Plant & Animal Tissue Culture

Experiment 2

Principle

Media preparation is one of the primary and most essential steps in tissue culture. Media is prepared based on the type of tissue being cultured. Most media differentiate from each other based on the requirements of the growth of the specimen it supports.

• To understand the basic process of preparing media for plant tissue culture to promote.

• Growth and differentiation of tissues and acknowledge the basic requirements for the plant Tissue culture.

• To prepare culture medium based on plant species requirements.

• To prepare stock solution consists of macronutrient, micronutrient and organic elements.

Materials required

• Chemicals

• Ready to use MS medium or Stock solutions for Macronutrients, Micronutrients, FeSO4 EDTA and Vitamins as listed below:-

• Stock Solution A (20 X solution 1000 ml)

• MgSO_4.7H₂0 : 7.4 g

• KH₂PO₄ : 3.4 g

• KNO₃ : 38 g

• NH₄NO₃ : 33 g

• CaCl₂.2H₂O : 8.8 g

• Stock Solution B (200 X solution 1000 ml)

• H₃BO₃ : 1.24 g

• MnSO₄.4H₂O : 4.46 g

• ZnSO₄.7H₂O : 1.72 g

• Na₂MoO₄.2H₂O : 0.05 g

• CuSO₄.5H₂O : 0.005 g

• CoCl₂.6H₂O : 0.005 g

• KI : 0.166 g

• Stock Solution C (20 X solution 1000 ml)

• FeSO_4.7H₂0 : 0.556 g

• Na₂EDTA : 0.746 g

• Stock Solution D Vitamins (200 X solution 1000 ml)

• Nicotinic acid : 0.1 g

• Thiamine : 0.02 g

• Pyridoxine HCl : 0.1 g

• Glycine : 0.4 g

• OTHER

• Sucrose

• Myoinositol

• Agar-agar or Gellangum (Gelrite/Phytagel/Clerigel).

• NaOH/KOH and HCl (for pH)

• PLANT GROWTH REGULATORS

• Either prepared as molar stock solution or mg/ml

• Molar solution: A 1 M solution contains 1 mole of the solute in 1 liter of the solution. A mole is the number of grams of the solute that equals its molecular weight.

• Auxin or auxin like compunds (50 mg/50 ml stock solution)

• 2,4-D

• IAA

• IBA

• NAA

• Picloram

• Dicamba

• Cytokinin or Cytokinin like compounds (50 mg/50 ml stock solution)

• Kinetin

• BA/BAP

• Zeatin (Trans or Cis) or Zeatin ribosides

• Thidiazuron

• Metatopoline

• Adenine sulphate

• Other plant growth regulators (50 mg/50 ml stock solution)

• Gibberellic acid

• Abscisic acid

• Tri-iodo-benzoic acid (TIBA)

• Other Chemicals

• Labolene/ Teepol

• Tween 20

• Chromic acid

• Nitric acid

• Potassium dichromate

• Formaldehyde

• Potassium permanganate

• Sterilization of explants

• Carbendazim

• Mencozeb

• Mercuric chloride

• Sodium hypochloride

• Ethanol and/or Isopropyl alcohol

• GLASSWARES

• Glass beakers

• Conical flasks

• Screw cap reagent bottles

• Centrifuge tubes: For storage of plant growth regulators

• Measuring cylinders

• Brown coloured bottles for storage of Stock solution of MS media components

• Borosilicate test tubes 25 x 150 mm: About 1000.

• Plant tissue culture bottles: Small and medium size.

• MISCELLANEOUS

• Cotton role (Absorbent and non-absorbent)

• Tissue role

• Aluminium foil

• Muslin cloth

• Test tube caps

• Plastic spray bottles

Procedure

For preparing stock solutions of media- glass, distilled or purified water and chemicals of high purity (Analytical grade) should be used.

• Outline the medium to be prepared and check off the ingredients as they are added to the flask in which the medium is being prepared. Keep records on the date of preparation and use.

• In media preparation always use sterile-distilled water—never tap water or tap-distilled water. Water (~500 ml for final 1-liter volume) must always be in your flask before the stock solutions are added; otherwise, concentrated stocks will co-react and precipitate out.

• Never pour excess stocks back into the original stock solution container and never put excess sucrose or agar back into the original container. Always clean up spills around balance and work areas.

• Packaged powders of the MS salts and other media are available by eliminating the need to prepare stocks and measure ingredients. Follow the manufacturer’s directions for their use.

Stock A (Macronutrients): Required quantities of salts were weighed and added one at a time in 750 ml of distilled water with constant stirring. CaCl₂.H₂O is dissolved in another flask and added to the previous flask containing remaining salts. The final volume is made up to 1000 ml with distilled water and store at 10˚C ± 1˚c.

Stock B (Micronutrients): Required quantities of micronutrients were weighed and dissolved in 450 ml distilled water. The final volume is made up to 1000 ml distilled water and store at 10˚C ± 1˚C.

Stock C (Iron stock): FeSO₄.7H₂O and Na₂.EDTA dissolved separately in 750 ml of distilled water by heating and constant stirring, mix the two solutions and adjust the pH to 5.5. Final volume is made up to 1000 ml with distilled water. It is stored in brown bottle.

Stock D (Vitamins): The required quantity of components were weighed and add one by one and dissolved completely in 450 ml of distilled water. The final volume is made up to 1000 ml. Store at 10˚c ± 1˚c.

Auxin: It is required by most plant cells for division and root initiation. At high concentrations, auxin can suppress morphogenesis. The auxin 2,4-D is widely used for callus induction: IAA, IBA, and NAA are used for root induction Auxin stocks are usually prepared by weighing out 50 mg of auxin into a 200-ml beaker, adding several drops of 1 N NaOH or KOH until the crystals are dissolved (not more than 0.3 ml), rapidly adding 50 ml of double-distilled water. Auxins can also be dissolved in 95% ethanol and diluted to volume; however, ethanol is toxic to plant tissues. Make IAA stocks fresh weekly; IAA is degraded within a few days by light and within several hours to a few days by plant tissues. Auxins are thermostable at 110–120°C for up to 1 h. However, IAA is destroyed by low pH, light, oxygen, and peroxides; NAA and 2,4-D, which are synthetic auxins, are more stable than IAA, which is the naturally occurring auxin.

Cytokinins: Promote cell division, shoot proliferation, and shoot morphogenesis. Cytokinin stocks are prepared in a fashion similar to that for auxin stocks, except that 1 N HCl and a few drops of water are used to dissolve the crystals. Gentle heating is usually required to completely dissolve crystals. Double- distilled water is rapidly added to avoid the crystals’ falling out of solution. Cytokinin stocks can be stored for several months in the refrigerator.

Result

Prepared stock solutions of MS medium, plant growth regulators are utilized for various types of plant tissue culture experiments like axillary shoot multiplication, callus culture, somatic embryogenesis etc.

Reference Material

• Bhojwani, S.S., Razdan, M.K., 1996a. Laboratory requirements and general techniques, in: Plant Tissue Culture Theory and Practice, a Revised Edition. pp. 19–38. https://doi.org/10.1016/S0928-3420(96)80004-8

• Bhojwani, S.S., Razdan, M.K., 1996b. Tissue culture media, in: Plant Tissue Culture: Theory and Practice, a Revised Edition. pp. 39–62. https://doi.org/10.1016/S0928-3420(96)80005-X

• Smith, R.H., 2013a. Setup of a Tissue Culture Laboratory, in: Plant Tissue Culture. Elsevier, pp. 23–29. https://doi.org/10.1016/B978-0-12-415920-4.00002-5

• Smith, R.H., 2013b. Media Components and Preparation, in: Plant Tissue Culture. Elsevier, pp. 31–43. https://doi.org/10.1016/B978-0-12-415920-4.00003-7

• MS media preparation:www.youtube.com/watch?v=eMv_PMNPYMc

Questions

1. What are macro-nutrients?

2. What are micro-nutrients?

3. What is the function of vitamins in growth of explants?

4. What is the function of sucrose in MS medium?

5. Write the name of substitute of Agar Agar (geling agent).

6. If you need 2.5 mg/liter, 2,4-D in 1 liter of medium and the stock solution of 2,4-D contains 10 mg/100 ml, how much stock solution do you need?

7. If you have a 15 mg/100 ml stock solution of GA3 and you need a 1 mg GA3 in 25 ml, how much stock solution would you add to 125 ml of medium?