In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.

https://www.genetics.org/content/genetics/165/1/387.full.pdf

A new opaque variant of maize by a single dominant RNA-interference-inducing transgene.

The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3–1, 4)-β-glucanase with the D hordein gene (Hor3–1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an α-amylase promoter and the code for the export signal peptide yielded ≈1 μg⋅mg−1 soluble protein, whereas 54 μg⋅mg−1 soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3–1, 4)-β-glucanase under the control of the Hor3–1 promoter.

https://www.pnas.org/content/pnas/97/4/1914.full.pdf

The production of recombinant proteins in transgenic barley grains.

A selectable marker system for plant transformation that does not require the use of antibiotics or herbicides was developed. The selectable marker consists of the manA gene from Escherichia coli under the control of a plant promoter that encodes for phosphomannose isomerase, pmi. Only transgenic plants were able to metabolize the selection agent, mannose, into a usable source of carbon, fructose. Transgenic plants were produced efficiently after delivery by Biolistics™ of the pmi gene into maize and wheat tissues, with mean transformation frequencies of 45% for maize and 20% for wheat. Adjustment of the sucrose and mannose levels in the selection medium essentially eliminated escapes. Transgenic events can be identified as early as 2 months for wheat and 4 months for maize. A simple test, a modified chlorophenol red assay, was used for early identification of transgenic events expressing the pmi gene. Transformation frequencies for both crops exceeded those obtained with the bar and pat genes with selection on either Basta® or bialaphos.

Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker.

Fungal wheat (Triticum aestivum) diseases greatly affect crop productivity and require the economically and ecologically undesirable application of fungicides in wheat agriculture. We have generated transgenic wheat plants constitutively expressing an antifungal barley-seed class II chitinase. The transgene was stably expressed and the chitinase properly localized in the apoplast of the transgenic lines. The engineered wheat plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis.

/pdf/genetic-engineering-of-wheat-for-increased-resistance-to-4r35thkc8p.pdf

Genetic engineering of wheat for increased resistance to powdery mildew disease

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose as a carbon source. Maize, wheat and barley plants, genetically modified to express the Escherichia coli manA gene (pmi) under the control of a plant promoter, were able to survive selection on mannose-containing medium. Transformation frequencies averaged 45% for maize transformation via Biolistics™ 35% for maize Agrobacterium-mediated transformation, 20% for wheat, 3% for barley, and 2% for watermelon transformation. Moreover, the frequencies exceeded those obtained for maize and wheat using the pat or bar gene with Basa® selection. A preliminary safety assessment has been conducted for PMI. Purified PMI protein demonstrates no adverse, effects in an acute mouse toxicity test. Purified PMI protein was readily digested in simulated mammalian gastric and intestinal fluids. Plants derived from surgar beet and corn cells that had been genetically modified to express the E. coli manA gene were evaluated for biochemical changes in mannose-associated pathways. No detectable changes in glycoprotein profiles were detected in PMI-transformed plants as compared to nontransgenic controls. The yield and nutritional composition of grain from PMI-transformed corn plants compared to their non-transformed isogenic counterparts were also determined and no statistically significant differences were found. The inherent safety of a system based on simple sugar metabolism coupled with high transformation frequencies for monocots make pmi and ideal selectable marker for plant transformation.

Phosphomannose isomerase: An efficient selectable marker for plant transformation

Phosphinothricin (PPT) inhibits glutamine synthetase in plant cells, resulting in an accumulation of ammonia that can be toxic. Bar, from Streptomyces hygroscopicus is a gene that codes for phosphinothricin acetyl-transferase, an enzyme that detoxifies PPT by acetylation. We used polyethylene-glycol-mediated transformation to introduce bar into protoplasts of maize (Zea mays L.). With a novel assay utilizing the pH indicator chlorophenol red we identified transformants after only two to four weeks of selection on PPT.

Selection of transformed protoplast-derived Zea mays colonies with phosphinothricin and a novel assay using the pH indicator chlorophenol red

Lactoferricin has been found to have antifungal activity against plant pathogens. Genetic constructs comprising a synthetic gene for bovine lactoferricin are prepared and used to provide transgenic disease-resistant plants. A method of transforming plants to express lactoferricin which is expressed in plants to provide disease resistance to those plants is also described.

Peptide with inhibitory activity towards plant pathogenic fungi

This invention describes genes encoding proteins which control resistance of plants to fungal pathogens. The invention also describes transgenic plants resistant to fungal pathogens and methods for making plants resistant to fungal pathogens. The invention further discloses a method to isolate additional genes coding for additional proteins controlling the resistance of plants to fungal pathogens.

Genes encoding mlo proteins and conferring fungal resistance upon plants

Mlo-genes controlling diseases

D'apres cette invention, il a ete prouve que la lactoferricine possede une activite antifongique a l'encontre de pathogenes de plantes. Des constructions genetiques, qui comprennent un gene synthetique pour la lactoferricine bovine, ont ete preparees et utilisees afin d'obtenir des plantes transgeniques resistant aux maladies. Cette invention concerne egalement un procede de transformation des plantes permettant d'exprimer la lactoferricine. Lorsqu'elle est exprimee dans des plantes, la lactoferricine leur confere une resistance aux maladies.

Catherine Mae Kramer

Papers

Selection of transformed protoplast-derived Zea mays colonies with phosphinothricin and a novel assay using the pH indicator chlorophenol red

Peptide with inhibitory activity towards plant pathogenic fungi

Genes encoding mlo proteins and conferring fungal resistance upon plants

Mlo-genes controlling diseases

Peptide possedant une action inhibitrice a l'encontre de champignons pathogenes de plantes