Agrobacterium-mediated direct transformation of wheat mature embryos through organogenesis.

Transgenic plant production in monocotyledonous species has primarily relied on embryogenic callus induction from both immature and mature embryos as the pathway for plant regeneration. We have efficiently regenerated fertile transgenic wheat plants through organogenesis after Agrobacterium-mediated direct transformation of mechanically isolated mature embryos from field-grown seed. Centrifugation of the mature embryos in the presence of Agrobacterium was found to be essential for efficient T-DNA delivery to the relevant regenerable cells. The inoculated mature embryos formed multiple buds/shoots on high-cytokinin medium, which directly regenerated into transgenic shoots on hormone-free medium containing glyphosate for selection. Rooted transgenic plantlets were obtained within 10-12 weeks after inoculation. Further optimization of this transformation protocol resulted in significant reduction of chimeric plants to below 5%, as indicated by leaf GUS staining and T1 transgene segregation analysis. Direct transformation of wheat mature embryos has substantial advantages over traditional immature embryo-based transformation systems, including long-term storability of the mature dry explants, scalability, and greatly improved flexibility and consistency in transformation experiments.

Commercial scale genetic transformation of mature seed embryo explants in maize.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

Site-directed integration of exogenous DNA into the soybean genome by LbCas12a fused to a plant viral HUH endonuclease.

High efficiency site-directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR-associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR-associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70-nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site-directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four-fold higher than in untethered controls. Integrations via canonical homology-directed repair or non-homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN-associated tethering to improve site-directed DNA integration in plants.

Demonstration of targeted crossovers in hybrid maize using CRISPR technology.

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.

Agrobacterium tumefaciens-mediated genetic transformation of cereals using immature embryos.

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.

Transcriptome analysis of nitrogen-efficient rice over-expressing alanine aminotransferase.

Crop 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.

Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase.

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.

Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers.

Cereal 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.

Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production?

Plant scientists have long recognized the need to develop crops that absorb and use nutrients more efficiently. Two approaches have been used to increase nutrient use efficiency (NUE) in crop plants. The first involves both traditional breeding and marker-assisted selection in an attempt to identify the genes involved. The second uses novel gene constructs designed to improve specific aspects of NUE. Here, we discuss some recent developments in the genetic manipulation of NUE in crop plants and argue that an improved understanding of the transition between nitrogen assimilation and nitrogen recycling will be important in applying this technology to increasing crop yields. Moreover, we emphasize the need to combine genetic and transgenic approaches to make significant improvements in NUE.