2.7. Cotyledon cell characteristics
The number of cotyledon cells was estimated by the improved
method of Wang et al. (2009) (Patent Number: ZL200910072894.4)
compared to Swank et al. (1987). Seeds were allowed to imbibe
water for 8–10 h in a beaker and then the testa and embryo axis
were carefully removed. Seeds were dried for 24 h (30–35 ◦C) and
were then weighed using a balance with 0.0001 g precision. Seeds
were then ground to fine powder in a mortar. A small amount
(approximately 50 mg) of seed powder was weighed and then put
into a 50 mL beaker. Then 10 mL chromic acid solution (concentration
10–20%) was added to the beaker for 48 h. A further 10 mL chromic acid solution was added for another 24 h, and then the
third 10 mL chromic acid solution was added for another 12 h
to completely dissolve the powder. The solution was constantly
stirred by a vortex device. After a uniform cell suspension was
obtained by glass rod stirring, a 5 L suspension was transferred to
a hemacytometer with a pipettor and the cells were counted under
a 100× magnification microscope. Fifteen replicates were made for
the counting. Given the known counting chamber volume in the
blood count plate and the total cell suspension volume, the cotyledon
cell number in total cell suspension was calculated as follows,
W:W1 = X:A, then X = A * (W/W1), where W is the dry soybean seed
weight, X is cotyledon cell number, W1 is the dry weight of a small
amount of seed powder, and A is the cotyledon cell number in total
cell suspension from this known weight. Cotyledon cell volume was
obtained by dividing seed volume by number of cotyledon cells.
Cell weight was obtained by dividing seed size by cotyledon cell
number.
Statistical analysis of data was performed by using the PROC
ANOVA of SAS, and mean comparison was made according to the
Duncan’s multiple range tests (SAS Institute, 1996). Experimental
figures were drawn by Sigma plot 2000 software.