<em>Brassica oleracea</em> Transformation

With easier access to genome sequences and genome editing technology over the last few years, transformation technologies are routinely being used now as a research tool for elucidating gene function. In this chapter, we outline a simple A. tumefaciens-mediated transformation method for the diploid brassica species B. oleracea, using a doubled haploid spring line called AG DH1012. This genotype has become our model genotype for training purposes and for most of our routine work, with a transformation efficiency of 25% (no. independent shoots/no. explants). This easy-to-follow protocol has made for a 100% technology transfer success rate to other researchers and laboratories around the globe. In this chapter, we detail our method using 4-day-old cotyledonary explants, with a step-by-step guide and supplemented with an online video to show the explant and subsequent shoot isolation stages. The video helps to remove any ambiguity that written protocols sometimes have. Primary transgenics ready for DNA extraction and early molecular analysis can be generated within 5–7 weeks from explant isolation

The status under EU Law of Organisms developed through novel genomic techniques

In a ruling on 25 July 2018, the Court of Justice of the European Union concluded that organisms obtained by means of techniques/methods of mutagenesis constitute GMOs in the sense of Directive 2001/18, and that organisms obtained by means of techniques/methods of directed mutagenesis are not excluded from the scope of the Directive. Following the ruling, there has been much debate about the possible wider implications of the ruling. In October 2019, the Council of the European Union requested the European Commission to submit, in light of the CJEU ruling, a study regarding the status of novel genomic techniques under Union Law. For the purpose of the study, the Commission initiated stakeholder consultations early in 2020. Those consultations focused on the technical status of novel genomic techniques. This article aims to contribute to the discussion on the legal status of organisms developed through novel genomic techniques, by offering some historical background to the negotiations on the European Union (EU) GMO Directives as well as a technical context to some of the terms in the Directive, and by analysing the ruling. The article advances that (i) the conclusion that organisms obtained by means of techniques/methods of mutagenesis constitute GMOs under the Directive means that the resulting organisms must comply with the GMO definition, ie the genetic material of the resulting organisms has been altered in a way that does not occur naturally by mating and/or natural recombination; (ii) the conclusion that organisms obtained by means of techniques/methods of directed mutagenesis were not intended to be excluded from the scope of the Directive is not inconsistent with the negotiation history of the Directive; (iii) whether an organism falls under the description of “obtained by means of techniques/methods of directed mutagenesis” depends on whether the genetic material of the resulting organisms has been altered in a way that does not occur naturally by mating and/or natural recombination. Finally, the article offers an analysis of the EU GMO definition, concluding that for an organism to be a GMO in the sense of the Directive, the technique used, as well as the genetic alterations of the resulting organism, must be considered

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

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.</p

The Status under EU Law of Organisms Developed through Novel Genomic Techniques

In a ruling on 25 July 2018, the Court of Justice of the European Union concluded that organisms obtained by means of techniques/methods of mutagenesis constitute GMOs in the sense of Directive 2001/18, and that organisms obtained by means of techniques/methods of directed mutagenesis are not excluded from the scope of the Directive. Following the ruling, there has been much debate about the possible wider implications of the ruling. In October 2019, the Council of the European Union requested the European Commission to submit, in light of the CJEU ruling, a study regarding the status of novel genomic techniques under Union Law. For the purpose of the study, the Commission initiated stakeholder consultations early in 2020. Those consultations focused on the technical status of novel genomic techniques. This article aims to contribute to the discussion on the legal status of organisms developed through novel genomic techniques, by offering some historical background to the negotiations on the European Union (EU) GMO Directives as well as a technical context to some of the terms in the Directive, and by analysing the ruling. The article advances that (i) the conclusion that organisms obtained by means of techniques/methods of mutagenesis constitute GMOs under the Directive means that the resulting organisms must comply with the GMO definition, ie the genetic material of the resulting organisms has been altered in a way that does not occur naturally by mating and/or natural recombination; (ii) the conclusion that organisms obtained by means of techniques/methods of directed mutagenesis were not intended to be excluded from the scope of the Directive is not inconsistent with the negotiation history of the Directive; (iii) whether an organism falls under the description of "obtained by means of techniques/methods of directed mutagenesis" depends on whether the genetic material of the resulting organisms has been altered in a way that does not occur naturally by mating and/or natural recombination. Finally, the article offers an analysis of the EU GMO definition, concluding that for an organism to be a GMO in the sense of the Directive, the technique used, as well as the genetic alterations of the resulting organism, must be considered