September 20, 2011
CYTOGENETIC TECHNIQUES

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.

There are many methods for handling plant and animal cells. This lecture will supply an overview and some basic principles. The techniques and the interpretation will be practised in the laboratory.
 

A. Collection of material for study

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.

somatic cells - differentiated cells specialized for a tissue, organ, or function. Somatic cells divide by mitosis
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.

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

http://www.cellimagelibrary.org/images/15797

http://www.cellimagelibrary.org/images/15798




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 potted plants.
 

   


Mitotic cells are circled in green. Interphase cells are indicated by blue arrows.

Image displayed by hypertext link from
http://www.vcbio.science.ru.nl/en/virtuallessons/cellcycle/intro/

The callus produced in tissue culture can be used but has the problem of culture-induced variations in chromosome number. Pollen tube mitosis is useful but hard to find.

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.

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. 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.
 

2. Meiosis. Chromosome morphology is not apparent but the stages can be used for establishing chromosome numbers. The most important use of meiotic cells is to examine pairing relationships between chromosomes.

 
a. Animal meiotic cells. The egg mother cell megasporocytes arrested at diplotene can be used. Spermatocytes are more abundant.

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.