MILAN, ITALY

University of Milan

Biochemical characterization of some dek mutants

--M. L. Racchi, M. Sturaro, S. Faranda1 and L. A. Manzocchi1

1C.N.R. Istituto Biosintesi Vegetali

The isolation of single gene mutants defective in hormone responses or biosynthetic pathways is a powerful tool to study plant hormones and their role in growth and development. With the aim of isolating hormone mutants in maize we considered mutants affecting seed and embryo development (dek mutants) with altered or suppressed germination isolated by means of EMS or X ray mutagenesis (Dolfini, F et al., Somatic Embryogenesis, IPRA, pp. 122-132, 1985) or somaclonal variation. dek mutants have already greatly contributed to the understanding of genetic programs underlying embryogenesis but not all their potential has been exploited. Biochemical characterization of this class of mutants can give further important information on the differentiation process.

We report a characterization of a group of defective endosperm mutants (ed41v, ed42v, ed45v and ed47v from G. Gavazzi's, G collection) selected on the basis of their different seed morphology and germination capability.

A screening has been done culturing immature and mature mutant embryos as well as the normal counterpart in cytokinine enriched MS media to estimate the response to the hormones in terms of morphogenic capability. Results are presented in Table 1.

Table 1. Development of mutant embryos (mature = M and immature = I) on media without (ER0) or supplemented with cytokinines (ER1 = 0.5 mg/l BAP); (ER2 = 1mg/l kinetin).
 
 
ER0
ER1
ER2
Mutant
I
M
I
M
I
M
ed41v
43
33
90
0
90
0
ed42v
47
50
59
45
55
45
ed45v
23
36
35
32
19
34
ed47v
6
nd
44
nd
7
nd
*Numbers refer to embryos developing seedling structures as percent of total cultured embryos. Mutant embryos, both immature or mature, are from the same ear.

One of the mutants analyzed (ed41v) showed a positive response to cytokinines. Immature embryos cultured in the presence of 0.5mg/l BAP or 1mg/l kinetin developed into normal fertile plants. The action exerted by the hormone is specific for the embryonic phase; in fact, no response is obtained on mature embryos.

To discriminate between mutants altered mainly in the germination process and those in embryogenesis we have determined on dormant and germinating mutant and wildtype embryos a series of biochemical markers: polypeptide patterns, catalase isozymes (by zymograms on polyacrylamide gels) and malate synthase (by western blot experiments with an antibody against malate synthase). The results obtained allowed us to define dek mutants in different classes according to their specific developmental deficiency.

Among the mutants analyzed two are particularly interesting in this regard: ed41v and ed45v. The former, characterized by absence of shoots during germination, gains normal embryo development in the presence of cytokinines. No alterations on the germination process have been observed in terms of differences in polypeptide patterns, catalase 2 or malate synthase expression. It is a developmental mutant blocked in the differentiation process, probably due to a hormonal defect. The latter mutant does not respond to cytokinines and does not show the activation of biochemical pathways related to germination; in fact, no catalase 2 and malate synthase expression has been found. The presence in this mutant of an apparently normal embryo suggests a defect in the control of dormancy.


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