Maize Callus Research Articles

The involvement of a PIG3 homolog quinone oxidoreductase gene in maize resistance to insects and fungi demonstrated through transgenic expression in maize callus

Insect and pathogen damage of maize inhibits sustainable production. Discovery of maize genes coding for products active against both classes of pests would significantly accelerate the rate of development of resistant varieties. A quinone oxidoreductase gene homologous to apoptosis related P53 inducible gene 3 (PIG3) in vertebrates was identified as a pest resistance candidate in a quantitative trait locus region for maize ear rot resistance. The quinone oxidoreductase gene was cloned from a Fusarium resistant inbred of maize and expressed in maize callus. The transformed callus had some significant resistance to the maize pathogen F. graminearum, compared to control transformants, and was often highly resistant to two major caterpillar pests of maize. A band of enhanced reactive oxygen species (ROS) generation in the presence of relevant substrates was noted when protein extracts from the transgenic callus compared to those from control callus were separated by polyacrylamide gel electrophoresis. Thus, presence or introduction of an optimally functional form of this gene should lead to enhanced resistance of maize and other crops to major insect and fungal pests.

Potential role of a maize metallothionein gene in pest resistance

Maize is grown worldwide and much of the world depends on its production, which is lessened by insect and fungal pests. Many maize genes with the potential to improve pest resistance exist in non-functional forms in several inbreds but are functional in those that show resistance. One such gene, encoding a metallothionein protein, was located from a resistance locus of maize inbred GE440, which shows resistance to some Fusarium spp. pathogens. The identified gene, encoding ZmMT10, is disrupted in many maize inbreds, including the commonly used inbred B73. When introduced into maize callus, transformants often significantly increased resistance to F. proliferatum, but were often less effective against F. graminearum. Some transformed callus with the ZmMT10 gene also retarded growth of two classes of insect pests, fall armyworms and corn earworms. Recombinant ZmMT10 was purified from Escherichia coli. The purified protein was found to bind zinc, copper, and nickel and scavenged reactive oxygen species in vitro, which are possible mechanisms for its antiinsect and antifungal activities. In bioassays, the purified protein retarded growth of fall armyworms and corn earworms, but did not show activity against fungi, suggesting that the antifungal activity observed in callus tissue is dependent on the interaction with other plant factors. The inclusion of the identified gene in new plant varieties should increase resistance to both insects and fungi.

Overexpression of maize GOLDEN2 in rice and maize calli improves regeneration by activating chloroplast development.

Golden2 (G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene (rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

A maize gene coding for a chimeric superlectin reduces growth of maize fungal pathogens and insect pests when expressed transgenically in maize callus

Plant breeding for pest resistance is a long process, and the rate of development of resistant varieties would be accelerated if resistance genes to multiple pests could be identified. A gene located in a resistance locus was cloned from a maize inbred with resistance to Fusarium spp. pathogens. This gene codes for an unusual lectin with regions of high histidine and proline frequency and two apparent sugar binding moieties. Binding studies with yeast-produced versions indicated N-acetyl glucosamine residues are a likely target. When the gene was expressed transgenically in maize callus, reduced growth of pathogenic Fusarium species (up to 71%) and caterpillar pests (up to 65%) was noted compared to control transformants, and the degree of growth inhibition was associated with levels of the protein detected. Available sequence of the gene from other maize inbreds indicated it was often disrupted and likely to code for proteins with reduced function. Incorporation of functional versions of the gene into crops will likely assist with sustainable crop production by promoting enhanced pest resistance.

Optimization of Callus Induction Conditions from Immature Embryos of Maize under Stress

The embryos of maize (Zea mays L.) inbred lines GS02, GS07, GS08, GS11 and GS15 were used as receptor materials to optimize the receptor system from the aspects of genotype, medium components and stress (PEG6000, mannitol, salt and low phosphorus). The results showed that GS07 had the highest induction rate (95.2%). Orthogonal test analysis showed that the best combination of medium components in induction was A2B3C1D3(2), namely, the concentration of 2, 4-dichlorophenoxy acetic acid (2,4-D) was 4 mg·mL-1, the concentration of L-Proline (L-Pro) was 0.8 mg·mL-1, and the concentration of silver nitrate (AgNO3) was 10 mg·mL-1 (or 5 mg·mL). Interestingly, we found that the optimal medium supplemented with 30 g·L-1 PEG6000 or 80 g·L-1 mannitol was suitable for antioxidant enzyme activity and malondialdehyde (MDA) content in GS07 callus. Exogenous 10 mmol·L-1 Ca2+ in the medium components with 100 mmol·L-1 sodium chloride (NaCl) could improve the activity of antioxidant enzymes in GS07 callus. Callus of GS07 could divide normally and grow well in medium components with 27 mg·L-1 KH2PO4. This study enhanced the adaptability of maize callus to stress and optimized the culture conditions.

Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures.

A Gram-stain positive, aerobic, motile, rod-shaped bacterium designated as strain CBP-2801T was isolated as a contaminant from a culture containing maize callus in Peoria, Illinois, United States. The strain is unique relative to other Cohnella species due to its slow growth and reduced number of sole carbon sources. Phylogenetic analysis using 16S rRNA indicated that strain CBP-2801T is a Cohnella bacterium and showed the highest similarity to Cohnella xylanilytica (96.8%). Genome-based phylogeny and genomic comparisons based on average nucleotide identity confirmed the strain to be a novel species of Cohnella. Growth occurs at 15-45°C (optimum 40°C), pH5-7 (optimum pH6) and with 0-1% NaCl. The predominant fatty acids are anteiso-15:0 and 18:1 ω6c. Genome mining for secondary metabolites identified a putative biosynthetic cluster that encodes for a novel lasso peptide. In addition, this study contributes five new genome assemblies of type strains of Cohnella species, a genus with less than 30% of the type strains sequenced. The DNA G + C content is 58.7mol %. Based on the phenotypic, phylogenetic and biochemical data strain CBP-2801T represents a novel species, for which the name Cohnella zeiphila sp. nov. is proposed. The type strain is CBP-2801T (= DSM 111598 = ATCC TSD-230).

A maizewin protein confers enhanced antiinsect and antifungal resistance when the gene is transgenically expressed in maize callus

Maize is a crop of worldwide importance, but insects and plant pathogens limit sustainable production. The development of maize varieties with improved resistance will be facilitated by identification of relevant resistance alleles through appropriate biological assays. A barwin-like gene (which we call maizewin) located in a QTL for Fusarium resistance annotated such that potential glucanase and chitinase activity could occur in the translated protein, was cloned from an ear rot resistant inbred and transgenically expressed in maize callus. Although limited increases in insect resistance occurred in the transgenic callus material containing the maizewin gene compared to a GUS control gene, resistance to Fusarium pathogens was common and associated with increased glucanase activity. The gene appeared to produce a protein with glucanase and chitosanase activity, consistent with an antifungal role. The allele that produces the functional resistant protein may be relatively uncommon in maize germplasm. Incorporation of a functional maizewin allele into susceptible germplasm should help increase production by contributing to control of both insect pests and plant pathogens.

Glyphosate selection of maize transformants containing CP4epsps gene

Aim. To study the selection conditions of maize transformants containing the CP4epsps gene using glyphosate as a selective agent. Methods. Tissue culture in vitro, Agrobacterium-mediated transformation, selection of transgenic plants, isolation of total plant DNA, analysis of plant DNA by polymerase chain reaction (PCR). Results. The morphogenic maize callus of immature embryos of the hybrid (PLS61)R2×PLS61 was produced, which had a high regeneration rate (up to 95%), that persisted over long cultivation. Agrobacterium mediated transformation of the morphogenic callus and selection of the transgenic material using glyphosate yielded maize transformants containing the CP4epsps gene at a frequency of 1%. Conclusions. Maize genotype (PLS61)R2×PLS61 is promising for studies on the maize genetic transformation, in particular for the production of transgenic maize resistant to glyphosate herbicide. The use of morphogenic maize callus (PLS61)R2×PLS61 and glyphosate as a selective agent at a concentration of 0.1 mM and 0.25 mM in media for callusogenesis and 0.01 mM in the medium for regeneration was effective for the selection of transgenic plants with the gene CP4epsps.
 Keywords: Zea mays L., morphogenic callus, Agrobacterium-mediated transformation, PCR, genetic engineering.

Effect of auxin type on the total phenols synthesis and some enzymatic proteins activity in maize callus from seeds irradiated to gamma radiation

Secondary metabolites of plants widely used in the medicinal and cosmetic industries, can be produced by tissue culture. In vitro production of these secondary metabolites is affected by various factors such as the type and concentration of phyto regulators. In this study, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methoxy-3,6-dichlorobenzoic acid (Dicamba) effect on phenolic compounds synthesis as well as Phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), Polyphenoloxidase (PPO), peroxidase (POD), Catalase (CAT) and Ascorbate Peroxidase (APX) activities from root explants have been studied. The root explants come from the seeds in vitro germination of maize irradiated (200 and 300 grays) or not irradiated (control) with gamma radiation. The results obtained showed that the auxin type had a very highly significant effect (p

Transgenic expression of a previously uncharacterized maize AIL1 gene in maize callus increases resistance to multiple maize fungal and insect pests

Large portions of the world are dependent on maize. Maize production is limited by insect and fungal damage, and some ear rotting fungi also contaminate grain with toxins. Plant resistance is part of a sustainable strategy for maize production, but genes involved in resistance still need to be identified. A previously uncharacterized AIL1 gene located in a resistance metaQTL was cloned from a Fusarium resistant maize inbred and transgenically expressed in maize callus. Transformed callus producing the protein inhibited growth of Fusarium maize pathogens and maize feeding caterpillar pests. The degree of enhanced resistance was correlated with expression levels of the protein. Additional proteins were upregulated in the transgenic callus as predicted by prior interactome information. The identification of this newly identified resistance gene should help the production of more pest resistant maize, resulting in higher quality and safer maize for humans and animals.

Effect of auxin type on the total phenols synthesis and some enzymatic proteins activity in maize callus from seeds irradiated to gamma radiation

Effect of auxin type on the total phenols synthesis and some enzymatic proteins activity in maize callus from seeds irradiated to gamma radiation

Establishment of a maize callus regeneration system from haploid shoot tips

For maize transgenic breeding, a major bottleneck is time need to produce homozygous transgenic plants by selfing of one or more additional generations. This study established an effective haploid maize callus regeneration system using haploid shoot tips of maize which enables to form homozygous transgenic insertions in a single generation. Haploid shoot tips were cultured for haploid embryonic callus induction, which was then transformed with a GFP-contained vector and generated transgenic haploid plants. We tested different seed sterilization methods, germination manners, shoot lengths, cutting patterns, medium components, light treatments in differentiation and particle bombardment distances. The results indicated that HgCl2 was better than H2O2 for decreasing seeds contamination rate. Seedlings with 2.0–6.0 cm in length was optimal for the high frequency of embryonic callus induction. Then, the best culture medium components were “MS + 2 mg/L 2,4-D + 1 mg/L 6-BA + 500 mg/L CH + 10 g/L Mannitol + 7 mg/L AgNO3 + 1 mg/L B5” and “MS + 1 mg/L 6-BA + 0.4 mg/L IBA + 500 mg/L CH + 1 mL/L Cephalothin”, respectively, for callus induction and regeneration. Callus with 4 mm in size and 4 generations of subculture was better than the others for particle bombardment to obtain positive transgenic plants. Among the six genotypes of haploids, haploid 18-599 W had the highest induction rate of embryonic callus. Interestingly, each of the haploids did not show different induction rate compared to its diploid, except haploid 18-599R. Our research provides new insights for rapid homozygosis of transformation events in maize. An effective haploid maize shoot tips callus regeneration system was established which enabled homozygous transgenic plants to be obtained in a single generation.

Global Profiling of Alternative Splicing in Callus Induction of Immature Maize Embryo

Callus induction in plants is similar to pluripotent stem cell induction in animals and can incite global changes in gene expression. Alternative splicing is considered a key factor underlying functional complexity and function in the induction of pluripotent stem cells in animals. However, the role of alternative splicing in plant callus induction remains unclear. We performed high-throughput RNA sequencing of maize callus induced on a callus induction medium (CIM) at different stages and recorded global alternative splicing changes. At every stage, over 20,000 alternative splicing events were detected, and the numbers slightly increased with callus induction. In total, 648 to 2580 genes were found to exhibit significant alternative splicing changes during callus induction—mainly those that belong to the spliceosome, metabolic pathways, and mRNA surveillance pathways. It was also found that alternative splicing can function alongside transcriptional regulation to contribute to callus induction. Especially, ZmWOX11, whose orthologous gene in Arabidopsis plays a vital role in the first-step cell fate transition of callus induction, was found to exhibit alternative splicing and expression level changes during callus induction. Our results establish a foundation from which researchers can further explore the molecular mechanism of callus induction in maize, especially at alternative splicing level.

Transcriptome Profiling Predicts New Genes to Promote Maize Callus Formation and Transformation.

Maize transformation is highly based on the formation of embryonic callus, which is mainly derived from scutellum cells of the immature maize embryo. However, only a few genes involved in callus induction have been identified in maize. To reveal the potential genes involved in the callus induction of maize, we carried out a high-throughput RNA sequencing on embryos that were cultured for 0, 1, 2, 4, 6, and 8 days, respectively, on a medium containing or lacking 2,4-dichlorophenoxyacetic acid. In total, 7,525 genes were found to be induced by 2,4-dichlorophenoxyacetic acid and categorized into eight clusters, with clusters 2 and 3 showing an increasing trend related to signal transmission, signal transduction, iron ion binding, and heme binding. Among the induced genes, 659 transcription factors belong to 51 families. An AP2 transcription factors, ZmBBM2, was dramatically and rapidly induced by auxin and further characterization showed that overexpression of ZmBBM2 can promote callus induction and proliferation in three inbred maize lines. Therefore, our comprehensive analyses provide some insight into the early molecular regulations during callus induction and are useful for further identification of the regulators governing callus formation.

Enhanced insect and fungal resistance of maize callus transgenically expressing a maize E2F regulatory gene

A maize gene coding for an E2F regulatory protein located in a quantitative trait locus (QTL) for Fusarium ear rot resistance was cloned and transgenically introduced into maize callus. Several of the transformants were more resistant to feeding by corn earworms and fall armyworms, and retarded growth of Fusarium proliferatum compared to GUS control transformants. More effective transformants contained higher levels of E2F product than GUS controls as indicated by antibody detection. Increased and decreased levels of regulated proteins were noted in E2F overexpressing compared to GUS expressing control transformants. These differentially produced proteins were previously reported to be interactors with the E2F protein, suggesting E2F is affecting the production of these proteins. Other proteins coded for by genes reported to interact with the relevant E2F protein and associated phenotypic effects that could be promoting resistance include those that would increase cell resistance to water stress, increase the presence of reactive oxygen species, and increase the density of indigestible components. Although the full complex of responsible proteins regulated by the E2F examined that promote resistance to insects and fungi remains to be determined, overproduction of the E2F in transgenic callus enhances resistance to some maize insect and fungal pests.

ITRAQ-Based Quantitative Proteomic Analysis of Embryogenic and Non-embryogenic Calli Derived from a Maize (Zea mays L.) Inbred Line Y423.

Somatic embryos (SE) have potential to rapidly form a whole plant. Generally, SE is thought to be derived from embryogenic calli (EC). However, in maize, not only embryogenic calli (EC, can generate SE) but also nonembryogenic calli (NEC, can’t generate SE) can be induced from immature embryos. In order to understand the differences between EC and NEC and the mechanism of EC, which can easily form SE in maize, differential abundance protein species (DAPS) of EC and NEC from the maize inbred line Y423 were identified by using the isobaric tags for relative and absolute quantification (iTRAQ) proteomic technology. We identified 632 DAPS in EC compared with NEC. The results of bioinformatics analysis showed that EC development might be related to accumulation of pyruvate caused by the DAPS detected in some pathways, such as starch and sucrose metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, fatty acid metabolism and phenylpropanoid biosynthesis. Based on the differentially accumulated proteins in EC and NEC, a series of DAPS related with pyruvate biosynthesis and suppression of acetyl-CoA might be responsible for the differences between EC and NEC cells. Furthermore, we speculate that the decreased abundance of enzymes/proteins involved in phenylpropanoid biosynthesis pathway in the EC cells results in reducing of lignin substances, which might affect the maize callus morphology.

Overexpression of a maize (Zea mays) defensin-like gene in maize callus enhances resistance to both insects and fungi

Identification of genes responsible for pest resistance in maize will assist with breeding attempts to reduce crop losses and hazards due to toxins produced by mold infecting ears. A gene coding for a defensin-like gene was cloned from an inbred reported to be resistant to Fusarium proliferatum and Fusarium verticillioides ear rot, based on its location in a QTL associated with resistance to those and other species of ear rot molds that produce mycotoxins. The gene was expressed transgenically in maize callus and the construct presence confirmed in transformants by PCR analysis and by detection with antibody made to a portion of the protein. Positive transformants were more resistant to corn earworms (Helicoverpa zea) and fall armyworms (Spodoptera fruigperda) as indicated by significantly lower weights compared to control callus expressing a β–glucuronidase (GUS) gene. Positive transformants also had significantly less visible growth of F. proliferatum and F. verticillioides, but not Fusarium graminearum, than controls. This indicates for the first time a defensin that is active against both insects and fungi, thereby allowing for more effective breeding for resistance to both major classes of pests attacking maize.

Alterations in Heterochromatic Knobs in Maize Callus Culture by Breakage-Fusion-Bridge Cycle and Unequal Crossing Over

The meiotic and mitotic behavior of regenerated plants derived from a long-term callus culture, designated 12-F, was analyzed. This culture was heterozygous for an amplification of the heterochromatic knob on the long arm of chromosome 7 (K7L). We aimed to investigate if the amplification resulted from a breakage-fusion-bridge (BFB) cycle or from unequal sister chromatid recombination. Therefore, C-banded mitotic metaphases and pachytene, diakinesis, and anaphase I of regenerated plants were analyzed. Additionally, the occurrence of alterations in K7L was investigated in C-banded metaphases from short-term callus cultures derived from lines related to the donor genotype of the 12-F culture. As a result, plants homozygous and heterozygous for the amplification were detected. Meiosis was normal with few abnormalities, such as a low frequency of univalents at diakinesis. In the callus cultures a chromosome 7 with knobs of different sizes in the sister chromatids was detected and interpreted as a result of unequal crossing over. Other chromosomal alterations were consistent with the occurrence of BFB cycles. The finding of unequal crossing over in the cultures supports the conclusion that the amplification in the culture 12-F would be derived from this mechanism. If the amplification was derived from a BFB cycle, the terminal euchromatic segment between knob and the telomere would be deleted, and possibly, homozygous plants would not be viable.

Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone isomerase 3-like (CI3), located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in maize callus. Transformants were often more resistant to feeding by the corn earworm and fall armyworm compared to glucuronidase (GUS) callus transformant controls. The transformed callus was also more resistant to colonization by the mycotoxin producing ear rot fungi Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides. Chemical analysis unexpectedly indicated increased production of several phenolic compounds, instead of flavonoids, compared to GUS controls. The levels of one of these, a glucosylated ferulic acid, were significantly correlated with the degree of growth inhibition of F. graminearum. Increased levels of a peroxidase isozyme previously associated with pest resistance also occurred in these CI3 transformants. The production of phenolics and higher levels of a peroxidase suggests this gene product is behaving as a fatty acid binding protein and enhancing transport of fatty acids involved in the production of jasmonic acid, which can induce the biosynthesis of resistance molecules. Breeding for enhanced expression of this gene can potentially contribute to resistance to both insects and plant pathogenic fungi, thereby contributing to sustainable production and better quality food.

Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens

Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait locus was cloned from an inbred with reported resistance to Fusarium proliferatum and Fusarium verticillioides ear rot. Transgenic expression of the gene in maize callus reduced colonization by these two Fusarium species and also Fusarium graminearum relative to a β-glucuronidase (GUS) transformant control. Some transformants were also more insect resistant. The more fungal resistant transformant lines produced higher levels of headspace ethanol which were significantly associated with antifungal activity, especially for F. verticillioides. Maize pyruvate decarboxylase appears to have a moiety capable of interacting with the geranyl geranyl transferase, suggesting ethanol production is enhanced due to more efficient transfer of pyruvate through the mitochondrial membrane. Other undetermined mechanisms may also be enhancing resistance of the transformants to the Fusarium fungus, however. This is the first report of the involvement of a geranyl geranyl transferase-like sequence in fungal resistance in plants, and represents a novel mechanism for producing higher yielding and better quality maize.