Agriculture Reference
In-Depth Information
• The root-tips were pre-treated with 0.02 % 8-Hydroxyquinoline at 4 °C for 4 h.
• Following which, the root-tips were fixed in a modified Carnoy's fluid contain-
ing absolute alcohol-chloroform-Glacial acetic acid-Methanol (7:3:1:1) for 48 h.
• Finally, the root-tips were preserved in 70 % ethanol at 4 °C.
5.2 Chromosome Staining
• The root-tips from fixed roots were excised and placed in a mixture of nine drops
of 2 % aceto-orcein and one drop of 1N HCl in a watch glass and warmed gently.
• After cooling, the individual root-tips were placed in a drop of aceto-orcein on a
glass slide, covered with a cover slip, warmed gently and squashed.
5.3 Flow Cytometric Analysis
Flow cytometry using DNA selective flourochromes has been considered to be a
fast and reliable method for the measurement of nuclear DNA content in recent
years (Muirhead et al.
1985
; Thorthwaite et al.
1985
; Dolezel et al.
2007
). Unfortu-
nately, its application in plant biology has been overdue, largely owing to the fact
that flow cytometry requires single cell suspension (Shapiro
1985
). As plant cells
usually exist in complex tissues, methods had to be developed for the preparation of
such suspensions. Although flow cytometry is an extremely efficient technique with
high degree of accuracy, the preparation of high quality plant samples for ploidy
analysis remains a vital issue. For determination and interpretation of the haploid
status of the regenerants, firstly tissue of known ploidy is analyzed followed by the
unknown tissue whose ploidy is to be analyzed.
5.4 Protocol for Flow-Cytometric Analysis in Tea
5.4.1 Preparation of Nuclear Sample
• Well developed calli obtained after two months of culture initiation were used
for ploidy analysis.
• Extraction of nuclei and the analysis were carried out via fine chopping of the
hard calli placed in 1 ml ice-cold woody plant buffer. The woody plant buf-
fer was prepared by mixing 0.2 M Tris HCl, 4mM MgCl
2
.6H
2
O, 2mM EDTA
Na
2
.2H
2
O, 86mM NaCl, 10 mM Sodium Metabisulfite, 1 % Triton X-100 (v/v),
and 2 % PVP-10 (w/v) according to the protocol of Loureiro et al.
2007
with
