Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression.

Selectable marker gene systems are vital for the development of transgenic crops. Since the creation of the first transgenic plants in the early 1980s and their subsequent commercialization worldwide over almost an entire decade, antibiotic and herbicide resistance selectable marker gene systems have been an integral feature of plant genetic modification. Without them, creating transgenic crops is not feasible on purely economic and practical terms. These systems allow the relatively straightforward identification and selection of plants that have stably incorporated not only the marker genes but also genes of interest, for example herbicide tolerance and pest resistance. Bacterial antibiotic resistance genes are also crucial in molecular biology manipulations in the laboratory. An unprecedented debate has accompanied the development and commercialization of transgenic crops. Divergent policies and their implementation in the European Union on one hand and the rest of the world on the other (industrialized and developing countries alike), have resulted in disputes with serious consequences on agricultural policy, world trade and food security. A lot of research effort has been directed towards the development of markerfree transformation or systems to remove selectable markers. Such research has been in a large part motivated by perceived problems with antibiotic resistance selectable markers; however, it is not justified from a safety point of view. The aim of this review is to discuss in some detail the currently available scientific evidence that overwhelmingly argues for the safety of these marker gene systems. Our conclusion, supported by numerous studies, most of which are commissioned by some of the very parties that have taken a position against the use of antibiotic selectable marker gene systems, is that there is no scientific basis to argue against the use and presence of selectable marker genes as a class in transgenic plants.

a b s t r a c tGenetic gains in quality traits were assessed in grain samples from 4 field experiments involving 16 bread wheat varieties representative of those most widely cultivated in Spain during the 20th century. The allelic composition at three glutenin loci (Glu-A1, Glu-B1, and Glu-D1) was obtained by PCR-based DNA markers and published references. From 1930 to 2000 grain protein content decreased by −0.030% y −1 , or in relative terms by −0.21% y −1 , but the protein produced per hectare increased by 0.39% y −1 . Alveographic tests revealed significant changes in dough rheological properties. Dough strength (W) and tenacity (P) increased at relative rates of 1.38% y −1 and 0.99% y −1 , respectively, while dough extensibility (L) decreased by −0.46% y −1 , resulting in an increase of 1.45% y −1 in dough equilibrium (P/L). The rise in protein quality could be related to the replacement of the null allele by subunits 1 or 2* at Glu-A1 and the prevalence of subunits 7 + 8 and 5 + 10 at Glu-B1 and Glu-D1 loci, respectively, in the most recent varieties. Dough extensibility was affected by water input during the crop cycle, this relationship being partially explained by the presence of the 5 + 10 HMW glutenin subunit. Fermentation tolerance was improved in the most modern varieties. Collapse during fermentation was avoided only in doughs with a W ≥ 159 J × 10 −4 and a P/L ≥ 0.56 mm H 2 O mm −1 , levels achieved by most of the modern varieties. The over-strong and unbalanced rheological properties of some modern varieties resulted in highly porous doughs, and no clear advances in dough maximum height during fermentation were attained.

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.

BackgroundLeaf pigment content is an important trait involved in environmental interactions. In order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant (Triticum turgidum L. var. durum) under contrasting water availability conditions.ResultsThe pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. Under well-watered (control) conditions the mutant had lower levels of chlorophylls and carotenoids, but higher levels of xanthophyll de-epoxidation compared to wild-type. Transcriptomic analysis under control conditions showed that defense genes (encoding e.g. pathogenesis-related proteins, peroxidases and chitinases) were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. Transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. We also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction.ConclusionsThis study provides insight on the effect of pigment content in the molecular response to drought. Identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat.