Plantlet regeneration from glume calli of maize (Zea mays L.)

Summary Totipotent callus cultures were established from anther-free glumes of Sweet corn, Seed corn, DHM 103 and DHM 101 on MS medium supplemented with 1–2 mg/l 2,4-D. The callusing response of the glumes was tested on six different media. Glumes at the uninucleate stage of pollen development callused with a high frequency compared to other stages. Organogenesis was observed in 40% of the cultures on media devoid of hormones. A total of 76 plantlets were regenerated on medium with 0.5–1.0 mg/l of both IAA and kinetin. Cytological observations in root tips indicated a diploid chromosome number (2n=20).     

Biochemical characterization of maize (Zea mays L.) for salt tolerance

The inherent differences for salt tolerance in two maize cultivars (Agatti-2002 and Sahiwal-2002) were evaluated in pot experiments. Plants were grown in half-strength of Hoagland nutrient solution added with 0, 80, 100, 120, 140 and 160 mM of NaCl. Salt stress markedly reduced the shoot and root lengths and fresh and dry masses. Reduction in growth attributes was more pronounced in cv. Agatti-2002 than cv. Sahiwal-2002. Both maize cultivars exhibited significant perturbations in important biochemical attributes being employed for screening the crops for salt tolerance. Cultivar Sahiwal-2002 was found salt tolerant as compared to cv. Agatti-2002 because it exhibited lower levels of H₂O₂, malondialdehyde (MDA) and higher activities of antioxidant enzymes. In addition, cultivar Sahiwal-2002 exhibited less salt-induced degradation of photosynthetic pigments, lower levels of toxic Na⁺ and Cl^?  and higher endogenous levels of K⁺ and K⁺/Na⁺ ratio. The results indicate that salt stress induced a marked increase in MDA, H₂O₂, relative membrane permeability, total soluble proteins and activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase andascorbate peroxidase). Moreover, increase in endogenous levels of Na⁺ and Cl^?  and decrease in K⁺ and K⁺/Na⁺ ratio and photosynthetic pigments were recorded in plants grown under salinity regimes.     

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 association analysis of seedling root development in maize (Zea mays L.)

Background

Plants rely on the root system for anchorage to the ground and the acquisition and absorption of nutrients critical to sustaining productivity. A genome wide association analysis enables one to analyze allelic diversity of complex traits and identify superior alleles. 384 inbred lines from the Ames panel were genotyped with 681,257 single nucleotide polymorphism markers using Genotyping-by-Sequencing technology and 22 seedling root architecture traits were phenotyped.

Results

Utilizing both a general linear model and mixed linear model, a GWAS study was conducted identifying 268 marker trait associations (p ≤ 5.3×10^-7). Analysis of significant SNP markers for multiple traits showed that several were located within gene models with some SNP markers localized within regions of previously identified root quantitative trait loci. Gene model GRMZM2G153722 located on chromosome 4 contained nine significant markers. This predicted gene is expressed in roots and shoots.

Conclusion

This study identifies putatively associated SNP markers associated with root traits at the seedling stage. Some SNPs were located within or near (<1 kb) gene models. These gene models identify possible candidate genes involved in root development at the seedling stage. These and respective linked or functional markers could be targets for breeders for marker assisted selection of seedling root traits.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1226-9) contains supplementary material, which is available to authorized users.     

Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)

Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10 days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (> two-fold change; t-test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals.     

Free proline accumulation in maize (Zea mays L.) subjected to prolonged waterlogging

Summary Free proline accumulation was measured in two maize genotypes (Ganga-2 and D747) subjected to waterlogging for three weeks at the knee high stage. The initial content of free proline was same in both the genotypes (0.1 micromole per gram fresh weight of leaves). The free proline content increased in both the genotypes when the plants were subjected to waterlogging. However, Ganga-2 accumulated more free proline than D747. Ganga-2 appeared to be more waterlogging tolerant than D-747.     

Genotypic effects on the amino acid relationships in maize (Zea mays L.) pollen and style

Summary The free amino acid content of the pollen grains and the style from three single cross hybrids (Wf9 x H55, Ky49 x Ky27, K64 x K55) and two inbred lines (Oh43, H55) was determined. No tyrosine was detected in the pollen grains of any genotype. Significant differences between pollen genotypes were found for aspartic acid, threonine, serine, lysine and histidine with no effect resulting from the vigor of the pollen source. No proline was found in the style of any genotype. Significant differences between style genotypes were obtained for threonine, serine, glutamic acid, leucine, tyrosine, ethanolanine, aminobutyric acid and histidine with a relationship between vigor of the style genotype present for tyrosine, ethanolanine and aminobutyric acid. The relationship between the pollen and style level for each amino acid as influenced by genotype was analyzed. A significant negative correlation was found only for threonine. Apparently, a complementary relationship between the pollen and style exists for some amino acids. Proline and tyrosine are available only from the pollen and style respectively. Threonine levels are balanced with varying contributions from both pollen and style depending on the genotype.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

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.     

Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil

In two pot-culture experiments with maize in a silty loam (P2 soil) contaminated by atmospheric deposition from a metal smelter, root colonization with indigenous or introduced arbuscular mycorrhizal (AM) fungi and their influence on plant metal uptake (Cd, Zn, Cu, Pb, Mn) were investigated. Soil was -irradiated for the nonmycorrhizal control. In experiment 1, nonirradiated soil provided the mycorrhizal treatment, whereas in experiment 2 the irradiated soil was inoculated with spores of a fungal culture from P2 soil or a laboratory reference culture, Glomus mosseae. Light intensity was considerably higher in experiment 2 and resulted in a fourfold higher shoot and tenfold higher root biomass. Under the conditions of experiment 1, biomass was significantly higher and Cd, Cu, Zn and Mn concentrations significantly lower in the mycorrhizal plants than in the nonmycorrhizal plants, suggesting a protection against metal toxicity. In contrast, in experiment 2, biomass did not differ between treatments and only Cu root concentration was decreased with G. mosseae-inoculated plants, whereas Cu shoot concentration was significantly increased with the indigenous P2 fungal culture. The latter achieved a significantly higher root colonization than G. mosseae (31.7 and 19.1%, respectively) suggesting its higher metal tolerance. Zn shoot concentration was higher in both mycorrhizal treatments and Pb concentrations, particularly in the roots, also tended to increase with mycorrhizal colonization. Cd concentrations were not altered between treatments. Cu and Zn, but not Pb and Cd root-shoot translocation increased with mycorrhizal colonization. The results show that the influence of AM on plant metal uptake depends on plant growth conditions, on the fungal partner and on the metal, and cannot be generalized. It is suggested that metal-tolerant mycorrhizal inoculants might be considered for soil reclamation, since under adverse conditions AM may be more important for plant metal resistance. Under the optimized conditions of normal agricultural practice, however, AM colonization even may increase plant metal absorption from polluted soils.     

Cadmium accumulation and its effects on metal uptake in maize (Zea mays L.)

The effects of different concentrations of Cd on growth of maize (Zea mays L.) and metal uptake were investigated. Cd accumulations in roots and shoots and the interactions among other metals (Mn, Fe, Cu and Zn) were analyzed using inductively coupled plasma atomic emission spectrometry (ICP-AES). The concentrations of cadmium chloride (CdCl(2).2.5H2O) used ranged from 10(-4) M to 10(-6) M. Cd had stimulatory effects during the first 5 days on root length of Nongda No. 108 at 10(-6) M and 10(-5) M Cd concentrations. Seedlings exposed to 10(-4) M Cd solution exhibited substantial growth reduction, and root growth even stopped. Root growth of Liyu No. 6 was stimulated at concentrations of 10(-5) M and 10(-6) M Cd during the entire experiment (15 days). Cadmium inhibited root growth of Liyu No. 6 at 10(-4) M Cd after 10 days of treatment. The Cd accumulation in roots and shoots of the two cultivars increased significantly (P < 0.05) with increasing Cd concentration and duration of treatment. Cadmium concentrated mainly in the roots, and small amounts were transferred to shoots. The proportion of Cd in the roots of Nongda No. 108 decreased with increases in Cd concentrations and duration of treatment, except for the group exposed to 10(-4) M Cd. In Liyu No. 6, the proportion of Cd in the root decreased progressively with an increase in Cd concentrations. Liyu No. 6 has a greater ability to remove Cd from solution and accumulate it when compared with Nongda No. 108. Liyu No. 6 can be considered a Cd-hyperaccumulator, according to the current accepted shoot concentration that defines hyperaccumulation as 0.01% (w/w) for cadmium. This cultivar, producing many roots and a high biomass and with great ability to accumulate Cd can play an important role in the treatment of soils stressed by Cd.     

Root cortical aerenchyma improves the drought tolerance of maize (Zea mays L.)

Root cortical aerenchyma (RCA) reduces root respiration in maize by converting living cortical tissue to air volume. We hypothesized that RCA increases drought tolerance by reducing root metabolic costs, permitting greater root growth and water acquisition from drying soil. To test this hypothesis, recombinant inbred lines with high and low RCA were observed under water stress in the field and in soil mesocosms in a greenhouse. In the field, lines with high RCA had 30% more shoot biomass at flowering compared with lines with low RCA under water stress. Root length density in deep soil was significantly greater in the high RCA lines compared with the low RCA lines. Mid‐day leaf relative water content in the high RCA lines was 10% greater than in the low RCA lines under water stress. The high RCA lines averaged eight times the yield of the low RCA lines under water stress. In mesocosms, high RCA lines had less seminal root respiration, deeper rooting, and greater shoot biomass compared with low RCA lines under water stress. These results support the hypothesis that RCA is beneficial for drought tolerance in maize by reducing the metabolic cost of soil exploration.     

Genotypic variation between maize (Zea mays L.) single cross hybrids in response to drought stress

Effects of soil drought on growth and productivity of 16 single cross maize hybrids were investigated under field and greenhouse experiments. The Drought Susceptibility Index (DSI) was evaluated in a three year field experiment by the determination of grain loss in conditions of two soil moisture levels (drought and irrigated) and in a pot experiment by the effects of periodical soil drought on seedling dry matter. In the greenhouse experiment response to drought in maize genotypes was also evaluated by root to shoot dry mater ratio, transpiration productivity index, indexes of kernel germination and index of leaf injury by drought and heat temperature. The obtained values of DSI enabled the ranking of the tested genotypes with respect to their drought tolerance. The values of DSI obtained in the field experiment allow to divide the examined genotypes into three, and in the greenhouse experiment into two groups of drought susceptibility. The correlation coefficients between the DSI of maize hybrids in the field and the greenhouse experiments was high and statistically significant, being equal to 0.876. The ranking of hybrids drought tolerance, identified on the basis of field experiments was generally in agreement with the ranking established on the basis of the greenhouse experiment. In the greenhouse experiment statistically significant coefficients of correlation with DSI values in hybrids were obtained for the ratio of dry matter of overground parts to dry matter of roots, both for control and drought treatments, whereas in the estimation of the transpiration productivity coefficient and total dry matter the correlation coefficients were not statistically significant. In this study several laboratory tests were carried out for the drought tolerance of plants (kernel germination, leaf injury) on 4 drought resistant and 4 drought sensitive maize hybrids. Statistically significant correlation coefficients between DSI and the examined parameter of grain germination and leaf injury were obtained for the determination of promptness index (PI), seedling survival index (SS) and leaf injuries indexes (IDS, ITS) as a result of exposure to 14 days of soil drought, osmotic drought −0.9 MPa and exposure to high temperature 45 ° or 50 °C. The results of laboratory tests show that in maize the genetic variation in the degree of drought tolerance is better manifested under severe conditions of water deficit in the soil.     

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.     

B chromosomes of maize (Zea mays L.) are positioned nonrandomly within sperm nuclei

We used fluorescence in situ hybridization to identify and map the position of B chromosomes (supernumerary chromosomes) within maize sperm cells. Observations on over 1,000 sperm cells from several genotypes show that, on average, the B chromosomes are positioned in the tip one-fourth of the sperm nucleus two-thirds of the time. In contrast, the centromeres and knobs of the A chromosomes (the normal set) are not restricted to the tip portion of the nucleus. To our knowledge, this is the first example of specific chromosome positioning within a plant gamete. Studies on nuclear architecture of somatic cells in both plants and animals suggest that chromosome behavior and gene expression may correlate with chromosome position within the nucleus. The functional significance of nonrandom positioning of the B chromosomes within maize sperm is as yet unclear. Received: 10 May 2000 / Revision accepted: 6 September 2000     

Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.)

In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeño and South American 5) maize genotypes. In a growth solution containing 6 M Al³⁺, Tuxpeño and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al³⁺ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.Abbreviations SA3 South American 3, an Al-tolerant maize cultivar - SA5 South American 5, an Al-sensitive maize cultivar The authors would like to express their appreciation to Drs. John Thompson, Ross Welch and Mr. Stephen Schaefer for their training and guidance in the use of the chromatography systems. This work was supported by a Swiss National Science Foundation Fellowship to Didier Pellet, and U.S. Department of Agriculture/National Research Initiative Competitive Grant 93-37100-8874 to Leon Kochian. We would also like to thank Drs. S. Pandey and E. Ceballos from the CIMMYT Regional office at CIAT Cali, Colombia for providing seed for the maize varieties and inbred line.     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm

The development of single nucleotide polymorphism (SNP) markers in maize offers the opportunity to utilize DNA markers in many new areas of population genetics, gene discovery, plant breeding and germplasm identification. However, the steps from sequencing and SNP discovery to SNP marker design and validation are lengthy and expensive. Access to a set of validated SNP markers is a significant advantage to maize researchers who wish to apply SNPs in scientific inquiry. We mined 1,088 loci sequenced across 60 public inbreds that have been used in maize breeding in North America and Europe. We then selected 640 SNPs using generalized marker design criteria that enable utilization with several SNP chemistries. While SNPs were found on average every 43 bases in 1,088 maize gene sequences, SNPs that were amenable to marker design were found on average every 623 bases; representing only 7% of the total SNPs discovered. We also describe the development of a 768 marker multiplex assay for use on the Illumina^® BeadArray? platform. SNP markers were mapped on the IBM2 intermated B73 × Mo17 high resolution genetic map using either the IBM2 segregating population, or segregation in multiple parent-progeny triplets. A high degree of colinearity was found with the genetic nested association map. For each SNP presented we give information on map location, polymorphism rates in different heterotic groups and performance on the Illumina^® platform.     

Salt tolerance of maize (Zea mays L.): the role of sodium exclusion

The influence of NaCl and Na₂SO₄ on growth of two maize cultivars (Zea mays cv. Pioneer 3906 and cv. Across 8023) differing in Na⁺ uptake was investigated in two green-house experiments. Na⁺ treatment with different accompanying anions (Cl^?/SO₄^2?) showed that ion toxicity was caused by Na⁺. While shoot growth of the two cultivars was markedly affected by salt in comparison to the control during the first 2–3 weeks, there were only slight differences between the cultivars. The shoot Ca²⁺ concentration was reduced in both cultivars, and the youngest leaves contained an even lower concentration compared with the rest of the shoot. During this first phase, Across 8023 tended to have higher concentrations of Ca²⁺ than Pioneer 3906. The Na⁺-excluding cultivar Pioneer 3906 showed continuous, although reduced, growth compared with the control, while the Na⁺ concentration in the shoot decreased until flowering. Cultivar Across 8023 accumulated Na⁺ until flowering: the reduction in the growth of stressed plants was greater than that for Pioneer 3906. Leaves of cultivar Across 8023 showed clear toxic symptoms, while those of the more salt-tolerant cultivar Pioneer 3906 did not. It is concluded that Na⁺ exclusion contributes to the salt tolerance of maize.