Genetic Screening for EMS-Induced Maize Embryo-Specific Mutants Altered in Embryo Morphogenesis

Abstract

We have previously identified embryo-specific (emb) mutations that resulted in maize kernels containing abnormal embryos with normal-appearing endosperm among the progeny of active Robertson's Mutator stocks. Our rationale for the mutant screen described here is that it should be possible to produce ethyl methane sulfonate (EMS)-induced emb mutations at a frequency higher than that obtained by transposon mutagenesis and with greater ease. This proved to be the case when we screened for mutations that are embryo-specific among progeny of materials generated with EMS-treated pollen. The EMS-induced emb mutation frequency reported here is nearly three times the 4.5% we obtained with the transposable element stocks. The 45 mutants reported here were all tested for germination capacity and nearly all were lethal. The embryo phenotypes of 34 mutations were examined by dissection of the mature embryos. All were found to be retarded in development and morphologically abnormal. Half of the mutants in this group were blocked in the proembryo and transition stages. They likely include mutations in nuclear genes coding for plastid proteins. The other 17 are mainly blocked in the coleoptilar stage, or in later stages with a low frequency. This group likely includes mutations in genes regulating the completion of shoot apical meristem (SAM) development and accompanying morphogenetic events. Most of the complementation tests using 19 of the mutations in 35 unique combinations complimented each other, except for two pairs of mutations with similar phenotypes. Our results provide additional evidence for the presence of many emb loci in the maize genome.

Keywords: EMS mutagenesis; embryo-specific; maize; morphogenesis; mutants.

Figure 1

The frequency of embryos blocked at each stage is shown for each of the 34 mutants under the embryo stage heading. For stages 1–6, values in “()” indicate the number of embryos that appear morphologically farther along in development beyond the coleoptilar stage; however, we have not confirmed the development of leaf primordia. co, coleoptilar stage; pro, proembryo stage; trans, transition stage.

Figure 2

Mutant emb embryos in fresh dissection at kernel maturity. (a) UND-17 transition stage embryo with swollen SU; (b) UND-14 transition stage embryo with an elongated swollen SU and light tan-colored EP indicating early NE; (c) UND-11 transition stage embryo with a necrotic EP; (d) UND-25 normal-appearing transition stage embryo; (e) UND-38 abnormal transition stage embryo with early signs of NE in the EP region and an elongated SU; (f) UND-22 abnormal transition stage embryo with a swollen SU; (g) UND-1 abnormal transition stage embryo with an unexpanded EP showing NE in its uppermost region; (h) UND-10 transition stage embryo with asymmetric EP region showing early NE; (i) UND-12 transition stage embryo with a twisted SU; (j) UND-16 transition stage embryo with a swollen EP showing early NE; (k) UND-28 abnormal transition stage embryo with a swollen SU and a swollen EP region bearing a dimpled region on its upper front surface; (l) UND-3 abnormal coleoptilar stage with an abnormal SC-like structure flanking a central cleft showing NE. AB, abnormal; D, dimple; EP, embryo proper; NE, necrosis; SAM, shoot apical meristem; SC, scutellum; SU, suspensor.

Figure 3

Mutant emb embryos in fresh dissection at kernel maturity. (a) UND-2 transition stage embryo with necrotic-appearing SU; (b) UND-51 abnormal transition stage with an elongated SU topped by necrotic-appearing elongated narrow EP region; (c) UND-52 transition stage embryo with a swollen SU and an EB at its base; (d) UND-6 late transition stage/early coleoptilar stage with a necrotic SAM nearly surrounded by a CR and unexpanded SC region showing NE in the upper most region; (e) UND-18 early transition stage embryo with NE in the region where the SU merges into the EP; (f) UND-29 transition stage embryo with a narrow healthy-appearing SU capped by a completely necrotic EP region; (g) UND-21 abnormal transition stage embryo with a truncated SU region and three EBs; (h) UND-8 abnormal transition stage with a healthy-appearing lower SU region subtending a necrotic upper region bearing a necrotic EP; (i) UND-4 abnormal transition stage embryo with a narrow elongated SU and an asymmetric EP; (j) UND-9 abnormal coleoptilar stage with a small healthy-appearing SU underlying an abnormal SC that has a frontal surface lacking an evidence of a SAM or CR but displays tan tissues at its edges indicating NE; (k) UND-39 abnormal late transition/early coleoptilar stage with normal-appearing SU merging into an asymmetric EP partially expanded; (l) UND-49 late transition/early coleoptilar stage embryo that is healthy-appearing with a partially expanded EP but no well-defined signs of a forming SAM or CR. AB, abnormal; CR, coleoptilar ring; EB, embryonic bulge; EP, embryo proper; NE, necrosis; SAM, shoot apical meristem; SC, scutellum; SU, suspensor.

Figure 4

Mutant emb embryos in fresh dissection at kernel maturity. (a) UND-23 abnormal coleoptilar stage with an expanded smooth surface SC showing no evidence of the initiation of a SAM or CR and subtended by a poorly defined SU region; (b) UND-24 abnormal coleoptilar stage with a greatly expanded healthy-appearing SU region subtending a reduced SC region bearing a small, well defined SAM and a necrotic abnormal SC apical region; (c) UND-20 abnormal coleoptilar stage with an expanded healthy-appearing SU and a smooth-faced SC bearing no features indicating initiation of a SAM or CR; (d) UND-7 fully formed coleoptilar stage with an expanded SU and a well-formed SC bearing a very small SAM but no well-defined CR; the entire embryo is healthy-appearing; (e) UND-13 abnormal coleoptilar stage with an abnormally large SAM and SU, a much reduced SC and no evidence of a CR; (f) UND-15 abnormal coleoptilar stage embryo with grossly enlarged healthy-appearing lobes of abnormal SC-like material surrounding a poorly defined central region lacking any well-defined structures; (g) UND-27 abnormal coleoptilar stage with an abnormal expanded SC partially covering the necrotic EA and subtended by a poorly defined SU region; (h) UND-40 abnormal coleoptilar stage with a darkened SC indicating NE and displaying no evidence of the initiation of a SAM or CR and partially covering a poorly defined SU; (i) UND-19 abnormal coleoptilar stage embryo with well-developed SC with a prominent vertical crevice in the middle of the SC in the region where the EA would normally form and showing no evidence of initiation of the SAM or CR, subtended by a broad, short SU; (j) UND-50 fully formed abnormal coleoptilar stage embryo with an abnormally shaped SC that is broader in the upper region than the bottom displaying a well-defined vertically aligned narrow band of protruding tissue where the EA would normally form and showing no evidence of initiation of a SAM or CR, and subtended by a healthy-appearing normal SU. AB, abnormal; EA, embryo axis; NE, necrosis; SAM, shoot apical meristem; SC, scutellum; SU, suspensor.