Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana.

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 = 1.6 µg/kg) and resulted in a mean time from inoculation to death of 18 and 180 min when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 min post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication.

Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize.

An amaranth ( Amaranthus hypochondriacus) 11S globulin cDNA, encoding one of the most important storage proteins (amarantin) of the seed, with a high content of essential amino acids, was used in the transformation of CIMMYT tropical maize genotype. Constructs contained the amarantin cDNA under the control of a tissue-specific promoter from rice glutelin-1 ( osGT1) or a constitutive ( CaMV 35S) promoter with and without the first maize alcohol dehydrogenase intron ( AdH). Southern-blot analysis confirmed the integration of the amarantin cDNA, and copy number ranged from one to more than ten copies per maize genome. Western-blot and ultracentrifugation analyses of transgenic maize indicate that the expressed recombinant amarantin precursors were processed into the mature form, and accumulated stably in maize endosperm. Total protein and some essential amino acids of the best expressing maize augmented 32% and 8-44%, respectively, compared to non-transformed samples. The soluble expressed proteins were susceptible to digestion by simulated gastric and intestinal fluids, and it is suggested that they show no allergenic activity. These findings demonstrate the feasibility of using genetic engineering to improve the amino acid composition of grain crops.

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos.

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests - corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta(TM) contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.

Type II callus production and plant regeneration in tropical maize genotypes.

A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally, four media combinations of 15 or 30 µM dicamba with or without 88 µM AgNO3 were used to study the effect of dicamba and AgNO3 on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 µM and when AgNO3 was added to the medium. A synergistic effect between 88 µM AgNO3 and 30 µM dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best tissue culture performance and plant regeneration capacity.

Plant regeneration from immature embryos of 48 elite CIMMYT bread wheats.

Forty-eight bread wheat (Triticum aestivum L.) released cultivars and elite advanced lines were evaluated for their ability to produce embryogenic callus using three different media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,4-D (E3) or MS medium containing 2,4-D plus different amino acids (E5) were used for callus initiation and maintenance. Plant regeneration was achieved on basal MS medium with indole-3-acetic acid (IAA) and 6-benzylamino purine (BAP) and rooting on MS with 1-naphthaleneacetic acid (NAA). Percentage regeneration varied widely with both genotype and initiation medium, with values ranging from 2% to 94%. The number of plantlets produced per embryo ranged from 6 to 42. Thirteen genotypes showed at least 50% regeneration after culture on E5 medium; 3 genotypes after culture on E3 initiation medium and 1 after initiation on E1. After four subcultures, over a 16-week period, 41 genotypes (85%) lost their ability to regenerate plants while the remaining 7 lines (15%) retained plant regeneration potential but at reduced levels. E3 medium was found to be the best for maintaining regeneration potential after four subcultures.

Molecular analysis of a new cytoplasmic male sterile genotype in sunflower.

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in the vicinity of the atpA locus, generated by various restriction enzymes. The organization of the mitochondrial genes 26S rRNA, 18S + 5S rRNA and coxII was investigated by Southern blot analysis. These genes have similar structures in fertile and all studied sterile sources. Using the atpA probe, 5 from the 6 investigated CMS genotypes showed identical hybridization patterns to the Petiolaris CMS line, which is used in all commercial sunflower hybrids. Only 1 cytoplasm derived from an open pollination of Helianthus annuus ssp. texanus, known as ANT1, contained a unique mitochondrial DNA fragment, which is distinguishable from the fertile and sterile Petiolaris genotypes and from all investigated CMS genotypes. Male fertility restoration and male sterility maintenance of the ANT1 line are different from the Petiolaris CMS system, which is a confirmation that a novel CMS genotype in sunflower has been identified.

Isolation, culture and callus regeneration of protoplasts of wild and cultivated Helianthus species.

Protoplasts were isolated from seedling roots, hypocotyls, and cotyledons of four cultivars of Helianthus annuus and from leaves of axenic shoot cultures of the wild species H. praecox, H. scaberimus and H. rigidus. Optimal culture conditions were established for the respective protoplast systems, using the agarose bead method of culture. Protoplast division was induced for all the species examined. In the case of the cultivars of H. annuus, hypocotyl and cotyledon protoplast division was sustained leading to callus formation, which in turn, could be induced to produce roots and organised meristematic regions in the presence of NAA and 6-BAP.