GRC title

Cytology

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Pollen Mother Cells

Fixation and Staining

  1. Fix panicles in fresh fixative, 6:3:1 ethanol: chloroform: acetic acid with 0.25g ferrous chloride per 100 ml fixative for 24 h at 4 °C.
  2. Store in 70% ethanol at 4 °C.
  3. Prior to staining, wash spikelets in 70% ethanol at least three times and blot with paper towel. Transfer to clean vials with sufficient amount of snow carmine to submerse the spikelets. Incubate in an oven at 50 °C for 24 h.
  4. Keep at room temperature for at least 3 d before squashing.

Slide preparation

  1. Remove anthers with fine forceps and add one drop of 45% acetic acid.
  2. Squash gently with a fine needle and remove debris.
  3. Place cover slip and heat slide gently.
  4. Apply enough pressure to flatten cells.
  5. For temporary mounting, add Hoeyer’s solution to one side of coverslip and let dry for an hour at room temperature. Do the same for other side.

Microscopic observation, analysis, and recording

  1. Examine slides using binocular microscope first under low power objectives, then shift to high power objective adding oil for oil immersion objectives.
  2. Perform meiotic analysis using appropriate form (see Appendix 9-1).
  3. Take photomicrographs of noteworthy materials.


Root tips

Germination, pre-treatment, and staining

  1. Germinate seeds in sterilized Petri dishes lined with moist filter paper at 30 °C in an incubator.
  2. Harvest 1 to 2 mm root tips and pre-treat with 002 M hydroxyquinoline for 3 h. Protect from light. (Pre-treatment is best started at about 9:00 A.M.).
  3. Fix with fresh 3:1 ethanol:acetic acid for 24 h at 4 °C.
  4. Store in 70% ethanol at 4 °C.
  5. For staining, submerse roots in 2% aceto orcein and store at room temperature for at least 5 days before squashing.

Slide preparation

  1. Transfer one root tip to a clean slide and remove root cap. Cut off a small piece and add a drop of 45% acetic acid.
  2. Place a cover slip with edge raised on top of a razor blade to allow for cell movement during squashing.
  3. Squash gently with the tip of a rod and remove the razor blade gently when cells are sufficiently squashed.
  4. Apply enough pressure to flatten cells.
  5. Temporarily seal slides with Hoeyer’s solution.


Stains and reagents

Snow carmine

4 g carmine

15 ml distilled water

1 ml HCL

95 ml 85% ethanol

  1. Mix 4 g carmine, 15 ml distilled water, and 1 ml HCL.
  2. Stir boil gently for 10 min.
  3. Cool and add 95 ml 85% ethanol.
  4. Filter.

Orcein

2 g orcein

100 ml 45% acetic acid

  1. Add 2 g orcein to 100 ml 45% acetic acid.
  2. Stir boil gently for 10 min.
  3. Cool and filter.

Hoeyer’s solution

30 ml distilled water

3 g gum arabic

25 g chloral hydrate

3 ml glycerin

  1. Dissolve 3 g of gum arabic in 30 ml distilled water for 24 h.
  2. Add 25 g chloral hydrate and dissolve for 24 h.
  3. Add 3 ml glycerin.
  4. Mix thoroughly and filter through cloth.

 

Pre-treating agent

8-hydroxyquinoline: .002 M solution in water (0.29 g l –2)

a-bromo-napthalene: used as saturated solution in water or 1% aqueous solution of stock solution of 1 ml bromonapthalene dissolved in 100 ml absolute ethanol for 30 min.

Fixative: Fixative should be freshly prepared for each fixation. It consists of:

absolute ethanol                       6 parts

chloroform                                3 parts

glacial acetic acid                    1 part

or

absolute ethanol                       3 parts

glacial acetic acid                    1 part