The Italian Research on the Molecular Characterization of Maize Kernel Development.

The study of the genetic control of maize seed development and seed-related pathways has been one of the most important themes approached by the Italian scientific community. Maize has always attracted the interest of the Italian community of agricultural genetics since its beginning, as some of its founders based their research projects on and developed their "schools" by adopting maize as a reference species. Some of them spent periods in the United States, where maize was already becoming a model system, to receive their training. In this manuscript we illustrate the research work carried out in Italy by different groups that studied maize kernels and underline their contributions in elucidating fundamental aspects of caryopsis development through the characterization of maize mutants. Since the 1980s, most of the research projects aimed at the comprehension of the genetic control of seed development and the regulation of storage products' biosyntheses and accumulation, and have been based on forward genetics approaches. We also document that for some decades, Italian groups, mainly based in Northern Italy, have contributed to improve the knowledge of maize genomics, and were both fundamental for further international studies focused on the correct differentiation and patterning of maize kernel compartments and strongly contributed to recent advances in maize research.

Drought-Responsive ZmFDL1/MYB94 Regulates Cuticle Biosynthesis and Cuticle-Dependent Leaf Permeability.

In all land plants, the outer surface of aerial parts is covered by the cuticle, a complex lipid layer that constitutes a barrier against damage caused by environmental factors and provides protection against nonstomatal water loss. We show in this study that both cuticle deposition and cuticle-dependent leaf permeability during the juvenile phase of plant development are controlled by the maize (Zea mays) transcription factor ZmFUSED LEAVES 1 (FDL1)/MYB94. Biochemical analysis showed altered cutin and wax biosynthesis and deposition in fdl1-1 mutant seedlings at the coleoptile stage. Among cutin compounds, ω-hydroxy fatty acids and polyhydroxy-fatty acids were specifically affected, while the reduction of epicuticular waxes was mainly observed in primary long chain alcohols and, to a minor extent, in long-chain wax esters. Transcriptome analysis allowed the identification of candidate genes involved in lipid metabolism and the assembly of a proposed pathway for cuticle biosynthesis in maize. Lack of ZmFDL1/MYB94 affects the expression of genes located in different modules of the pathway, and we highlighted the correspondence between gene transcriptional variations and biochemical defects. We observed a decrease in cuticle-dependent leaf permeability in maize seedlings exposed to drought as well as abscisic acid treatment, which implies coordinated changes in the transcript levels of ZmFDL1/MYB94 and associated genes. Overall, our results suggest that the response to water stress implies the activation of wax biosynthesis and the involvement of both ZmFDL1/MYB94 and abscisic acid regulatory pathways.

The Role of Brassinosteroids in Controlling Plant Height in Poaceae: A Genetic Perspective.

The most consistent phenotype of the brassinosteroid (BR)-related mutants is the dwarf habit. This observation has been reported in every species in which BR action has been studied through a mutational approach. On this basis, a significant role has been attributed to BRs in promoting plant growth. In this review, we summarize the work conducted in rice, maize, and barley for the genetic dissection of the pathway and the functional analysis of the genes involved. Similarities and differences detected in these species for the BR role in plant development are presented. BR promotes plant cell elongation through a complex signalling cascade that modulates the activities of growth-related genes and through the interaction with gibberellins (GAs), another class of important growth-promoting hormones. Evidence of BR-GA cross-talk in controlling plant height has been collected, and mechanisms of interaction have been studied in detail in Arabidopsis thaliana and in rice (Oryza sativa). The complex picture emerging from the studies has highlighted points of interaction involving both metabolic and signalling pathways. Variations in plant stature influence plant performance in terms of stability and yield. The comprehension of BR's functional mechanisms will therefore be fundamental for future applications in plant-breeding programs.

Characterization of the Biogenic Volatile Organic Compounds (BVOCs) and Analysis of the PR1 Molecular Marker in Vitis vinifera L. Inoculated with the Nematode Xiphinema index.

Upon pathogen attack, plants very quickly undergo rather complex physico-chemical changes, such as the production of new chemicals or alterations in membrane and cell wall properties, to reduce disease damages. An underestimated threat is represented by root parasitic nematodes. In Vitis vinifera L., the nematode Xiphinema index is the unique vector of Grapevine fanleaf virus, responsible for fanleaf degeneration, one of the most widespread and economically damaging diseases worldwide. The aim of this study was to investigate changes in the emission of biogenic volatile organic compounds (BVOCs) in grapevines attacked by X. index. BVOCs play a role in plant defensive mechanisms and are synthetized in response to biotic damages. In our study, the BVOC profile was altered by the nematode feeding process. We found a decrease in ß-ocimene and limonene monoterpene emissions, as well as an increase in α-farnesene and α-bergamotene sesquiterpene emissions in nematode-treated plants. Moreover, we evaluated the PR1 gene expression. The transcript level of PR1 gene was higher in the nematode-wounded roots, while in the leaf tissues it showed a lower expression compared to control grapevines.

Novel DICER-LIKE1 siRNAs Bypass the Requirement for DICER-LIKE4 in Maize Development.

Dicer enzymes function at the core of RNA silencing to defend against exogenous RNA or to regulate endogenous genes. Plant DICER-LIKE4 (DCL4) performs dual functions, acting in antiviral defense and in development via the biogenesis of trans-acting short-interfering RNAs (siRNAs) termed tasiR-ARFs. These small RNAs play an essential role in the grasses, spatially defining the expression domain of AUXIN RESPONSE FACTOR3 (ARF3) transcription factors. However, contrary to tasiR-ARFs' essential function in development, DCL4 proteins exhibit strong evidence of recurrent adaptation typical of host factors involved in antiviral immunity. Here, we address how DCL4 balances its role in development with pressures to diversify in response to viral attack. We show that, in contrast to other tasiR-ARF biogenesis mutants, dcl4 null alleles have an uncharacteristically mild phenotype, correlated with normal expression of select arf3 targets. Loss of DCL4 activity yields a class of 22-nucleotide tasiR-ARF variants associated with the processing of arf3 transcripts into 22-nucleotide secondary siRNAs by DCL1. Our findings reveal a DCL1-dependent siRNA pathway that bypasses the otherwise adverse developmental effects of mutations in DCL4. This pathway is predicted to have important implications for DCL4's role in antiviral defense by reducing the selective constraints on DCL4 and allowing it to diversify in response to viral suppressors.

The maize lilliputian1 (lil1) gene, encoding a brassinosteroid cytochrome P450 C-6 oxidase, is involved in plant growth and drought response.

Background and Aims: Brassinosteroids (BRs) are plant hormones involved in many developmental processes as well as in plant-environment interactions. Their role was investigated in this study through the analysis of lilliputian1-1 (lil1-1), a dwarf mutant impaired in BR biosynthesis in maize (Zea mays). Methods: We isolated lil1-1 through transposon tagging in maize. The action of lil1 was investigated through morphological and genetic analysis. Moreover, by comparing lil1-1 mutant and wild-type individuals grown under drought stress, the effect of BR reduction on the response to drought stress was examined. Key Results: lil1-1 is a novel allele of the brassinosteroid-deficient dwarf1 (brd1) gene, encoding a brassinosteroid C-6 oxidase. We show in this study that lil1 is epistatic to nana plant1 (na1), a BR gene involved in earlier steps of the pathway. The lill-1 mutation causes alteration in the root gravitropic response, leaf epidermal cell density, epicuticular wax deposition and seedling adaptation to water scarcity conditions. Conclusions: Lack of active BR molecules in maize causes a pleiotropic effect on plant development and improves seedling tolerance of drought. BR-deficient maize mutants can thus be instrumental in unravelling novel mechanisms on which plant adaptations to abiotic stress are based.

The maize fused leaves1 (fdl1) gene controls organ separation in the embryo and seedling shoot and promotes coleoptile opening.

The fdl1-1 mutation, caused by an Enhancer/Suppressor mutator (En/Spm) element insertion located in the third exon of the gene, identifies a novel gene encoding ZmMYB94, a transcription factor of the R2R3-MYB subfamily. The fdl1 gene was isolated through co-segregation analysis, whereas proof of gene identity was obtained using an RNAi strategy that conferred less severe, but clearly recognizable specific mutant traits on seedlings. Fdl1 is involved in the regulation of cuticle deposition in young seedlings as well as in the establishment of a regular pattern of epicuticular wax deposition on the epidermis of young leaves. Lack of Fdl1 action also correlates with developmental defects, such as delayed germination and seedling growth, abnormal coleoptile opening and presence of curly leaves showing areas of fusion between the coleoptile and the first leaf or between the first and the second leaf. The expression profile of ZmMYB94 mRNA-determined by quantitative RT-PCR-overlaps the pattern of mutant phenotypic expression and is confined to a narrow developmental window. High expression was observed in the embryo, in the seedling coleoptile and in the first two leaves, whereas RNA level, as well as phenotypic defects, decreases at the third leaf stage. Interestingly several of the Arabidopsis MYB genes most closely related to ZmMYB94 are also involved in the activation of cuticular wax biosynthesis, suggesting deep conservation of regulatory processes related to cuticular wax deposition between monocots and dicots.

Biochemical Characterization of the Seed Quality of a Collection of White Lupin Landraces from Southern Italy.

Lupin species provide essential nutrients and bioactive compounds. Within pulses, they have one of the highest contents of proteins and fibers and are among the poorest in carbohydrates. The Mediterranean region is an important cradle area of the origin and domestication of cultivated white lupin (Lupinus albus L.). In this work, we present the characterization of 19 white lupin landraces collected from several sites in southern Italy, characterized by different pedoclimatic conditions. The protein contents and electrophoretic patterns, total polyphenols, phytic acid, lipids and phosphorous content, and reducing and anti-tryptic activities have been determined for each landrace. The relationships of the compositional characteristics, the area of origin of landraces and between compositional characteristics and thermo-pluviometric trends that occurred in the genotype comparison field during the two-year period between 2019 and 2020 are compared and discussed. From a nutritional point of view, some of the analyzed landraces differ from the commercial reference. The panel of molecular analyses performed can help in building an identity card for the grain to rapidly identify those varieties with the desired characteristics.

Whole-genome resequencing-based characterization of a durum wheat landrace showing similarity to 'Senatore Cappelli'.

Durum wheat (Triticum turgidum spp. durum) is a major cereal adopted since antiquity to feed humans. Due to its use, dating back several millennia, this species features a wide genetic diversity and landraces are considered important repositories of gene pools which constitute invaluable tools for breeders. The aim of this work is to provide a first characterization of a wheat landrace, referred to as 'TB2018', that was collected in the Apulia region (Southern Italy). 'TB2018' revealed, through visual inspection, characters reminiscent of the traditional variety 'Senatore Cappelli', while exhibiting a distinctive trait, i.e., reduced stature. Indeed, the comparison with a set of Italian durum wheat cultivars conducted in this study, in which 24 CPVO plant descriptors were adopted, placed the 'TB2018' landrace in proximity to the 'Senatore Cappelli' cultivar. In addition, the close similarity between the two genotypes was confirmed by the analysis of the seed protein pattern. A relative reduction was detected for 'TB2018' root elongation in the early stages of plant growth. The 'TB2018' genome sequence, obtained through low-coverage resequencing and comparison to the reference 'Svevo' cultivar is also reported in this study, followed by a genome-wide comparison against 259 durum wheat accessions that placed 'TB2018' close to the 'Cappelli' reference. Hundreds of genes putatively affected by variants that possess Gene Ontology descriptors were detected, among which some were shown to be putatively linked to the morphological traits that distinguish 'TB2018' from 'Senatore Cappelli', Overall, this study poses the basis for a possible exploitation of 'TB2018' per se in cultivation or as a source of alternative alleles in the breeding of traditional cultivars. This work also presents a genomic methodology that exploits the information contained in a low-depth, whole-genome sequence to derive genotypic data useful for cross-platform (chip data) comparisons.

Roles of the MYB94/FUSED LEAVES1 (ZmFDL1) and GLOSSY2 (ZmGL2) genes in cuticle biosynthesis and potential impacts on Fusarium verticillioides growth on maize silks.

Maize silks, the stigmatic portions of the female flowers, have an important role in reproductive development. Silks also provide entry points for pathogens into host tissues since fungal hyphae move along the surface of the silks to reach the site of infection, i.e., the developing kernel. The outer extracellular surface of the silk is covered by a protective hydrophobic cuticle, comprised of a complex array of long-chain hydrocarbons and small amounts of very long chain fatty acids and fatty alcohols. This work illustrates that two previously characterized cuticle-related genes separately exert roles on maize silk cuticle deposition and function. ZmMYB94/FUSED LEAVES 1 (ZmFDL1) MYB transcription factor is a key regulator of cuticle deposition in maize seedlings. The ZmGLOSSY2 (ZmGL2) gene, a putative member of the BAHD superfamily of acyltransferases with close sequence similarity to the Arabidopsis AtCER2 gene, is involved in the elongation of the fatty acid chains that serve as precursors of the waxes on young leaves. In silks, lack of ZmFDL1 action generates a decrease in the accumulation of a wide number of compounds, including alkanes and alkenes of 20 carbons or greater and affects the expression of cuticle-related genes. These results suggest that ZmFDL1 retains a regulatory role in silks, which might be exerted across the entire wax biosynthesis pathway. Separately, a comparison between gl2-ref and wild-type silks reveals differences in the abundance of specific cuticular wax constituents, particularly those of longer unsaturated carbon chain lengths. The inferred role of ZmGL2 is to control the chain lengths of unsaturated hydrocarbons. The treatment of maize silks with Fusarium verticillioides conidia suspension results in altered transcript levels of ZmFDL1 and ZmGL2 genes. In addition, an increase in fungal growth was observed on gl2-ref mutant silks 72 hours after Fusarium infection. These findings suggest that the silk cuticle plays an active role in the response to F. verticillioides infection.

PPR8522 encodes a chloroplast-targeted pentatricopeptide repeat protein necessary for maize embryogenesis and vegetative development.

The pentatricopeptide repeat (PPR) domain is an RNA binding domain allowing members of the PPR superfamily to participate in post-transcriptional processing of organellar RNA. Loss of PPR8522 from maize (Zea mays) confers an embryo-specific (emb) phenotype. The emb8522 mutation was isolated in an active Mutator (Mu) population and co-segregation analysis revealed that it was tightly linked to a MuDR insertion in the first exon of PPR8522. Independent evidence that disruption of PPR8522 caused the emb phenotype was provided by fine mapping to a region of 116kb containing no other gene than PPR8522 and complementation of the emb8522 mutant by a PPR8522 cDNA. The deduced PPR8522 amino acid sequence of 832 amino acids contains 10 PPR repeats and a chloroplast target peptide, the function of which was experimentally demonstrated by transient expression in Nicotiana benthamiana. Whereas mutant endosperm is apparently normal, mutant embryos deviate from normal development as early as 3 days after pollination, are reduced in size, exhibit more or less severe morphological aberrations depending on the genetic background, and generally do not germinate. The emb8522 mutation is the first to associate the loss of a PPR gene with an embryo-lethal phenotype in maize. Analyses of mutant plantlets generated by embryo-rescue experiments indicate that emb8522 also affects vegetative plant growth and chloroplast development. The loss of chloroplast transcription dependent on plastid-encoded RNA polymerase is the likely cause for the lack of an organized thylakoid network and an albino, seedling-lethal phenotype.

A mutational approach for the detection of genetic factors affecting seed size in maize.

KEY MESSAGE: Genes influencing seed size. The designation emp (empty pericarp) refers to a group of defective kernel mutants that exhibit a drastic reduction in endosperm tissue production. They allow the isolation of genes controlling seed development and affecting seed size. Nine independently isolated emp mutants have been analyzed in this study and in all cases longitudinal sections of mature seeds revealed the absence of morphogenesis in the embryo proper, an observation that correlates with their failure to germinate. Complementation tests with the nine emp mutants, crossed inter se in all pairwise combinations, identified complementing and non-complementing pairs in the F1 progenies. Data were then validated in the F2/F3 generations. Mutant chromosomal location was also established. Overall our study has identified two novel emp genes and a novel allele at the previously identified emp4 gene. The introgression of single emp mutants in a different genetic background revealed the existence of a cryptic genetic variation (CGV) recognizable as a variable increase in the endosperm tissue. The unmasking of CGV by introducing single mutants in different genetic backgrounds is the result of the interaction of the emp mutants with a suppressor that has no obvious phenotype of its own and is present in the genetic background of the inbred lines into which the emp mutants were transferred. On the basis of these results, emp mutants could be used as tools for the detection of genetic factors that enhance the amount of endosperm tissue in the maize kernel and which could thus become valuable targets to exploit in future breeding programs.

The maize pentatricopeptide repeat gene empty pericarp4 (emp4) is required for proper cellular development in vegetative tissues.

The empty pericarp4 (emp4) gene encodes a mitochondrion-targeted pentatricopeptide repeat (ppr) protein that is involved in the regulation of mitochondrial gene expression and is required for seed development. In homozygous mutant emp4-1 kernels the endosperm is drastically reduced and the embryo is retarded in its development and unable to germinate. With the aim of investigating the role of emp4 during post-germinative development, homozygous mutant seedlings were obtained by cultivation of excised immature embryos on a synthetic medium. In the mutants both germination frequency as well as the proportion of seedlings reaching the first and second leaf stages were reduced. The anatomy of the leaf blades and the root cortex was not affected by the mutation, however severe alterations such as the presence of empty cells or cells containing poorly organized organelles, were observed. Moreover both mitochondria and chloroplast functionality was impaired in the mutants. Our hypothesis is that mitochondrial impairment, the primary effect of the mutation, causes secondary effects on the development of other cellular organelles. Ultra-structural features of mutant leaf blade mesophyll cells are reminiscent of cells undergoing senescence. Interestingly, both structural and functional damage was less severe in seedlings grown in total darkness compared with those exposed to light, thus suggesting that the effects of the mutation are enhanced by the presence of light.

The CC-NB-LRR-type Rdg2a resistance gene confers immunity to the seed-borne barley leaf stripe pathogen in the absence of hypersensitive cell death.

BACKGROUND: Leaf stripe disease on barley (Hordeum vulgare) is caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea. Race-specific resistance to leaf stripe is controlled by two known Rdg (Resistance to Drechslera graminea) genes: the H. spontaneum-derived Rdg1a and Rdg2a, identified in H. vulgare. The aim of the present work was to isolate the Rdg2a leaf stripe resistance gene, to characterize the Rdg2a locus organization and evolution and to elucidate the histological bases of Rdg2a-based leaf stripe resistance. PRINCIPAL FINDINGS: We describe here the positional cloning and functional characterization of the leaf stripe resistance gene Rdg2a. At the Rdg2a locus, three sequence-related coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NB-LRR) encoding genes were identified. Sequence comparisons suggested that paralogs of this resistance locus evolved through recent gene duplication, and were subjected to frequent sequence exchange. Transformation of the leaf stripe susceptible cv. Golden Promise with two Rdg2a-candidates under the control of their native 5' regulatory sequences identified a member of the CC-NB-LRR gene family that conferred resistance against the Dg2 leaf stripe isolate, against which the Rdg2a-gene is effective. Histological analysis demonstrated that Rdg2a-mediated leaf stripe resistance involves autofluorescing cells and prevents pathogen colonization in the embryos without any detectable hypersensitive cell death response, supporting a cell wall reinforcement-based resistance mechanism. CONCLUSIONS: This work reports about the cloning of a resistance gene effective against a seed borne disease. We observed that Rdg2a was subjected to diversifying selection which is consistent with a model in which the R gene co-evolves with a pathogen effector(s) gene. We propose that inducible responses giving rise to physical and chemical barriers to infection in the cell walls and intercellular spaces of the barley embryo tissues represent mechanisms by which the CC-NB-LRR-encoding Rdg2a gene mediates resistance to leaf stripe in the absence of hypersensitive cell death.

A mutational approach to the study of seed development in maize.

The maize seed comprises two major compartments, the embryo and the endosperm, both originating from the double fertilization event. The embryogenetic process allows the formation of a well-differentiated embryonic axis, surrounded by a single massive cotyledon, the scutellum. The mature endosperm constitutes the bulk of the seed and comprises specific regions containing reserve proteins, complex carbohydrates, and oils. To gain more insight into molecular events that underlie seed development, three monogenic mutants were characterized, referred to as emp (empty pericarp) on the basis of their extreme endosperm reduction, first recognizable at about 12 d after pollination. Their histological analysis reveals a partial development of the endosperm domains as well as loss of adhesion between pedicel tissues and the basal transfer layer. In the endosperm, programmed cell death (PCD) is delayed. The embryo appears retarded in its growth, but not impaired in its morphogenesis. The mutants can be rescued by culturing immature embryos, even though the seedlings appear retarded in their growth. The analysis of seeds with discordant embryo-endosperm phenotype (mutant embryo, normal endosperm and vice-versa), obtained using B-A translocations, suggests that emp expression in the embryo is necessary, but not sufficient, for proper seed development. In all three mutants the picture emerging is one of a general delay in processes related to growth, as a result of a mutation affecting endosperm development as a primary event.

empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize.

The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35-amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4-green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.

Genetic analysis as a tool to investigate the molecular mechanisms underlying seed development in maize.

BACKGROUND: In angiosperms the seed is the outcome of double fertilization, a process leading to the formation of the embryo and the endosperm. The development of the two seed compartments goes through three main phases: polarization, differentiation of the main tissues and organs and maturation. SCOPE: This review focuses on the maize kernel as a model system for developmental and genetic studies of seed development in angiosperms. An overview of what is known about the genetic and molecular aspects underlying embryo and endosperm formation and maturation is presented. The role played by embryonic meristems in laying down the plant architecture is discussed. The acquisition of the different endosperm domains are presented together with the use of molecular markers available for the detection of these domains. Finally the role of programmed cell death in embryo and endosperm development is considered. CONCLUSIONS: The sequence of events occurring in the developing maize seed appears to be strictly regulated. Proper seed development requires the co-ordinated expression of embryo and endosperm genes and relies on the interaction between the two seed components and between the seed and the maternal tissues. Mutant analysis is instrumental in unravelling the genetic control underlying the formation of each compartment as well as the molecular signals interplaying between the two compartments.

Programmed cell death during embryogenesis in maize.

Programmed cell death (PCD) in plants is considered an integral part of development. Evidence of DNA fragmentation, occurring at specific sites and times during embryo formation in maize (Zea mays L.), was obtained using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labelling (TUNEL) and by genomic DNA ladder detection. During the crucial period of elaboration of the primary shoot and root axis (14-20 d after pollination), TUNEL-positive nuclei are present in the scutellum, coleoptile, root cap and principally in the suspensor. Additional evidence of a form of programmed cell death occurring in these tissues comes from the detection of a DNA ladder. Upon completion of the differentiation process, all embryonic cells are TUNEL-negative, indicating that possible programmed cell death events during maize embryogenesis are confined to structures or organs that do not contribute to the adult plant body.

Mutations in two independent genes lead to suppression of the shoot apical meristem in maize.

The shoot apical meristem (SAM), initially formed during embryogenesis, gives rise to the aboveground portion of the maize (Zea mays) plant. The shootless phenotype (sml) described here is caused by disruption of SAM formation due to the synergistic interaction of mutations at two genetic loci. Seedlings must be homozygous for both sml (shootmeristemless), and the unlinked dgr (distorted growth) loci for a SAM-less phenotype to occur. Seedlings mutant only for sml are impaired in their morphogenesis to different extents, whereas the dgr mutation alone does not have a recognisable phenotype. Thus, dgr can be envisaged as being a dominant modifier of sml and the 12 (normal):3 (distorted growth):1 (shoot meristemless) segregation observed in the F(2) of the double heterozygote is the result of the interaction between the sml and dgr genes. Other segregation patterns were also observed in the F(2), suggesting instability of the dgr gene. Efforts to rescue mutant embryos by growth on media enriched with hormones have been unsuccessful so far. However, mutant roots grow normally on medium supplemented with kinetin at a concentration that suppresses wild-type root elongation, suggesting possible involvement of the mutant in the reception or transduction of the kinetin signal or transport of the hormone. The shootless mutant appears to be a valuable tool with which to investigate the organization of the shoot meristem in monocots as well as a means to assay the origins and relationships between organs such as the scutellum, the coleoptile, and leaves that are initiated during the embryogenic process.