The culture media used for in vitro cultivation ofexplants are composed of complex mixture of salts, vitamins, carbon source,solidifying agents, plant growth regulators and antibiotics.
The complexmixture of salts is divided into three groups which are macroelements, microelementsand iron source. The examples of macroelements are N, K, P, Ca, S and Mg andthey are building blocks of amino acid, proteins, nucleic acid, cell wall and enzymecofactors. However, the micronutrients like zinc, copper and cobalt are usuallypart of certain enzymes. The vitamins usually added to promote plant’s growthwhereas the carbon source is needed as the explants have insufficient chlorophyllor maturity to produce food by itself.
The carbon source that commonly used issucrose which is readily assimilated and relatively stable. Thesolidifying agent in this experiment is agar. It plays supporter rolefor cultures and it has many advantages such as it does not react with mediaconstituents, it does not digested by plant enzymes and it can remain stable atall incubation feasible temperature. The antibiotics that prevent growth ofmicrobes are not used because it may inhibit cell growth that is why in thefirst week of observation there is already contaminated by fungus.
There is several precaution steps needed to be carriedout during experiment. For examples, the working areas and equipment that should besterilized with alcohol such as 70% ethanol solution before performing thetissue culture. These protocols describe basic procedures for aseptic techniquein cell culture technology. Firstly, personal hygiene is the one basic concernfor a successful aseptic technique. This is because human skin harbors anaturally occurring and vigorous population of bacterial and fungal inhabitantswhich is shed microscopically and ubiquitously on ours skins which maycontaminated ours experiments.
Most unfortunately for cell culture work, cellculture media and incubation conditions provide ideal growth environments forthese potential microbial contaminants. This procedure outlines steps toprevent introduction of human skin flora during aseptic culture manipulations. Everyitem that comes into contact with a culture must be sterile in order to avoidany contamination on ours experiments.
This includes direct contact as well asindirect contact too. Ideally, all aseptic work should be conducted in alaminar cabinet .However; work space preparation is essentially the same for workingat the bench. Flame sterilization is used as a direct, localized means ofdecontamination in aseptic work at the open bench. It is most often used toeliminate potential contaminants from the exposed openings of media bottles,culture flasks, or test tubes during transfers. Besides, to sterilize small instrumentssuch as forceps or to sterilize wire inoculating loops and needles before andafter transfers. Where possible, flame sterilization should be minimized inlaminar-flow environments as the turbulence generated by the flame cansignificantly disturb the sterile air stream.
In this experiment, cutting methods also the things thatwe need to concern about in order to avoid contamination and to make us easy tohandle the garlic embryos. This is because in laminar flowhood there is notenough space for us to cut the embryo like we cut the garlic in the kitchen. So,we must cut the garlic in order to get the embryo by using forceps and scalpel.
The positions of the garlic must be horizontal because it is easier to dissectthe garlic in horizontal positions. When the garlic is cut in horizontalpositions, it is easy and more comfortable for us to cut and avoid from thegarlic to jump out from the paper that we use to cover up the laminar flowhoodfloor. On the other hand, our lab coat cannot touch the laminar flowhood whencutting the garlic embryo because it may conduct a contamination if our labcoat touches the floor. From the results above, the culturing explants in petridishes are contaminated with fungus from the first week but the explants inculture vials (callus induction) are not contaminated with any fungus orbacteria.
The reason is due to the different source of explants. The source offormer is from outside plantlet while the latter is from the in vitro plantlet.Hence, although the outside plantlet’s was washed and sterilized with Clorox andTween 20, there are may be some dusts or spores hidden inside the cracks ofplant leaf.
In contrast, the in vitro was already in aseptic condition,therefore there will be no contaminants inside the vial.