II. 15. Relative DNA contents of Hannchen barley (C.I. 531) megagametes at the pre- and peri-fertilization stages.
Christy MacKinnon and L. W. Mericle, Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824, U.S.A. "R"
Cytological and genetic data obtained during earlier radiosensitivity studies on developing embryos of Hordeum distichum cv. Hannchen, C.I. 531, raised a number of questions concerning the nuclear DNA constituency of the barley zygotes and early proembryo stages. Unexpected chimeric capability of the zygote following acute irradiation (Mericle and Mericle, 1967), coupled with cytological implications of cell component partitioning during the first few mitoses after fertilization (Mericle and Mericle, 1969) were evidence for suggesting that the zygotic nucleus possessed an unusually high amount of DNA associated with a state of endopolyploidy or polyteny, or that its behavior reflected an unusual structural state of the chromosomes.
Initial investigations determined (microspectrophotometrically) the
relative DNA complements of megagametes at peri-fertilization, zygotes,
and 2- 4-celled stage proembryos.* The values obtained were significantly
higher than that predicted by classical genetics and plant embryology:
microgametes and synergids measured 1C, as expected, but megagametes measured
4C, and zygotes measured from 4C up to as high as 16C (Mericle and Mericle,
1970, 1973). Gene amplification, comparable to that observed in amphibian
oocytes, was considered a possible basis for such high values, but it was
determined later that the megaspore mother cell and its resulting megaspores
had relative DNA complements close to classically expected amounts - 4C
and 1C, respectively (MacKinnon and Mericle, 1979). In order to confirm
initial findings of "excess" DNA amounts, as well as to identify the stage
of DNA elaboration, we recently measured approximately a dozen "eggs" in
mature embryo sacs at the pre- and peri-fertilization stages. The relative
DNA contents of megagametes (based on an average 2C "baseline" value of
ca. 40 ovule wall telophase nuclei) have been determined to range from
1C up to 6C. It appears that the elaboration of "excess" DNA can be correlated
with the maturation and dehiscence of the pollen (see Table 1.). Other
cells of the embryo sac do not appear to synthesize excessive amounts of
DNA: approximately 20 synergids were measured and determined to have DNA
complements of 1-2C, while four fused polar nuclei were found to have relative
values from ca. 2C up to 5C.
[*All DNA measurements were made on Feulgen stained nuclei using either
Leitz or Zeiss microspectrophotometers. Relative DNA values were calculated
via the two-wavelength method of Patau (Patau, 1952). It is worth noting
that it is extremely difficult to secure usable material for microspectrophotometric
DNA quantitation. Only about 30% of the material prepared is quantifiable
since complete serial sections of the embryo sac with the egg apparatus
nuclei intact are essential for reliable measurements.]
Table 1. Distribution of relative DNA content of
megagametes at varying fertilization stages.**
References:
MacKinnon, Christy and L. W. Mericle. 1979. DNA complements of megaspore mother cells and megaspores in Hannchen barley. Genetics 91:s72.
Mericle, L. W. and R. P. Mericle. 1967. Mutation induction as influenced by developmental stage and age. Erwin Baur Memorial Lectures IV, 1966. Abhandl. Deut. Akad. Wiss. Berlin. pp. 65-77.
Mericle, L. W. and R. P. Mericle. 1969. Cytological consequences of proembryo irradiation. Radiation Botany 9:269-282.
Mericle, L. W. and R. P. Mericle. 1970. Nuclear DNA complements of young proembryos of barley. Mutation Research 10:515-518.
Mericle, L. W. and R. P. Mericle. 1973. Confounding the quandary of zygotic DNA. Barley Genetics Newsletter 3:39-42.
Patau, K. 1952. Absorption microphotometry of irregularly-shaped objects. Chromosoma 5:341-362.
