Production of transgenic maize plants and progeny by bombardment of hi-II immature embryos

Summary Production of transgenic maize (Zea mays L.) callus, plants, and progeny from microprojectile bombardment of 2–5-d cultured Hi-II immature embryos is described. Histological evidence indicates that these tissues are amenable to transformation due to surface layer cell division of the scutellum. Two out of every 100 bombarded embryos produced transgenic callus and R₀ transgenic plants were both male and female fertile. Expected segregation of transgenes was observed in progeny. The primary advantage of bombarding these tissues is increased male and female fertility of transgenic plants compared with those produced using long-term callus or suspension cultures.     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.     

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants

Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from microspore-derived cell suspension cultures. Protoplasts were electroporated in the presence of plasmid DNA containing the gus A and npt II genes encoding ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II), respectively. Transformed calli were selected and continuously maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA. analysis. Stably transformed tissue was transferred to regeneration medium and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.     

Cleavage polyembryony in maize

Summary Two types of cleavage polyembryony are described in the inbred line VIR 17 of maize. Suspensorial embryony was observed to occur spontaneously. Typical cleavage of the zygotic proembryo occurred spontaneously, but could also be induced by treating the developing caryopses with 2,4-Dichlorophenoxyacetic acid (2,4-D) on the second day after pollination. 2,4-D was active as a decorelative factor also evoking the expression of totipotency in individual proembryonal cells.     

Production of fertile transgenic maize by electroporation of suspension culture cells

Fertile, transgenic maize plants were generated by electroporation of suspension culture cells that were treated with a pectin-degrading enzyme. Electroporation of cells from two different suspension cultures, one derived from A188 X B73 and one derived from a B73-related inbred, with a plasmid containing the bar gene, resulted in high-frequency recovery of stably transformed callus lines. Plants were regenerated from thirteen transformed callus lines and transmission of bar to progeny was demonstrated.     

Production of herbicide-resistant transgenic maize plants using electroporation of seed-derived embryos

The improvement of commercial maize lines via biotechnological approaches is limited by the lack of a transformation system that is tissue culture free. In this paper, the development of a genetic transformation system is presented using electroporation for gene delivery and seed-derived embryo as the gene target. Plasmid DNA (pBARGUS), which contained the selectablebar gene for resistance to the herbicide Basta and the screenablegus gene, was delivered into enzymatically wounded mature maize embryos via electroporation. Transformed plants were identified by their ability to grow on a selective medium containing 30 mg/L of phosphinothricin. Southern hybridization, plant resistance to the application of Basta, GUS expression, and segregation analysis indicated that a functionalbar gene had integrated into the maize genome and was inherited in a mendelian fashion by the progeny.     

Estimates of gene frequencies in two heterotic maize populations

Summary The present study was undertaken to obtain information on average gene frequency in two heterotic populations of maize (Zea mays L.), Mezcla Amarillo Selection (MAS) and J607. Sixty-four male plants were taken in each of the populations and each of these were crossed to a different set of eight plants, four of which belonged to the same population and four to the other population. This resulted in two groups of intra-population (within MAS and within J607) and two groups of inter-population (MAS X J607 and J607 X MAS) progenies. Each group consisted of 256 full-sib progenies on the pattern of the North Carolina Design I mating system. The male plants were selfed to produce 64 S₁ prgenies in each population. The materials were evaluated at two diverse locations, Ludhiana and Gurdaspur, for grain yield, ear length, ear girth, number of kernel rows, plant height, ear height and days to silk. An incomplete block design with two replications were used. The plot consisted of a 5 m long row. Ratios of estimated genetic components of variance and covariance were compared with corresponding theoretical ratios computed for a single locus for various gene frequencies and levels of dominance, and approximate ranges of the gene frequencies and their relative magnitude were worked out in the two populations. The average frequency of favourable genes for plant height was estimated as 0.6 in MAS and 0.8 in J607. For grain yield the average gene frequency was 0.8 to 0.9 in MAS and 0.7 to 0.8 in J607 whereas for ear height it was 0.5 to 0.7 in MAS and 0.4 to 0.6 in J607. The gene frequency in the two populations seemed to be similar for days to silk, ear length, ear girth and kernel rows.     

Tillage effects on nitrogen uptake by maize from fine textured soils in the northwestern Corn Belt,USA

Summary Nitrogen (N) accumulation data from a replicated field study were fitted to a tanh (time) function and the derivate obtained to determine relative maximum rates of accumulation by maize. Both positive and negative effects of tillage on N accumulation rates were observed. Most of the N accumulation occurred over a 30-day period and time of N accumulation was not affected by tillage. Tilled profiles tend to contain greater NO₃–N, greater aeration, and lower moisture contents than untilled profiles, and these characteristics interact to affect plant N accumulation.     

Anther culture of maize and the visualization of embryogenic microspores by fluorescent microscopy

Summary Three maize genotypes previously shown in the literature to respond to anther culture were tested under various conditions. Studies indicated that embryogenic response ranged from 0 to 100 embryos per 1,000 anthers plated and was significantly lower without cold pretreatment of the anthers. Culture in liquid media tended to produce more embryos than in semi-solid as did the addition of activated charcoal to either liquid or solid culture media. Most results were confounded by plant-to-plant variation which tended to obscure significant differences. In one study, germination rate of androgenetic embryos averaged about 20%, but only 26% of those embryos that germinated completed their reproductive cycle and formed seed albeit through sibpollination since plants could not be selfed. Chromosome counts using root tip squashes indicated that regenerated plants were either haploid or diploid but plants scored as non-diploid yielded as much seed as scored diploids. This suggests that progeny can be recovered even from putative haploids, presumably as a result of sectoring in the developing ear. A DNA-specific fluorescent dye was used to visualize the presence of putative embryogenic microspores (PEMs) during the culture period. PEM counts were a function of time in culture and were apparently greater than the number of embryos obtained for a given treatment. The data indicate that, as previously reported for other species, both induction and survival phases also exist in maize anther culture.     

Treatment with salicylic acid decreases the effect of cadmium on photosynthesis in maize plants

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting photosynthesis from cadmium (Cd) toxicity. Seeds of maize (Zea mays L., hybrid Norma) were sterilized and divided into two groups. Half of the seeds were presoaked in 500muM SA solution for only 6h, after which both groups were allowed to germinate for 3d and were then grown for 14d in Hoagland solution at 22/18 degrees C in a 16/8-h light/dark period and 120mumolm(-2)s(-1) PAR. All seedlings (without H(2)O and SA controls) were transferred to Cd-containing solutions (10, 15, and 25muM) and grown for 14d. The rate of CO(2) fixation and the activity of ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were measured. Changes in the levels of several important parameters associated with oxidative stress, namely H(2)O(2) and proline production, lipid peroxidation, electrolyte leakage, and the activities of antioxidative enzymes (superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase (POD, EC 1.11.1.7)) were measured. Exposure of the plants to Cd caused a gradual decrease in the shoot and root dry weight accumulation, with the effect being most pronounced at 25muM Cd. Seed pretreatment with SA alleviated the negative effect of Cd on plant growth parameters. The same tendency was observed for the chlorophyll level. The rate of CO(2) fixation was lower in Cd-treated plants, and the inhibition was partially overcome in SA-pretreated plants. A drop in the activities of RuBPC and PEPC was observed for Cd-treated plants. Pretreatment with SA alleviated the inhibitory effect of Cd on enzyme activity. Proline production and the rates of lipid peroxidation and electrolyte leakage increased in Cd-treated plants, whereas the values of these parameters were much lower in SA-pretreated plants. Treatment of plants with Cd decreased APX activity, but more than doubled SOD activity. Pretreatment with SA caused an increase in both APX and SOD activity, but caused a strong reduction in CAT activity. The data suggest that SA may protect cells against oxidative damage and photosynthesis against Cd toxicity.     

Inhibition of maize root growth by high nitrate supply is correlated with reduced IAA levels in roots

The plant root system is highly sensitive to nutrient availability and distribution in the soil. For instance, root elongation is inhibited when grown in high nitrate concentrations. To decipher the mechanism underlying the nitrate-induced inhibition of root elongation, the involvement of the plant hormone auxin in nitrate-dependent root elongation of maize was investigated. Root growth, nitrogen and nitrate concentrations, and indole-3-acetic acid (IAA) concentrations in roots and in phloem exudates of maize grown under varying nitrate concentrations were analyzed. Total N and nitrate concentrations in shoots and roots increased and elongation of primary, seminal and crown roots were inhibited with increasing external nitrate from 0.05 to 5 mM. High nitrate-inhibited root growth resulted primarily from the reduced cell elongation and not from changes in meristem length. IAA concentrations in phloem exudates reduced with higher nitrate supply. Inhibition of root growth by high nitrate was closely related to the reduction of IAA levels in roots, especially in the sections close to root tips. Exogenous NAA and IAA restored primary root growth in high nitrate concentrations. It is concluded that the inhibitory effect of high nitrate concentrations on root growth may be partly attributed to the decrease in auxin concentrations of roots.     

The effect of oxygen deficiency on uptake and distribution of nutrients in maize plants

Young maize (Zea mays L.) plants, 7 days after germination were exposed to nutrient solutions which were either aerated or not aerated for 14 days. Nutrients were supplied as 50% strength Hoagland’s solution or, in the case of the four ‘low nutrient’ treatments, N, P, K or Ca were supplied at the equivalent of 10% strength Hoagland’s solution. Shoot fresh weight was decreased by 25% due to lack of aeration; O₂ deficiency also impaired leaf elongation but not dry weights, suggesting that lack of O₂ in the roots impaired cell expansion in shoots more than dry weight accumulation. The distribution of N, P, K and Ca within shoots was consistent with their relative mobilities in the phloem; at least 7% of Ca in plants after 14 days of treatments was found in the oldest leaf whereas N, P and K were rapidly remobilised to younger tissues. Between 33 and 49% of the total N, P and K in the shoot was found in the 40 mm of tissue at the base of the growing leaves in plants grown for 14 days at low nutrient concentrations. Concentrations (dry weight basis) of phloem-mobile nutrients were also greatest in the growing zones of the leaves, especially in the case of N and P. Calcium, on the other hand, was found in relatively low concentrations in the youngest tissue and as with the other nutrients, concentrations declined due to low external supply, non-aeration or a combination of both. In spite of the failure of Ca to move from old to young leaves, the effect of the deficiencies of N, P and K was probably as severe as that of Ca in the youngest tissues of treated plants. Calcium uptake by the whole shoot appeared to be slightly less sensitive to O₂ deficits than that of N, P and K. This compensated for the failure of Ca to move to growing tissues during periods of low external Ca supply.     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

Two-locus theory in recurrent selection for general combining ability in maize

Summary Two-locus theory for recurrent selection for general combining ability in maize was developed. The theory featured: (a) recombination of the selfed progeny of selected parents; and (b) linkage disequilibrium in the initial gametic array. The theory indicated: (a) that initial linkage disequilibrium exerts a permanent influence upon selection progress; (b) that interposition of one or more generations of random mating before each cycle reduces the permanent effect in ensuing cycles; and (c) that random mating done before initiation of selection is more efficient in removing the influence of linkage disequilibrium on selection progress than random mating done between subsequent cycles.     

Heritable targeted mutagenesis in maize using a designed endonuclease

The liguleless locus ( liguleless1 ) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I- Cre I homing endonuclease. A single-chain endonuclease, comprising a pair of I- Cre I monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 ( LG1 ) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium -mediated transformation of immature embryos, and transgenic T₀ plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T₀ plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant.     

Effects of High Temperature on Chlorophyll Fluorescence Induction and the Kinetics of Far Red Radiation-Induced relaxation of apparent F0 in maize leaves

Temperature dependence (25–50 °C) of chlorophyll (Chl) fluorescence induction, far-red radiation (FR)-induced relaxation of the post-irradiation transient increase in apparent F₀, and the trans-thylakoid proton gradients (pH) was examined in maize leaves. Temperatures above 30 °C caused an elevation of F₀ level and an enhancement of F₀ quenching during actinic irradiation. Millisecond delayed light emission (ms-DLE), which reflects the magnitude of pH, decreased strikingly above 35 °C, and almost disappeared at 50 °C. It indicates that the heat-enhanced quenching of F₀ under actinic irradiation could not be attributed mainly to the mechanism of pH-dependent quenching. The relaxation of the post-irradiation transient increase in apparent F₀ upon FR irradiation could be decomposed into two exponential components (₁ = 0.7–1.8 s, ₂ = 2.0–9.9 s). Decay times of both components increased with temperature increasing from 25 to 40–45 °C. The bi-phasic kinetics of FR-induced relaxation of the post-irradiation transient increase in apparent F₀ and its temperature dependence may be related to plastoquinone (PQ) compartmentation in the thylakoid membranes and its re-organisation at elevated temperature.     

Reversion of Texas male-sterile cytoplasm maize in culture to give fertile,T-toxin resistant plants

Summary Plants carrying Texas male-sterile (Tms) cytoplasm are normally sensitive to Drechslera maydis T-toxin. Tissue cultures were initiated from immature embryos of maize carrying Tms-cytoplasm, and plants were regenerated after selection for resistance to T-toxin. Fertile, T-toxin resistant plants were obtained from the unselected control cultures as well as from the selected material. In addition, one regenerant from an unselected culture was fertile and T-toxin sensitive. The progeny of the regenerants showed the phenotype of the female parent with respect to pollen-fertility, and T-toxin resistance. The data are consistent with the heritable changes observed being the result of the expression of an altered mitochondrial genome.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

The ultrastructural development of plastids in leaves of maize plants exposed to continuous illumination

Summary Chloroplast differentiation in relation to increasing leaf age has been investigated in maize plants exposed to continuous illumination. In the young leaves the proplastids differentiate into chloroplasts containing well organized grana as well as prolamellar bodies. In the older leaves, while plastids differentiate, the prolamellar bodies are no longer detectable. Chloroplast ability to build up prolamellar bodies does not seems so much a light dependent process as it is affected by cell differentiation rate.Supported by a grant of C.N.R.