Cytogenetic abnormalities in callus and plants derived from one maize embryo after 60 months in culture
--M. del C. Molina and M. D. Garcia**
**Also affiliated with Fac. Ing. y Cs. Agrarias (UNLZ)

The occurrence of numerical and structural chromosome variation in cell and tissue culture and regenerated plants is now a widely accepted component of the general phenomenon of somaclonal variation (Larkin , Band Scowcroft, Theor. Appl. Genet., 1981). Many types of cytogenetic alterations have been described in tissue cultures and regenerated plants, for example polyploidy, aneuploidy, chromosomal rearrangements, deficiencies, dicentric chromosomes, deletions, duplications, inversions and translocations. The genotype and culture medium, mainly the kind and concentrations of plant growth regulators, influence the chromosomal aberration frequencies and types (Edallo et al., Maydica 26:39-56, 1981; McCoy et al., Can. J. Genet. Cytol. 24:37-50, 1982; Puolimatka and Karp, Heredity 71:138-144). Otherwise, Lee and Phillips (Genome 29:122-128, 1987) observed no chromosomal abnormalities in maize plants regenerated from 3 or 4 month old cultures, but 50% of plants regenerated from 8 or 9 month old cultures showed chromosomal alterations. Of these, 96% had changes in the chromosomal structure, 42% deficiencies and 19% heteromorphic pairs. This indicates that somaclonal variation is influenced not only by the medium and genotype stability, but also by the time in culture.

The objectives of this research were to determine frequency and types of chromosomal aberrations among maize somaclones, and whether cytogenetic abnormalities could explain the extreme phenotypic variation among somaclones after 60 months in culture.

Organogenic callus cultures were initiated from one Colorado Klein embryo on media supplemented with 0.5 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D). Calli have been maintained by monthly subculturing on media containing 1 or 2 mg l-1 2,4-D (Garcia et al., Rev. Fac. Agron. La Plata 68:15-25, 1992). For cytogenetic studies, callus was pretreated for 3 hours in 1-4-dichlorobenzene saturated solution. Callus and tassels were fixed in an acetic acid-ethanol (1:3) solution, then stained with acetic haematoxylin.

The frequencies of cytogenetically abnormal plants from 17, 32 and 52 month old callus were 30%, 95% and 100%, respectively. The frequencies of phenotypically abnormal plants from 17, 32 and 52 month old cultures were 0%, 92% and 100%, respectively. Pollen fertility also decreased with the age of cultures (Garcia et al., Rev. Fac. Agron. La Plata 68:15-25, 1992).

Cytogenetic analysis of 60 month old tissue cultures revealed great variability in chromosome number with 2n=18, 19, 20, 21, 22 and 23. Some cells showed 2 or 4 chromosomes with satellite, heteromorphic pairs and a ring of chromosomes. However, 90% of plants regenerated from these tissues had 2n=21 and 10% 2n=20. The types of chromosomal alterations observed in these plants were deficiencies, duplications, high number of univalents, one or more extra chromosomes and inversions, chromosomes with two nucleolus organizer regions and translocations. The analysis of anaphase showed 30% normal plants and 70% with a different chromosome number at each pole and one or more chromosome bridges. All regenerated plants were phenotypically abnormal and completely sterile.

In conclusion, after 60 months on 2,4-D containing media, maize tissues showed changes in chromosome number (2n=18, 19, 20, 21, 22 or 23) and other alterations. We were only able to regenerate plants from tissues with cells with 2N=10 or 21. These plants exhibit also phenotypic and chromosome aberrations, which increased with the age of cultures. 

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