Phenotype of the mutant seed. Immature (16 DAP) mutant seeds obtained by selfing +/abs7065 heterozygous plants, are easily distinguishable from normal sibs by a reduction in size and a pale, translucent appearance.

Their endosperm has a soft and fluid consistency while the embryo, not different in size from that of wild-type, appears retarded in its morphogenesis. Mature seeds are completely collapsed but retain a reduced amount of endosperm tissues.

Mutant embryo rescue. Immature embryos of mutant and normal sibs segregating on a selfed +/abs7065 ear were cultured on minimal or enriched media to assay their germination and growth capacity.

Starting at 13 DAP, germination of excised wt embryos can be obtained, while mutant embryos do not germinate until 25 DAP (Table 1). This observation may indicate a delay in mutant development, that affects its germination capacity. Furthermore the percentage of germination is significantly lower (13.2%) in comparison to wt (100%) and seedlings obtained from homozygous abs7065 embryos are retarded and impaired in their growth. No promoting effect on germination or growth is observed by culturing mutant embryos on enriched media.

A plausible interpretation of these observations that takes into account the close relationship between endosperm and embryo development is that the primary effect of the mutation is impairment of endosperm development leading to a retarded morphogenetic potential of the embryo as a secondary effect, likely due to lack of component(s) elaborated by the endosperm and supplied to the embryo to accomplish its regular development.

The failure to observe a complete phenotypic repair of mutant seedlings obtained by immature embryos would suggest impairment of an early effect of the endosperm upon the embryogenetic process.

Molecular analysis. Since these mutants have been isolated from a progeny derived from an active Robertson�s Mutator maize stock, its origin can be ascribed to an insertional event. To verify the association between the mutant phenotype and a molecular polymorphism, cosegregation analysis was performed. Heterozygous plants were crossed to the W64A line and the genotype (+/- or +/+) of individuals from the progenies was determined by selfing. Genomic DNA was extracted from single plants (110 individuals) and analysed by Southern blot. A 12 Kb EcoRI fragment was found in association with all heterozygous plants tested using an internal fragment of Mu3 as a probe. By digesting with PstI, a restriction enzyme internal to Mu3, two polymorphic fragments have been detected of 9Kb and 2kb respectively (Fig. 1). The 2kb fragment was cloned; sequence data confirm the presence of the 5� portion of the Mu3 element and of 1115 bp flanking genomic DNA. A 390 bp Xho-Mlu1 internal fragment was used as a probe on Southern analysis: this probe confirms the presence of the 12kb EcoRI and 2kb PstI fragments in +/- plants, previously highlighted in the Mu3 profile. Isolation of the genomic clones is in progress.

Table 1. Growth of mutant and normal sib embryos at two developmental stages on a minimal (MS) medium or on media supplemented with amino acids, vitamins or hormones.
 

Developmental stage	Cultured embryos		Germination %		Seedling growth:
					M M		M V		M A		M M(1)
	+	m	+	m	+	m	+	m	+	m	+	m
13 DAP	146	42	100	0	8.3	-	10,7	-	3,7	-	ND	ND
25 DAP	218	83	100	13.2	15.6	1.7	6.6	1.0	ND	ND	6.0	0.5

(1) gibberellic acid and benzyladenine

Figure 1. Identification of the Mu3-hybridizing Pst1 fragments that cosegregate with the abs*-7065/+ genotype. Pst1 digested DNA from sibling plants was analyzed by Southern blot, using an internal EcoRI/HindIII fragment of Mu3. The arrows indicate the Mu3-hybridizing fragments that cosegregate with the abs*-7065/+ genotype.

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