Transgene expression in cowpea (Vigna unguiculata (L.) Walp.) through Agrobacterium transformation of pollen in flower buds

Pollen transformation shows potential as a fast and easy means of obtaining transformed plants carrying desirable transgenes. Agrobacterium tumefaciens has been suggested as the best natural plant genetic engineering system. Laboratory and screenhouse studies were undertaken to investigate the possibility of obtaining transformed progeny in cowpea by Agrobacterium-mediated transformation of pollen. Flower buds of selected cowpea accessions were inoculated the evening before opening with a late log phase culture of A. tumefaciens strain pGV 2260 carrying the transgene vector ptjk 142. The vector is a disarmed Ti plasmid carrying a chimeric fusion of the UidA and Bar genes for β-glucuronidase expression and bialaphos resistance respectively. Seedlings resulting from inoculated flowers were screened for expression of the genes by β-glucuronidase (GUS) assay and Basta spraying. GUS positive plants were analyzed by polymerase chain reaction and Southern hybridization. Up to 90% of inoculated flowers and developing pods aborted. Pod set from inoculated flower buds ranged between 8.92 and 10.51% with no significant difference in pod set among accessions. Nine seedlings showed positive GUS expression. None was tolerant to Basta. Seven seedlings showing positive GUS expression also showed positive signals under PCR and Southern analysis giving indicative evidence of transgene presence. Overall transformation frequency was 0.36%.Key words: Agrobacterium tumefaciens, b-glucuronidase, bialaphos resistance, cowpea, pollen transformation

Transgenic wheat plants: a powerful breeding source

Plant breeders are always interested in new genetic resources. In the past, the sources have been limited to existing germplasm. Genetic engineering now provides the opportunity for almost unlimited strategies to create novel resources. As a first stage, the Applied Biotechnology Center (ABC) at CIMMYT developed a method for the mass production of fertile transgenic wheat (Triticum aestivum L.) that yields plants ready for transfer to soil in 13–14 weeks after the initiation of cultures, and, over the course of a year, an average production of 5–6 transgenic plants per day. CIMMYT elite cultivars are co-bombarded with marker gene and a gene of interest with co-transformation efficiencies around 25–30%. The reliability of this method opens the possibility for the routine introduction of novel genes that may induce resistance to diseases and abiotic stresses, allow the modification of dough quality, and increase the levels of micronutrients such as iron, zinc, and vitamins. The first group of genes being evaluated by the ABC are the pathogenesis related (PR) proteins, such as the thaumatin-like protein (TLP) from barley, chitinase, and 1–3 β-glucanase. Stable integration of the genes in the genome and inheritance in the progeny were determined by phenotypical analyses that challenged the plants against a wide range of pathogens. Using these genes, we have recovered more than 1200 independent events (confirmed by PCR and Southern blot analyses) that show responses to the pathogens that range from tolerance to hypersensitive reactions. The quantity and antifungal activity of the endogenous thaumatin-like proteins were analyzed in T 1 and T2 progeny plants.Western blot analyses showed different protein patterns of the wheat endogenous TLPs. Preliminary results indicated that some patterns increased the resistance of transgenic wheat plants to Alternaria triticina. This relationship is being further investigated

Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants

The efficiency of wheat biolistic transformation systems strongly depends on the bombardment parameters, the condition of the donor plant, and the plant genotype chosen for the transformation process. This paper analyzes the transformation efficiency of the 129 wheat sister lines generically called 'Bobwhite', originally obtained from the cross 'Aurora'//'Kalyan'/'Bluebird 3'/'Woodpecker'. A number of factors influencing the transformation were examined, such as the ability to produce embryogenic callus, regeneration in selection medium, and overall transformation performance. Of the 129 genotypes evaluated, eight demonstrated transformation efficiencies above 60% (60 independent transgenic events per 100 immature embryos bombarded). Among the eight genotypes identified, we studied agronomic characteristics such as earliness to identify the most adaptable line(s) for different lab conditions. 'Bobwhite' SH 98 26 was identified as a super-transformable wheat line.Key words: wheat transformation, 'Bobwhite', genotype effect

Testing public Bt maize events for control of stem borers in the first confined field trials in Kenya

Transgenic maize (Zea mays L), developed using modified genes from the bacterium Bacillus thuringiensis (Bt), controls stem borers without observable negative effects to humans, livestock or the environment, and is now sown on 134 million hectares globally. Bt maize could contribute to increasing maize production in Kenya. Nine public Bt maize events of cry1Ab and cry1Ba genes were tested in confined field trials site (CFTs) to assess the control of four major Kenyan stem borer species. Leaf damage rating, number of exit holes and tunnel length were scored in the field evaluations. Leaf area consumed and mortality rates among stem borers were scored in the leaf bioassays in a Biosafety Level II laboratory, located at the Kenya Agricultural Research Institute (KARI), National Agricultural Research Laboratories (NARL). Field evaluations showed that Bt maize controlled Chilo partellus with mean damage scores of 1.2 against 2.7 for the non-Bt CML216 control. Laboratory bioassays showed high control for Eldana saccharina and Sesamia calamistis, with mean larval mortality of 64 and 92%, respectively. However, substantial control was not observed for Busseola fusca. These results showed that Bt maize could control three of the four major stem borers in Kenya with mortality records of 52.7% for B. fusca, 62.3% for E. saccharina and 85.8% for S. calamistis. Additional Bt genes need to be sought and tested for effective stem borer control in all maize growing ecologies in Kenya.Key words: Maize, Bt, stem borers, confined field trials

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), ‘MPB-Bobwhite 26’, and an elite durum wheat (Triticum turgidum var. durum L.), ‘Mexicali’, were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5mg l−1 2,4-D for 45 days produced callus and plant regeneration in ‘MPB-Bobwhite 26’, but lower callus yield and plant regeneration in ‘Mexicali’, indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in ‘Mexicali’ embryos that were incubated in media containing 2mg l−1 2,4-D and 2 mg l−1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use

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’

Testing public Bt maize events for control of stem borers in the first confined field trials in Kenya

Transgenic maize (Zea mays L), developed using modified genes from the bacterium Bacillus thuringiensis (Bt), controls stem borers without observable negative effects to humans, livestock or the environment, and is now sown on 134 million hectares globally. Bt maize could contribute to increasing maize production in Kenya. Nine public Bt maize events of cry1Ab and cry1Ba genes were tested in confined field trials site (CFTs) to assess the control of four major Kenyan stem borer species. Leaf damage rating, number of exit holes and tunnel length were scored in the field evaluations. Leaf area consumed and mortality rates among stem borers were scored in the leaf bioassays in a Biosafety Level II laboratory, located at the Kenya Agricultural Research Institute (KARI), National Agricultural Research Laboratories (NARL). Field evaluations showed that Bt maize controlled Chilo partellus with mean damage scores of 1.2 against 2.7 for the non-Bt CML216 control. Laboratory bioassays showed high control for Eldana saccharina and Sesamia calamistis, with mean larval mortality of 64 and 92%, respectively. However, substantial control was not observed for Busseola fusca. These results showed that Bt maize could control three of the four major stem borers in Kenya with mortality records of 52.7% for B. fusca, 62.3% for E. saccharina and 85.8% for S. calamistis. Additional Bt genes need to be sought and tested for effective stem borer control in all maize growing ecologies in Kenya

Daily heliotropic movements assist gas exchange and productive responses in DREB1A soybean plants under drought stress in the greenhouse.

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A genetic network of flowering-time genes in wheat leaves, in which an APETALA1/FRUITFULL-like gene, VRN1, is upstream of FLOWERING LOCUS T

To elucidate the genetic mechanism of flowering in wheat, we performed expression, mutant and transgenic studies of flowering-time genes. A diurnal expression analysis revealed that a flowering activator VRN1, an APETALA1/FRUITFULL homolog in wheat, was expressed in a rhythmic manner in leaves under both long-day (LD) and short-day (SD) conditions. Under LD conditions, the upregulation of VRN1 during the light period was followed by the accumulation of FLOWERING LOCUS T (FT) transcripts. Furthermore, FT was not expressed in a maintained vegetative phase (mvp) mutant of einkorn wheat (Triticum monococcum), which has null alleles of VRN1, and never transits from the vegetative to the reproductive phase. These results suggest that VRN1 is upstream of FT and upregulates the FT expression under LD conditions. The overexpression of FT in a transgenic bread wheat (Triticum aestivum) caused extremely early heading with the upregulation of VRN1 and the downregulation of VRN2, a putative repressor gene of VRN1. These results suggest that in the transgenic plant, FT suppresses VRN2 expression, leading to an increase in VRN1 expression. Based on these results, we present a model for a genetic network of flowering-time genes in wheat leaves, in which VRN1 is upstream of FT with a positive feedback loop through VRN2. The mvp mutant has a null allele of VRN2, as well as of VRN1, because it was obtained from a spring einkorn wheat strain lacking VRN2. The fact that FT is not expressed in the mvp mutant supports the present model