In-vitro morphogenesis of corn (Zea mays L.)

The objective of this research was to study the in-vitro morphogenetic pattern of corn (Zea mays L.) shoot tips excised from aseptically-grown seedlings, and of expiants of axillary shoot buds, immature tassels and ears (staminate and pistillate inflorescences) obtained from greenhouse-grown corn plants. The seedling shoot tips and immature ears first regenerated clumps of multiple shoots within four weeks of culture on Murashige and Skoog (MS) basal medium supplemented with 500 mg/L casein hydrolysate (CH) and 9.0 M N⁶-benzyladenine (BA). Multiple shoot clumps were also differentiated from spikelets of immature tassels cultured on MS medium containing 500 mg/L CH, 4.5 M BA and 0.45 M 2,4-dichlorophenoxy acetic acid (2,4-D). All these multiple shoot clumps in turn differentiated clusters of ears after further four subcultures at four-week intervals under light on MS medium supplemented with 500 mg/L CH and 2.25, 4.5, 9.0 or 18 M BA. Axillary shoot buds readily differentiated clusters of ears within four weeks of the initial culture on these media. Secondary and tertiary ear clusters were initiated following subculture of primary ears on MS medium containing 500 mg/L CH and 4.5 or 9.0 M BA. Most of the ear primordia developed into ears with well-developed ovaries and styles on subculture on MS medium containing 500 mg/L CH and 1.0 M BA. Corn kernels were obtained after pollination of in-vitro-formed ears with pollens collected from greenhouse-grown corn. These kernels germinated in vitro and developed into mature corn plants in the greenhouse. Clusters of tassels were also differentiated in darkness from the multiple shoot clumps after six months successive subcultures but the spikelet primordia of tassels failed to develop fully under the in-vitro conditions tested. Somatic embryos arose directly from spikelet primordia of young tassels or ears on MS medium containing 500 mg/L CH and 4.5 M 2,4-D, or indirectly from calli derived from spikelets of young tassels and ears on MS medium containing 500 mg/L CH and 9.0 M 2.4-D.Abbreviations BA N⁶-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (basal medium) Heng Zhong is a Rockefeller Foundation Fellow on leave from the Institute of Botany, Academia Sinica, Beijing, P.R. China. This work was supported by a grant from the MidWest Plant Biotechnology Consortium and U.S.-A.I.D. grant No. DAN-4197-A-00-1126-00 to M.B. Sticklen. Thanks are due to Illinois Foundation Seeds, Champaign, USA for the supply of Honey N Pearl sweetcorn seeds and the Services of Center for Electron Optics, Michigan State University, for the electromicroscopic work as related to this publication.     

Polyamine-stimulated phosphorylation of proteins from corn (Zea mays L.) coleoptiles

The effect of polyamines (spermine, spermidine and putrescine) on in vitro phosphorylation of proteins from corn coleoptiles was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine promoted the phosphorylation of several membrane and soluble proteins and most of the proteins phosphorylated were different from those phosphorylated in the presence of calcium. Spermidine promoted the phosphorylation to a lesser extent and putrescine had very little stimulatory effect. Spermine-promoted phosphorylation of soluble proteins was dependent upon the presence of Mg2+ and was discernible at 100 microM spermine concentration.     

A re-examination of corn (Zea mays L.) ear volatiles

Helicoverpa zea (Boddie) is a major insect pest of corn and other agricultural crops. An improved understanding of semiochemcials that control adult behavior is needed to develop alternative control measures. In this study, overnight SPME collection of volatiles from corn ears enclosed in Teflon bags in the field at two stages of development were made. C₈–C₁₀ aldehydes, a C₈-alcohol, C₆–C₉ alcohol acetates, and numerous monoterpenes, sesquiterpenes, sequiterpene alcohols, and geosmin were identified after thermal desorption and GC/MS. Structural assignments of the alcohol acetates, Z-3-hexenyl acetate, 2-heptyl acetate, 2-nonyl acetate, and 4-nonyl acetate, the monoterpenes, α- and β-ocimene, and geosmin were made by analysis of standards that were purchased or prepared in the laboratory. All other assignments were based on published Kovat’s retention time indices (KI) and mass spectra. Pair-wise comparison of the relative amounts of each component between two groups of corn ears defined by silk weight did not identify significant differences, thus it is unknown whether or not silk weight impacted volatile emission composition and rate. To our knowledge three compounds detected in SPME collections, 2-heptyl acetate, 2-nonyl acetate, and 4-nonyl acetate have not been previously reported in corn ear or silk volatiles. Their impact on the flight response of gravid earworm females was evaluated in a flight chamber. No significant response to the individual compounds or a blend of all three was observed. Thus, their impact on moth behavior remains uncertain.     

Callus formation from cell culture protoplasts of corn (Zea mays L.)

Summary Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the quality of the culture used for protoplast isolation.     

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.     

Genetic studies of corn (Zea mays L.) anther culture response

Summary Anthers of two maize (Zea mays L.) inbred lines, DBTS (P₁) and B73 (P₂), their F₁, F₂ and first backcross generations — F₁ x DBTS (B₁), and F₁ x B73 (B₂) — were float cultured in YP medium to study the inheritance of corn anther culturability using generation mean analysis. Significant effects of generation were observed for the three traits measured: anther response (%), frequency of embryos (%) and anther productivity. Variation among the generations was similar for anther response and frequency of embryos: no significant differences were found among the P₁, F₁, F₂ and B₁ means, but the means of P₂ and B₂ were significantly lower than those of the other generations. For anther productivity, the F₂ generation tended to have a slightly higher tendency for multiple embryo formation. A simple additive-dominance model was adequate in explaining the inheritance of anther response and frequency of embryos, but digenic epistasis (additive x dominance) was involved in the inheritance of anther productivity. Additive genetic variance was higher than non-additive genetic variance for all the traits; however, only environmental variance was significant. Narrow-sense heritability estimates were 65% and 75% for anther response and frequency of embryos, respectively. Significant inter-plant variation was observed within generations, even for the inbred line DBTS, but isozymic analysis involving five enzyme loci did not reveal any genotypic variability within the inbred lines DBTS and B73.     

Nematodes associated with the rhizosphere of corn (Zea mays L.)

The research was carried out during 1999 in 8 different localities in Northern Italy. The nematodes were extracted from soil samples of rhizosphere of corn plants (Zea mays L.). The objective of the study was to investigate plant-parasite nematodes associated with maize. Some phytophagous genera are common pests of this crop and its yield-loss are often due to their high densities. In addition the nematode community was investigated for the genus composition, the trophic structure and its biodiversity. After the extraction from soil with a Bearmann funnel and Ludox centrifugation, nematodes were identified at genus level. They belonged to 22 families and 45 different genera. The genus Rhabditis, Pratylenchus, Helicotylenchus and Acrobeloides made up more than 70% of the total nematodes collected. The dominant trophic group was the bacterial feeders (61%) in particular Rhabditis, that was the most abundant and often the dominant one. Phytophagous represented in almost all fields more than 30% of the total nematodes. In all the examined sites biodiversity was quite low, being the H' values no more than 1.08. The data indicates a high level of disturbance. In some localities high densities of Helicotylenchus and Pratylenchus were found. While these nematodes have been identified as being potentially harmful for corn plants in our latitudes, especially in light soils, this research could give an indication for further monitoring studies regarding plant parasitic nematodes of corn crops. This data is particularly important considering that methyl bromide, often used in Italian agriculture against soil pathogens, has been banned since the beginning of 2005.     

Biochemical characterization of elongase activity in corn (Zea mays L.) roots

Chemical analysis of 4-day-old corn (Zea mays L.) root cell walls revealed that the lipophilic biopolymer suberin forms an important constituent of rhizodermal and hypodermal cell walls. Identified aliphatic monomers had chain lengths ranging from C16 to C26 and they belonged to 5 substance classes (omega-hydroxycarboxylic acids, 1,omega-dicarboxylic acids, 2-hydroxycarboxylic acids, carboxylic acids and alcohols) by which suberin is characterized. Biochemical experiments proved the occurrence of elongase activities in corn roots. Highest enzymatic activities were found in corn root microsomes, and major products synthesized by root elongases were elongated fatty acids with chain lengths ranging from C20 to C24. Preferred substrates of root elongases were acyl-CoAs of the chain length C18 and C20, whereas monounsaturated acyl-CoAs (C16:1 and C18:1) and acyl-CoAs of lower (C12-C16) and higher chain lengths (C22-C24) were rarely elongated. Elongase activities significantly decreased over the length (40 cm) of 10-day-old corn roots going from the young tip to the older base of the root. Thus, results presented here show the presence and activity of elongases in roots of plants.     

Biochemical and molecular characterization of corn (Zea mays L.) root elongases

Root surfaces are protected against the soil environment by the deposition of lignin and suberin. In order to obtain more insight into the regulation of root suberin biosynthesis, elongases from primary roots of corn (Zea mays L.) seedlings were characterized. Elongase activities (acyl-CoA and ATP-dependent) were located in the microsomal fraction of the root cells. C(20), C(22) and C(24) fatty acids were detected as primary products of elongases. Preferred substrates of the acyl-CoA elongases were C(18:0)-CoA and C(20:0)-CoA. Applying a molecular approach, using PCR and degenerate primers derived from the sequences of known leaf and seed 3-ketoacyl-CoA synthases (KCSs), catalysing the first step of very-long-chain fatty acid synthesis, the cDNA of a putative root KCS was obtained showing high homology to known leaf and seed KCSs at the DNA and amino acid levels. Thus, our approach provides the first direct evidence for the presence and the activity of root elongases in Z. mays. Ongoing research is focusing on the molecular analysis and the regulation of KCS expression in roots in reaction to different environmental stimuli.     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

Lipid composition and fluidity of plasma membranes isolated from corn (Zea mays L.) roots

Although the results of lipid analyses from several plant species have been available for many years a complete characterization of the corn root plasma membrane is still lacking. The present study provides a detailed analysis of individual lipids and a characterization of the membrane fluidity of corn (Zea mays L.) root plasma membranes isolated by phase-partitioning. Phospholipids (43.9 mol%), sterols (40.8 mol%), and sphingolipids in the form of glucocerebroside (6.8 mol%) constitute the major lipid classes. Stigmasterol (19.8 mol%), campesterol (13.0 mol%), phosphatidylcholine 15.8 mol%), and phosphatidylethanolamine (14.2 mol%) represent the most ubiquitous individual lipids. Hydroxy fatty acids make up 80.9 mol% and very long chain fatty acids are almost 78% of fatty acids in glucocerebroside. Hydroxy arachidic acid (20:0 h) and hydroxy lignoceric acid (24:0 h) are most prominent and glucocerebroside from corn root plasma membranes contains virtually no unsaturated fatty acids. Among the phospholipids only phosphatidylserine displayed a high proportion of very long chain fatty acids (e.g., behenic and lignoceric acid). Membrane fluidity was estimated by fluorescence anisotropy. Due to the high sterol content the plasma membrane of corn roots is relatively rigid.     

Genetic studies in an exotic population of corn (Zea mays L.) grown under two plant densities

Summary Progenies of a Design II [Comstock and Robinson (1948)] using random S ₁ lines from an exotic population of corn (Zea mays L.) were evaluated in a randomized incomplete block design with two replications at two plant-population densities (1 7,222 plants/ha and 68,888 plants/ha) in 1970 and 1971, at Lincoln, Nebraska. Five traits were studied i.e. grain weight, number of ears, days to flower, plant height and ear height.Under both densities the estimates of additive genetic variance were much larger than those of dominance genetic variance for all traits. The ratio of dominance to additive genetic variance estimates was less than 0.5 suggesting that for the majority of loci controlling the traits, partial to complete dominance is likely.The estimates of additive genetic x year interaction variance were high and significantly different from zero under both densities, indicating that estimates of additive genetic variance in this population obtained from experiments conducted in only one year may be seriously biased. The estimates of dominance genetic x year interaction variance were not significant and most of them were negative.Under both densities high genetic inter-relationships were indicated between grain weight and number of ears, days to flower and plant height, days to flower and ear height, and plant height and ear height.Even though there was a large difference between the two densities used in the study, the differences between the estimates of genetic parameters were not significant in all cases.The sample size of S ₁ plants representing each S₀ parent in the crossing nursery used in the present study (11.75) caused a small upward bias in the estimates of additive genetic variance, but it caused an upward bias in the estimates of dominance genetic variance of 6–7% of the total genetic variance.It is suggested that a trait such as grain weight should be expressed on a unit area basis when genetic parameters (except for correlation and the ratio between two values) obtained from experiments with different plant-population densities are to be compared.Published as Paper Number 3542, Journal Series, Nebraska Agricultural Experimental Station. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the Ph. D. degree.A. I. D. Participant.The work was supported in part by a grant from the Rockefeller Foundation.     

Rate of dehydration of corn (Zea mays L.) pollen in the air

The water content of corn (Zea mays L.) pollen directly affects its dispersal in the atmosphere through its effect on settling speed and viability. Therefore, the rate of water loss from pollen after being shed from the anther is an important component of a model to predict effective pollen transport distances in the atmosphere. The rate of water loss from corn pollen in air was determined using two methods: (1) by direct weighing of samples containing approximately 5 x 10(4) grains, and (2) by microscopic measurement of the change in size of individual grains. The conductance of the pollen wall to water loss was derived from the time rate of change of pollen mass or pollen grain size. The two methods gave average conductance values of 0.026 and 0.027 cm s-1, respectively. In other experiments, the water potential, psi, of corn pollen was determined at various values of relative water content (dry weight basis), either by using a thermocouple psychrometer or by allowing samples of pollen to come to vapour equilibrium with various saturated salt solutions. Non-linear regression analysis of the data yielded psi (MPa) = -3.218 theta(-1.35) (r2 = 0.94; for -298 < or = psi < or = -1 MPa). This result was incorporated into a model differential equation for the rate of water loss from pollen. The model agreed well (r2 approximately 0.98) with the observed time-course of the decrease of water content of pollen grains exposed to a range of temperature and humidity conditions.     

Purification and properties of alanine aminotransferase from maize (Zea mays L.) leaves

Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg protein)⁻¹ at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated the presence of arginine residue as anionic binding site in the active centre of AlaAT. This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510     

Cadmium and zinc influx characteristics by intact corn (Zea mays L.) seedlings

Summary Cadmium and zinc uptake parameters were determined for intact corn (Zea mays L.) seedlings grown for 15 and 22 in nutrient solutions containing levels of Cd and Zn that were similar to those found in soil solutions. Uptake of both elements was assumed to follow Michaelis-Menten kinetics. Calculations were based on the concentrations of free ionic Cd (Cd²⁺) and Zn (Zn²⁺) rather than the total solution concentration. Rates of Zn uptake were measured by determining depletion of Zn for periods of up to 30 h from solutions containing initial concentrations of 1.5 and 10μmol Zn 1⁻¹. Depletion curves suggested that Zn uptake characteristics were similar at both levels of Zn in solution. The Imax for Zn uptake decreased from 550 to 400 pmol m⁻² root surface s⁻¹ between 16 and 22 d of growth while Km decreased from 2.2 to 1.5 μmol Zn²⁺ 1⁻¹. Cadmium uptake parameters were measured by controlling Cd²⁺ activities in nutrient solution betwen 6.3 to 164 nmol l⁻¹ by continuous circulation of nutrient solution through a mixed-resin system. Imax for Cd uptake was 400 pmol m⁻² root surface s⁻¹ at 15 and 22 d of growth. The magnitude of Km increased from 30 to 100 nmol Cd²⁺ 1⁻¹ during this time period. The Km value suggests that corn is efficient for Cd uptake. The results of these uptake studies are consistent with the observed uptake of Zn and Cd by corn seedlings in soils.     

Plant Regeneration from Protoplasts of Corn(Zea mays L.)

Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one.     

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.