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

In order to isolate novel delta-endotoxins from Bacillus thuringiensis, a total of 426 native isolates (in varying numbers for each pest) were screened against four major maize pests: Helicoverpa zea, Spodoptera frugiperda, Diatraea grandiosella and 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 II-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 maize

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

Susceptibility of Four Tropical Lepidopteran Maize Pests to Bacillus thuringiensis CryI-Type Insecticidal Toxins

The relative susceptibility of 4 tropical maize pests, Spodoptera frugiperda (J. E. Smith),Diatraea grandiosella Dyar, D. saccharalis (F.), and Helicoverpa zea (Boddie), to the lepidopteran-specific CryI-type proteins produced by B. thuringiensis is presented. The toxin with the highest potency against H. zea larvae was the CryIAc toxin. S. frugiperda larvae were susceptible to CryID and CryIF toxins. The CryIB toxin showed to be highly toxic against D. grandiosella and D. saccharalis. This information will establish a basis for selecting B. Thuringiensis strains producing the appropriate CryI proteins to be used for the biological control of these tropical pests

Tissue culture response of CIMMYT elite bread wheat cultlvars and evaluation of regenerated plants

Six elite CIMMYT bread wheat genotypes Pavon 76, Seri M82, Opata M85, Mochis T88, Baviacora M93, and the advanced line Attila were evaluated for their response to in vitro tissue culture. Donor plants were grown at EI Batan and CIANO experiment stations in Mexico in 1992. Immature embryos, about 1.0 mm in length, were excised and placed scutellum side up on callus initiation media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,40 (E3), or MS medium containing 2,40 plus different amino acids (E5) was used for callus initiation and maintenance. Plant regeneration and rooting were achieved on basal MS medium with IAA and BAP. Embryogenic calli with regeneration potentials of 10-92% were obtained on E3 and E5 media; each embryo produced 5-50 plants. A total of 800 regenerated plants were transferred to pots in the greenhouse for evaluation and seed production. Of the regenerated plants, 85% were morphologically normal. reached full maturity, and produced seeds. Seeds (R1) of 360 plants regenerated from Mochis T88, Baviacora M93, and Altila were planted in the field. Field observations indicated that embryos cultured on E3 medium resulted in 29% of the progeny producing deviating plants, compared with 21 % of those grown on E5 medium. These results will form the basis for future efforts aimed at transforming CIMMYT bread wheat varieties. IntroductionSix elite CIMMYT bread wheat genotypes Pavon 76, Seri M82, Opata M85, Mochis T88, Baviacora M93, and the advanced line Attila were evaluated for their response to in vitro tissue culture. Donor plants were grown at EI Batan and CIANO experiment stations in Mexico in 1992. Immature embryos, about 1.0 mm in length, were excised and placed scutellum side up on callus initiation media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,40 (E3), or MS medium containing 2,40 plus different amino acids (E5) was used for callus initiation and maintenance. Plant regeneration and rooting were achieved on basal MS medium with IAA and BAP. Embryogenic calli with regeneration potentials of 10-92% were obtained on E3 and E5 media; each embryo produced 5-50 plants. A total of 800 regenerated plants were transferred to pots in the greenhouse for evaluation and seed production. Of the regenerated plants, 85% were morphologically normal. reached full maturity, and produced seeds. Seeds (R1) of 360 plants regenerated from Mochis T88, Baviacora M93, and Altila were planted in the field. Field observations indicated that embryos cultured on E3 medium resulted in 29% of the progeny producing deviating plants, compared with 21 % of those grown on E5 medium. These results will form the basis for future efforts aimed at transforming CIMMYT bread wheat varieties. Introductio

The application of biotechnology to wheat improvement

Today, the world’s population is increasing at the most rapid rate ever. Two hundred people are being added to the planet every minute. It is forecast that by the year 2050, the world’s population will double to nearly 12 billion people. To feed this population, these people will require a staggering increase in food production. In fact, it has been estimated that the world will need to produce more than twice as much food during the next 50 years as was produced since the beginning of agriculture 10 000 years ago. How will researchers continue to develop improved wheat varieties to feed the world in the future? At least for the foreseeable future, plant breeding as it is known today will play a primary role. What will change are the tools that can be employed. This chapter focuses on current approaches for the use of modern molecular-based technologies to develop improved varieties and discusses areas for future applications. Biotechnology can be defined in many different ways, but for the purpose of this chapter, all areas that use molecular approaches to understand and manipulate a plant genome will be considered. However, for the sake of discussion, the techniques are divided between those that make use of molecular markers for studying the genetic material already present within the wheat plant and genetic engineering aimed at the introduction of novel genetic material. It is the latter that often raises concern and that many believe represents ‘modern biotechnology’

Morphogenetic responses of embryo culture of wheat related to environment culture conditions of the explant donor plant

Availability of immature embryos as explants to establish wheat (Triticum aestivum L.) by tissue culture can be limited by climatic factors and the lack of high quality embryos frequently hampers experimentation. This study evaluates the effects of rainfall, various temperature-based variables and sunshine duration on tissue culture response (TCR) traits including callus formation (CF), regenerating calli (RC), and number of plants per embryo (PPE) for 96 wheat genotypes of worldwide origin. The objectives of this study were to evaluate the significance of a particular climatic factor on TCR traits and to determine the period of wheat growth during which these factors were the most effective. The genotypes were grown in an experimental field during three seasons differing in meteorological conditions. The relationships between TCR traits and climatic factors within three time periods of wheat growth: 2, 6 and 10 weeks prior to embryo sampling were analysed by biplot analysis. The tissue culture traits were influenced at very different degrees by climatic factors: from 16.8% (RC) to 69.8% (CF). Donor plant environment with high temperatures and low rainfalls reduced (p lt 0.05) the tissue culture performance of wheat genotypes. Callus formation was most sensitive to the temperature based factors. The environmental conditions between flowering and the medium milk stage were the most important for CF, while RC and PPE were not particularly related to any period

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 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 expressio

The Present and Future Role of Insect-Resistant Genetically Modified Maize in IPM

Commercial, genetically-modified (GM) maize was first planted in the United States (USA, 1996) and Canada (1997) but now is grown in 13 countries on a total of over 35 million hectares (\u3e24% of area worldwide). The first GM maize plants produced a Cry protein derived from the soil bacteriumBacillus thuringiensis (Bt), which made them resistant to European corn borer and other lepidopteran maize pests. New GM maize hybrids not only have resistance to lepidopteran pests but some have resistance to coleopteran pests and tolerance to specific herbicides. Growers are attracted to the Btmaize hybrids for their convenience and because of yield protection, reduced need for chemical insecticides, and improved grain quality. Yet, most growers worldwide still rely on traditional integrated pest management (IPM) methods to control maize pests. They must weigh the appeal of buying insect protection “in the bag” against questions regarding economics, environmental safety, and insect resistance management (IRM). Traditional management of maize insects and the opportunities and challenges presented by GM maize are considered as they relate to current and future insect-resistant products. Four countries, two that currently have commercialize Bt maize (USA and Spain) and two that do not (China and Kenya), are highlighted. As with other insect management tactics (e.g., insecticide use or tillage), GM maize should not be considered inherently compatible or incompatible with IPM. Rather, the effect of GM insect-resistance on maize IPM likely depends on how the technology is developed and used