Maize Genetics Cooperation Newsletter 80. 2006.

PRESIDENTE PRUDENTE, BRAZIL

University of Western Sao Paulo

Changes in chromosomes in highly embryogenic cultured cells and in germinating stored seeds of maize

--Scandolieri, RF; Koyanagui, AP; Takahashi, FT; Fluminhan, A

In higher plants, an increased frequency of genetic and chromosomal changes is usually observed during the germination and development of plants derived from aged seeds and regenerated from in vitro cultured cells. Much evidence supports a close relationship between the age of stored seeds or cell cultures and the loss of vigour and germinability of seeds, and the regeneration ability of cultures and number of chromosome aberrations that are observed at the first mitoses of root meristems in surviving plants.

In vitro culture of plant cells has led to several useful approaches for biotechnology in the agricultural sciences, such as: the selection and clonal propagation of promising genotypes and the production of transgenic cultivars. However, it is necessary that the cell cultures maintain their ability to regenerate fertile plants without showing any genetic variation, even after long periods of in vitro culture. Cytogenetic analysis of regenerated plants and their progenies and in germinating aged seeds has allowed the identification of several chromosome number variations and changes in chromosome structure.

We have investigated the occurrence, nature and frequency of chromosome abnormalities in mitotic anaphases in both systems: highly friable and embryogenic (type II) callus cultures and aged seeds of a maize synthetic cultivar obtained from a breeding program developed at our university. In vitro performance of the Unoeste 101 cultivar was evaluated following standard protocols for callus induction from immature embryo, and the embryogenic callus cultures have been maintained for more than 32 months, and continuously evaluated for their ability to regenerate complete plants. Samples of root meristems collected from regenerated plants and from germinating aged seeds (stored in culture rooms at 25^o C, for 3 years, with moisture content brought to approximately 8% by drying over regularly regenerated silica gel) were taken for cytogenetic analysis, and the squash preparations were stained by Feulgen�s and C-banding methods.

The results demonstrate the occurrence of extensive mitotic abnormalities in both systems (cultured cells and aged seeds), mainly as a result of the formation of bridges that may lead to chromosome breakages (Fig. 1). Previously, we have demonstrated that the primary chromosome breakages occur preferentially within knobs or at junctions between the euchromatin and a heterochromatic knob (Fluminhan et al., Ann. Bot. 78:73-81, 1996; Fluminhan and Kameya, Theor. Appl. Genet. 92:982-990, 1996; Fluminhan and Kameya, Genome 40:91-98, 1997).

Figure 1. Photomicrographs of mitotic anaphases observed in cultured cells (a and b), and at first mitoses of germinating aged seeds (c and d) of the maize cultivar Unoeste 101. 1a. Typical anaphase showing the primary event, characterized by a delay in segregation of sister chromatids, resulting in a lagging chromosome (arrow). 1b. Typical anaphase showing double bridges. 1c. Chromosome breakage giving rise to a fragment (arrow). 1d. Typical anaphase with multiple bridges, with and without heterochromatic knob, apparently resulting from successive breakage-fusion-bridge cycles. Magnification: x100 objective; x10 ocular; x1,2 additional lens.

These findings are consistent with our previous observation of an increased presence of methylated cytosine at knob heterochromatin (Fluminhan et al., MNL 71:75-77, 1997) and with the proposition that changes in the degree and pattern of DNA methylation could be an underlying cause of chromosomal abnormalities observed in cultured cells and regenerated plants (Phillips et al., Proc. Intl. Cong. Plant Tissue Cell Cult. 7:131-141, 1990).

Our observations indicate that all the phenomena described above resemble each other in both systems analysed, and represent an important step aiming the proposition of mechanisms related to their occurrence. One interesting issue to be analysed is that both systems could be under the influence of common or related mechanisms of cellular senescence, leading to the occurrence of similar abnormalities at mitosis.

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