Paul A. Lazzeri

Biotechnology of breadmaking: unraveling and manipulating the multi-protein gluten complex.

Breadmaking is one of humankinds oldest technologies, being established some 4,000 years ago. The ability to make leavened bread depends largely on the visco-elastic properties conferred to wheat doughs by the gluten proteins. These allow the entrapment of carbon dioxide released by the yeast, giving rise to a light porous structure. One group of gluten proteins, the high molecular weight (HMW) subunits, are largely responsible for gluten elasticity, and variation in their amount and composition is associated with differences in elasticity (and hence quality) between various types of wheat. These proteins form elastomeric polymers stabilized by inter-chain disulphide bonds, and detailed studies of their structures have led to models for die mechanism of elasticity. This work has also provided a basis for direct improvement of wheat quality by transformation with additional HMW subunit genes.

Biotechnology of Wheat Quality

Wheat gluten proteins are largely responsible for the visco-elastic properties that allow doughs to be processed into bread and various other food products including cakes, biscuits (cookies), pasta and noodles. Detailed biochemical and biophysical studies are revealing details of the molecular structures and interactions of the individual gluten proteins, and their roles in determining the functional properties of gluten. In particular, one group of gluten proteins, the high molecular weight (HMW) subunits of glutenin, have been studied in detail because of their role in determining the strength (elasticity) of doughs. The development of robust transformation systems for bread wheat is now allowing the role of the HMW subunits to be explored experimentally, by manipulating their amount and composition in transgenic plants. Such studies should lead to improvement of the processing properties of wheat for traditional end uses and the development of novel end uses in food processing or as raw material for other industries.

Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues

Abstract The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to wheat (Triticum aestivum L.) scutellum and inflorescence tissue. The main factors studied were the DNA/gold precipitation process, bombardment parameters and tissue culture variables. Efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS expression in bombarded tissues. Of the parameters analysed, amount of plasmid DNA, spermidine concentration, presence of Ca++ ions, calcium chloride concentration, amount of gold particles, gold particle size, acceleration pressure, chamber vacuum pressure, bombardment distance, osmotic conditioning of tissues and type of auxin had a clear influence on transient gene expression. A bombardment procedure suitable for elite wheat varieties was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.

Biotechnology of Cereals

This latest volume surveys opportunities and applications of biotechnology in improvement of cereal production, protection, and end use quantity. It will appeal to a broad readership, from academics to industry. It includes explanations of the technologies involved and discusses their applications. It is the only current volume which covers the full range of applications of biotechnology to cereals, thus providing the best available coverage of the topic in a single volume. The chapters it contains on transformation and end use quality are very topical and likely to attract great interest. Advances in Botanical Research is a multi-volume publication that brings together reviews by recognized experts on subjects of importance to those involved in botanical research. First published in 1963, Advances in Botanical Research has earned a reputation for excellence in the field for more than thirty years. In 1995, Advances in Botanical Research was merged with Advances in Plant Pathology to provide one comprehensive resource for the plant science community, with equal coverage of plant pathology and botany in both thematic and mixed volumes. Now edited by J.A. Callow (University of Birmingham, UK), supported by an international Editorial Board, Advances in Botanical Research publishes in-depth and up-to-date reviews on a wide range of topics which will appeal to post-graduates and researchers in plant sciences including botany, plant biochemistry, plant pathology and plant physiology. Eclectic volumes in the serial are supplemented by thematic volumes on such topics as Plant Protein Kinases, and Plant Trichomes. In 1999, the Institute for Scientific Information released figures showing that Advances in Botanical Research has an Impact Factor of 4.378, placing it 8th in the highly competitive category of Plant Sciences.

A facile method for screening for phosphinothricin (PPT)-resistant transgenic wheats

A bioassay was developed for identifying transgenic wheat plants based on De Block et al.s [8] ammonium-multiwell assay which allows qualitative and quantitative evaluation of the expression of the enzyme phosphinothricin acetyl transferase (PAT). Important parameters in the assay are the use of young leaf tissues, short incubation period (6 h) and a high light intensity during incubation (250 μmol s−1m−2). The assay is quick and results are obtained within a day. Ammonium measurements based on a colourimetric (modified Berthelot) reaction are conducted using sodium salicylate to avoid the use of phenol. Results of the assay show high correlation with Basta leaf painting tests and polymerase chain reaction (PCR) results. Thus, the assay may be used as a facile screen for bar-expressing transgenic cereals.

Transient gene expression in cassava somatic embryos by tissue electroporation

Abstract Embryogenic cassava ( Manihot esculenta Crantz) tissues were transformed with the β-glucuronidase (GUS) gene by electroporation. Physiological and developmental state of the tissues, electric field strength and incubation temperature and duration were observed to be the most important factors influencing transient expression. Optimised frequencies of GUS expression were obtained when tissue pieces bearing torpedo-stage embryos were preincubated at 37°C for 1 h, in sodium aspartate buffer containing plasmid DNA and electroporated with a single electric pulse (750 V/cm, 960 μF capacitance). The system was efficient and reproducible and, under these conditions, it was possible to obtain expression in 75% of the tissue pieces electroporated, with an average of 174 (±35.6) GUS spots per replicate (of approximately 20 pieces). These results suggests that tissue electroporation could have a general application as a facile gene transfer method.

Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize.

Abstract. A novel constitutive promoter from the maize histone H2B gene was recently identified. In this study, we characterised H2B promoter activity in both wheat and maize tissues using the gusA reporter gene and two synthetic versions of the pat (phosphinothricin acetyl transferase) selectable marker gene, namely mopat and popat. Analyses of transgenic plants showed that the H2B promoter is able to drive the expression of gusA to strong, constitutive levels in wheat and maize tissues. Using an H2B:mopat construct and phosphinothricin selection, we recovered transgenic wheat plants at efficiencies ranging from 0.3% to 7.4% (mean 1.6%), and the efficiency of selection ranged from 40% to 100% (mean 77.7%). In another application, H2B was combined with the maize Ubi-1 or the maize Adh-1 intron to drive the expression of mopat and popat. Transformation efficiencies with the Ubi-1 intron were between 1.4- to 16-fold greater than with the Adh-1 intron. However, the use of either of the introns was necessary for the recovery of transgenic plants. Mopat gave higher transformation efficiencies and induced higher levels of PAT protein in maize tissues than popat.

Transgenic Wheat, Barley and Oats: Production and Characterization

Ever since the first developments in plant transformation technology using model plant species in the early 1980s, there has been a body of plant science research devoted to adapting these techniques to the transformation of crop plants. For some crop species progress was relatively rapid, but in other crop groups such as the small grain cereals, which were not readily amenable to culture in vitro and were not natural hosts to Agrobacterium, it has taken nearly two decades to develop reliable and robust transformation methods.In the following chapters of this book, transformation procedures for small grain cereals are presented, together with methods for gene and protein expression and the characterization of transgenic plants. In this introductory chapter we try to put these later chapters into context, giving an overview of the development of transformation technology for small grain cereals, discussing some of the pros and cons of the techniques and what limitations still exist.

Morphogenic cell and protoplast cultures of tritordeum

Abstract Immature embryo- and anther-derived suspension and protoplast cultures were established in hexaploid tritordeum. The morphogenic capability of these cultures was studied under different nitrogen regimes. Both embryo- and anther-derived suspensions showed morphogenic capability. Suspension and protoplast cultures established from immature embryos gave rise to green shoots and green plants, whereas albino plants were regenerated when anther cultures were used as suspension-starting material. Chromosomal stability of suspensions and regenerated plants was analysed, revealing considerable instability in these cultures.

Morphological and Agronomic Variation in Transgenic Tritordeum Lines Grown in the Field

Summary Progenies produced by self-pollinating transgenic tritordeum plants containing the β-glucuronidase ( uid A) and neomycin phosphotransferase ( neo ) genes were grown under field conditions at the Instituto de Agricultura Sostenible, Cordoba, Spain. Various morphological and agronomic traits were studied in the transgenic plants and compared with plants from two controls, one derived from non-transformed tissue cultures and another one derived from standard seeds. Significant differences between transgenic lines and controls were detected in both trials. Transgenic lines with simple integration patterns did not appear to be conspicuously influenced on their agronomic performance, whereas the line with the most complex pattern of integration clearly performed differently from the controls. Somaclonal variation and physiological disturbances were observed among the transgenic lines; both were clearly reduced when plants were selected on the basis of their fertility. Aneuploidy was observed which might have contributed to the observed variation.