REFERENCE: Chapter 2, The Handling of Plant Chromosomes .pp.7.-24 Singh.R.J. 1993. Plant Cytogenetics. CRC Press.
KEY CONCEPTS FOR TODAY
Know the main steps for observing chromosomes:
1. Collection of material to optimize number of mitotic or meiotic cells ie. high Mitotic Index
2. Pretreatment to prevent unwanted changes
3. Fixation to stabilize cellular structures
4. Staining to create optical contrast between cellular structures
5. Slide preparation for optimal viewing
6. Data recording and micrometry
The reason that cytological techniques were developed is to obtain information on chromosome- number, structures and behaviour during cell division. The basic principles applied are generally similar for all species. However, some procedures are modified for different species and to highlight particular features of the chromosomes.
Good preparation of the cells is required for good results. Attention to detail is necessary at every step, starting with the selection and collection of material. Seeing anything at all requires good technique.
Chromosomes are only visible during cell division. The first step in a cytological preparation is the choice of material from which to obtain actively dividing cells.
cells - differentiated cells specialized
for a tissue, organ, or function. Somatic cells divide by
germline cells - cells that give rise to gametes. Germline cells divide by mitosis except during gametogenesis, at which time they divide by meiosis. In some species, such as flowering plants, haploid gametes can also undergo mitotic divisions.
Why not use cells from
tissue culture? Cells in tissue culture frequently
undergo drastic duplications or losses of chromosomes, so
chromosome numbers would be incorrect if counted in cultured
The best material has a high Mitotic Index (MI). MI is the ratio of the number of actively dividing cells to the number of cells in the sample. In plants, there is a diurnal variation in the cell cycle and different species respond to day-length and temperature conditions. It is best to do a literature review to determine optimum time for collection of a particular species.
1. Mitosis. In order to view chromosomes at mitosis, you need to obtain healthy somatic tissue with dividing cells.
a. Animal mitotic cells. The most common sources are skin, bone marrow, and white blood cells. Embryos also provide a good source of cells with a high mitotic index.
Example: Flourescent images of mitosis in sand dollar embryos
Early embryogenesis - http://www.cellimagelibrary.org/images/15784
Slightly later in embryogenesis
b. Plant mitotic cells. The meristematic tissue found in
expanding leaf tips, cambium and root tips is the best
source of actively dividing cells. Root tips are readily
available and can be sampled from germinating seeds or
See image at http://www.vcbio.science.ru.nl/image-gallery/show/print/PL0097
by hypertext link from
Example: Technique of sampling root tips
The best material is obtained from actively growing roots of either germinating seeds or healthy plants. The key to success is to have an abundance of dividing cells from which to select the desired cell stage. Generally, root tips are collected from germinating seeds for ease of handling. The seeds are placed on moistened Filter paper in a Petri dish. The seeds of cereal species such as wheat or barley are germinated in a dark cold room or refrigerator at 0 - 4 °C for 3 to 5 days. This cold treatment produces uniform and rapid seed germination. The Petri plates are then removed from the cold and maintained at room temperature until the seeds have germinated.
The best roots for collection are 1 to 2 cm long, with rounded thick ends tipped with white. The root tips are then transferred to a vial of cold water.
|Germination of seeds on wet paper towels
Displayed from: http://campuscropsmcgill.blogspot.ca/2014/05/seeds-germination-and-seedlings.html
a. Animal meiotic cells. -
b. Plant meiotic cells. The Pollen Mother Cell (PMC) is most commonly used. Sampling at the correct stage is required. Fortunately, in plants it is possible to collect the entire inflorescence for sampling.
The meiotic divisions in
each anther are synchronised and there are anthers at
different stages in the same flower. In a wheat head or
spike there are approximately 40 florets. If one floret
is in anaphase, the next youngest may be in metaphase.
It is possible to sample up and down the spike from a
central point to obtain the desired stage.
Correct Stage for Applying Fungicides
for Scab Control in Wheat and Barley (6/23/11)
North Dakota State University
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