Staining Procedure

1. Remove root tips (1/8 ins.)of rapidly growing roots with a sharp knife and place in a small vial containing 0.002 M 8-oxyquinoline solution for 3-4 hrs. at about 65 F

2. Wash in water and place the roots in 45 % acetic acid at 60F for ten minutes to an hour . For overnight fixation , transfer roots ot Carnoy's solution and place at 45 F .

3. Treat roots with IN HCI at 140 F (60C) for 30 seconds and proceed with staining or treat with 1: 1 mixture of Conc. HCI and ethyl alcohol at room temperature for 3-5 minutes. Wash with water and proceed with staining.

4. Place a root tip on a blotting paper, separate root cap and transfer root on to a clean slide. Add a drop of aceto-orcein and gently macerate with a dissecting needle . Place a cover glass over the material and heat the slide over the flame of an alcohol lamp for 10-30 seconds. Do not allow the stain to boil over . Heating will darken the stain on the chromosomes and clear the cytoplasm. Cover the slide with a bloating paper or a paper towel and press down on the cover slip with the thumb. This will flattern the cells and remove excess stain. Care should be taken to avoid sideways movement when pressing on the cover slip.

In order to study meiotic chromosomes of orchids the pollinia do not require any pretreatment , but can be directly fixed in Carnoy's fluid. In species where the buds mature simultaneously , as for example Thrixspermum , Ephemerantha etc., as well as in species with singleflowered inflorescences , as for example , Paphiopedilum , Bulbophyllum aureum , Trias stocksii, etc., it is difficult to obtain correct stages of division . On the other hand , in species where the buds mature successively , the various stages of division could easily be obtained . In all spurred forms , stages of meiosis could be expected in buds with the spur just emerging . Mc Quade (1949) working on Paphiopedilum and Kamemoto and Randolph (1949) on Cattleya could predict the stage of division going on inside the bud by measuring the length of the bud .

Even though the mitotic chromosomes are difficult to obtain in an evenly spread out condition from teh root cells, the remarkably early occurrence of the first pollen mitosis which takes place in all the four cells of the tetrads simultaneously and is characteristic of all buds , facilitates the study of somatic chromosomes in their haploid state. This was first reported in detail by Barber(1942) in Listera ovata , where he found all cells in a packet of pollinium undergoing mitosis simultaneously . Treatment of pollinia at room temperature with 1N HCI for 1-2 minutes dissolves away the thickenings on the walls of the pollen trends . Transfer them to fresh fixative to which a trace of iron acetate has been added and squash after five minutes . The tetrades will spread out easily revealing the mitotic chromosomes in all the four cells, spread out evenly. But the morphology of chromosomes of pollen mitotic metaphase is seldom as clear as in normal root mitosis

.