Two novel meiotic restitution mechanisms in haploid maize (Zea mays L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

Epistasis in maize (Zea mays L.)

Summary Three flint and three dent maize (Zea mays L.) inbred lines, their possible F₁ crosses, F₂ and backcross progenies, and all possible three-way crosses were evaluated in a three-year experiment for yield, ear moisture, and plant height. The purpose was to estimate genetic parameters in European breeding materials from (i) generation means analysis, (ii) diallel analysis of generation means, and (iii) analysis of F₁ and three-way cross hybrids. Method (i) was based on the F-metric model and methods (ii) and (iii) on the Eberhart-Gardner (1966) genetic model; both models extended for heterotic maternal effects.Differences among generation means for yield and plant height were mainly attributable to dominance effects. Epistatic effects were significantly different from zero in a few crosses and considerably reduced heterosis in both traits. Additive x additive and domiance x dominance effects for yield were consistently positive and negative, respectively. Significant maternal effects were established to the advantage of generations with a heterozygous seed parent. In the diallel analysis, mean squares for dominance effects were greater than for additive effects for yield and plant height but smaller for ear moisture. Though significant for yield and plant height, epistatic variation was small compared to additive and dominance variation. Estimates of additive x additive epistasis for yield were significantly negative in 11 of 15 crosses, suggesting that advantageous gene combinations in the lines had been disrupted by recombination in the segregating generations. The analysis of hybrids supported the above findings regarding the analysis of variance. However, the estimates of additive x additive epistasis for yield were considerably smaller and only minimally correlated with those from the diallel analysis. Use of noninbred materials as opposed to materials with different levels of inbreeding is considered the main reason for the discrepancies in the results.     

Genome-wide isolation of resistance gene analogs in maize (Zea mays L.)

Conserved domains or motifs shared by most known resistance (R) genes have been extensively exploited to identify unknown R-gene analogs (RGAs). In an attempt to isolate all potential RGAs from the maize genome, we adopted the following three methods: modified amplified fragment length polymorphism (AFLP), modified rapid amplification of cDNA ends (RACE), and data mining. The first two methods involved PCR-based isolations of RGAs with degenerate primers designed based on the conserved NBS domain; while the third method involved mining of RGAs from the maize EST database using full-length R-gene sequences. A total of 23 and 12 RGAs were obtained from the modified AFLP and RACE methods, respectively; while, as many as 109 unigenes and 77 singletons with high homology to known R-genes were recovered via data-mining. Moreover, R-gene-like ESTs (or RGAs) identified from the data-mining method could cover all RACE-derived RGAs and nearly half AFLP-derived RGAs. Totally, the three methods resulted in 199 non-redundant RGAs. Of them, at least 186 were derived from putative expressed R-genes. RGA-tagged markers were developed for 55 unique RGAs, including 16 STS and 39 CAPS markers.     

The mouse Spo11 gene is required for meiotic chromosome synapsis

The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.     

A low-pressure gene gun for genetic transformation of maize (Zea mays L.)

We have successfully used the low-pressure BioWare gene gun, developed for gene transfer in animal cells, for plant tissues. The BioWare device is easy to manipulate. Just 50 psi helium pressure was sufficient to transfer foreign genes into the aleurone layer and embryo of maize without causing tissue damage in the impact area. As shown by expression signals from invasive histochemical β-glucuronidase (GUS) activity, the foreign reporter gene expressed well in bombarded tissues. This successful GUS-transient expression extends the application of this low-pressure gene gun from animal cells to plant tissues.     

The nucleolus organizer region of maize (Zea mays L.)

Maize plants were produced partially triploid for the heterochromatic segment of the nucleolus organizer region (NOR) or partially triploid or tetraploid for the site giving rise to the secondary constriction of the NOR. These partially hyperploid plants were characterized in terms of chromosome and/or nucleolar constitution by light microscopy at pachytene, diakinesis, and quartet stages of microsporogenesis. DNA's of the various partially hyperploid plants and appropriate controls were extracted and hybridized with ³H-rRNA. The heterochromatic segment of the NOR was found to contain most of the rRNA cistrons, but has little or no interaction with the nucleolus. In contrast with the heterochromatic segment, the site giving rise to the secondary constriction contains few rRNA cistrons but is active in nucleolar formation as viewed at pachytene, diakinesis and quartet stages.     

Thiols in cadmium- and copper-treated maize (Zea mays L.)

Maize plants (Zea mays L. cv. Honeycomb F-1) were grown on quartz sand containing amounts of Cd or Cu which resulted in comparable internal contents in the roots. Fresh and dry weights and the content of Cd or Cu were measured in roots and shoots after eight weeks. In addition, cysteine, γ-glutamylcysteine (γEC), glutathione (GSH) and the thiols in heavy-metal-binding peptides (HMBPs) were determined in the roots. At low internal contents, Cd and Cu inhibited root growth to the same extent. Inhibition by Cu was enhanced, however, at high internal contents, indicating that Cu was more toxic than Cd. Separation of extracts from roots of Cd- and Cutreated plants on a Sephadex G-50 column resulted in HMBP complexes with relative molecular masses (M_rs) of 6200 and 7300, respectively. Separation of these HMBP-complexes using HPLC resulted in a distinct pattern of thiol compounds for each heavy metal. The accumulation of HMBPs was linearly dependent on the content of Cd at all values examined. In Cu-treated roots, HMBP accumulation was linearly dependent on the internal Cu content only up to 7.1 μmol·g^?1 dry weight. At internal contents which caused an enhanced inhibition of root growth, no further significant increase in the HMBP content was detected. At these internal Cu contents an increased transport of Cu to the shoot was measured. This result indicates that HMBPs are involved in reducing heavy-metal transport from roots to shoots.     

Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.)

We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5 untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron).     

Plot size for progeny selection in maize (Zea mays L.)

Summary Six progeny trials that included 147 half-sib progenies of maize (Zea mays L.) population ESALQ PB-5 were conducted for the purpose of studying plot size and its consequences in recurrent selection programs. The progenies were evaluated in three 7x7 duplicate simple lattice experiments using one-row plots of 5 m². At harvest each plot was partitioned into five sub-plots (sampling units), and data was collected from each sampling unit. At the same time and place the same progenies were evaluated in three 7x7 duplicate simple lattice experiments using 1-m² (linear row with 5 plants) plots. Data were collected for plant and ear height, ear diameter, total ear weight, and total grain yield. The data were combined by using adjacent sampling units, and the analyses were performed by considered five plot sizes in addition to those of the independent trials with 1-m² plots. The experiments with 1-m² plots were less efficient in discriminating for yield traits among progenies than those with 5-m² plots. The combination of plot size and number of progenies evaluated indicated that an optimum plot size for yield was between 3 and 4 m², or 15–20 plants per plot. With such sizes the expected gain was maximized for the four replications used in this study. If the total area covered by each progeny is constant, the maximum gain from selection, however, is attained by decreasing plot size and increasing the number of replications. The minimum size of plots is, however, limited by practical or theoretical criteria. Plot size affected the estimates of additive genetic variance, coefficient of heritability, and genetic coefficient of variation for all of the traits. No practical limitation was observed for conducting experiments with 1-m² plot.     

Biochemical composition of maize (Zea mays L.) pollen

Summary Pollen grains containing either the Wx, wx, Su ₁, su ₁, Sh ₂ or sh ₂ alleles were stored at 0, 1, 2, 3, 4 and 5 days at 2 °C. After each storage period, a portion of pollen from each genotype was analyzed for free amino acid content. Over all genotypes, storage significantly altered the content of all 16 amino acids measured. With increasing storage, a relatively consistent increase in aspartic acid, isoleucine, leucine, phenylalanine, ethanolanine, aminobutyric acid, NH₃ and lysine was found. A relatively consistent decrease in glutamic acid, proline, glycine and alanine occurred with increasing storage. No consistent response to storage was obtained with threonine-serine, valine, histidine and the unknown. Apparently, storage or stage of viability loss has a pronounced effect on amino acid metabolism in maize pollen grains. The experiment was designed so that comparisons free of genetic background effects could be made between alleles at each locus. Significant allele X storage interactions at each locus were found as follows: at the waxy locus, aspartic acid, glycine, alanine and ethanolanine; at the sugary locus, aspartic acid, alanine, ethanolanine and aminobutyric acid; and at the shrunken locus, aspartic acid, alanine, valine, leucine and ethanolanine. Amino acid metabolism is apparently influenced by the action of the alleles at these loci. The differences between the loci in the amino acids affected indicate the different areas of amino acid metabolism are influenced by each locus.Journal Series Paper No. 4425, Florida Agricultural Experiment Station.     

Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero

The construction of maize genotypes with high haploid induction capacity made it possible to study the effect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine added to the induction medium had no negative influence on the androgenic responses (anther induction, induction of structures of microspore origin and their regeneration ability) of the genotypes examined. However, significantly higher fertility was observed in plants originating from colchicine-treated microspores, especially at 0.03%. Cytological examinations showed that colchicine treatment before the first microspore division efficiently arrested mitosis and resulted in homozygous doubled-haploid microspores. Under the experimental conditions, the antimitotic drug had no later effect on the division symmetry of the microspore nucleus, and unequal divisions remained dominant. Callus formation from the induced microspores seemed to be more typical (ranging between 60–70%), but embryo frequency was increased by approximately 10%, especially at the higher colchicine concentration. These results suggest that the mechanism of colchicine action in premitotic maize microspores may differ from that previously observed in wheat. Received: 15 June 1998 / Revision received: 17 September 1998 / Accepted: 3 December 1998     

Biochemical composition of maize (Zea mays L.) pollen

Summary Proline was the most abundant amino acid with a mean value of 186.28 moles/mg dry pollen. The other amino acids tested were below 33 moles/mg dry pollen. The mutant wx significantly increased aspartic acid, valine, histidine and an unknown but significantly decreased aminobutyric acid. The mutant sh ₂ significantly increased glutamic acid, proline, lysine, histidine and an unknown but significantly decreased aspartic acid and aminobutyric acid. The effect of su ₁ was altered by the genetic background involved. In one genetic background, su ₁ did not significantly increase any amino acid but significantly decreased alanine and aminobutyric acid. However, in a distinctly different background, su ₁ significantly increased aminobutyric acid but significantly decreased aspartic acid and glutamic acid. Apparently the genetic background is capable of producing major shifts in the amino acid pattern in addition to the action of these mutants.The fatty acids, palmitic and linolenic were the most common with percentages of 54.1 and 34.4 respectively. The mutants tested did not affect the fatty acid distribution.

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Zusammenfassung Prolin war die am reichlichsten vorkommende Aminosäure mit einem mittleren Gehalt von 186,28 Mikromol per mg trockenen Pollen. Die anderen Aminosäuren erreichten weniger als 33 Mikrogramm per mg trockenen Pollen.Die Mutante wx zeigte einen signifikant erhöhten Gehalt an Asparaginsäure, Valin, Histidin, sowie einer nicht identifizierten Komponente, während der Gehalt an -Aminobuttersäure signifikant erniedrigt war. Die Mutante sh ₂ ist gekennzeichnet durch einen signifikant erhöhten Gehalt an Glutaminsäure, Prolin, Lysin, Histidin, sowie einer unbekannten Fraktion; der Gehalt an Asparaginsäure und -Aminobuttersäure war dagegen signifikant erniedrigt. Die Wirkung des mutierten Gens su ₁ wurde durch das übrige Genom, in dem es sich befand, geändert. In dem einen genetischen Milieu verursachte su ₁ keine signifikante Erhöhung des Gehaltes irgend einer Aminosäure, während der Gehalt an Alanin und -Aminobuttersäure signifikant erniedrigt war. In einem anderen genetischen Milieu jedoch zeigte su ₁ eine signifikante Erhöhung der -Aminobuttersäure; Asparaginsäure und Glutaminsäure waren signifikant erniedrigt.Offensichtlich ist das übrige Genom zusätzlich zu der Wirkung der genannten Mutanten in der Lage, wesentliche Verschiebungen im Verteilungsmuster der Aminosäuren zu verursachen.Von den Fettsäuren wurden am häufigsten Palmitin- und Linolen-Säure mit einem Gehalt von 54,1 bzw. 34,4% gefunden. Die untersuchten Endosperm-Mutanten zeigten keinen Einfluß auf die Fettsäureverteilung im Pollen.

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Journal Series Paper No. 3468, Florida Agricultural Experiment Station.     

Biochemical composition of maize (Zea mays L.) pollen

Summary Mean percentages of sugars, water-soluble polysaccharides, starch, total carbohydrates and lipids were 40.1, 7.4, 28.6, 76.1, and 1.8 respectively. Differences among the mutants were found only for water-soluble polysaccharides with both wx and sh ₂ decreasing the percentage a small but significant amount. In terms of the various carbohydrates measured, no correlation was found between the expression of these mutants in the pollen and the kernel.

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Zusammenfassung Die Untersuchung des Kohlenhydrat- und Lipid-Gehaltes im Pollen der Endosperm-Mutanten waxy, shrunken und sugary vom Mais ergab einen gemittelten Gehalt an Zucker von 40,1%, wasserlöslichen Polysacchariden 7,4%, Stärke 28,6, Gesamtkohlenhydraten 76,1% und Lipiden 1,8%.Unterschiede zwischen den Mutanten wurden lediglich hinsichtlich der wasserlöslichen Polysaccharide gefunden, und zwar bei wx und sh ₂, die einen signifikant niedrigeren Gehalt aufwiesen.Hinsichtlich der verschiedenen analysierten Kohlenhydrate wurde keine Korrelation zwischen der Expression der Mutanten im Pollen und in den Karyopsen gefunden.

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Herrn Professor Dr. J. Straub (Köln-Vogelsang) zu seinem 60. Geburtstag gewidmet.

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Journal Series Paper No. 3621, Florida Agricultural Experiment Station.     

A comparison of methods for direct gene transfer into maize (Zea mays L.)

Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers. Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery.     

Hydroxylation reactions in roots of maize (Zea mays L.)

The authors succeeded in demonstrating accumulation of tyrosine and p-coumaric acid in three day-old roots of maize (Zea mays L.) fed with L-phenylalanine and cinnamic acid. Phenylpyruvic acid applied under the same conditions gave rise to phenylalanine, indicating the presence of the corresponding transferase activity. Even simultaneous application of inhibitors of transaminase activity—hydroxylamine and isonicotinic acid hydrazide—did not result in the formation of p-hydroxyphenylpyruvic acid.     

Hydroxylation reactions in roots of maize (Zea mays L.)

The authors succeeded in demonstrating accumulation of tyrosine and p-coumaric acid in three day-old roots of maize (Zea mays L.) fed with L-phenylalanine and einnamie acid. Phenylpyruvic acid applied under the same conditions gave rise to phenylalanine, indicating the presence of the corresponding transferase activity. Even simultaneous application of inhibitors of transaminase activity — hydroxylamine and isonicotinic acid hydrazide — did not result in the formation of p-hydroxyphenylpyruvic acid.     

Induced cytomictic diversity in maize (Zea mays L.) inbred

Mutation breeding has been used for improving oligogenic and polygenic characters, disease resistance and quantitative characters including yielding ability. The cytological stability of maize inbred lines is an important consideration in view of their extensive use in genetics and plant breeding research. Investigation in Zea mays L. confirms that the migration of chromosomes is a real event that cannot be misunderstood as an artifact produced by fixation or mechanical injuries. During present investigation, we found that out of six inbred lines of Zea mays L. viz. CM-135, CM-136, CM-137, CM-138, CM-142 and CM-213 at various treatment doses of gamma irradiations viz. 200, 400 and 600 Gy, some of the plants of inbred line CM- 138 at 200 Gy dose displayed characteristic cytoplasmic connections during all the stages of meiosis. Four plants from this treatment set were found to be engaged in a rare phenomenon reported as "Cytomixis". It elucidates that in inbred of Zea mays L., induced cytomixis through gamma rays treatment may be considered to be a possible source of production of aneuploid and polyploid gametes. This phenomenon may have several applications in Zea mays L. improvement in the sense of diversity and ever yield potential.