Plant tissue culture is a process whereby small pieces of living tissues explants from the plant organs such as embryos, shoots, roots and flower or parts of the plant tissues like the cells, callus and protoplast are isolated from an organisms and grown aseptically on a nutrient medium under controlled conditions (in vitro). The aseptic refer to the growth of plant tissues under condition that is free from microbial contaminants either from the bacteria, yeast or fungi. A proper sterilization technique is important in order to produce a friable and healthy callus as well as to make sure the successful in the tissue culture. All work must be done in the laminar flow and ...view middle of the document...
, 2003). In order to provide an ideal medium for growth of plant cells it is necessary to sterilize the media, culture vessels, tools and instruments. Surface area also need to disinfect and the explants as well. The most commonly used means of sterilizing equipment and media is by autoclaving at 121ºC with a pressure of 15Psi for 15 minutes or longer for large volumes (Trigiano & Gray, 2000). Then, the success in plant tissue culture is depends on the media preparation technique and the uses of the media components. The media components or medium for the growth and development of the explants consists of 95% of water, macro and micronutrients, plant growth regulators which are the hormones, vitamins, sugar and sometimes various other simple to complex organic materials (Dahleen & Bregitzer, 2002). A high efficiency in sterilization technique and media preparation that contains important elements for the growth and development of the explants in vitro will contribute to the formation or induction of the callus on the media.
1) To learn about the sterilization technique and how to make a sterile environment, explants, instruments and media in plant tissue culture.
2) To learn and practice the technique on how to prepare the media for plant tissue culture in a proper and correct way.
3) To induce the formation of callus on the media from the explants.
MATERIALS AND METHODS
Practical 1: Sterilization of tools, instruments and surface of the explants
Explants (seed), Laminar air flow, Clorox, sterile/deionized distilled water, 70% ethanol, conical flask, petri dish, parafilm
1) The explants (bean) were obtained and washed first with tap water.
2) Hand was washed with soap before the work begins and wears gloves.
3) All work must be done in the laminar flow and hands must be sprayed first with ethanol in order to produce a sterile environment and explants.
4) The explants were placed in a sterile conical flask, 70% ethanol was added and the explants were allowed for 1-3 minutes.
5) Then, the explants were rinsed with sterile/deionized distilled water.
6) Clorox was used to sterilize the explants and make sure that the explants were fully covered with the Clorox.
7) Next, the explants were allowed for 20-25 minutes by vigorous shaking.
8) After that, the explants were then rinse with sterile distilled water for several times.
9) The explants were later transferred into a sterile petri dish that contains filter paper which had been moisture with sterile distilled water.
10) Finally, the petri dish was sealed with parafilm and labeled.
Practical 2: Media Preparation
Conical flask, pipette, pH meter, magnetic stirrer, electronic balancer, aluminium foil
1) For the preparation of 100ml media MS, a...