BERGAMO, ITALY
Istituto Sperimentale per la Cerealicoltora

Chromosome endoreduplication in endosperm cells of IHP and ILP maize strains grown in vitro
--Cavallini, A, Bosio, D, Reali, A, Natali, L*, Balconi, C, Motto, M
*Universitý di Pisa - Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica

An important feature of cell differentiation in maize endosperm is nuclear enlargement through chromosome endoreduplication (Duncan and Ross, J. Heredity 41:259-268, 1950). This process may be related to many differentiation events, such as protein and starch synthesis and storage, accumulation of nucleotides, enzyme activation, and hormone synthesis (D'Amato, Caryologia 42:183-211, 1989), and consists of subsequent cycles of DNA replication without entering mitosis until high ploidy levels are attained. Variations in chromosome endoreduplication frequency in endosperm parenchyma have been described among maize populations (Kowles and Phillips, Int. Rev. Cytol. 112:97-136, 1988): in particular, different mean ploidy levels were reported in the endosperm of the strains used in these experiments, IHP (36.4C) and ILP (25.8C) (Cavallini et al., Protoplasma 189:156-162, 1995). To establish if an in vitro culture method may mimic the development of maize endosperm in vivo, we analyzed zein and starch content and chromosome endoreduplication in IHP and ILP strains, excised at 4 or 9 days after pollination (DAP) and cultured until 25 DAP. Ears of IHP and ILP maize strains were harvested at 4 and 9 DAP and were cut into blocks containing 10 kernels per block as described by Gengenbach (Planta 134:91-93, 1977). The blocks were cultured on agar media containing salts as described in Nitsch and Nitsch (Science 163: 85-87, 1969), 150 g/l sucrose and 0 or 4 g/l glutamine. For endoreduplication analysis, after fixation in ethanol/acetic acid 3:1 (v/v), the endosperms at 25 DAP were macerated in 5% pectinase and squashed; slides were then stained by Feulgen reaction and DNA content of nuclei was measured cytophotometrically.

At 25 DAP, both IHP and ILP kernels explanted at 4 or 9 DAP and grown in vitro with or without glutamine, showed a phenotype similar to that observed in kernels grown to maturity in field conditions; in particular at 4 DAP the ILP strain showed, in both media, a reduced accumulation of the zein fraction and a higher starch content in comparison to the IHP strain. IHP showed a higher zein content (%/d.w.) in both media than ILP (4.18% vs.1.87% without glutamine; 3.22% vs. 1.60% with glutamine in the media); on the other hand the ILP strain showed a higher starch content (%/d.w.) in both media than IHP (40.00% vs.28.00% without glutamine; 40.50% vs. 28.43% with glutamine in the media).

For endoreduplication analysis we observed that mean ploidy level in endosperms cultured after excision at 4 DAP was similar to that found in vivo: IHP showed a higher ploidy level than ILP (34.08C ( 0.71 vs. 24.18C ( 0.54 for endosperm cultured without glutamine, 30.89C ( 0.92 vs. 24.97C ( 0.72 for those cultured in 4 g/l glutamine). Variability in chromosome endoreduplication was observed in endosperms excised at 9 DAP, probably related to the higher level of differentiation in these organs compared to those excised after 4 DAP.

In summary the results of this research suggest that the in vitro culture method tested mimics the development of maize endosperm grown in vivo.


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