Plant & Animal Tissue Culture

Experiment 1

Introduction

An ideal tissue culture laboratory should have at least two big rooms and a small room. One big room is for general laboratory work such as preparation of media, autoclaving, distillation of water etc. The other big room is for keeping cultures under controlled light, temperature and humidity. The small room is for aseptic work and for keeping autoclaved articles.

A tissue culture laboratory should be provided with the following equipment, lab wares and facilities:

1. A Washing Area: This is very important for a tissue culture laboratory. It should be provided with a large sink, running hot and cold tap water, brushes of various sizes, detergent and a bucket of single distilled water for a fine rinse of the washed glass goods. A number of plastic buckets are required for soaking the glass goods to be washed. Another separate bucket with lid is also required for disposing off the used or infected media before cleaning. Only this bucket should be kept outside of the room or cleaning area and should be cleaned twice in a week.

2. Hot Air Oven: For drying washed glass goods.

3. Refrigerator: Essential for storing various thermo labile chemicals like vitamins, hormones, amino acids, casein hydrolysate, yeast extract, coconut milk etc. Stock solutions of salts are also kept to prevent contamination.

4. Distillation Unit: Two big carboys are required for storing the distilled water.

5. Weighing Balance: Pan balance, chemical balance, electric balance and electronic balances

6. pH Meter: Adjustment of pH of the nutrient medium and solutions

7. Autoclave: Moist sterilization unit

8. Working Tables: Preparation of the medium

9. Heater: Needed for heating or warming the medium to dissolve agar or to melt the agarified medium.

10. Microscopes: Simple, compound, inverted binocular stereo dissection microscopes, some attached with camera and computer

11. Glass Stands: For keeping chemicals

12. Inoculation Room: This room should be without any window or ventilator in order to make it dust free. The rooms should be provided with double doors. The doors should have an automatic door closer. Before entering the room shoes should be kept outside. Also the room provided with air curtains if there is no double door. Laminar air flow cabinet: Horizontal Maintains sterile working area. Air blows directly at the investigator Enough to protect your cultures but not you. Air is recycled and goes through HEPA filter before being returned to the room. Such filters remove particles larger than 0.3µm. The ultraclean air which is free from fungal and bacterial contaminants, flows at the velocity of about 27±3m/minute through the working area. All the contaminants are blown away by the ultra-clean air and thereby an aseptic environment is maintained over the working area. Before starting work, laminar air flow is put on for 10-15 minutes. The flow of air does not put out the flame of a spirit lamp. Therefore, a spirit lamp can be used conveniently during the work.

13. Culture Room: For incubation of cultures under controlled temperature, light and humidity. The culture room with double doors in order to make it dust free and to maintain a constant room temperature. One should enter the room keeping their shoes outside the door. Maintain the temperature 25±2^oC inside the culture room. Air coolers are used. Specially designed stand to keep culture vessels. A thermometer and a hygrometer are fixed on the wall at the safety corner of the room to check temperature and relative humidity, respectively. The relative humidity of the culture room is maintained above 50%.

Procedure

Culture of Organized Structures

Organ culture is used as a general term for those types of culture in which an organized form of growth can be continuously maintained. It includes the aseptic isolation from whole plants of such definite structures as leaf primordia, immature flowers and fruits, and their growth in vitro. The most important kinds of organ culture are:

1. Meristem cultures: They are grown as very small excised shoot apices, each consisting of the apical meristematic dome with or without one or two leaf primordia. The shoot apex is typically grown to give one single shoot.

2. Shoot tip or shoot cultures: It starts from excised shoot tips or buds, larger than the shoot apices employed to establish meristem cultures, having several leaf primordia. These shoot apices are usually cultured in such a way that each produce multiple shoots.

3. Node cultures: The separate lateral buds, each carried on a small piece of stem tissue or stem section carrying either single or multiple nodes can be cultured. Each bud is grown to provide a single shoot.

4. Isolated root cultures: The growth of roots, unconnected to shoots through which a branched root system may be obtained.

Initiating Tissue Cultures

Explants: Tissue culture starts from pieces of whole plants. The small organs or pieces of tissue that are used are called explants. The part of the plant (mother plant) from which explants are obtained depends on the kind of culture to be initiated, the purpose of the proposed culture and the plant species to be used.

Plants growing in the external environment are invariably contaminated with micro-organisms and pests. These contaminants are mainly confined to the outer surfaces of the plant, although, some microbes and viruses may be systemic within the tissues. Since they start from small explants and to be grown on nutritive media that are also favorable for the growth of microorganisms they must be free from microbial contaminants when they are first placed on a nutrient medium. This usually involves growing mother plants in ways that will minimize infection, treating the explant material with disinfecting chemicals to kill superficial microbes and sterilize the tools used for dissection and the vessels and media in which cultures are grown.

Isolation and incubation: The work of isolating and transferring cultured planting material is usually performed in special rooms or Laminar air flow cabinets from which microorganisms can be excluded. Cabinets used for isolation are placed in a special inoculation room reserved for the purpose. Cultures, once initiated are placed in incubators or growth rooms where lighting, temperature and humidity are controlled. The rate of growth of a culture will depend on the temperature regime adopted.

The cultural environment: Plant tissue cultures are commenced by placing one or more explants into a pre- sterilized container of sterile nutrient medium. Initiate several cultures at the same time, each being started from an identical organ or piece of tissue. Explants taken from mother plants at different times of the year may not give reproducible results in tissue culture. This may be due to variation in the level of external contaminants or because of seasonal changes in endogenous (internal) growth regulator levels in the mother plant.

Pattern of Growth and Differentiation: A typical unorganized plant callus, initiated from a new explant or a piece of a previously-established culture has three stages of development, namely: the induction of cell division; a period of active cell division during which differentiated cells lose any specialized features they may have acquired and become dedifferentiated; a period when cell division slows down or ceases and when, within the callus, there is increasing cellular differentiation.

Conclusion

Plant tissue culture is technique helps to produce exact copies of same plant which lead to many desired chracters.

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

Questions

1. What is in vitro micro propogation?

2. what is direct organogenesis?

3. What is indirect organogenesis?

4. Name different factors affecting growth of explant in plant tissue culture?