1DIFCA and Dept. of Genetics and Microbiology
University of Milan
2Minoprio Foundation, Vertemate - Como

Maize mutants defective in embryogenesis
--Giulini, AP, Busti, E1, Consonni, G, Dolfini, AS1, Furini, A2, MacCabe, AP, Gavazzi, G

The data discussed in this note refer to the characterization of mutants with defective embryogenesis: emb (embryo specific) mutants, exhibiting a phenotype suggestive of specific defects in embryogenesis and abs (aborted seed) mutants affected in an early stage of embryo and endosperm development. Table 1 reports the list of mutants included in this study and their prominent features, while Table 2 refers to their immature embryo growth in vitro. The mutants have been generated by Mu or Ac transposon mediated mutagenesis. At the molecular level, the presence of a co-segregating polymorphism may provide a means by which the disrupted genes may be cloned.

Characteristics of emb and abs mutants: The general picture emerging from the data of Table 1 is that the abs mutants, even though dramatically disrupted in their development, maintain their morphogenetic potential with shoot and root primordia recognizable while emb mutants appear specifically blocked at an early embryogenic step. The two abs mutants (abs*-7065 and abs*-8075), tested as immature embryos on appropriate media (Table 2), yield callus and slow growing seedlings.

As to the emb mutants the following observations were obtained :

emb*-7182: mutant embryos are recognizable at 8 DAP as small proembryos while at 15 DAP they show an abnormally long suspensor that disappears at later stages (25 DAP) leaving an apparently blocked embryo, whose developmental arrest might be related to the abnormal suspensor development.

Histological analysis of emb*-7917 mature seeds reveals absence of embryo while the endosperm shows an abnormal aleurone consisting of two or more cell layers, particularly evident in the basal portion of the seed, probably due to alterations in the orientation of the cell division plane.

Immature emb*-7065 embryos, recovered in the selfed progeny of heterozygous +/emb plants, can be rescued if grown on regeneration MS media where they produce small albino seedlings while normal sibs yield green seedlings. This observation suggests an involvement of the Emb gene in the control of components of the photosynthetic apparatus.

The emb*-7190 mutant was detected in a line obtained by selfing F1 plants heterozygous for Ac and r-scm3 and originally selected by Dr. S. Dellaporta as a putative nongerminating mutant. Further analysis of F3 progeny indicated that the mutant produces primary root while lacking shoot primordia. The ìshootlessî character segregates in a 1 to 3 ratio indicating that it is a monogenic Mendelian trait. Lack of shoot/leaf primordia organization is observed on immature (17 DAP) embryos. Another shootless mutant in our collection, ed*-41v, analyzed by Racchi et al. (Maydica 41:271-277, 1996) has been shown to respond to the addition of kinetins to the growth medium by producing shoots while a similar mutant (dks8) has been studied by Dr. C. Rivin. Allelism tests between the three mutants is under way and their study should be revealing in terms of the shoot apical meristem organization.

Co-segregation analysis: Co-segregation analysis has been performed on individual seedlings obtained by outcrossing heterozygous +/emb (or +/abs) male parents to W64A inbred females or F2 segregating progenies with the aim of identifying molecular fragments segregating with the mutant phenotype. Southern blots of restriction digests of DNA extracted from single +/+, m/+ and, when possible, m/m seedlings, have been probed with internal Ac and/or Mu fragments. Restriction length polymorphism was observed in the case of abs*-7065 (DNA digested with EcoRI, Southern blotted and probed with a Mu3 fragment). This result is based on the analysis of 40 individuals obtained by crossing +/abs*-7065 male parents to W64A females and it was confirmed with DNA isolated from homozygous mutant seedlings recovered through immature ìin vitroî rescued embryos.

A test of gametophytic selection of the mutants: An assay of the occurrence of gametophytic selection is possible if we consider the distribution of homozygous mutants at the tip and bases of selfed +/m heterozygous plants.

The results obtained (Table 3) refer to eight different mutants. Of the eight mutants tested only one (abs*-8075) shows a significant shortage of mutant segregation over the expected, while the remaining seven show a segregation fitting the 1:3 ratio. In two cases (abs*-7065 and emb*-7192), the heterogeneity c2 test indicates a significantly higher proportion of mutants in the basal portion of the ear, suggesting that they exhibit a slight selective advantage over the corresponding wt allele.

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