We have generated transgenic rice plants expressing the Datura stramonium adc gene and investigated their response to drought stress. We monitored the steady-state mRNA levels of genes involved in polyamine biosynthesis (Datura adc, rice adc, and rice samdc) and polyamine levels. Wild-type plants responded to the onset of drought stress by increasing endogenous putrescine levels, but this was insufficient to trigger the conversion of putrescine into spermidine and spermine (the agents that are believed to protect plants under stress). In contrast, transgenic plants expressing Datura adc produced much higher levels of putrescine under stress, promoting spermidine and spermine synthesis and ultimately protecting the plants from drought. We demonstrate clearly that the manipulation of polyamine biosynthesis in plants can produce drought-tolerant germplasm, and we propose a model consistent with the role of polyamines in the protection of plants against abiotic stress.A biotic stresses such as drought represent some of the most significant constraints to agricultural productivity. Transgenic approaches can be used in combination with conventional breeding strategies to create crops with enhanced drought tolerance, and one way in which this can be achieved is through the manipulation of polyamine metabolism. Polyamines are small, ubiquitous, nitrogenous compounds that have been implicated in a variety of stress responses in plants (1). The link between polyamines and abiotic stress was first documented through putrescine accumulation in response to suboptimal potassium levels in barley (2). Since then, a connection has been suggested between increased putrescine levels and abiotic stress (3). Similar phenomena have been described in animals, e.g., during ischemic and postischemic responses in neurons (4). The physiological role of putrescine in abiotic stress responses is a matter of controversy. It has been very difficult to establish a direct cause-and-effect relationship between increased putrescine levels in plants and abiotic stress. Elevated putrescine might be the cause of stress-induced injury or, alternatively, a protective response resulting from stress (5).The genetic manipulation of polyamine metabolism has become a valuable tool for studying their physiological roles in plants (6). Plant polyamine content has been modulated by the overexpression͞down-regulation of arginine decarboxylase (adc), ornithine decarboxylase (odc), and S-adenosylmethionine decarboxylase (samdc) (6-10). Overexpression of heterologous adc or odc cDNAs in plants generally results in the production of high levels of putrescine (11-13). In most cases, this is accompanied by a relatively small increase in spermidine and spermine concentrations (7,14). Such findings suggest that the levels of spermidine and spermine are under strict homeostatic regulation (15). Therefore, the study of plants transformed with genes involved in polyamine biosynthesis may shed light on the importance of polyamines, their role in the acquisition of stress ...

Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of ␤-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.folic acid ͉ metabolic engineering ͉ transgenic maize ͉ vitamin A fortification ͉ vitamin C

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.

SummaryThe CRISPR/Cas9 system and related RNA‐guided endonucleases can introduce double‐strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on‐target mutations), the targeting accuracy (likelihood of off‐target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species‐dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome‐editing tools.

email christou@pvcf.udl.es) † These authors contributed equally to this work.Keywords: biofortification, antioxidants, genetic engineering. SummaryAntioxidants are protective molecules that neutralize reactive oxygen species and prevent oxidative damage to cellular components such as membranes, proteins and nucleic acids, therefore reducing the rate of cell death and hence the effects of ageing and ageing-related diseases. The fortification of food with antioxidants represents an overlap between two diverse environments, namely fortification of staple foods with essential nutrients that happen to have antioxidant properties (e.g. vitamins C and E) and the fortification of luxury foods with healthpromoting but non-essential antioxidants such as flavonoids as part of the nutraceuticals/ functional foods industry. Although processed foods can be artificially fortified with vitamins, minerals and nutraceuticals, a more sustainable approach is to introduce the traits for such health-promoting compounds at source, an approach known as biofortification. Regardless of the target compound, the same challenges arise when considering the biofortification of plants with antioxidants, that is the need to modulate endogenous metabolic pathways to increase the production of specific antioxidants without affecting plant growth and development and without collateral effects on other metabolic pathways. These challenges become even more intricate as we move from the engineering of individual pathways to several pathways simultaneously. In this review, we consider the state of the art in antioxidant biofortification and discuss the challenges that remain to be overcome in the development of nutritionally complete and health-promoting functional foods.