Plant & Animal Tissue Culture

Experiment 3

Introduction

Contamination that results from improperly sterilized tissue will generally arise from the explant and be located in the medium adjacent to the explant. Contamination due to poor technique generally will appear over the entire agar surface. Contamination of cultures by fungi appears as a fuzzy growth. Bacterial contamination appears as smooth pink, white, or yellow colonies. If contamination is due to poor technique, contaminated transfer hood filters, or improperly sterilized media it will be scattered on or in the medium. Contamination from insects will generally appear as tracks across the medium, which are visible due to bacterial or fungal growth on the insect tracks.

The establishment of sterile cultures can be a major challenge with some plant materials. Additionally, the initial process of cleaning and disinfecting the plant material, especially if the parent plant is rare and the supply is limited, can be time-consuming and expensive. Not only can the establishment of clean cultures be a problem, but also subsequent loss of cultures due to insect infestations, which bring in fungal and bacterial contamination, can be devastating.

Materials required

• MS Medium (ready to use)

• Stock solutions of Hormone 2,4-D, BAP and IAA

• For sterilisation: Mercuric chloride, alcohol, chlorine bleach solution, Tween-20

Sodium hypochlorite (NaOCl), found in laundry bleach, is approximately a 5.25% (v/v) solution. Calcium hypochlorite (Ca(OCl)2) at 0.8% (w/v) (8 g calcium hypochlorite in 1 litre water, stir for 10–15 min, allow to settle, decant solution, and mix with equal parts of water), add 2 drops of Tween-20/100 ml, treat tissue for 5–30 min.

• Ethyl or isopropyl alcohol at 70% (v/v)

• Mercuric chloride (HgCl2)(0.1%) is a very hazadorous chemical that is often reported to be used as a topical explant sterilant.

• Sucrose

• Agar agar or Gelrite

• Autoclaved deionized water

GLASSWARES

• Glass beakers

• Conical flasks

• Measuring cylinders

• Borosilicate test tubes 25 x 150 mm or plant tissue culture bottles or 150 ml capacity flask

MISCELLANEOUS

• Metal wired (Corrosion free) tray For incubation of plant tissue culture bottle in PTC lab.

• Metal wired (Corrosion free) test tube racks capacity 24-25 test tubes per rack

• Cotton role (Absorbent and non-absorbent)

• Tissue role

• Aluminium foil

• Muslin cloth

• Test tube caps

• Plastic spray bottles

• Cotton plugs

INSTRUMENTS

• Magnetic stirrer with hotplate

• pH meter

• Refrigerator for media storage

• Micropipets (Variable range)

• Digital balance

mg accuracy for measurement of plant growth regulators and micronutrients

Gm accuracy for measurement of macronutrients and general media preparation

• Forceps 15 cm plus for inoculation of explant

• Surgical blades (Specification 23/24) and Blade holder (Specification 3 and 4).

• Microwave oven

• Autoclave

• Laminar cabinet:

A laminar air flow hood is extremely useful for aseptic transfer work. A laminar flow hood creates a positive air pressure toward the worker and creates a flow of clean air over the work area. High efficiency particulate air (HEPA) filters are “absolute” or 99.97% free of 0.3-µm particles.

• Flame and glass bead sterilizers

• Plant Growth Chambers (With Photoperiod, temperature and humidity controller).

• Plant tissue culture incubation room with tissue culture racks (Photoperiod control; With attached AC, and regular power supply).

• Hot-air Oven

Procedure

Medium Preparation: 1-litre equivalent to Seed Germination Medium.

1. Into a 2000-ml Erlenmeyer flask pour 500 ml of deionized, distilled water.

2. Prepare MS medium from stock solution or ready to use powder

3. Adjust volume to 1000 ml.

4. Use combination of 2 mg/L of 2,4-D with 0.5 to 1 mg/L of BAP or Thidiazuron or Kinetin

5. Adjust pH to 5.7-5.8 using 1 N HCl or NaOH.

6. Add 8 g/litre agar or agar brand of choice. Hydrolyse it in Microwave oven.

7. Distribute 10 ml/culture tube (25 × 150 mm). Cap them.

8. Prepare sterile rinse water: 250-ml Erlenmeyer flask with ~100 ml distilled water. Cap them using cotton plugs.

9. Autoclave for 15 min at 121°C and 15 psi.

Explant Preparation (In laminar): (Garlic clove, Vinca rosea leaves, Mint leaf, leaves of any plant with midrib).

1. Take explant and wash them thoroughly under tap water

2. Wash them with mild detergent.

3. Later, bring them to laminar air flow hood and wash them with distilled water.

4. Rinse 2–3 min in freshly prepared 0.1% mercuric chloride aqueous solution. Decant mercuric chloride. and wash them twice with sterile distilled water.

5. Rinse 2–3 min in 70% alcohol. Decant alcohol.

6. Wash 2–3 times with autoclaved distilled water.

7. Cut leaves ~2 cm x 2 cm disc (quadrate) with midrib. Cut the end of garlic cloves and inoculate them.

8. Inoculate on medium and keep cultures in dark condition for one week for callus induction.

Observations

Observe those tube for development of callus.

(after one month)

Plant name and date of inoculation___________________ Date of observation________________

Add % response of callus formation in the observation table for e.g. 2 tubes out of 10 tubes shows callus regeneration indicates 20% callus formation. Also indicates the morphology of callus for e.g. colour (White, Yellow, Green), Appearance (Compact, embryogeneic, friable etc).

Results

Reference Material

1. 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

2. 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

3. 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

4. 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

Questions

1. Types of surface sterilizing agents.

2. Why 70% alcohol is used for disinfection?

3. Dose callus use for secondary metabolite production?