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FIXATION OF PLANT METAPHASE CHROMOSOMES
(a) Metaphase arresting agents: Choose one of the following (Note 2) and shake vigorously to aerate before putting in living plant material; except for ice water, the solution should be the same temperature as that where the plants grow to avoid shock.
(i) ice water (for cereals and temperate grasses): put distilled or deionized water in a clean plastic bottle, shake to aerate and keep at –20°C until the water starts to freeze, shake again. (b) Alcohol:acetic acid fixative: 3 parts 96% ethanol (or 100% methanol) to 1 part glacial acetic acid (prepare immediately before use; do not store for more than 30 min as the compounds degrade).
(ii) 0.05% (w/v) colchicine (for most plant tissues). Can be stored in the dark at 4°C for several days to weeks.
(iii) 2 mM 8-hydroxyquinoline (for dicotyledonous plants, particularly those with small chromosomes such as Arabidopsis thaliana). Can be stored in the dark at 4°C for several days to weeks.
(iv) a -bromonaphthalene: store water above liquid a -bromonaphthalene in a bottle (typically 100 ml over 50 ml). Shake, allow to separate, and then pipette out aliquots of the a -bromonaphthalene-saturated water-phase into small vials for treatment of material. Can be stored in a dark bottle indefinitely.
For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable. Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used (Notes 2 and 3).
For germination of many seeds, put onto filter paper saturated with distilled water at 20-25°C in the dark and leave until roots are about 10-20 mm long (Note 1). Seeds of trees that grow long single roots are best germinated in a pot of vermiculite, while small seeds are best germinated under sterile conditions on agar minimal medium, e.g. Murashige and Skoog without sugar (Notes 3 and 4). Seed suppliers will give advice about germination of difficult species; moving between 4 °C and 25 °C every 3-14 days often assists germination.
For hydroponic growth, suspend plantlets or bulbs cleaned from soil above an aerated plant nutrient solution (commercial ‘complete’ plant fertilizers, used at 1/10 the strength recommended as a plant feed are suitable; e.g. Phostrogen); existing roots should be immersed in the solution, but not the plant itself. Root growth is normally initiated within a few days.
Plants established in soil (e.g. trees such as oil palm) may produce actively growing roots within a few weeks when compost is applied on the surface around the stem (Note 4).
The following steps are carried out in small glass or plastic containers (5-10 ml) or 1.5 ml microcentrifuge tubes. Use generous amounts of solutions: typically 1 ml per specimen. Material is transferred carefully by clean forceps or a pipette (Note 2).
1. To accumulate metaphases, treat excised root tips (5-20mm long) or other material with one of the metaphase arresting agents as follows (Note 3):
(i) ice water for 24 h
(ii) colchicine for 3-6 h at room temperature or 10-24 h at 4°C
(iii) 8-hydroxyquinoline for 1-2 h at room temperature, then 1-2 h at 4°C
(iv) a -bromonaphthalene saturated water for 2-6 h at room temperature.
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- Quickly blot material and transfer to fixative (Note 5).
- Fix for at least 16 h at room temperature. If fixed material is to be kept (up to several months), leave for 2 h at room temperature and then transfer to new fixative (or 70% or 96% ethanol) and store at -20°C.
The response to the metaphase accumulation reagents is different from species to species and has to be established by trial and error. Some guidelines for choosing are given; Dolezel et al. (1996) discusses alternative reagents, including spindle poisons used a herbicides.
- It is very important not to expose seedlings, roots and plants during germination and metaphase-arrest to chemicals and fumes, particularly fixatives (e.g. in a cold room also used for chemical storage) and to use clean labware with tight lids (disposable plastic is ideal), clean forceps, and distilled water.
- Root tips from germinating seeds, and plants grown in controlled conditions, often show waves of cell division that may follow internal or environmental rhythms (e.g. light) or correlate with root length. At certain times, there may be no divisions at all, so it may be helpful to make several fixations.
- Representative times are given. For best results fix material after different times of treatment, experiment with different reagents and check the mitotic index by making chromosome preparations. Treating material for too long in arresting agents, particularly colchicine, results in over-condensation of the metaphase chromosomes which might be desirable for counting chromosomes, but not for in situ hybridization where spatial resolution along chromosomes is wanted.
- Fixative should not be contaminated with water, so careful blotting or an extra rinse in fixative is advised.