The protocols for C-banding (Jewell et al. 1994, Maize Handbook pp. 484:492) and silver staining (Howell and Black 1980, Experientia 36:1014-1015) were modified so that spread cell preparations could be C-banded and then silver stained to examine the arrangement of the knobs on 6S and 7L. Binucleate tapetal cells were used because they are easily identified in interphase, and the stage of the PMCs can be used to ensure that all of the tapetal cells selected are approximately at the same stage of development.
Tassel branches from inbred Mo17 grown
in our nursery were fixed for 24 h (3:1 ethanol to acetic acid); anthers
were staged by examining propriocarmine stained cells. Anthers with PMCs
in diplotene to diakinesis were rinsed in water for 10 min, digested in
5 % [v/v] cellulase (Sigma), 0.5 % betaglucuronidase (Sigma) in 0.01 M
citrate buffer, pH4.7 with 10 % [v/v] pectinase (Sigma) for 2 h at 37 C.
The anthers were spread in a drop of fixative on a microscope slide, and
air dried for 30 min. The slides were stored in 100% ethanol overnight.
The slides were immersed in 0.2 M HCl at 60 C for 90 to 120 sec, rinsed
in water twice, immersed in filtered 5 % [w/v] BaOH for 7 min at room temperature,
rinsed three times in water and incubated in 2 X SSC for 1 h at 60 C. The
slides were stained in 5 % giemsa [v/v] in phosphate buffer for 60 min.
Photographs of wet mounted slides were taken at a magnification of 400
with a photomicroscope. The coordinates were recorded at each location
where photographs were taken. The C-banding was removed by immersion in
100% ethanol, water, 0.2 M HCl, and water, each for 15 sec and air dried
for 30 min. The slides were silver stained with 2 drops of silver nitrate
(0.5g / ml) and two drops of colloidal developer (2 % gelatin [w/v], 10%
formic acid) under a coverslip for 60 to 90 sec at 70 C on a slide warmer.
After staining, the coverslip was removed with warm flowing water, and
the slides were air dried overnight. The slides were mounted in permount
under a coverslip and photographed again. Figure 1 presents the same binucleate
tapetal cell a) C-banded, (four dsrs were observed) and b) silver stained,
(two dsrs were present) which allows the two C-bands to be distinguished
from each other.
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