Nucleic Acid Metabolism in Germinating Onion

Abstract

Nucleic acid synthesis in the GI cell population of the 1-millimeter apex of the Allium cepa embryo was studied during the initial 73 hours of germination. Quantitative data indicate that the total amount of RNA per cell began to increase after 18 hours of germination while the initial DNA per cell increase did not occur until some 20 hours later. Polyacrylamide gel electrophoresis patterns of 3H-uridine-labeled total nucleic acid samples indicated that synthesis of all detectable RNA fractions present in the pre-emergent 1-millimeter apex (i.e., cytoplasmic and "chloroplast-like" RNA) (3,4,6,15,37) and few if any biochemical determinations have been made.The Allium root tip offers a unique opportunity to study a population of higher plant cells which, while in the early stages of germination, are in an extended G1 phase (3).This report presents the results of experiments designed to map the pattern of nucleic acid synthesis in the 1 mm apex of the A llium root tip during the initial 73 hr of germination.The apical 1 mm of the A llium embryo contains several tissues including the leaf primordium, cotyledon, hypocotyl, promeristem, provascular tissue, and root cap (11). These tissues are associated with the apical 1 mm of the axis throughout germination until the emergent axis is approximately 5 mm in length, by which time the leaf primordium and cotyle- Philadelphia, Pa.) were sown on moist filter paper in covered glass Petri dishes and placed in a dark incubator at 24 C. At the appropriate time seeds were removed and taken to a 24 C darkroom where they were harvested under dim green light. Care was taken to keep the seeds in the dark at all times prior to harvesting. All samples were sown to be harvested between the hours of 8 and 10 AM.Seeds were sown in the manner described. At 36 hr the lengths of all emergent roots were estimated to the nearest 0.1 mm. All those roots between 0.8 mm and 1.2 mm in lengths were transferred to new dishes and measured at subsequent 24-hr intervals. The mean length and standard deviation for each root was recorded and the data used to plot a standard 1-mm growth curve.The pre-emergent intervals analyzed were defined by the length of time the seeds were exposed to germinative conditions and included 4-, 12-, 18-, and 24-hr seed. Postemergent intervals included 1-, 2-, 5-, and 10-mm roots, all of which were harvested at the optimal time designated by the growth curve. In all cases the apical 1 mm of the root was used as sample. After being subjected to the bacterial decontamination step outlined below, the pre-emergent samples were harvested by gently squashing the seeds with a spatula (thereby forcing the embryo from the seed at the micropylar end), and then excising the apical 1 mm of the root with a scalpel. The postemergent samples were treated with the decontamination procedure, and the apical 1 mm of the root was excised directly.Extraction of the Tips for TNA3 and Subsequent Prepara-

DNA Synthesis and Cell Division in Germinating Onion: III. Variation in Apparent Feulgen-DNA Content with Nuclear area Change

“…Biochemical studies from this laboratory, (MELERA 1971), have now verified the results of the cytochemical analysis, showing a constant 11.8 !J. !-Lg of DNA per nucleus during the first 30-36 hours of exposure to germinative conditions.…”

“…Second, it is a system which has the capacity to involve the complete histological complement of the organism, as indicated in the next paper, and may therefore be extended to other tissues and comparisons made. Finally, it is a system for which good groundwork in the biochemical area has been laid, (MELERA 1971;MAL-LERY 1971 ), and more is being prepared.…”

“…Further biochemical studies (MELERA 1971;MALLERY 1971), have defined the points of initiation of both RNA and protein synthesis as approximately 18 hours of hydration. More precise definition of times and sites of RNA and protein synthesis awaits the completion of the correlative cytochemical investigation now in progress.…”

Elimination of Bacterial Contamination from Onion Seed

“…Radicle tips (1 mm) of pre-and postemergent sample periods were collected as described previously (7,9). Labeling, counting, and electrophoretic procedures for nucleic acid and protein precursor incorporation and fractionation were as previously described (7,9 Figure 1.…”

“…The pre-emergent intervals analyzed were defined by the length of exposure to germinative conditions and included 4-, 12-, 18-, and 24-hr seeds. Postemergent intervals were taken at 1-, 2-, 3-, 5-, and 10-mm lengths, all of which were collected at the optimal time designated by a standard 1-mm growth curve (1, 7, 9).Radicle tips (1 mm) of pre-and postemergent sample periods were collected as described previously (7,9). Labeling, counting, and electrophoretic procedures for nucleic acid and protein precursor incorporation and fractionation were as previously described (7,9 Figure 1.…”