CRISPR/Cas9-mediated targeted T-DNA integration in rice.

Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize.

Heritable site-specific mutagenesis using TALENs in maize.

Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus.

Rice, Japonica (Oryza sativa L.).

The importance of rice, as a food crop, is reflected in the extensive global research being conducted in an effort to improve and better understand this particular agronomic plant. In regard to biotechnology, this has led to the development of numerous genetic transformation protocols. Over the years, many of these methods have become increasingly straightforward, rapid, and efficient, thereby making rice valuable as a model crop for scientific research and functional genomics.

Maize (Zea mays L.).

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector.

Genetic transformation using maize immature zygotic embryos.

Epidermal and subepidermal cells in the abaxial, basal region of the maize (Zea mays L.) immature zygotic embryo (IZE) scutellum can be induced by exogenous auxin to proliferate and undergo somatic embryogenesis. Successful genetic transformation of IZEs depends not only on optimizing transformation parameters for these totipotent cells, but also on achieving high embryogenic callus induction frequency (ECIF) in a population of targeted explants.

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts.

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos.