Assignment – 2 ( Plant Tissue Culture) | DESIGN INNOVATION CENTER

Tissue Culture

http://vadic.vigyanashram.blog/2021/11/17/tissue-culture/

Plant tissue culture is defined as culturing plant seeds, organs, explants, tissues, cells, or protoplasts on a chemically defined synthetic nutrient media under sterile and controlled conditions of light, temperature, and humidity.
Plant tissue culture is a technique that has been around for more than 30 years.

DAY -1

Clean lab by using Dettol and ethanol.
Rearrange all the needed instruments.
Washing of all glass bottles and tubes which is use in tissue culture.
Stored the Distilled water and sterile the water in autoclave for the media preparation.
Fumigate the lab by using Potassium permanganate and formaldehyde. This method produce a violent chemical reaction that generates considerable heat and releases formaldehyde gas.

Day – 2

Preparation of stock culture-

MS stock solution –

MS media well know as basal media for plant tissue culture studies, supplies all the essential elements such as macro, micro nutrients in highly balanced composition. So it supports invitro growth of plants

2. PGR stock solution –

Plant. growth regulators. ( PGR) are numerous chemical substances that profoundly influence the growth and differentiation of plant cells, tissues and organs.

Day – 3

Media Preparation

MS Basal Media-Basal media contain amino acids, glucose, and ions (calcium, magnesium, potassium, sodium, and phosphate) essential for cell survival and growth. l-glutamine is an amino acid that is essential for protein and nucleic acid synthesis and energy production in cell culture.

MS basal media for seed germination (1000 ml) –
MS+ 0.55 mg/liter BAP (500 ml)
MS + 1 mg/liter BAP ( 500 ml)

Precaution should be taken when prepare the media-

Maintain the pH of the media
pH of media should be 5.8
Autoclave should be done properly

pH adjust at 5.8

Day – 4

Mother plant collection –

Combretum indicum ( Madhumalati)
Frerea indica ( Shiv suman )
Clitoria ternatea ( gokarna)

Clitoria ternatea ( gokarna)

Combretum indicum (Madhumalati)

Explant surface sterilization –

UV treatment for sterilization

An explant is the part of a plant which has got the regeneration potential and is capable to give rise to the whole plant.

Tap water washing with tween 20 or Bavistin
Washing with distilled water inside laminar
Sterile distilled water, glasswares,culture water and miscellaneous and UV treatment 20-30 min.
Wash explant with distilled water twice
Deep explant in 0.1% HgCl2 and wait for 2-3 min
3-5 times wash with D/W
Deep explant in 70% alcohol, for seeds wait 2-3 min and for leaf or nodule section wait 30 sec-1 min
3-5 times wash with D/W
Drain out sterilized explant for inoculation

Inoculation and labelling –

Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions.
All parts of plants can be used as explants for tissue culture purposes: leaves, stems, a portion of shoots, flowers, anthers, ovary, single undifferentiated cells, or mature tissues.

Tissue culture of Tobacco plant

As a explant of tobacco, we were used tobacco seeds which are collected by Dr Lokhande sir from Uttarakhanda

Explant surface sterilization –

An explant is the part of a plant which has got the regeneration potential and is capable to give rise to the whole plant.

Seeds were put in 1.5 ml centrifuge tube.
Sterile distilled water, glassware’s, culture water and miscellaneous and UV treatment 20-30 min.
Wash explant with distilled water twice
Deep explant in 0.1% HgCl2 and wait for 2-3 min
3-5 times wash with D/W
Deep explant in 70% alcohol, for seeds wait 2-3 min and for leaf or nodule section wait 30 sec-1 min
3-5 times wash with D/W
Drain out sterilized explant for inoculation