The method for counting chromosomes in daylilies from one of the recent research articles.
"Chromosome number of root tips was counted using conventional squash method
(Yan et al., 1994). Plants were watered in the morning, and root tips of about 1 cm long
were collected around 9:30 am next day. The materials were treated with saturated
solution of p-dichlorobenzene for 5 h, fixed for 22 h in fresh Cannoy’s I fixative, and then
transferred to 70% ethanol for preservation. Root tips were softened with 1 M
hydrochloric acid at 60°C for 8 min and stained for 15 min with basic fucshin before
being squashed between a glass slide and a cover slip. For each genotype, 10 cells with
good chromosomal dispersion were selected, counted, and photographed under the
microscope of 400× magnification."
Yan et al. 1994 is
Yan, G., Ferguson, A.R. and McNeilage, M.A. 1994. Ploidy races in Actinidia chinensis.
Euphytica 78:175-183.
Their method for counting chromosomes in kiwi vine (Actinidia)
"Chromosome spreads were prepared according to a procedure modified from Chen et al. (1982), Geber &
Schweizer (1988) and Zhuang (1990). Five tight young shoot tips (or occasionally ten root tips) were collected and held in saturated aqueous p-dichlorobenzene for 3-4 h. Samples were then fixed in Carnoy's I fixative and left for up to 24 h. After being washed with 0.075 M KCl, the samples were digested in 4% cellulase (Onozuka R-10) + 1% pectinase (Macerozyme R-10), both from Yakult Honsha Co. Ltd.. Japan, in 0.1 M citrate buffer. pH 4.8, at 25° C for 3 h. After being washed in distilled water, the digested samples were refixed in glacial acetic acid:methanol, 1:3, and then smeared on microscope slides prechilled at - 20° C. Slides were dried quickly over an alcohol flame, left overnight and then stained in 2.5% Giemsa (Gurr's Improved Stain R 66, Gurr, BDH) in 0.067 M phosphate buffer, pH 7.2, for 30 min."
