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Key message Our Induced Somatic Sector Analysis and protein–protein interaction experiments demonstrate that Eucalyptus grandis IAA13 regulates xylem fibre and vessel development, potentially via EgrIAA13 modules involving ARF2, ARF5, ARF6 and ARF19. Abstract Auxin is a crucial phytohormone regulating multiple aspects of plant growth and differentiation, including regulation of vascular cambium activity, xylogenesis and its responsiveness towards gravitropic stress. Although the regulation of these biological processes greatly depends on auxin and regulators of the auxin signalling pathway, many of their specific functions remain unclear. Therefore, the present study aims to functionally characterise Eucalyptus grandis AUX/INDOLE-3-ACETIC ACID 13 (EgrIAA13), a member of the auxin signalling pathway. In Eucalyptus and Populus, EgrIAA13 and its orthologs are preferentially expressed in the xylogenic tissues and downregulated in tension wood. Therefore, to further investigate EgrIAA13 and its function during xylogenesis, we conducted subcellular localisation and Induced Somatic Sector Analysis experiments using overexpression and RNAi knockdown constructs of EgrIAA13 to create transgenic tissue sectors on growing stems of Eucalyptus and Populus. Since Aux/IAAs interact with Auxin Responsive Factors (ARFs), in silico predictions of IAA13-ARF interactions were explored and experimentally validated via yeast-2-hybrid experiments. Our results demonstrate that EgrIAA13 localises to the nucleus and that downregulation of EgrIAA13 impedes Eucalyptus xylem fibre and vessel development. We also observed that EgrIAA13 interacts with Eucalyptus ARF2, ARF5, ARF6 and ARF19A. Based on these results, we conclude that EgrIAA13 is a regulator of Eucalyptus xylogenesis and postulate that the observed phenotypes are likely to result from alterations in the auxin-responsive transcriptome via IAA13-ARF modules such as EgrIAA13-EgrARF5. Our results provide the first insights into the regulatory role of EgrIAA13 during xylogenesis.

Microtubules are directly and indirectly involved in guiding cellulose synthase complexes (CSCs) through the plasma membrane. The angle of cellulose deposition is a critical response to environmental signals and/or stress conditions, and particularly crucial during reaction wood formation, a process in which woody plants deposit additional cell wall material to counteract gravitational forces. Tubulin genes are upregulated in response to gravitational stimulus during reaction wood formation, which can result in changes to microtubule assembly. In this study, microtubules were visualised in three woody tree species (two angiosperms: Eucalyptus globulus Labill., Populus alba L., and one gymnosperm: Pinus radiata D. Don.) using immunofluorescence to quantitatively evaluate microtubule organisation during reaction wood formation. Our results suggest that reorientation of the cortical microtubule array affects secondary cell wall deposition, even across different types of reaction wood, by ensuring context-appropriate orientation of cellulose microfibrils and determining MFA in wood cells. Pharmacological studies conducted on in vitro cultured stem segments or in vivo during reaction wood formation corroborated these important roles for microtubules during wood development. This study starts to unveil the role of tubulins during wood formation by exploring cortical microtubule array organisation in trees subjected to gravitational stimulus and it sheds light on cellular and molecular mechanisms behind cellulose deposition in tree species.

The products of secondary xylem are of significant biological and commercial importance, and as a result, the biology of secondary growth and how intrinsic and extrinsic factors influence this process have been the subject of intense investigation. Studies into secondary xylem range in scale from the cellular to the forest stand level, with phenotypic analyses often involving the assessment of traits relating to cell morphology and cell wall chemical composition. While numerous techniques are currently available for phenotypic analyses of samples containing abundant amounts of secondary tissue, only a few of them (microanalytical techniques) are suitable when working with limiting amounts of secondary tissue or where a fine-scale resolution of morphological features or cell wall chemical composition is required. While polarised light microscopy, scanning electron microscopy, field emission-scanning electron microscopy and X-ray scattering and micro-tomography techniques serve as the most frequently used microanalytical techniques in morphotyping, techniques such as scanning ultraviolet microspectrophotometry, X-ray photoelectron spectroscopy, gas chromatography, Fourier-transform infrared spectroscopy and matrix-assisted laser desorption ionisation mass spectrometry serve as the most commonly used microanalytical techniques in chemotyping. Light microscopy, fluorescence microscopy, confocal laser scanning microscopy, transmission electron microscopy and Raman spectroscopy serve as dual micro morphotyping and chemotyping techniques. In this review, we summarise and discuss these techniques in the light of their applicability as microanalytical techniques to study secondary xylem.

Myrtle rust (caused by Austropuccinia psidii Beenken) is exotic to Australia, yet specific resistance is present in a range of naive myrtaceous host plants. Resistance to myrtle rust is primarily quantitative in nature and controlled by multiple interacting loci. We undertook a genome-wide association study (GWAS) to discover relationships between nuclear genomic sequence variation and multiple aspects of resistance to A. psidii in Eucalyptus obliqua, a representative species from subgenus Eucalyptus. Sequence variation was assessed with respect to numeric severity, binary symptomatic, hypersensitive, and pustulation responses to inoculation of seedlings with A. psidii. A total of 1.13 million, single-nucleotide polymorphisms (SNPs) were tested for association with the rust resistance responses of 637 phenotyped E. obliqua seedlings, each from different, single mother tree seedlots. Thirty-three highly significant SNP-trait associations were detected, of which 26 associated with the binary symptomatic/asymptomatic response to A. psidii. Comparison of the genomic position of these SNPs with rust resistance loci (Ppr1-5), previously reported in species from subgenus Symphyomyrtus, suggested that several were positioned near the major Ppr1 locus and other Ppr loci. This study provides the first integrated genomic view of A. psidii resistance across the Eucalyptus subgenera and provides the foundation for discovering key resistance genes for use in marker-based resistance breeding.

Myrtle rust poses a significant biosecurity threat to Australia with potential for long-term damaging impacts on nativeflora and plant industries. This study describes the disease cycle of Austropuccinia psidii, the myrtle rust pathogen, in Eucalyptus globulus and Eucalyptus obliqua, two commercially and ecologically important species from different sub-genera of Eucalyptus. Ontogeny and morphology of infection structures of A. psidii on plants of both Eucalyptus species with different rust response phenotypes, i.e. completely resistant, hypersensitive and highly susceptible, were investigated. Plants were inoculated with single-uredinium-derived urediniospores and examined by scanning electron microscopy. No differences between rust response phenotypes were observed in germination of urediniospores, formation of appressoria or length of germ tubes. The growth of germ tubes had no affinity towards stomata of either species. Histological observations indicated direct penetration by infection pegs through the leaf cuticle and no penetration beyond the epidermis on rust-resistant E. obliqua.Eucalyptus obliqua plants that were identified as susceptible to A. psidii at 3- and 6-months-old showed no disease when reinoculated with A. psidii at 12-months-old; this indicated possible early acquisition of adult plant resistance to A. psidii in this species. In the susceptible phenotype of E. globules rust inoculation led to rapid colonization of leaf parenchyma cells with the disease cycle completed within 10 days. These findings provide valuable insights into host–pathogen interactions in the Eucalyptus–A. psidii pathosystem,which might be useful for the development of effective rust control strategies across Eucalyptus subgenera.

Recent advances in our understanding of the molecular control of secondary cell wall (SCW) formation have shed light on molecular mechanisms that underpin domestication traits related to wood formation. One such trait is the cellulose microfibril angle (MFA), an important wood quality determinant that varies along tree developmental phases and in response to gravitational stimulus. The cytoskeleton, mainly composed of microtubules and actin filaments, collectively contribute to plant growth and development by participating in several cellular processes, including cellulose deposition. Studies in Arabidopsis have significantly aided our understanding of the roles of microtubules in xylem cell development during which correct SCW deposition and patterning are essential to provide structural support and allow for water transport. In contrast, studies relating to SCW formation in xylary elements performed in woody trees remain elusive. In combination, the data reviewed here suggest that the cytoskeleton plays important roles in determining the exact sites of cellulose deposition, overall SCW patterning and more specifically, the alignment and orientation of cellulose microfibrils. By relating the reviewed evidence to the process of wood formation, we present a model of microtubule participation in determining MFA in woody trees forming reaction wood (RW).

We used sector analysis to study cambium development and dynamics and to test whether fundamental developmental and functional differences exist between cambial initials as true 'stem cells' and more differentiated mother cells. In many higher plants, a cylindrical lateral meristem, the vascular cambium, forms along the plant axis. Most notably in stems of perennial tree species, this meristem gives rise to xylem (wood) towards the inside of the trunk and phloem (bark) towards the outside. As such, the vascular cambium is responsible for the production of most of the planet's forest biomass, significantly contributing to the global carbon cycle. Using the bacterial uidA reporter gene in Agrobacterium-based in vivo stem transformation experiments in poplar trees, we created 379 cambium sectors that originated from the transformation of individual cells. Results from our analysis of sector frequency and patterns are consistent with the poplar cambium featuring a single layer of true cambial initials (being able to divide both anti- and periclinally). We show that initials are frequently lost from the cambium, that such cell loss rarely occurs at mother cell level, that phloem and xylem differentiation are controlled independently, and that the frequency of mother cell replenishment is not pre-determined.

Myrtle rust, caused by the pathogen Austropuccinia psidii, is a disease affecting numerous species of Myrtaceae around the globe. Many Australian ecosystems are dominated by Myrtaceae, making them, along with the industries that rely on them, particularly vulnerable to this disease. With over 800 endemic species, Eucalyptus is a major genus within the Myrtaceae in Australia. Wide variation in response to A. psidii infection, from extreme susceptibility to resistance, has been reported among Eucalyptus species in which any pre-formed resistance to this invasive pathogen is unexpected. This study aims to define and contrast geographical patterns of variation in rust susceptibility within the overlapping, natural ranges of Eucalyptus globulus and Eucalyptus obliqua, two commercially and ecologically important species from different Eucalyptus subgenera. Phenotypic disease screening of seedlings of E. globulus races and E. obliqua forest districts (defined geographically) showed E. obliqua to be more susceptible than E. globulus with significant differences in disease susceptibility and symptomatic trait expression. Eucalyptus globulus showed a trend for decreased susceptibility to A. psidii from south- to north-eastern Tasmania, eastwards along the Otway Ranges and southward from the Strzelecki Ranges to the Wilson Promontory Lighthouse in Victoria, but no such geographical patterns were observed within E. obliqua. No significant correlations were found between climatic conditions (i.e. rainfall, temperature and elevation) and rust susceptibility at provenance levels in either species. Taken together, these results support a hypothesis that population divergence in resistance to A. psidii has not been driven by climate.

Climate change can negatively affect sensitive tree species, affecting their acclimation and adaptation strategies. A common garden experiment provides an opportunity to test whether responses of trees from different provenances are genetically driven and if this response is related to factors at the site of origin. We hypothesized that antioxidative defence systems and leaf mass area ofAcacia melanoxylonR. Br. samples collected from different provenances will vary depending on local rainfall. Thirteen provenances ofA. melanoxylonoriginating from different rainfall habitats (500-2000 mm) were grown for 5 years in a common garden. For 2 years, phyllode samples were collected during winter and summer, for measurements of leaf mass area and concentrations of glutathione and ascorbic acid. Leaf mass area varied between seasons, years and provenances ofA. melanoxylon, and an increase was associated with decreasing rainfall at the site of origin. Ascorbic acid and glutathione concentrations varied between seasons, years (i.e., environmental factors) and among provenances ofA. melanoxylon In general, glutathione and ascorbic acid concentrations were higher in winter compared with summer. Ascorbic acid and glutathione were different among provenances, but this was not associated with rainfall at the site of origin.

Eucalyptus foliar terpenes have been reported widely to function as chemical defence agents against mammalian herbivores and defoliating insects both via direct toxicity and/or indirect priming of systemic defence mechanisms. Less frequently they have been reported as disease resistance biomarkers. The present study compares composition of foliar essential oils in Eucalyptus globulus Labill. and Eucalyptus obliqua L’Her. between distinct myrtle rust (causal pathogen Austropuccinia psidii Beenken) response phenotypes i.e. completely resistant, hypersensitive and highly susceptible, using canonical discriminant analysis. Stepwise forward variable selection identified six terpene compounds (bicyclogermacrene, globulol, geraniol, β-pinene, cis-p-menth-2-en-1-ol and δ-terpinene) in E. globulus and four terpenes (δ-cadinene, caryophyllene oxide, longifolenaldehyde and α-humulene) in E. obliqua, which in combination, significantly discriminated between rust response phenotypes. The discriminating compounds varied between E. globulus and E. obliqua and were different from those previously identified as rust resistance biomarkers in Eucalyptus grandis W.Hill ex Maiden × Eucalyptus urophylla S.T.Blake hybrids (1,8-cineole and α-terpinyl acetate). While the discriminant functions were statistically significant within each species, cross validation of the marker sets between Eucalyptus species was not significant. These findings indicate that either (a) terpene composition or amount is not a driver of resistance to myrtle rust or (b) that terpene contribution to resistance is through combined synergistic effects of variable terpenes, likely in combination with other plant metabolites or resistance mechanisms.

KEY MESSAGE : Two species from warmer climates, but not the corresponding congeneric species from relatively cooler ones, decreased stomatal conductance upon heatwaves, and one of them showed significant decrease in the efficiency of open reaction centres of PSII in the light. In contrast, responses of major antioxidants ascorbic acid and glutathione to heatwaves were more similar between congeneric species than between species from similar climates. According to climate change predictions, heatwaves will increase in frequency and intensity. This can threaten survival of sensitive tree species. Heatwaves affect photosynthetic capacity and cause an imbalance between light driven electron transport and carbon fixation. This can increase the concentration of reactive oxygen species, and can cause photo-oxidative stress. Heat dissipation and antioxidants are crucial in plant defence against photo-oxidative stress. We hypothesised that stomatal regulations, heat dissipation as measured by chlorophyll fluorescence and responses of major antioxidants ascorbic acid and glutathione to heatwaves will vary according to the climate at the site of origin of tree species. Tree seedlings from warmer (Eucalyptus grandis, Acacia aneura) and cooler climates (Eucalyptus tricarpa, Acacia melanoxylon) were exposed to air temperatures about 5 °C above control levels for 5 days. The two species from warmer climates responded to heatwaves with stomatal closure restricting transpiration and carbon fixation, and E. grandis also significantly increased heat dissipation (as judged by decreased efficiency of open reaction centres of PSII in the light). Species from cooler climates kept stomata open allowing continuing carbon assimilation and transpirational cooling. Heatwaves did not reduce maximum quantum efficiency of PSII in Acacia species, with insignificant decreases in Eucalyptus species. Glutathione concentrations increased significantly upon heatwave in E. tricarpa (from cooler climates), showed a time-dependent response to heatwave in E. grandis (from warmer climates) with an increase at later stages, and showed a tendency to decrease upon heatwave in both Acacia species (non-significant only for A. melanoxylon). Ascorbic acid concentrations increased significantly in E. tricarpa (cooler climates), and did not change significantly in other species. The redox state of ascorbic acid or glutathione only changed significantly and transiently in response to heatwave in A. aneura (warmer climates; GSSG % and ASC %) and A. melanoxylon (cooler climates; GSSG %), but not Eucalyptus species. Therefore, differences in stomatal regulations upon heatwaves aligned with the origin of species in warmer or cooler climates, whereas responses of ascorbic acid and glutathione to heatwaves were more aligned with the two genera.

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING-H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild-type plants. Following an RNA-seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development.

The moderate to high levels of nucleotide diversity and low linkage disequilibrium found in many forest tree species make them ideal candidates for association mapping. Here, we report candidate gene-based association mapping results for complex wood quality and growth traits in Eucalyptus globulus Labill. ssp. globulus, the most widely grown eucalypt in temperate regions of the world. Ninety-eight single nucleotide polymorphisms (SNPs) from 20 wood quality candidate genes were assayed in a discovery population consisting of 385 trees sourced from a provenance-progeny trial. Twenty-five selected SNPs with significant associations (P < 0.05) in the discovery population were assayed for validation in 296 trees sourced from an independent second-generation breeding trial. To account for background genetic structure, mixed models were used in the association analyses. Two associations identified in the discovery population were independently supported in the validation testing. However, combining the discovery and validation results in a combined analysis, we discovered nine stable marker-trait associations for seven traits. These associations link underlying complex wood and growth phenotypes to earlier putative selection signatures opening new avenues to accelerate the dissection of these traits.

We present a phylogenetic analysis and comparison of structural features of chloroplast genomes for 39 species of the eucalypt group (genera Eucalyptus, Corymbia, Angophora, and outgroups Allosyncarpia and Stockwellia). We use 41 complete chloroplast genome sequences, adding 39 finished-quality chloroplast genomes to two previously published genomes. Maximum parsimony and Bayesian analyses, based on >7000 variable nucleotide positions, produced one fully resolved phylogenetic tree (35 supported nodes, 27 with 100% bootstrap support). Eucalyptus and its sister lineage Angophora+Corymbia show a deep divergence. Within Eucalyptus, three lineages are resolved: the 'eudesmid', 'symphyomyrt' and 'monocalypt' groups. Corymbia is paraphyletic with respect to Angophora. Gene content and order do not vary among eucalypt chloroplasts; length mutations, especially frame shifts, are uncommon in protein-coding genes. Some non-synonymous mutations are highly incongruent with the overall phylogenetic signal, notably in rbcL, and may be adaptive. Application of custom informatics pipelines (GYDLE Inc.) enabled direct chloroplast genome assembly, resolving each genome to finished-quality with no need for PCR gap-filling or contig order resolution. Analysis of whole chloroplast genomes resolved major eucalypt clades and revealed variable regions of the genome that will be useful in lower-level genetic studies (including phylogeography and geneflow).

Due to their long reproductive cycles and the time to expression of mature traits, marker-assisted selection is particularly attractive for tree breeding. In this review, we discuss different approaches used for developing markers and propose a method for application of markers in low linkage disequilibrium (LD) populations. Identification of useful markers for application in tree breeding is mainly based on two approaches, quantitative trait locus (QTL) mapping and association genetic studies. While several studies have identified significant markers, effect of the individual markers is low making it difficult to utilize them in breeding programs. Recently, genomic selection (GS) was proposed for overcoming some of these difficulties. In GS, high density markers are used for predicting phenotypes from genotypes. Currently small effective populations with high LD are being tested for GS in tree breeding. For wider application, GS needs to be applied in low LD populations which are found in many tree breeding programs. Here we propose an approach in which the significant markers from association studies may be used for developing prediction models in low LD populations using the same methods as in GS. Preliminary analyses indicate that a modest numbers of markers may be sufficient for developing prediction models in low LD populations. GS based on large numbers of random markers or small numbers of associated markers is poised to make marker-assisted selection a reality in forest tree breeding.

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree's lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant's secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

Information on public awareness and acceptance issues regarding the use of Genetically Modified (GM) trees in forestry is lacking, although such information is available for GM organisms in agriculture. This is mainly due to the fact that in Europe there is no authorization for commercial planting of GM forest trees. To address this issue and within the frame of a European COST Action on the Biosafety of Transgenic Forest Trees (FP0905), a KAP (Knowledge Attitude Practice ) cross-country pilot survey was conducted among university students of different disciplines as sampling subjects. In total, 1920 completed questionnaires from 16 European and non-European countries were evaluated. The results provided novel cross-country insights into the level of public knowledge, particularly of young people and their perceptions on safety issues related to the use of GM forest trees , as well as on their attitude towards the acceptance of GM forest trees cultivation. The majority of the respondents, which was more than 60 % in all countries, approved the use of GM forest trees for commercial plantations , excluding natural forests. The majority of respondents also appeared willing to buy products from such plantations, such as wood products, pulp and paper. Over 80 % of the respondents from all countries were in favour of using labelling to identify products of GM origin, while more than 80 % of those would prefer that this labelling be legally mandatory. The top three benefits that were rated as very important in all countries involved the potential lower demand of the GM forest plantations for pesticides, the potential of GM forest trees for restoration of contaminated soils and the potential higher GM forest tree productivity. The top three GM forest tree risks that were perceived as serious hazards in all countries included the potential loss of biodiversity due to gene flow between transgenic and wild trees, the adverse effects of biotrophic processes on host ecosystems and the cultural adaptation to changing biodiversity conditions due to transgene escape. Overall, lack of knowledge regarding the potential benefits and potential risks of the cultivation of GM forest trees was observed in almost all surveyed countries.

Eucalyptus globulus (Labill.) is the most widely planted eucalypt for pulpwood in temperate regions of the world. Breeding to improve pulp properties of this species has been hampered by the long time between planting and pulp trait assessment and the high cost of estimating pulp traits. Identifying and employing allelic variants that associate with superior pulp yield and quality has the potential to assist breeding programs. Before this strategy can deliver benefits, detailed knowledge of population structure, nucleotide diversity, haplotype diversity and linkage disequilibrium (LD) must be collected. To address this, 20 wood quality candidate genes were sequenced in 8 to 28 Eucalyptus globulus individuals. Relative to other tree species where such studies have been conducted, single nucleotide polymorphism (SNP) frequencies were high. Decay of linkage disequilibrium was rapid at all loci tested, with linkage rarely extending beyond 500 base pairs. Regions within many candidate genes exhibited significant positive or negative selection signatures, indicative of purifying or balancing selection, respectively. Our findings have implications for association mapping in Eucalyptus species. The potential for E. globulus pedigree reconstruction and whole-genome association approaches in eucalypts in general are discussed.

Aim There is a need for more Southern Hemisphere phylogeography studies, particularly in Australia, where, unlike much of Europe and North America, ice sheet cover was not extensive during the Last Glacial Maximum (LGM). This study examines the phylogeography of the south-east Australian montane tree species Eucalyptus regnans. The work aimed to identify any major evolutionary divergences or disjunctions across the species’ range and to examine genetic signatures of past range contraction and expansion events. Location South-eastern mainland Australia and the large island of Tasmania. Methods We determined the chloroplast DNA haplotypes of 410 E. regnans individuals (41 locations) based on five chloroplast microsatellites. Genetic structure was examined using analysis of molecular variance (AMOVA), and a statistical parsimony tree was constructed showing the number of nucleotide differences between haplotypes. Geographic structure in population genetic diversity was examined with the calculation of diversity parameters for the mainland and Tasmania, and for 10 regions. Regional analysis was conducted to test hypotheses that some areas within the species’ current distribution were refugia during the LGM and that other areas have been recolonized by E. regnans since the LGM. Results Among the 410 E. regnans individuals analysed, 31 haplotypes were identified. The statistical parsimony tree shows that haplotypes divided into two distinct groups corresponding to mainland Australia and Tasmania. The distribution of haplotypes across the range of E. regnans shows strong geographic patterns, with many populations and even certain regions in which a particular haplotype is fixed. Many locations had unique haplotypes, particularly those in East Gippsland in south-eastern mainland Australia, north-eastern Tasmania and south-eastern Tasmania. Higher haplotype diversity was found in putative refugia, and lower haplotype diversity in areas likely to have been recolonized since the LGM. Main conclusions The data are consistent with the long-term persistence of E. regnans in many regions and the recent recolonization of other regions, such as the Central Highlands of south-eastern mainland Australia. This suggests that, in spite of the narrow ecological tolerances of the species and the harsh environmental conditions during the LGM, E. regnans was able to persist locally or contracted to many near-coastal refugia, maintaining a diverse genetic structure.

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

Treating pollen with mutagens prior to controlled pollination may facilitate the production of mutant trees for developmental studies and eventual plantation improvement. To establish a suitable dose of the chemical mutagen ethyl methanesulfonate (EMS) for the testing of this hypothesis, pollen of Eucalyptus globulus ssp. globulus and E. grandis was studied in vitro. Pollen germination, pollen tube elongation and generative cell division were examined after 48 h of culture, following exposure to between 0 and 1,000 ppm EMS. Doses of 600 to 1,000 ppm EMS reduced pollen germination in vitro in both species. Doses of up to 1,000 ppm EMS were not observed to significantly impact on either pollen tube length, or generative cell division in vitro of either species. A dose of 600 ppm EMS in paraffin oil is predicted to induce mutation in Eucalyptus species whilst impacting minimally on seed production based on the effect on pollen germination.

ARBORKNOX1 (ARK1), a poplar functional homolog of the Arabidopsis Class-I KNOTTED- like homeobox (KNOXI) transcription factor SHOOT MERISTEMLESS (STM), has been shown to have a role in maintaining meristematic cells in an undifferentiated state. Studies in which this gene was ectopically overexpressed identified a delay in the differentiation of cambial derivatives. Overexpression of ARK1 in poplar stem sectors, using the induced somatic sector analysis (ISSA) experimental system, was undertaken to further investigate its role in the differentiation of xylem fibre cells. ISSA involves the in vivo transformation of a small number of cambial cells in the stem with a gene of interest giving rise to a discreet transgenic tissue sector that can be directly compared to adjacent non-transgenic fibres to elucidate gene function. Phenotypes obtained were comparable with the proposed role of ARK1, where a delay in the development of the cell wall in differentiating fibre cells of active cambia was observed. Additional evidence suggests a role for ARK1 in the positive regulation of cell viability in the cambium. The significance of dormancy signals in the context of differentiation was also highlighted, as some transgenic fibres failed to fully differentiate prior to the onset of dormancy. Our findings are discussed in the context of wood formation and the use of ISSA as an experimental system.

International audience; AbstractKey messageTwoAcaciaspecies adapted to contrasting habitats showed different response of photoprotective and antioxidative defence systems to imposed drought and heatwave.ContextPredicted increases in drought frequency and intense heatwaves are expected to lead to dieback of sensitive tree species. Stomatal closure restricts CO2 input into the leaf, resulting in imbalances between light energy-driven electron transport rate and electron consumption in the Calvin cycle. Reactive oxygen species formed under these circumstances have to be kept under control by photoprotective and antioxidative defence systems.AimsWe hypothesised that these defence systems behave differently in tree species from contrasting habitats.MethodsAcacia aneura (adapted to arid habitats) and Acacia melanoxylon (adapted to humid habitats) were exposed to two water treatments for 50 days including two short heatwave periods. Responses were assessed by gas exchange, chlorophyll fluorescence and concentrations of antioxidants (phyllodes, roots).ResultsPhotosynthesis and quantum yield of photochemistry decreased significantly in both Acacia species, especially after water was withheld in combination with the second heatwave episode. In phyllodes, the concentration of antioxidants remained unchanged until exposure to severe drought and heatwave conditions (except for A. melanoxylon where changes in glutathione concentration were observed prior to exposure to severe stress), but after water was withheld and the second heatwave occurred, oxidised forms of glutathione increased. After exposure to the second heatwave, well-watered seedlings of A. melanoxylon but not A. aneura increased ascorbic acid concentration in phyllodes. Under well-watered conditions, Acacia species also showed increased concentration of antioxidants in roots following heatwaves.ConclusionsBoth Acacia species showed photodamage to photosystem II (PSII) after water was withheld and the second heatwave imposed, but with more gradual response in A. aneura. Total concentration of investigated antioxidants increased in response to the first (A. melanoxylon) and second (A. aneura) heatwaves rather than drought stress alone.

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.

A series of mutants of Arabidopsis thaliana was selected in which the inflorescence stem elongates but loses the ability to produce flower primordia on its flanks. Mutants fell into two classes, further occurrences of pin‐formed mutants and mutations at a new locus named pinoid. As well as causing inflorescence defects, pinoid mutations result in pleiotropic defects in the development of floral organs, cotyledons and leaves. Most changes involve the number of organs produced rather than their differentiation suggesting that PINOID controls an early general step in meristem development. pinoid mutant defects are similar to those seen in pin‐formed mutants for inflorescences and flowers, but different for cotyledons and leaves indicating that the two genes have separate but overlapping functions. A defect in polar auxin transport is implicated in the pin‐formed mutant phenotype, but in young inflorescence stems of even the strongest pinoid mutants it occurs at close to wild‐type levels. It is markedly reduced only after stems have ceased elongating. Thus, it is likely that polar auxin transport is secondarily affected in pinoid mutants rather than being directly controlled by the PINOID gene product. Even so, double mutant studies indicate that the process controlled by PINOID overlaps with that specified by the AUXIN RESISTANT1 gene, suggesting that PINOID plays some role in an auxin‐related process. Copyright © 1995, Wiley Blackwell. All rights reserved

The increasing focus on plantation forestry as a renewable source of cellulosic biomass has emphasized the need for tools to study the unique biology of woody genera such as Eucalyptus, Populus and Pinus. The domestication of these woody crops is hampered by long generation times, and breeders are now looking to molecular approaches such as marker-assisted breeding and genetic modification to accelerate tree improvement. Much of what is known about genes involved in the growth and development of plants has come from studies of herbaceous models such as Arabidopsis and rice. However, transferring this information to woody plants often proves difficult, especially for genes expressed in woody stems. Here we report the use of induced somatic sector analysis (ISSA) for characterization of promoter expression patterns directly in the stems of Populus and Eucalyptus trees. As a case study, we used previously characterized primary and secondary cell wall-related cellulose synthase (CesA) promoters cloned from Eucalyptus grandis. We show that ISSA can be used to elucidate the phloem and xylem expression patterns of the CesA genes in Eucalyptus and Populus stems and also show that the staining patterns differ in Eucalyptus and Populus stems. These findings show that ISSA is an efficient approach to investigate promoter function in the developmental context of woody plant tissues and raise questions about the suitability of heterologous promoters for genetic manipulation in plant species.

Detailed knowledge of the tissue specificity of gene expression is of central importance not only for our understanding of developmental processes during wood formation, but also is a prerequisite for the deliberate manipulation of xylogenesis candidate genes. Today, much of our knowledge about specific gene expression is based on annual model plants in part because perennial tree systems are often seen as too cumbersome for detailed promoter studies. Here we address this issue in a novel way with the investigation of a number of well characterised cellulose synthase (CesA) genes in the stems of perennial tree species using the in vivo Induced Somatic Sector Analysis (ISSA) system. Three primary and four secondary cell wall associated CesA gene promoters from Eucalyptus grandis and Arabidopsis thaliana were previously isolated and cloned in front of the GUS reporter gene then, using Agrobacterium-mediated transformation, transformed into cambial initials in stems of eucalypt and poplar plants. A CAMV35S promoter::GUS vector was also used as a control. After a period of growth, stems were harvested and GUS-staining patterns were analysed and interpreted. Results from this work show that in eucalypts the staining patterns are consistent with observations in other species whereas in poplar conflicting results were observed. These findings and the general utility of ISSA for these studies are discussed.

Background The identification of polymorphisms that underlie complex phenotypic traits presents new and exciting challenges to molecular genetics. Association mapping, which uses linkage disequilibrium (LD) to map trait variation with nucleotide polymorphisms, has proved suited for this purpose in outcrossed tree species. Association mapping can be undertaken either at a candidate gene level or at the whole-genome level however; until sufficiently dense marker assays are developed the candidate gene approach will remain the most effective way of dissecting complex traits in tree species. To date, this approach has led to the identification of several quantitative trait nucleotides (QTN) that associate with a variety of breeding traits including, early wood specific gravity, percentage latewood [1], microfibril angle [2], cellulose [3]and carbon isotope discrimination [4]. Aim

Plant chimaeras and mosaics are not only found in our gardens as variegated ornamentals. They also constitute valuable research tools with a range of applications in developmental and molecular biology. For example, much of our knowledge about the development of the shoot and root apical meristems is derived from studying patterns of variegation, and where patterns persist throughout the plant, deductions can be made as to the fate of individual meristematic cells and the number and arrangement of initial cells giving rise to a variety of plant organs or structures. At a molecular level, defined sectors can shed light on the cell autonomy of a certain trait, inform us about the propagation of molecular signals and indeed allow us to directly compare genetically distinct tissue sectors in an attempt to determine specific gene function. Key Concepts: Plant chimaeras comprise cells of distinct genomes, whereas in genetic mosaics, cells of different genotypes derived from the same zygote. Cells with different genotypes or even from different species can communicate to form functional meristems and other tissues. Continuous persistent sectors throughout a plant can be explained by a mosaic shoot apical meristem and can be used to create ‘developmental fate maps’. The fate of most plant cells is determined late in development by their position rather than by their ancestry. Sector boundary analysis is used to identify the number of cells set aside as progenitors of an organ. Mosaic plants are used to analyse where a molecular signal originates and how it is propagated. Sector analysis provides clues about the cell autonomy of a trait as well as specific gene function. Keywords: chimaeras; genetic mosaics; shoot apical meristem; fate maps; cell lineage; sector analysis

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

The technique of animal vascular perfusion fixation was adapted for in situ fixation of the fragile and difficult to access cells of the ray parenchyma system in stems of 10-year-old Eucalyptus nitens trees. In situ fixation enabled tissue to be safely dissected for histological processing without risk of damage to microstructure or initiation of wound response. Fixative was perfused through the active vascular system under hydrostatic and transpiration pressure directly to vessel-associated ray cells. Diffusion from vessels allowed fixative to access nonvessel-associated ray cells. Acrolein was included to aid diffusion fixation and safranin dye was included to define fixed regions within the xylem. Sections prepared for light and electron microscopy from samples cut from regions showing intense safranin staining showed good microstructural preservation and were free of artefacts caused by mechanical injury or wound response.

In an attempt to devise a method for the rapid creation of somatic transgenic wood sectors of sufficient size that would allow us to detect and analyse altered wood characteristics within them, we have explored the manual wounding and subsequent infection with Agrobacterium of dormant lateral buds in poplar. Following treatment and transformation with a 35S–GUS construct, frequent stable transformation was found in the form of distinct and specific GUS staining patterns in the outer cortex, cambial region (including primary and secondary xylem and phloem) and pith. Sector frequency and size were consistent with anatomical features of dormant lateral buds at the time of manual wounding and Agrobacterium-infection. The suitability of somatic sector analysis for functional genomic studies as well as for studies investigating pattern formation and the developmental fate of various cell-types within poplar stems is discussed.

Sector boundary analysis has been used to deduce the number and orientation of cells initiating flower and floral organ development in Arabidopsis thaliana. Sectors were produced in transgenic plants carrying the Ac transposon from maize inserted between the constitutive 35S promoter and the GUS reporter gene. Excision of the transposon results in a blue-staining sector. Plants were chosen in which an early arising sector passed from vegetative regions into the inflorescence and through a mature flower. The range of sector boundary positions seen in mature flowers indicated that flower primordia usually arise from a group of four cells on the inflorescence flank. The radial axes of the mature flower are apparently set by these cells, supporting the concept that they act as a structural template. Floral organs show two patterns of initiation, a leaf-like pattern with eight cells in a row (sepals and carpels), or a shoot-like pattern with four cells in a block (stamens). The petal initiation pattern involved too few cells to allow assignment. The numbers of initiating cells were close to those seen when organ growth commenced in each case, indicating that earlier specification of floral organ development does not occur. By examining sector boundaries in homeotic mutant flowers in which second whorl organs develop as sepal-like organs rather than petals, we have shown that their pattern of origin is position dependent rather than identity dependent.

Background NAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored. Results We overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area. Conclusions This study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.

Background Cellulose is an important biopolymer produced by all plants and is used in a number of different industries, including for pulp and paper production. Cellulose is deposited into the plant cell wall by a large membranebound protein complex, which is composed of different cellulose synthase (CESA) proteins. The cellulose content and pattern of deposition in plant cell walls is highly variable depending on the function of the cell. All plant cells have a thin primary cell wall, but a number of plant cell types, including xylem cells, also deposit a secondary cell wall to give these tissues mechanical strength required to perform their function. Different cellulose synthase (CesA) genes have been shown to be involved in the deposition of primary and secondary walls. In Arabidopsis, three CesA (AtCesA4, 7 and 8) genes have consistently been associated with cells depositing secondary cell walls, while a different set of CesA genes have been shown to function during primary cell wall formation [Reviewed in 1]. These findings have been mirrored by studies of CesA gene orthologs in Populus and Eucalyptus[2-4]. While there have been a number of studies on CesA genes and their functions, much less is known about the regulation of these genes. In a previous study, we investigated the promoters of CesA genes involved in primary and secondary cell wall formation by performing a phylogenetic footprinting analysis to identify cis-elements conserved in the promoters from orthologous Arabidopsis, Populus and Eucalyptus cellulose synthase genes [5]. We identified a number of putative cis-regulatory elements that may play a role in the regulation of cellulose biosynthesis during primary and secondary cell wall formation. In the current study our aim is to further validate the ciselements identified in the CesA gene promoters by investigating their conservation across different Eucalyptus species and to determine the regulatory function of these promoter regions and the proteins which bind to them.

A midribless (mbl) mutant of Panicum maximum Jacq. induced by ethylmethanesulfonate treatment was characterized with respect to morphological and physiological features. The absence of the midrib was accompanied by a reduction of the large veins in the leaf lamina from 25% of total veins found in the wildtype to 13%. The florets of the mutant had no carpel but one or two extra stamens were observed. Similar phenotypic changes have been described for an ovaryless (ovl) mutant of barley. This supports the suggestion that a common morphogenetic determinant in wildtype plants induces both the development of the main midrib in leaves and the carpel in florets. Photosynthetic rate, CO2-compensation point and export of photosynthates, however, were similar in leaves of mutant and wild type plants.

Large-scale functional analysis of genes and transgenes suspected to be involved in wood development in trees is hindered by long generation times, low transformation and regeneration efficiencies and difficulties with phenotypic assessment of traits, especially those that appear late in a tree’s development. To avoid such obstacles many researchers have turned to model plants such as Arabidopsis thaliana (L.) Heynh., Zinnia elegans Jacq. and Nicotiana ssp., or have focused their attention on in vitro wood formation systems or in vivo approaches targeting primary meristems for transformation. Complementing such efforts, we report the use of Agrobacterium to introduce transgenes directly into cambial cells of glasshouse-grown trees in order to create transgenic somatic tissue sectors. These sectors are suitable for phenotypic evaluation and analysis of target gene function. In our experiments the wood formation zone containing the cambium of Eucalyptus, Populus and Pinus species of varying age was inoculated with Agrobacterium containing a CaMV 35S::GUS construct. Following an initial wound response, frequent and stable transformation was observed in the form of distinct GUS-staining patterns (sectors) in newly formed secondary tissues. Sector size and extent depended on the cell type transformed, the species and the length of time treated plants were allowed to grow (more than two years in some cases). Induced somatic sector analysis (ISSA) can now be efficiently used to study cell fate and gene function during secondary growth in stems of forest tree species.

Mutation induction has played an integral role in the improvement of most commercially important crop species but has not been successfully applied to tree species because of their long reproductive cycles which hinder the use of the traditional seed-treatment approaches. Treatment of pollen with a chemical mutagen prior to pollination will, theoretically, allow stable, heterozygous mutant trees to be produced in a relatively short time and might facilitate mutagenesis of tree species. As the first step in testing this hypothesis, a controlled-pollination trial with chemically treated pollen was conducted in Eucalyptus globulus ssp. globulus (Labill.). Assessment of fruit, seed and seedlings from more than 500 pollinations associated mutagenic treatment of pollen with a significant reduction in seed set. Non-significant increases in capsule (fruit) abortion, the inhibition of seed germination and the incidence of aberration in seedlings were also noted. We argue that pollen treatment may be a useful means of producing Eucalyptus mutants with variation in flowering time, salinity and frost tolerance, lignification and other traits of scientific and economic importance.

The Xylem Xapper is a prototype device capable of pulverizing xylem samples from stems of forest trees to a fine powder suitable for molecular biological investigations. Freezing of stem disks and pulverizing of xylem chips take place under liquid nitrogen, ensuring maximum retention and preservation of biologically active material. A modified technique enables extraction of sufficient quantities of high-quality RNA for preparation of cDNA libraries.

In maize, glossy (gl) mutants lack the wax layer normally present on the epidermis of young leaves. By insertion mutagenesis, unstable alleles (gl1‐m) have been induced at the Gl1 locus. In the gl1‐m8 strain, somatic reversions to wild‐type frequently result in the formation of large sectors occupying predictable positions in all seedlings' leaves. In studies of 230 gl1‐m8 seedlings with large reverted sectors covering around 50% of the first leaf, four patterns of sectoring were recognized: one large sector ending in all leaves at the main midrib (32.6% of cases); one central sector on leaves 1, 3 and 5 (or 2 and 4), corresponding to lateral stripes of Gl1 tissue on the other leaves (7.9%); a sector decreasing or increasing in successive leaves (9.1%); other types with one sector covering a leaf surface between 33 and 50% (19.1%) or with complex variegations (31.3%). Based on leaf sectoring, the pattern and stability of cell lineages during shoot apex establishment and embryonic activity leading to leaf primordia, are inferred from the genetic state (Gl or gl1‐m) of leaf founder cells present in the apex at the ring of primordia insertion. A genetic experiment indicates that the large somatic reversions considered derived from both the LI and the LII layers of the apex. A large majority of the observed patterns of reversion can be interpreted as due to a single event of transposition. The data are discussed and relevant conclusions proposed in relation to the age of the proembryo at the time of apex formation, the permanent or impermanent state of initial cells of the apex, the polarization of cell divisions and the plane of early apex cell division as a mechanism leading to the bilateral symmetry of the maize seedling. Copyright © 1992, Wiley Blackwell. All rights reserved

Two groups of mutants that affect the morphology of the lemma, a floral bract of barley, are described. The first comprises phenotypes associated with mutant alleles of calcaroides loci. On the lemma of these mutants, a well-organized neomorphic structure is formed, termed the sac. We provide a morphological description of wild-type (WT) and mutant lemmas, based on scanning electron microscopy (SEM), showing that both consist of similar tissues, but that the mutant is characterized by reversed growth polarity. The sac is a unique structure among grasses, and it is remarkable that recessive mutations at five different genetic loci lead to the same organ. The second group of mutants carry recessive alleles of two leafy lemma genes, both of which are necessary to cause the transformation of the lemma into a structure having all characteristics of a vegetative leaf, as shown by SEM analysis. The presence of sheath, blade, and ligule in the mutant lemma suggests that wild-type lemma development is interrupted at a leaf-like stage. The genes cal a, b, C, d, 23, lel1, and lel2 have now been mapped at precise positions on linkage groups 2, 7, 7, 3, 7, 5, and 7, respectively. The mutants considered in this article are unaffected in other floral organs. A model for lemma development is suggested.