Genome Editing in Brassica juncea Using CRISPR/Cas9 Technology.

Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely. Here we describe the protocol for editing the genome of a field crop Brassica juncea (mustard), an allotetraploid and important oilseed crop of the Indo-Pak Subcontinent region. This protocol is based on the Agrobacterium-mediated transformation for the delivery of CRISPR components into the plant genome using cotyledon as explants. We elaborate on steps for recovering genome-edited knockouts, for validation of the edits, as well as recovering the transgene-free edited plants through a commonly used segregating approach.

Overexpression of Brassica napus COMT1 in Arabidopsis heightens UV-B-mediated resistance to Plutella xylostella herbivory.

UV-B radiation regulates numerous morphogenic, biochemical and physiological responses in plants, and can stimulate some responses typically associated with other abiotic and biotic stimuli, including invertebrate herbivory. Removal of UV-B from the growing environment of various plant species has been found to increase their susceptibility to consumption by invertebrate pests, however, to date, little research has been conducted to investigate the effects of UV-B on crop susceptibility to field pests. Here, we report findings from a multi-omic and genetic-based study investigating the mechanisms of UV-B-stimulated resistance of the crop, Brassica napus (oilseed rape), to herbivory from an economically important lepidopteran specialist of the Brassicaceae, Plutella xylostella (diamondback moth). The UV-B photoreceptor, UV RESISTANCE LOCUS 8 (UVR8), was not found to mediate resistance to this pest. RNA-Seq and untargeted metabolomics identified components of the sinapate/lignin biosynthetic pathway that were similarly regulated by UV-B and herbivory. Arabidopsis mutants in genes encoding two enzymes in the sinapate/lignin biosynthetic pathway, CAFFEATE O-METHYLTRANSFERASE 1 (COMT1) and ELICITOR-ACTIVATED GENE 3-2 (ELI3-2), retained UV-B-mediated resistance to P. xylostella herbivory. However, the overexpression of B. napus COMT1 in Arabidopsis further reduced plant susceptibility to P. xylostella herbivory in a UV-B-dependent manner. These findings demonstrate that overexpression of a component of the sinapate/lignin biosynthetic pathway in a member of the Brassicaceae can enhance UV-B-stimulated resistance to herbivory from P. xylostella.

Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.

CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.

Enabling Trade in Gene-Edited Produce in Asia and Australasia: The Developing Regulatory Landscape and Future Perspectives.

Genome- or gene-editing (abbreviated here as 'GEd') presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world's growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Regulation of Molecular Farming Products.

The regulation of molecular farming is a complex topic because plants and plant-based systems are relative newcomers among the many production platforms available for recombinant proteins. The regulations specific for different types of product (human/veterinary pharmaceuticals and medical devices, cosmetics, diagnostics, and research reagents) must therefore be overlaid with the regulations governing hitherto unfamiliar production platforms, and this must be achieved in different jurisdictions that handle genetically modified organisms (and genetically modified plants in particular) in very different ways. This chapter uses examples of different product types and production methods in three different jurisdictions (the USA, the EU, and Canada) to demonstrate some of the challenges facing the regulatory authorities.

Beyond the gene: epigenetic and cis-regulatory targets offer new breeding potential for the future.

For millennia, natural and artificial selection has combined favourable alleles for desirable traits in crop species. While modern plant breeding has achieved steady increases in crop yields over the last century, on the current trajectory we will simply not meet demand by 2045. Novel breeding strategies and sources of genetic variation will be required to sustainably fill predicted yield gaps and meet new consumer preferences. Here, we highlight that stepping up to meet this grand challenge will increasingly require thinking 'beyond the gene'. Significant progress has been made in understanding the contributions of both epigenetic variation and cis-regulatory variation to plant traits. This non-genic variation has great potential in future breeding, synthetic biology and biotechnology applications.

CRISPR-Cas9-Mediated Gene Editing of MYB28 Genes Impair Glucoraphanin Accumulation of Brassica oleracea in the Field.

Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of MYB28 in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated sulfur metabolism in field-grown Brassica oleracea with the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene-editing technology. We describe the first myb28 knockout mutant in B. oleracea, and the first CRISPR field trial in the United Kingdom approved and regulated by the UK Department for Environment, Food & Rural Affairs after the reclassification of gene-edited crops as genetically modified organisms by the European Court of Justice on July 25, 2018. We report that knocking out myb28 results in downregulation of A-GSL biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants, whereas accumulation of sulfate, S-methyl cysteine sulfoxide, and indole glucosinolate in leaf and floret tissues remained unchanged. These results demonstrate the potential of gene-editing approaches to translate discoveries in fundamental biological processes for improved crop performance.

Investigating the In Vitro Regeneration Potential of Commercial Cultivars of Brassica.

In vitro regeneration is a pre-requisite for developing transgenic plants through tissue culture-based genetic engineering approaches. Huge variations among different genotypes of the genus Brassica necessitate the identification of a set of regeneration conditions for a genotype, which can be reliably used in transformation experiments. In this study, we evaluated the morphogenesis potential of four commercial cultivars (Faisal canola, Punjab canola, Aari canola, Nifa Gold) and one model, Westar, from four different explants namely cotyledons, hypocotyls, petioles and roots on three different Brassica regeneration protocols, BRP-I, -II and -III. The regeneration efficiency was observed in the range of 6-73%, 4-79.3%, 0-50.6%, and 0-42.6% from cotyledons, petioles, hypocotyls and roots, respectively, whereas, the regeneration response in terms of average shoots per explant was found to be 0.76-10.9, 0.2-3.2, 0-3.4 and 0-2.7 from these explants. Of the commercial varieties tested, almost all varieties showed poorer regeneration than Westar except Aari canola. In comparison to Westar, its regeneration frequency from cotyledons was up to 7.5-fold higher on BRP-I, while it produced up to 21.9-fold more shoots per explant. Our data show that the explant has strong influence on the regeneration response, ranging from 24% to 92%. While the growth of commercial cultivars was least affected by the regeneration conditions provided, the effect on Westar was twice that of the commercial cultivars. After determining the optimal explant type and regeneration conditions, we also determined the minimum kanamycin concentration levels required to selectively inhibit the growth of untransformed cells for these cultivars. Regenerated shoots of Aari canola could be successfully grown to maturity within 16-18 weeks, with no altered phenotype noted and normal seed yields obtained. Therefore, the commercial variety, Aari canola, could be a good candidate for future genetic transformation studies.

Impacts of the EU GMO regulatory framework for plant genome editing.

New plant breeding technologies, such as genome editing, are enabling new crop varieties to be developed far quicker and with greater precision and scope than achievable using conventional methods. These advances could help farmers address the challenges of climate change, sustainability, and global food security. However, despite their potential, the uptake of these new technologies has been slowed down due to the uncertainty associated with the regulation of genome edited crops. For many European consumers, their view of new breeding technologies is influenced by many factors. Those who have never faced a major food crisis may not sufficiently appreciate the challenges posed by a projected rise of 2 billion in the human population by 2050. In addition, consumers with a regular and plentiful supply of food may not have to consider how their food is produced, or appreciate the challenges EU farmers are already facing to meet future demand. Misleading online articles, questioning the safety and ethics of these "new" biotech foods, can also lead consumers to be reluctant to accept them. Consequently, Europe's mixed view on biotech crops may also be hindering their adoption in countries who have even more to gain from the technology. In this review, we discuss the current data on global and EU GM crop adoption and the potential impact a new wave of crop development may have for agriculture. We reflect on how the EU has viewed GM crops, and we consider the future of both genetic modification (GM) and genome editing (GE) in the EU. We explore lessons learnt from the adoption of GM crops and examine the potential impact the recent decision not to exempt genome edited crops from the EU GMO Directive, will have on EU farmers, scientists, consumers, trading countries, and the rest of the world.

Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9.

While public and political views on genetic modification (inserting "foreign" genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing offers a faster and simpler approach to gene knockout in both single and multiple genetic locations, within a single or small number of generations, in a way that has not been possible through alternative breeding methods. Here we describe an Agrobacterium-mediated delivery approach to deliver Cas9 and dual sgRNAs into 4-day-old cotyledonary petioles of Brassica oleracea. Mutations are detected in approximately 10% of primary transgenic plants and go on in subsequent T1 and T2 generations to segregate away from the T-DNA. This enables the recovery of non-transgenic, genome-edited plants carrying a variety of mutations at the target locus.