Callose and homogalacturonan epitope distribution in stomatal complexes of Zea mays and Vigna sinensis.

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function.

De-Esterified Homogalacturonan Enrichment of the Cell Wall Region Adjoining the Preprophase Cortical Cytoplasmic Zone in Some Protodermal Cell Types of Three Land Plants.

The distribution of highly de-esterified homogalacturonans (HGs) in dividing protodermal cells of the monocotyledon Zea mays, the dicotyledon Vigna sinensis, and the fern Asplenium nidus was investigated in order to examine whether the cell wall region adjoining the preprophase band (PPB) is locally diversified. Application of immunofluorescence revealed that de-esterified HGs were accumulated selectively in the cell wall adjacent to the PPB in: (a) symmetrically dividing cells of stomatal rows of Z. mays, (b) the asymmetrically dividing protodermal cells of Z. mays, (c) the symmetrically dividing guard cell mother cells (GMCs) of Z. mays and V. sinensis, and (d) the symmetrically dividing protodermal cells of A. nidus. A common feature of the above cell types is that the cell division plane is defined by extrinsic cues. The presented data suggest that the PPB cortical zone-plasmalemma and the adjacent cell wall region function in a coordinated fashion in the determination/accomplishment of the cell division plane, behaving as a continuum. The de-esterified HGs, among other possible functions, might be involved in the perception and the transduction of the extrinsic cues determining cell division plane in the examined cells.

Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape.

The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells.

IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation.

Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanol-choline oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 ω-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize.

Low temperature caused modifications in the arrangement of cell wall pectins due to changes of osmotic potential of cells of maize leaves (Zea mays L.).

The cell wall emerged as one of the important structures in plant stress responses. To investigate the effect of cold on the cell wall properties, the content and localization of pectins and pectin methylesterase (PME) activity, were studied in two maize inbred lines characterized by different sensitivity to cold. Low temperature (14/12 °C) caused a reduction of pectin content and PME activity in leaves of chilling-sensitive maize line, especially after prolonged treatment (28 h and 7 days). Furthermore, immunocytohistological studies, using JIM5 and JIM7 antibodies, revealed a decrease of labeling of both low- and high-methylesterified pectins in this maize line. The osmotic potential, quantified by means of incipient plasmolysis was lower in several types of cells of chilling-sensitive maize line which was correlated with the accumulation of sucrose. These studies present new finding on the effect of cold stress on the cell wall properties in conjunction with changes in the osmotic potential of maize leaf cells.

Verification and characterization of chromosome duplication in haploid maize.

Doubled haploid technology has been used by various private companies. However, information regarding chromosome duplication methodologies, particularly those concerning techniques used to identify duplication in cells, is limited. Thus, we analyzed and characterized artificially doubled haploids using microsatellites molecular markers, pollen viability, and flow cytometry techniques. Evaluated material was obtained using two different chromosome duplication protocols in maize seeds considered haploids, resulting from the cross between the haploid inducer line KEMS and 4 hybrids (GNS 3225, GNS 3032, GNS 3264, and DKB 393). Fourteen days after duplication, plant samples were collected and assessed by flow cytometry. Further, the plants were transplanted to a field, and samples were collected for DNA analyses using microsatellite markers. The tassels were collected during anthesis for pollen viability analyses. Haploid, diploid, and mixoploid individuals were detected using flow cytometry, demonstrating that this technique was efficient for identifying doubled haploids. The microsatellites markers were also efficient for confirming the ploidies preselected by flow cytometry and for identifying homozygous individuals. Pollen viability showed a significant difference between the evaluated ploidies when the Alexander and propionic-carmin stains were used. The viability rates between the plodies analyzed show potential for fertilization.

Anther development of maize (Zea mays) and longstamen rice (Oryza longistaminata) revealed by cryo-SEM, with foci on locular dehydration and pollen arrangement.

Key message: Pollen maturation in Poaceae. Another development has been extensively examined by various imaging tools, including transmission electron microscopy, scanning electron microscopy, and light microscopy, but none is capable of identifying liquid water. Cryo-scanning electron microscopy with high-pressure rapid freeze fixation is excellent in preserving structures at cellular level and differentiating gas- versus liquid-filled space, but rarely used in anther study. We applied this technique to examine anther development of Poaceae because of its economic importance and unusual peripheral arrangement of pollen. Maize and longstamen rice were focused on. Here, we report for the first time that anthers of Poaceae lose the locular free liquid during late-microspore to early pollen stages; the majority of pollen grains arranged in a tight peripheral whorl develops normally and reaches maturity in the gas-filled loculus. Occasionally, pollen grains are found situated in the locular cavity, but they remain immature or become shrunk at anthesis. At pollen stage, microchannels and cytoplasmic strands are densely distributed in the entire pollen exine and intine, respectively, suggesting that nutrients are transported into the pollen from the entire surface. We propose that in Poaceae, the specialized peripheral arrangement of pollen grains is crucial for pollen maturation in the gas-filled loculus, which enables pollen achieving large surface contact area with the tapetum and neighboring grains to maintain sufficient nutrient flow. This report also shows that the single aperture of pollen in Poaceae usually faces the tapetum, but other orientation is also common; pollen grains with different aperture orientations show no morphological differences.

Identifying the allergenicity of maize pollen in Iran.

BACKGROUND: Maize is a member of the Poaceae family, capable of producing large amounts of pollen grains which may constitute important allergens in spring and summer. The aim of this study was to determine the protein content of maize pollen and its allergenicity in guinea pigs. METHODS: The morphology of maize pollens was determined using light microscopy and scanning electron microscopy. The size of separated proteins was obtained by SDS-PAGE. A group of animals were immunized with maize pollen extract and the others were kept as control. After 40 days, the skin prick test was done in animals after blood sampling for counting the eosinophils. The allergenisity of proteins was identified by immunoblotting of transferred bonds using sera from sensitized guinea pigs. RESULTS: Pollen grains showed a spherical, monoporate structure with the scabrate exine surface. The SDS-PAGE indicated a major band of about 50 kD.We also showed increase in flare and wheal diameter following skin prick test in sensitized guinea pigs along with an elevated number of eosinophils. The presence of group 13 allergen (Zea m13) with molecular weight of ~ 50 kD was found in immunoblotting results. CONCLUSION: This study showed one protein in maize pollen extract that could be considered as an allergen belonging to group 13 of allergen categories. However, further investigations should be scheduled for precise analysis of the proteins. This allergen can be used for diagnostic or therapeutic purposes (vaccination approaches) in allergic asthma patients.

The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility.

Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.

Function of isoamylase-type starch debranching enzymes ISA1 and ISA2 in the Zea mays leaf.

Conserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement. Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined. Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf. The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form.

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

BACKGROUND AND AIMS: The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-ß-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. METHODS: Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. RESULTS: Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. CONCLUSIONS: The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.

The maize tapetum employs diverse mechanisms to synthesize and store proteins and flavonoids and transfer them to the pollen surface.

In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage, and transfer of these pollen-coat materials in maize (Zea mays) differs completely from that reported in Arabidopsis (Arabidopsis thaliana), which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, three proteins, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease (ZmPCP), are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid developmental stage, whereas protease transcript emerged at a late developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanase transcript was distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative amino-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All three proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid to late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the three proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or the pollen surface.

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.

[Cortical cytoskeletal ring in prophase II leads to correction of abnormalities of the first meiotic division and to meiotic restitution of pollen mother cell nucleus].

The deviation of prophase cytoskeletal ring formation was determined during meiotic division in 50% of pollen mother cells (PMCs) in maize haploid No 1498 (Zea mays). At prophase in both meiotic divisions the cytoskeletal ring is formed in cortical region of cytoplasm instead of perinuclear. Sometimes formation of both perinuclear and cortical rings is observed in the same cell. It has been shown that in multinucleate PMCs the cortical ring leads to the consolidation of chromosomes into common spindle and to meiotic restitution.

Effects of external phosphorus on the cell ultrastructure and the chlorophyll content of maize under cadmium and zinc stress.

Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis, it was found that the ultrastructure of chloroplasts were changed, the shape of the chloroplasts altered and the numbers of grana that were asymmetrical increased; the numbers of grana and thylakoids decreased under the stress of Cd and Zn. The results indicated that the complex pollution involving Cd and Zn resulted in the membrane system of chloroplasts being damaged. When external phosphorus was applied, the numbers of damaged chloroplasts were significantly reduced and the nucleoli were better formed than those that did not receive phosphorus treatment. Moreover, many phosphate deposits were found in the vacuoles and on the surface of the roots, which were formed by phosphorus complexing with Cd (K(sp)=2.53x10(-33)) and Zn (K(sp)=9.00x10(-33)), respectively. Treatment with phosphorus conduced an increased chlorophyll content in plants compared with those that did not receive external phosphorus.

Localization of group-1 allergen Zea m 1 in the coat and wall of maize pollen.

The pollen surface consists of an outermost coat and an underlying wall. It makes the initial contact with the stigma surface during sexual reproduction. To date, only two proteins have been identified from the maize pollen coat. Zea m 1 (beta-expansin 1) is the major group-1 allergen in maize pollen, but its presence and localization in the pollen coat and wall has not yet been explored. In the present study, immunoblot analysis using an antibody directed against group-1 allergen revealed that a small amount of Zea m 1 exists in the pollen coat fraction prepared using a diethyl ether wash. Immunogold labeling also showed that the extracellular localization of Zea m 1 was mainly restricted to the tectum and the foot layer of the exine (the outer pollen wall), and gold particles immunolabelling Zea m 1 were unevenly dispersed throughout the pollen coat and wall. Moreover, a substantial amount of Zea m 1 was localized in the cytoplasm of the pollen interior. The presence of Zea m 1 in the pollen coat and wall suggests that Zea m 1 may play a potential role in pollen germination on the stigma.

Fractionation of corn stover by hot-water and aqueous ammonia treatment.

The efficiency of biomass utilization can be significantly improved by fractionation of biomass. A two-stage percolation process was investigated for pretreatment and fractionation of corn stover. The two-stage process is composed of hot water treatment followed by treatment with aqueous ammonia, both applied in a flow-through (percolation) reactor. The first stage processing is intended for hemicellulose removal whereas the second stage is intended for delignification. The pretreated material was nearly pure cellulose and both reagents are cheap and environmentally friendly. The conditions that achieve satisfactory level of biomass fractionation and acceptable enzymatic hydrolysis were identified in terms of reaction temperature, flow rate (retention time) and reaction time for each stage. With proper operation of two-stage treatment, fractionation of biomass was achieved to the extent that the xylan fraction is hydrolyzed with 92-95% conversion, and recovered with 83-86% yields; and the lignin removal is 75-81%. The remaining solid after two-stage treatment contained 78-85% cellulose. The two-stage treatments enhanced the enzymatic digestibility to 90-96% with 60 FPU/g of glucan, and 87-89% with 15 FPU/g of glucan. In two-stage treatment, the composition and digestibility data indicate that the lignin content in the biomass is one of the major factors controlling the enzymatic digestibility.

Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis.

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.

In-Situ probing of the biotic-abiotic boundary of plants by laser desorption/ionization time-of-flight mass spectrometry.

Laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry was applied for the direct analysis of cuticular waxes on intact plant tissues. Cuticular wax compounds were ionized by laser desorption in the presence of colloidal silver. Silver-adduct ions were detected on samples from Arabidopsis thaliana and from maize. Good spot-to-spot reproducibility indicated homogeneous coverage of the sample by the fine colloidal material. The results were consistent with GC-MS analyses of cuticular extracts, thus confirming the feasibility of direct analysis based on this protocol. Molecular masses of the adduct ions correspond well with the known composition of cuticular waxes. Moreover, LDI-TOF gave good estimates of the relative local abundances of a given compound. However, bias was found in cases where compounds with different ionization efficiencies were analyzed.

Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis.

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7 day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.