In previous reports (MGCNL 58:136, 1984 and 59:77, 1985) we have outlined our attempts to characterize molecular changes occurring during embryogenesis. Results obtained over the past 3 years permit a preliminary assessment of the amount of variation attributable to certain parameters inherent in this project. Ears were collected from field grown plants at 5 day intervals between 20 and 40 days post-pollination in 1983 and between 20 and 50 days post-pollination in 1984 and 1985. An additional planting in a controlled environment with minimum temperature of 60 F and maximum temperature of 85 F was made during the spring of 1985 with samples collected between 20 and 40 days post-pollination. Two inbreds, Oh43 and M14, and their reciprocal hybrids were included in this study. At each sampling date embryos were collected for determination of dry weights and individual kernels were labelled with 35S-methionine as described by Kriz (MGCNL 56:14,1982).
The profile of change in dry weight as a function of age appeared similar for all plantings and genotypes. A 7.5 fold average increase was observed between 20 and 40 days post-pollination which subsequently leveled off between 45 and 50 days post-pollination. Mean maximum dry weight values per embryo of all plantings for each genotype were Oh43 14.5 ± 4.0 mg, M14 15.2 ± 3.2 mg, Oh43/M14 16.2 ± 5.3 mg, M14/Oh43 14.8 ± 5.2 mg. The largest range observed for all samples represented a 2.3 fold difference between values obtained for M14/Oh43 from field grown material in 1984 and greenhouse grown material in 1985. No two plantings, however, consistently yielded the greatest interseasonal range for all genotypes. Embryos produced on plants grown in the greenhouse in 1985 were invariably larger than those obtained from other plantings at all sampling dates. These plants also appeared to mature more rapidly, possibly indicating an accelerated rate of development under these near optimal conditions.
Incorporation of 35S-methionine was calculated as a function of dry weight with values ranging between 16,200 cpm/mg and 483,900 cpm/mg. Variation on the order of 85% of the mean was observed among replicate samples, making difficult any determination of genotypic or developmental differences on this basis. This variation may be attributable in part to the method of label delivery (injection using a Hamilton syringe). For example, label may seep from the opening in the kernel following withdrawal of the syringe, possibly affecting the concentration to which individual embryos are exposed. Values for peak incorporation were found to vary across plantings by a factor of 4 and to a lesser extent between genotypes, by a factor of 2.5. Variation was also encountered with respect to the age at which maximum incorporation was observed. Samples collected from field grown material in 1983 and 1985 showed peak values between 35 to 40 days post-pollination and 20 to 30 days post-pollination respectively for all genotypes. Maximum incorporation for field grown material in 1984 occurred between 20 and 40 days post-pollination, varying among genotypes. This increased variation may have been a result of different degrees of damage suffered by individual plants following a severe hailstorm 17 days after the first pollinations were made. The most consistent results were obtained from the plants grown under controlled environmental conditions in 1985: all genotypes showed maximum incorporation at 25 days post-pollination. This variation between plants for the age at which peak incorporation is observed may indicate subtle differences, due to environmental conditions, in the metabolic state of the embryos at the time of collection.
Analysis of newly synthesized proteins completed for material collected in 1983 and 1984 also reveals some degree of seasonal variability in development. The order and types of changes which are observed on two-dimensional fluorograms during the course of embryogenesis appear to be consistent for both plantings. The specific age at which these changes are observed may vary by as much as 5 days. Completion of the analysis of the patterns of newly synthesized proteins for material for both plantings in 1985 should provide further insight into the extent of seasonal variability as reflected in protein synthesis and its relationship to dry weight and 35S-methionine incorporation.
J. G. Boothe and D. B. Walden
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