The results of embryological investigations of AT-1 line (16 plants) and 6 hybrids (19 plants) are presented. Two hybrids were produced by crosses of AT-1 line as female with DPL-1 and Mangelsdorf's tester (MT) lines. Four hybrids were derived from crosses of AT-1 plants as males with plants of DPL-1, DPL-2, DPL-3, and MT lines. The DPL-1, DPL-2, DPL-3 and MT lines were shown in preliminary investigations to have no apomictic traits. The ears, previously isolated by parchment bags, were fixed in ethanol-acetic acid, 1:3, 7-8 days after silk emergence. Embryo sacs (ESs) were extracted under a stereo microscope from ovules stained with acetocarmine and macerated with cytase. 2025 ESs were examined in the course of the investigation.
In AT-1 plants proembryos were observed in a significant part of ESs without signs of pollen tube penetration. The proembryo stages varied from two-cell to globular. The cytological structure of proembryos in most cases was similar to normal zygotic ones, though in some ESs abnormal proembryos were found. The most frequent irregularity was that the egg cell divided not transversely but longitudinally resulting in two equal cells. These two cells gave rise to two equal proembryos. In some cases the following divisions in each of the "cleavage" embryos proceeded asynchronously, and as a result the two proembryos differed in size. "Cleavage" twin proembryos also were found in which one of the proembryo initials failed to divide. This cytological proembryo type is presumed to be a prerequisite of twin seedlings (2n-n), occurring rather often in offspring of the AT-1 line under pollination.
Other abnormalities of haploid embryogenesis also were observed: lack of cytokinesis after first or second egg cell division; oblique wall formation after first mitosis; an asymmetrical first division resulting in a large apical cell and a small basal one. The frequency of ESs with parthenogenetic embryogenesis varied from 17.1 to 82.6%. In 10 of 16 plants studied the frequency of such ESs comprised more than 50%.
In ESs of 11 plants an autonomous endosperm was registered. In the most cases it was accompanied by autonomous embryogenesis. The cytological course of endospermogenesis varied significantly. The first and second divisions, as a rule, happened typically, but further development passed abnormally. Disturbances were expressed in irregular nuclear location (nuclei were concentrated in several regions), in different nuclear sizes, and in premature cellularization in the presence of a low number of nuclei. The cells were of different size and contained several nuclei. In no case was the autonomous endospermogenesis found to be normal. Later this endosperm degenerated. The frequency of ESs with autonomous endosperm development in AT-1 plants ranged from 0 to 59.4%.
The analysis of hybrids derived from crosses when AT-1 plants were used as females showed that in 6 of 7 hybrids analyzed, autonomous embryogenesis occurred. In one hybrid plant autonomous endospermogenesis was revealed. The greatest amount of ESs with embryo or with endospermogenesis per plant was found to be 13.2% and 1.0% respectively. In hybrids derived from crosses when AT-1 line was used as males, parthenogenetic embryo development was observed in all 12 hybrid plants, and autonomous endospermogenesis in 5 plants. The greatest amount of ESs with proembryo or with endosperm per plant was 12.3% and 1.4% respectively. The cytoembryological patterns of embryo and endospermogenesis in hybrids were similar to the ones described above for AT-1 plants.
The results allow us to reach the following conclusions:
(1) AT-1 line is characterized by constant expression of autonomous haploid embryogenesis. Since its frequency in some plants amounted to up to 82.6%, the fundamental possibility of increasing the average percentage exists.
(2) The genetic system controlling autonomous embryogenesis can be transmitted either through female or male gametes, and can be expressed in different genetic backgrounds.
(3) Because of the lack of essential difference in frequency of embryogenesis between reciprocal hybrids, the cytoplasm obviously does not take part in determination of this phenomenon.
(4) The expression of autonomous endospermogenesis in AT-1 line is less frequent, and characterized by strong cytological abnormalities. It is not inconceivable that divisions both of the egg cell and of the central cell are induced by the same factor. However, owing to specific peculiarities of these cells, the cytological effects of induction are different. The reason for endospermogenesis abnormality is most likely related to a diploid chromosome set in the primary endosperm nucleus. It is known that for many species including Zea mays L., the endosperm develops normally under the condition that its chromosome set number is divisible by three.
Therefore, it appears to be promising to transfer the AT-1 line genome at the triploid level. This work was supported by a grant from the Russian Foundation for basic Research.
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