minutes.
2. After this the explants were washed with running tap water for removal of excess
Bavistin solution.
3. Then the explants were washed with the autoclaved distilled water inside the laminar
air flow and transfer the explants were transfer into the UV sterilize beaker.
4. Then the explants were surface sterilized with the Hgcl2 for 2 to 4 minutes.
5. Then the explants were washed with autoclaved water for 3 times.
6. The excess part of the explants was cut with the sterilized forceps and blade.
7. Then it was inoculated inside the test tube containing media.
Fig: initiation of Rauvolfia serpentina

Conclusion: The inoculation of the explants was successfully done

Expt3

Aim of the experiment:

Multiplication and rooting of sugarcane plant to another fresh media.

Requirements:
 Given sample
 Petri plates
 Forceps
 Blade
 Autoclaved tissue papers
 Culture tube containing media
 Gas burner
 70% ethanol
Principle:
Due to the depletion of nutrients present in media we have to transfer the plant into another fresh
media for better growth and development. Then the plant is again transfer into the rooting media
for root development.

Procedure:
1. The sugar cane bunch was taken out from the old media and placed on the tissue paper.
2. Then the bunch was cleaned with the help of forcep and blade.
3. The bunch was taken by the help of forcep and was placed into the culture tube
containing fresh media.

Conclusion:

The plant is placed in the culture room after transferring into the fresh media for further growth.

Fig:1 and fig:2- rooting and multiplication of sugarcane plant.

Expt: 4

Aim of the experiment:

To extract the essential oil from the leaves of Ocimum gratissimum.

Requirements:
 Leaves of Ocimum gratissimum
 Chopping board
 Knife
 Weighing balance
 Tray
 Clevenger apparatus
 Distilled water

Principle:
Essential oils are the pure and volatile extraction from plant. It can be extracted from the seeds,
flowers, leaves, fruits and root also. They are having low boiling temperature and volatile at
room temperature. We can use this oil in cosmetics and also in pharmaceutical industries. We
can extract the oil by the help of Clevenger apparatus. This apparatus is also known as
hydrodisstilation apparatus because in this we boil our sample with the water to collect the
essential oil. The principle of Clevenger apparatus is on the basis of the method commonly used
in obtaining the volatile oil from plant product. This is carried out either by passing steam or
water through a suitable vessel containing the plant material and condensing the steam or by
boiling the material with water in a suitable vessel distilling and collecting the distillate. The
volatile oil is carried over with the steam and condenses with it. Being only slightly soluble in
water it subsequently separates from the aqueous portion of the distillate in layers.

Procedure:
1. 75gm of leaf samples were taken and finely chopped with the help of knife.
2. Then the Clevenger apparatus was washed properly and set accordingly.
3. The finely chopped samples were taken and placed inside the apparatus with some
amount of water.
4. Sample was boiled and the oil was collected.
 Fig1: Clevenger apparatus

Observation:
400µl of essential oil is collected.

Calculation:

%yield = ( yield ÷ raw content )× 100÷ 1000

Yield = 400µl, raw content = 75gm

( 400 ÷ 75 ) ×100 ÷1000 = 0.533%

Conclusion:
400µl of essential oil is collected from 75gm of given sample and the yield is 0.533%.
Expt: 5

Aim of the experiment:

Solvent extraction from the given sample.

Requirements:

Chemicals requirement:
 Methanol

Apparatus and other requirements:

 Cotton
 Tap water
 Chopping board
 Knife
 Sample(Zingiber officinale)

Principle:
For non volatile secondary metabolites, we use an apparatus know as soxhlet apparatus.
Secondary metabolites are extracted semi continuously with organic solvent like methanol,
acetone, hexane etc. solvent is heated and volatized then is condensed above the sample. Solvent
drips onto the sample and soaks it to extract the secondary metabolite constituents at 15-20 min
interval the solvent is siphoned to the heating flask to start the process again.

Procedure:
1. 100gm of given sample was taken and chopped with the help of knife.
2. Then the chopped sample was put inside the apparatus along with the solvent (methanol)
and distillation unit was fit above the apparatus.
3. Then the whole system was switched on and running for next 72 hours. Then the extract
was collected and filtered. Again the reflux was set and run for two complete siphon and
the reflux methanol was collected.
4. The extract was dried and measure the amount of content.
 Fig1: Soxhlet apparatus, fig2 and fig3: extract collected from Curcuma
amada

Observation:
Wt. of blank beaker = 51.99gm

Wt. of beaker containing the extract = 52.74gm

Calculation:
Amount of extract from 10gm of given sample = 52.74- 51.99 = 0.75gm

Conclusion:

0.75gm of extract is collected from the given sample.

Expt: 6

Aim of the experiment:

Antimicrobial activity of C. amada essential oil by agar well diffusion method.

Requirements:
 Bacterial culture
 Nutrient media
 Distilled water
 Conical flask
 Cotton
 Gas burner
 Petri plate
 Weighing balance

Procedure:
1. 1.3gm of nutrient broth powder and 1.5gm of agar powder was weighed. Then mixed
with 100ml of distilled water to make the media and the media was autoclaved for
15minutes.
2. Then the media was poured into the petriplate and leave it for solidification.
3. The bacterial culture was spread on the media containing petriplate and the well was
made by the well maker.
4. Then the essential oil was poured into the well and the plate was stored for incubation.

Observation:

The inhibition zone was observed.

Conclusion:
From this above experiment we can know the antimicrobial activity of C. amada essential oil.

fig: antimicrobial activity of essential oil

Expt: 7

Aim of the experiment:

To prepare the vermicompost

Requirements:

 Container
 Bricks
 Sand
 Green leaves and dry leaves
 Paper wastes
 Cardboards
 Water
 Fresh cow dung
 Straws
 Gunny bag

Procedure:

The brick pieces were placed on bottom of the container, and then the sand was
placed on the top of the brick. Then the layer was prepared by putting the cow
dung, paper waste, green leaves, straws and dry leaves respectively. Then this was
covered by the gunny bag and was stored for few months.
 Bioinformatics:
Bioinformatics is a inter disciplinary field of science in which we can study the biological
problem by the help of the information technology, he computer science, mathematics and the
statistic.

It is a field that holds great potential for revolutionizing biological research in the coming
decades. Development of tools for elucidation of the function and interaction of all genes
products in cell by integration of disparate fields of biological knowledge and a variety of
complex mathematical and statistical tool and techniques.

Database:
A structured collection of data held in computer storage especially one that incorporates software
to make it accessible in a variety of ways; transfer any large collection of information.

Two types of databases are there in bioinformatics

1. Primary database
2. Secondary database
3. Composite database

Primary database:

 Swiss- prot and PIR for protein

 Gene bank and DDBJ for genome sequence
 PDB for protein structure

Secondary database:
  COGS and CluSTRr for protein
 FlyBase for genome sequence
 CATH and SCOP for protein structure

Composite database:

 NCBI
 OWL
 nr

Swiss prot+ Tr- EMBL + PIR= uniprot Kb

we can align the sequences by different method

Based on the number of sequence it is of two type

1. pair wise sequence alignment: compare two sequences

2. multiple sequence alignment : compare multiple sequences

Based on the site it is of two type

1. Global alignment: compare the entire length of the sequence.

2. Local alignment: compare certain region , avoid gaps

Methods of pair wise alignment:

1. Dynamic programming
2. Heuristic method: that is able to produce an acceptable solution to the problem. Example-
BLAST
3. Probabilistic method

BLAST (Basic Local Alignment Search Tool ) :

 Nucleotide blast: search nucleotide sequence with nucleotide sequence

 Blast x: search translated nucleotide with protein
 Protein blast: search protein sequence with protein sequence
 T blastn: search protein with translated nucleotide

Scoring of alignment:
For DNA- DNA identify matrix

For protein- BLOUSM, PAM

Phylogenetic Tree:
We can analysis the Phylogenetic of the sequences by different method

 Similarity of the sequence is more then we should go for maximum

parsimony.
 Sequence similarity is average then we should go for distance method.
(NGA and UPGMA)
 Sequence similarity is low then we should go for maximum likelihood
method.

(Q)How to do this?

Ans: uniprot

Sequence

Copy and paste (text folder)

Install mega software

Create a FAST file

Open the file

Edit (select all align with clustalW)

Data (expert alignment)

Mega format file

Open (phylogeny)

Compute the sequence

Tree