Phenotypic and chromosomic aberrations in plants regenerated from callus

--Maria del Carmen Molina, M. D. Garcia1 and O. Caso2


Maize plant regeneration from callus gives the possibility of obtaining genetic variability such as aneuploidy, polyploidy and chromosomic deficiencies, duplications, inversions and translocations. Nutrient media, time in culture, explant and genotype affect the occurrence of these aberrations (D'Amato, F, Frontiers of Plant Tissue, pp. 287-295, 1978; McCoy, TL and Phillips, RL, Can. J. Genet. Cytol. 24:559-565, 1982; Lee, M and Phillips, Genome 29:109-112, 1987).

The object of this work is the phenotypic and cytogenetic study of maize plants regenerated from organogenic callus after 32 months in culture (Fig. 1).

Figure 1. Meiotic chromosomes in maize plants regenerated from callus after 32 months in culture. 1a. Metaphase with an extra chromosome. 1b. Pachynema showing an extra chromosome (10 and deficiencies (2-3). 1c. Anaphase showing inversion bridges. 1d. Metaphase with 5 monovalents.

The callus was initiated from one immature embryo (1mm length) of Zea mays ssp. mays cv. Colorado Klein on nutrient medium containing 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). After 4 weeks the callus was cut into small pieces and maintained by subculturing on media with 1 or 2mg/L 2,4-D every 30 days (Garcia et al., MNL 64:72-73, 1990). Shoots arose from callus on maintenance medium near the end of each subculture period. Adventitious roots arose from shoots on nutrient medium containing N6 mineral salts (Chu, CC, Proc. Symp. Plant Tissue Cult., pp. 43-45), N6 vitamins, L-asparagine 150mg/L, 0.5 or 1mg/L naphthalenacetic acid, sucrose 5%, agar 0.8% and pH 5.8. Callus still regenerates plants after 50 months in culture.

Tassels were fixed in a solution composed by acetic acid and ethanol (1:3), then stained with acetic haematoxylin.

The cytogenetic analysis of maize plants regenerated after 12 months in culture revealed that 70% of them had normal karyotype and the remaining 30% showed some alterations (Garcia et al., MNL 64:72-73, 1990).

Fifty plants obtained from callus after 32 months of subcultures showed the following characteristics: a) normal plants with different ear insertions; b) plants of 1m height with many stems, leaves and ears; c) plants of 35-40cm height with short internodes and many leaves--these individuals are similar to shrunken mutants; d) plants of 15-30cm height, small stems, wrinkled leaves, sterile and small tassels and ears--similar to dwarf mutation.

Only 8% of the plants showed a normal phenotype, 92% showed different characteristics if compared to their progenitors (Graphic 1).

Graphic 1. Phenotypic frequency of regenerated plants.

Cytogenetic analysis revealed that 20% of the plants had a normal karyotype (2n=20), and 80% had an extra chromosome (2n=21).

The chromosomic configuration 10ii + 1i was the most frequently observed, although some plants showed a high number of monovalents, deficiencies, inversions and translocations. These phenomena have never been observed in plants regenerated from callus after 12 months in culture.

Chromosomic aberrations were not correlated with phenotypic abnormalities found in regenerated plants.

The conclusions of this work are: 1) Tissue culture in the presence of 2,4-D causes chromosomic and phenotypic aberrations in maize regenerated plants. 2) Chromosomic and phenotypic aberrations increase with the period of culture on medium containing 2,4-D.

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