Sjur Sandgrind
Research Scientist
Authors
Johanna Eva Bodin Paul Ragnar Berg Knut Tomas Dalen Nur Duale Erik Georg Granquist Anne-Marthe Ganes Jevnaker Tor Atle Mo Truls Nesbakken Kaare Magne Nielsen Kristian Prydz Espen Rimstad Eli Knispel Rueness Monica Sanden Sjur Sandgrind Guro Katrine Sandvik Line Elisabeth Breivik Sundt-Hansen Amin Sayyari Ville Erling Sipinen Eva Bonsak ThorstadAbstract
The Norwegian Environment Agency has tasked The Norwegian Scientific Committee for Food and Environment (VKM) with developing generic guidance documents for environmental risk assessment of genetically modified organisms (GMOs) to be used in field trials. GMOs used in limited field trials may have effect on the environment, and human and animal health. All experiments involving the use of GMOs in field trails for research require approval under the Norwegian Gene Technology Act. The Norwegian Environment Agency is the decision-making authority for deliberate release of GM plants in field trials. VKM performs health and environmental risk assessments (ERAs) of GMOs for the Norwegian Environment Agency. This guidance document is intended as a support for applicants seeking approval for field trials under the Gene Technology Act and identifies the scientific documentation and data necessary to facilitate an ERA of a genetically modified animal (GM animal) conducted by VKM. The risk assessments conducted by VKM generally follow the step-by-step approach outlined in EU Directive 2001/18/EC on the deliberate release of genetically modified organisms into the environment, starting with hazard identification, hazard characterization, exposure characterization and risk characterization. The Gene Technology Act and the Regulations on impact assessment under the Gene Technology Act implements directive 2001/18/EC into Norwegian legislation. Appendix 2 of the Regulation contain the principles for environmental risk assessment, which corresponds to the principles of Annex II in the Directive. The European Food Safety Authority (EFSA) has developed further guidance on the risk assessment of genetically modified organisms based on Annex II. ERAs of GM animals involve the collection, assessment and, where appropriate, generation of information on a GM animal to determine its potential impacts on the environment and on human and animal health, compared with non-GM animals or appropriate comparators. VKM performs risk assessments that include the following six steps: hazard identification, hazard characterisation, exposure characterisation, risk characterisation, risk reducing measures, and overall risk evaluation of the use of the GM animal in a field trial. Applicants should identify the scientific documentation and data necessary for VKM to perform an ERA based on these steps. Applicants should also consider general risk reducing measures in relation to the identified risks, taking into consideration the type of GM animal, the intended management regime (confinement), the scale of the field trial, the characteristics of the genetic modification, the characteristics of the identified hazard(s), and the potential consequences for the environment and for human and animal health. This document provides guidance for the assessment of the potential environmental effects of GM animals, based on data from the required molecular characterisation, consideration of the potential impacts of the modified characteristics of the GM animal, and any risk mitigating measures implemented in the event of escape of GM animals into the environment during field trials. The guidance is intended as a dynamic document to be amended in accordance with future scientific and regulatory developments.
Authors
Oda Eline Sandmo Ånesland Eline Seim Magne Nordang Skårn Arti Rai Sjur Sandgrind May Bente Brurberg Tage ThorstensenAbstract
No abstract has been registered
Abstract
Brassica carinata is an important oil crop with significant potential for food and industrial production. The application of the CRISPR/Cas9 genome editing tool in B. c arinata could accelerate its breeding cycle. However, no efficient DNA-free gene editing method currently exists for this species. Protoplast-based CRISPR editing presents a promising solution, though it is often challenging for many crop species. In this study, we investigated several critical factors influencing in vitro shoot regeneration, including genotype, sugar type, selection and combination of plant growth regulators (PGRs), and culture duration on different media throughout various stages of protoplast development. As a result, we developed a highly efficient, five-stage protoplast regeneration protocol for B. carinata based on specific stages of protoplast development. Key findings of this study include the requirement for high concentrations of NAA and 2,4-D in the initial medium (MI) for cell wall formation, while a lower auxin concentration relative to cytokinin was necessary for active cell division (MII). For callus growth and shoot induction, a high cytokinin-to-auxin ratio was essential (MIII), and an even higher cytokinin-to-auxin ratio was optimal for shoot regeneration (MIV). For shoot elongation, low levels of BAP and GA 3 were sufficient (MV). Our results also demonstrated that the duration of culture on different media and maintaining appropriate osmotic pressure at the early stages were crucial for successful protoplast regeneration. With this optimized protocol, we achieved an average regeneration frequency of up to 64% and a transfection efficiency of 40% using the GFP marker gene. This efficient protoplast regeneration protocol is now being employed for genome editing in our lab and is expected to significantly enhance the application of the CRISPR system in both basic research and the genetic improvement of B. carinata over the long term.