680 . PLANT CHROMOSOMES 



anthers or of whole buds. Anthers are dissected out from buds 

 and phinged immediately into the fixative, preferably of a 

 Flemming type. The Flemming fixatives when used for anthers 

 require a higher percentage of acetic and osmic acids than in the 

 standard formulae. If the anthers are larger, cut them into small 

 pieces with a sharp scalpel under the fixative. Exhaust all air 

 from the tissues with a vacuum pump. 



KiHARA {Bot. Mag. Tokyo, Ixi, 1927, p. 124) finds that cooling 

 of pollen mother-cells before fixing leads to preparations with 

 better-spaced chromosomes. Cool selected buds, put in glass 

 tubes, in running water at 12-5° C. for six hours ; or, place the 

 whole plant or shoot in water in an ice chest at 7° to 8° C. for 

 twelve to twenty-four hours. More prolonged treatment (for 

 forty-eight hours) has an injurious effect. 



In fixing whole buds, strip off bracts and as much of the perianth 

 as possible, opening up the structure so that the fixative has 

 ready access to the anthers. 



KiHARA {Journ. Genet., xx, 1928, p. 105) merely removes the 

 bracts and leaves the perianth covering the anthers. The buds are 

 then placed in Carnoy for one to two minutes according to their size 

 and nature ; the Carnoy is poured off and is replaced immediately 

 by Bouin, Navashin or a Flemming fixative. If possible the 

 sepals are removed just before washing, to aid infiltration and 

 section cutting. The Carnoy-Flemming method is also given 

 by Tahara (Bot. Mag. Tokyo, xxix, 1914, p. 491). 



For division of the generative nucleus in the pollen tubes 

 remove them from the style and fix and stain on the slide (O'Mara, 

 Bot. Gaz., xciv, 1933, p. 567). 



1362. Ovaries, for megasporogenesis, should have the ovary 

 wall dissected away. Slices may also be cut away from the row 

 of ovules to expose the deeper layers of the nucellus. Cut large 

 ovaries into thin slices. Split those with axile placentation longi- 

 tudinally and imbed the rows of ovules separately to obtain the 

 correct orientation of each row. 



1363. Dehydration and Imbedding. Washing is better done in 

 several changes of tepid tap-water than in running water. After 

 fixation is complete, pour off the fixing fluid and replace by several 

 changes of tap-water. Then stand the material, in tap-water, on 

 a piece of cardboard on top of the paraffin oven, or on a hot plate 

 at about 30° C. Change the water half-hourly. Washing is 

 complete in two to three hours, though flower buds may need 

 four to five hours. Careful dehydration is important. La Cour 

 recommends a series from 10 per cent, by 10 per cent, grades to 

 80 per cent., 95 per cent, and absolute. Chamberlain suggests a 

 closer grading at the lower end, 2|, 5, 7|, 10, 15, 20, 30 per cent., 

 etc. It is important that the lower grades up to 40 per cent, alcohol 

 should be passed through rapidly. Material may be left in 70 



