Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

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Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve Discosoma sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of Catharanthus roseus using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for Arabidopsis thaliana. We also demonstrate a successful method to cross Camelina sativa and provide qRT-PCR results to prove its effectiveness.

This paper sets out to investigate the origins and legacy of a significant 1960s modernist public housing estate in the North East of England. This estate at Kenton Bar remains virtually intact, and as such represents a unique example in Newcastle upon Tyne. Its architects, Ryder and Yates, had personal contact with Le Corbusier, Georges Braque, Berthold Lubetkin, Ove Arup and Clive Entwistle; and the influence and mentorship of these individuals is clearly evident in the design. Being selected to join the small team at Peterlee New Town in 1948, provided the opportunity for Ryder and Yates to work together for the first time. The experience enriched their architectural vocabulary and modernist values; and ultimately led to the establishment of their business partnership in 1953. By the time of Kenton Bar, it was by far the largest project that their architectural practice had undertaken. The office was greatly appreciative of Eric Lyons and Span Developments, and their work appears as a distinct precedent in the layout. With a pyramid in its central square, and an assortment of innovative design installations, the estate resonated strongly and immediately with residents.Today, even the pyramid that was removed some years ago, lives on in the collective memory of residents, past and present. Recently, weblogs have been established; and collages and models produced by artists, as well as adults and children from the Estate, in the style of the original design presentations. Fifty years after it was conceived, the interest of local artists and galleries, the tenants’ association, former residents, and pupils of the primary school specifically designed to be at the heart of the estate may not be unprecedented but it is certainly rare. Ryder and Yates have created a living monument to twentieth-century ideas.

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