Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.

Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

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Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

Crop improvements can help us to meet the challenge of feeding a population of 10 billion, but can we breed better varieties fast enough? Technologies such as genotyping, marker-assisted selection, high-throughput phenotyping, genome editing, genomic selection and de novo domestication could be galvanized by using speed breeding to enable plant breeders to keep pace with a changing environment and ever-increasing human population.

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Breeding crops to feed 10 billion.

Genome editing technologies have progressed rapidly and become one of the most important genetic tools in the implementation of pathogen resistance in plants. Recent years have witnessed the emergence of site directed modification methods using meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Recently, CRISPR/Cas9 has largely overtaken the other genome editing technologies due to the fact that it is easier to design and implement, has a higher success rate, and is more versatile and less expensive. This review focuses on the recent advances in plant protection using CRISPR/Cas9 technology in model plants and crops in response to viral, fungal and bacterial diseases. As regards the achievement of viral disease resistance, the main strategies employed in model species such as Arabidopsis and Nicotiana benthamiana, which include the integration of CRISPR-encoding sequences that target and interfere with the viral genome and the induction of a CRISPR-mediated targeted mutation in the host plant genome, will be discussed. Furthermore, as regards fungal and bacterial disease resistance, the strategies based on CRISPR/Cas9 targeted modification of susceptibility genes in crop species such as rice, tomato, wheat, and citrus will be reviewed. After spending years deciphering and reading genomes, researchers are now editing and rewriting them to develop crop plants resistant to specific pests and pathogens.

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The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology.

Shoot architecture is determined by the organization and activities of apical, axillary, intercalary, secondary, and inflorescence meristems and by the subsequent development of stems, leaves, shoot branches, and inflorescences. In this review, we discuss the unifying principles of hormonal and genetic control of shoot architecture including advances in our understanding of lateral branch outgrowth; control of stem elongation, thickness, and angle; and regulation of inflorescence development. We focus on recent progress made mainly in Arabidopsis thaliana, rice, pea, maize, and tomato, including the identification of new genes and mechanisms controlling shoot architecture. Key advances include elucidation of mechanisms by which strigolactones, auxins, and genes such as IDEAL PLANT ARCHITECTURE1 and TEOSINTE BRANCHED1 control shoot architecture. Knowledge now available provides a foundation for rational approaches to crop breeding and the generation of ideotypes with defined architectural features to impro...

Genetic Regulation of Shoot Architecture.

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

An improved
 Agrobacterium
 -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds.
 This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line

The current study describes a robust, high-frequency Agrobacterium-mediated transformation protocol suitable for multiple recalcitrant modern elite commercial maize inbreds employing media modifications with glucose, cupric sulfate and a cytokinin, 6-benzylaminopurine (BAP). An optimal combination of these three key elements in the co-cultivation, resting, and selection media resulted in 4- to 14-fold improvements in transformation frequencies at the T0 plant level of 9.7, 31.9, 9.6 and 10.0 % for PH4CN, PH12BN, PHW0V and PH17R8, respectively. Transformation frequency in PH1CP1 was also improved at the T0 tissue level from 2.5 to 8.3 %. The addition of cupric sulfate and BAP in the co-cultivation medium improved transformation frequency in all inbreds except PH4CN. The use of cupric sulfate and BAP in combination with additional glucose in the selection medium was especially important, significantly improving the transformation frequency in 3 (PH4CN, PHW0V and PH1CP1) out of 5 inbreds by increasing the proliferation of high quality regenerable tissue. It was observed that the amount/ratio of these three components needed to be optimized for each inbred. The results in this study can be applied to optimize the tissue culture response and improve transformation frequency in other recalcitrant elite commercial maize inbreds.

Improvement of Agrobacterium -mediated transformation frequency in multiple modern elite commercial maize ( Zea mays L.) inbreds by media modifications

Jenny Banh

Papers

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line

Improvement of Agrobacterium -mediated transformation frequency in multiple modern elite commercial maize ( Zea mays L.) inbreds by media modifications