Stage 1 involves the surface sterilization of the original shoot explant from a stock plant, and its establishment in culture ("in vitro" is the term commonly used). The goal of Stage 1 is not increase in plant numbers but simply survival under aseptic (uncontaminated) conditions. This may sound simple, but death of the exlant may occur simply from the trauma of the wounding involved in severing the original explant, which is a challenge with very tiny explants such as the apical meristems used for virus elimination. Another source of loss in Stage 1 is contamination of the culture by microorganisms (bacteria or fungi) due to incomplete surface sterilization. The microorganisms are generally not pathogenic (disease causing) to the explant per se, but rather they can easily overgrow the explant because their growth is accelerated by the abundance of sugar and other nutrients in the plant tissue culture medium.
Growth of the explant obviously presumes its survival, which unfortunately is by no means assured. Survival is an issue mainly because the conditions severe enough to kill all microorganisms on the surface of an explant (soaking in bleach solution) may be nearly severe enough to kill the explant itself. In other words, the micropropagator must walk the fine line between soaking the explant in a bleach solution long enough to kill all the surface microbes but not so long as to damage or kill the explant. The other goal of Stage I, stable growth refers to the fact that initially an aseptic, living explant may be slow to initiate growth, and even require several to many months before sufficient growth occurs for it to be moved on to Stage II. This delayed initiation of growth is especially characteristic of woody plants.
Preparing for this exercise
Read through this writeup and view
the integrated videos from the course CD you have received. In normal commercial
practice, a special piece of equipment called a laminar flow hood [link to slide
66] is used to filter sterilize the air stream in order to minimize the likelihood
of airborn microorganism contaminating your sterile culture vessels and defeating
your attempt to achieve aseptic culture. Although it is possible to construct
a laminar flow hood in your home if you are skilled at such things [link to
Burger plans for hood, slide 81, get permission to use it, with URL, and link
to his site], most of you will probably do the next best thing which is to use
a simplified dust cabinet [**what's a better name for this?] to minimize but
not eliminate air born contaminents. If you follow the procedures covered in
the section on aseptic culture carefully, this dust cabinet, which is nothing
more than a modified aquarium [link to picture of your aquarium dust cabinet
set up in your home**], can be used to achive sufficiently clean conditions
to maintain aseptic cultures. Go to the section on setting up your dust cabinet
[link**] if you haven't already done so.
Note that in the video the technician, Joe Lardner, is not using either a laminar flow hood or a dust cabinet because it would obscure your view of what he is doing. The imaginary boundry of his dust cabinet/hood is the rectangle created by the blue tape.
Materials
Greenhouse grown stock plant of Kalmia latifolia
Hood (delineated by blue lines)
Inside hood (delineated by blue lines) : These items have all been sterilized in an autoclave or pressure cooker.
Procedure
It is preferable to use a vegetative (entirely leafy) shoot tip if some are present on your stock plant, but in the video what is being removed is a flowering shoot tip with a cluster of undeveloped fower buds. This explant will develop vegetative lateral shoots in culture, but don't be surprised if it also develops a few flowers as well.
Sterilize shoot tips in 10% Clorox for 20 minutes. This process frees the explant of the pathogens (fungal or bacterial) that could possibly proliferate on the culture medium and ultimately destroy the explants.
Rinse 3x's in sterile distilled water.
Cut off the basipetal end of the shoot tip with a sterile scapel and place the remaining 1cm apical portion on Stage 1 culture medium (one tip per container).
Place jars/containers under light.
Sketch or otherwise record the size and condition of the explants at the time of setting up this experiment.
Results
Record weekly observations (text, sketches, photos); look for signs of growth, necrosis, contamination, etc.
Subculture to Stage II medium.
Note the composition of the nutrient media used for each stage of this experiment. Media differ between species and stage of micropropagation.