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Summary Nitrogen is quantitatively the most essential nutrient for plants and a major factor limiting crop productivity. One of the critical steps limiting the efficient use of nitrogen is the ability of plants to acquire it from applied fertilizer. Therefore, the development of crop plants that absorb and use nitrogen more efficiently has been a long‐term goal of agricultural research. In an attempt to develop nitrogen‐efficient plants, rice (Oryza sativa L.) was genetically engineered by introducing a barley AlaAT (alanine aminotransferase) cDNA driven by a rice tissue‐specific promoter (OsAnt1). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. The development of crop plants that take up and assimilate nitrogen more efficiently would not only improve the use of nitrogen fertilizers, resulting in lower production costs, but would also have significant environmental benefits. These results are discussed in terms of their relevance to the development of strategies to engineer enhanced nitrogen use efficiency in crop plants.

SummaryCereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium -mediated transformation of other major cereals.In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium -mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.

SummaryCrop plants require nitrogen for key macromolecules, such as DNA, proteins and metabolites, yet they are generally inefficient at acquiring nitrogen from the soil.Crop producers compensate for this low nitrogen utilization efficiency by applying nitrogen fertilizers. However, much of this nitrogen is unavailable to the plants as a result of microbial uptake and environmental loss of nitrogen, causing air, water and soil pollution. We engineered rice over-expressing alanine aminotransferase ( AlaAT ) under the control of a tissue-specific promoter that showed a strong nitrogen use efficiency phenotype. In this study, we examined the transcriptome response in roots and shoots to the over-expression of AlaAT to provide insights into the nitrogen-use-efficient phenotype of these plants.Transgenic and control rice plants were grown hydroponically and the root and shoot gene expression profiles were analysed using Affymetrix Rice GeneChip microarrays.Transcriptome analysis revealed that there was little impact on the transgenic transcriptome compared with controls, with 0.11% and 0.07% differentially regulated genes in roots and shoots, respectively. The most up-regulated transcripts, a glycine-rich cell wall ( GRP ) gene and a gene encoding a hypothetical protein ( Os8823 ), were expressed in roots. Another transgenic root-specific up-regulated gene was leucine rich repeat ( LRR ). Genes induced in the transgenic shoots included GRP , LRR , acireductone dioxygenase ( OsARD ), SNF2 ATP-translocase and a putative leucine zipper transcription factor. This study provides a genome-wide view of the response to AlaAT over-expression, and elucidates some of the genes that may play a role in the nitrogen-use-efficient phenotype.

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Naturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding.

A critical step in the development of a robust Agrobacterium tumefaciens-mediated transformation -system for cereal crop plants is the establishment of optimal conditions for efficient T-DNA delivery into target tissue, from which plants can be regenerated. Although, Agrobacterium-mediated transformation of cereals is an important method that has been widely used by many laboratories around the world, routine protocols have been established only in specific cultivars within a species and with specific tissues of high regeneration potential. Cocultivation of highly embryogenic callus tissue or healthy immature embryos with A. tumefaciens is considered one of the critical factors in successful genetic transformation of crop plants. Immature embryos collected only from vigorously growing healthy and green plants grown in the field or in the well-conditioned greenhouse are the ideal target for genetic transformation of recalcitrant crop species. Here, we describe an Agrobacterium-mediated transformation method that uses immature embryos as the starting material for inoculation with Agrobacterium. The aim of this chapter is to provide the key steps/components involved in Agrobacterium-mediated transformation of cereal crops. However, these steps or components often vary between protocols and from laboratory to laboratory, and can be optimized or modified based on the requirement of a specific cultivar or species.