N. Bohorova

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.

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.

Novel synthetic Bacillus thuringiensiscry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize

Abstract  In order to develop a resistance management strategy to control tropical pests based on the co-expression of different toxins, a fully modified Bacillus thuringiensiscry1B gene and the translational fusion cry1B-cry1Ab gene have been developed. Both constructs were cloned under the control of a maize ubiquitin-1 or a rice actin-1 promoter and linked to the bar gene driven by the CaMV 35S promoter. Immature embryos from the tropical lines CML72, CML216, and their hybrids, were used as the target for transformation by microprojectile bombardment. Twenty five percent of the transformed maize plants with cry1B expressed a protein that is active against southwestern corn borer and sugarcane borer. Ten percent of the transgenic maize expressed single fusion proteins from the translational fusion gene cry1B-1Ab and showed resistance to these two pests as well as to the fall armyworm. Transgenic maize plants that carried the cry1B gene in T1 to T3 progenies transmitted trangenes with expected Mendelian segregation and conferred resistance to the two target insects. Molecular analyses confirmed the cry genes integration, the copy number, the size of protein(s) expressed in maize plants, the transmission, and the inheritance of the introduced cry gene. These new transgenic products will provide another recourse for reducing the build-up of resistance in pest populations.

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

Abstract 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 BastaTM 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.

Selection and characterization of Mexican strains of Bacillus thuringiensis active against four major lepidopteran maize pests.

In order to isolate novel delta-endotoxins fromBacillus thuringiensis Berliner, a total of 426 native isolates (in varying numbers for each pest) were screened against four major maize pests: corn earworm,Helicoverpa zea; fall armyworm,Spodoptera frugiperda; southwestern corn borer,Diatraea graridiosella, and sugarcane borer,Diatraea saccharalis. Spore-crystal complexes from the isolates were integrated into semi-artificial diets of each pest and mortality was assessed 7 days after treatment. A total of 25 isolates were selected on the basis of highest larval mortality against at least one insect species. There was no correspondence of the most toxic isolates when tested against the four different insect species. Most of the 25 selected isolates caused higher toxicities against all four pests than the standard strain HD-1, regardless of not achieving 100% mortality in any bioassay.H. zea demonstrated the highest level of mortality (96%) and was susceptible to the largest number of isolates (98). None of the other insect species were found susceptible at levels greater than 60%. All the selected active strains were isolated from stored grain dusts (except for LBIT-167), and had bipyramidal crystals with Cry I-like proteins. Most isolates also formed an associated square (cubic) inclusion, with Cry Il-like proteins according to SDS-PAGE analysis of their parasporal bodies. The most active isolates will be subjected to further studies, in order to identify putative novel genes to be expressed in transgenic maizeRésuméLes 426 souches deBacillus thuringiensis Berliner isolées ont été testées sur quatre ravageurs importants du maïs,Helicoverpa zea, Spodoptera frugiperda, Diatraea grandiosella, Diatraea saccharalis dans le but d’identifier de nouvelles delta endotoxines. Le complexe spore-cristal obtenu à partir de chacun des isolats a été incorporé dans un milieu nutritif artificiel pour insecte et la mortalité a été relevée après sept jours de traitements. Vingt-cinq isolats ont été sélectionnés sur la base d’une toxicité élevée sur les larves d’au moins une espèce. Aucun des isolats actifs ne s’est révélé actif à la fois contre les quatre espèces d’insectes étudiées. La plupart des 25 isolats sélectionnés ont montré une toxicité supérieure à celle de la souche de reférence HD- 1, que la mortalité ait atteint ou non 100 %.H. zea s’est révélée être l’espèce la plus sensible (96 % de mortalité) au plus grand nombre d’isolats (98). Aucune des trois autre espèces n’a montré plus de 60 % mortalité. Toutes les souches actives ont été isolées à partir de poussières de graines stockées (excepté l’isolat LBIT-167) et produisaient des cristaux bipyramidaux. L’analyse du contenu protéique des cristaux en gel de polyacrylamide-SDS a montré que les cristaux bipyramidaux contenaient des protéines de type Cryl et que les cristaux cubiques associés produits par la plupart des isolats contenaient des protéines de type Cryll. Les souches les plus actives seront analysées de façon plus approfondie afin de détecter la présence potentielle de nouveaux gènes pouvant être exprimés chez les maïs transgéniques

Transgenic Tropical Maize with cryLAb and cryLAc Genes from Microprojectile Bombardment of Immature Embryos

Maize has been one of the prime targets for genetic manipulation in monocotyledonous grains. The first demonstrations of progress were the successful production of transgenic plants by microprojectile bombardment (Klein et al. 1989; Fromm et al. 1990; Gordon-Kamm et al. 1990; Genovesi et al. 1992; Walter et al. 1992; Frame et al. 1994; Wan et al. 1995, Brettschneider et al. 1997); then successful hygromycin (Walters et al. 1992), bialaphos (Spencer et al. 1990), and glyphosate (Howe et al. 1992) selection of stable transformants, and recent Agrtobacterium — mediated gene insertion in maize plants (Ishida et al. 1996). Most studies on maize transformation have utilized genotypes adapted to temperate zones (Fromm et al. 1990; Gordon-Kamm et al. 1990; Walter et al. 1992; Armstrong et al. 1995) and plants regenerated from these lines were shown to transmit the recombinant DNA to their progeny. Little or no attention, however, has been focused on the transformation potential of maize germplasm and inbred lines adapted to tropical and subtropical regions. Production of genetically transformed plants depends both on the ability to integrate foreign genes into target cells and the efficiency with which plants are regenerated from genetically transformed cells. Embryogenic calli and plant regeneration were obtained from 50% of tropical and subtropical lines, 87% of midaltitude lines, and 75% of highland lines tested (Bohorova et al. 1995) and type II callus with high potential for plant regeneration from tropical maize was produced (Prioli and Silva 1989; Carvalho et al. 1997). These studies serve as the basis for developing transgenic technology for maize inbreds adapted to tropical conditions.