Callus Culture Technique

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Culture of plant tissue under very control and hygienic condition with sufficient supply of nutrients to develop a whole plantlet is termed as plant tissue culture technology. Plant species serving as all major land plant groups have been shown to be capable of generating callus in tissue culture. It also promotes the amplification of limiting plant material. Callus induction medium contains agar and a mixture of macronutrients and micronutrients for the given cell type.3 There are so many basal salt mixtures used in plant tissue culture, but most noticeable modified Murashige and Skoog medium and White’s medium. Gamborg B5, inositol such vitamins are also provided to enhance the growth. For plant cells, enrichment with nitrogen, phosphorus, and potassium is especially important. Plant callus is a growing mass of unorganized plant cells, i.e. usually derived from somatic tissues. The cells that give rise to callus and somatic embryos usually undergo rapid division or are partially undifferentiated such as meristematic tissue.1

The aim of the experiment is to study the callus formation in a culture medium.

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Sample:              Carrot root

Medium:            Murashige and Skoog (MS) medium (Powdered form)

Apparatus:         Volumetric flask

  Culture tubes

Petri dishes

Pair of forceps

Scissors or scalpel blades


pH meter

Chemicals:        Sodium hydroxide

Hydrochloride acid

Sterilized double-distilled water


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Preparation of MS basal medium

Prepare 1 litre of medium by using one packet of MS (in powdered form). A beaker was filled up of 800 ml of distilled water. Slowly add 4.4 g of MS powdered medium into the beaker. Then add adequate amount of growth regulator and 30 g of sucrose in to it. Adjust the pH of the medium 5.7 to 5.8 by adding 1N HCL and 1N NAOH. Add 8 g per liter of the agar to the beaker and boiled it until the agar mixed fully.

Pour the medium immediately to the volumetric flask and close it by the non absorbent cotton or polypropylene caps. Then autoclave the media at 1210c at 150lb pressure for 15 minutes. Remove the closure (cotton plug) from a culture tube and flame the uppermost 20 mm of the open end. While holding the tube at an angle of 45°, transfer an explant using forceps onto the surface of the agarified nutrient medium. Nutrient medium is Gamborg’s B5 or MS medium supplemented with 0.5 mg/l 2, 4-D.

After inoculation take the culture tubes to the culture room where they can be placed in the racks. Incubate the cultures in dark at 25°C. After 2, 4-D the whole callus mass was taken out aseptically on a sterile petridish and should be divided into two or three pieces and transferred in to a fresh medium.2

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Callus creation is dependent on the growth regulators composition of the media and the transfer of root on fresh medium containing low concentration of auxin – cytokinin. Callus initiation has been implemented for all major groups of land plants.

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  1. Titouh K, Nazim B, Lakhdar K (2017) Microcalli induction in protoplasts isolated from embryogenic callus of date palm. In: JM AK, Jain SM, Johnson DV (eds) Date palm biotechnology protocols volume I. Part of the methods in molecular biology book series (MIMB, volume 1637). Springer, New York, pp 227–237.
  2. Larkin, P. J. and Scowcraft, W. R. (1981) Somaclonal variation – a novel source of variability from cell culture for plant improvement. Theor. Appl. Genet. 60, 197-214.
  3. Al-Mizory LSM, Yaseen SA, Hassan AM (2014) Effect of different explant and different concentrations of plant growth regulators on micropropagation of (Dianthus caryophyllus L.). IOSR J Agric Vet Sci 7:1–9.