LLAVALLOL, ARGENTINA

Universidad Nacional de La Plata

BUENOS AIRES, ARGENTINA

Centro de Ecofisiol. Vegetal, CONICET1

Regeneration in callus cultures and cytological analysis of regenerated plants --Dina Garcia, Maria del C. Molina and Osvaldo Caso1 Many investigators have obtained maize plant regeneration by organogenesis or somatic embryogenesis in callus cultures initiated from immature embryos (Green and Phillips, Crop Sci. 15:417, 1975; Lu et al., Theor. Appl. Genet. 62:109, 1982; Tomes and Smith, Theor. Appl. Genet. 70:505, 1985; among others), but some inbreds, such as B73, showed a very low percentage of response.

The object of this work is to evaluate the plant regeneration capacity of maize cv. Colorado Klein cultured in vitro. This cultivar is often used in the IFSC to obtain hybrids with other species of the genus Zea. The method used in this work is an adaptation from that of Lowe et al. for B73 (Plant Sci. 41:125, 1985). Cytogenetic analysis will show any chromosomal change in regenerated plants.

Table 1. Relationship between embryo length and days after pollination. Plant material: immature embryos of Colorado Klein.
 
Days after pollination
Average embryo length (mm)
11
1.15
14
2.08
16
3.03

Embryos were aseptically excised and placed on the culture media with the plumule radical axis side in contact with the medium. Cultures were incubated at 30-32 C in darkness for 15 days. Cultures were transferred to maintenance media and were incubated with a 16 hour photoperiod (I=2500 Lx) and subcultured every 30 days. Tissues like leaves or roots were excised in each subculture. After 7 months in culture, callus was transferred to regeneration medium. Plantlets were subcultured to rooting medium. Cultures were incubated in the same environment as in maintenance. Callus which did not regenerate plants was discarded.

The embryos germinated from 1 to 3 days after isolation, but they did not continue their normal development. About 7 days after isolation, white to pale yellow callus arose from 1 to 2 mm embryo scutellum. Embryos larger than 2 mm gave callus which turned brown and died.

Figure.

Structures like small leaves arose from green areas of callus initiated in N medium from 11-day-old embryos when these were transferred to N1 medium with 1 mg.L-1 2,4-D. These structures arose especially near the end of each culture period. No regeneration was observed from callus initiated in M medium. Plantlets originated when the green areas with structures like leaves were transferred to N2 medium. No adventitious roots were observed to appear from plantlets in this medium. Adventitious roots arose from plantlets in N3 medium.

Six percent of plated embryos gave callus capable of plant regeneration. These calli still regenerate plants after 20 months in culture.

Cytogenetic analysis of 10 regenerated plants revealed that 7 of them had a somatic chromosome number of 2n=20. Three plants showed alterations in the chromosome number, one of them had 2n=21, another one had 2n=23 and the third had 2n=40, with characteristic tetraploid cells.


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