Periodic Reporting for period 3 - Newcotiana (Developing Multipurpose Nicotiana Crops for Molecular Farming using New Plant Breeding Techniques)
Période du rapport: 2021-01-01 au 2021-12-31
NEWCOTIANA is a research project that employs New Breeding Techniques to develop new plant varieties of the genus Nicotiana producing end-value chemicals including proteins and metabolites. Plants can be used as biofactories of biopharmaceuticals and other added-value chemicals at an agricultural scale, offering a sustainable solution for present and future manufacturing needs. The species within the genus Nicotiana, more specifically N. tabacum (cultivated tobacco) and N. benthamiana (an Australian tobacco relative), are among the most widely used plant biofactories because of their favourable attributes, which include metabolic versatility, ease of cultivation and high yield, availability of genetic tools for trait manipulation, and their non-food status, which minimizes the possibility of contamination of the food supply.
In stark contrast with its high appreciation as biofactories, traditional tobacco cultivation is in continuous decline in Europe due, among other reasons, to the bad reputation of a crop that is used mainly for manufacturing cigarettes and other smoking products. The aim of NEWCOTIANA is to offer an alternative to tobacco farmers by breeding competitive Nicotiana biofactory varieties for high value non-smoking products. This would create a suitable production platform for the EU bioeconomy and at the same time would bring sustainability to a traditional crop. However, to become fully competitive and scalable biofactories, Nicotiana plants need to incorporate unconventional traits such as protein stability or metabolite fortification, which are not accessible to traditional crop breeding. Recently, New Plant Breeding Techniques (NPBTs) have emerged that bring unprecedented opportunities for Plant Biotechnology. The driving hypothesis in the NEWCOTIANA project is that breeding tobacco biofactory varieties for high value non-smoking products is now possible with the use of NPBTs. Four of those NPBTs, namely sequence-directed nuclease-mediated genome editing (known as CRISPR), intragenesis, agroinfiltration and grafting, will be employed in the project.
Most importantly, NEWCOTIANA goals include the improvement of the awareness and public understanding of NPBTs. Our aim is to create a two-way dialogue between science on the one hand and stakeholders, policymakers and the general public on the other, through provision of unbiased information and reliable data arising from the project, through innovative and highly participative channels. In this way, NEWCOTIANA will provide industry, policy-makers and consumers with experimental evidence and social communication channels to facilitate the decision-making process for the adoption of NPBTs.
In stark contrast with its high appreciation as biofactories, traditional tobacco cultivation is in continuous decline in Europe due, among other reasons, to the bad reputation of a crop that is used mainly for manufacturing cigarettes and other smoking products. The aim of NEWCOTIANA is to offer an alternative to tobacco farmers by breeding competitive Nicotiana biofactory varieties for high value non-smoking products. This would create a suitable production platform for the EU bioeconomy and at the same time would bring sustainability to a traditional crop. However, to become fully competitive and scalable biofactories, Nicotiana plants need to incorporate unconventional traits such as protein stability or metabolite fortification, which are not accessible to traditional crop breeding. Recently, New Plant Breeding Techniques (NPBTs) have emerged that bring unprecedented opportunities for Plant Biotechnology. The driving hypothesis in the NEWCOTIANA project is that breeding tobacco biofactory varieties for high value non-smoking products is now possible with the use of NPBTs. Four of those NPBTs, namely sequence-directed nuclease-mediated genome editing (known as CRISPR), intragenesis, agroinfiltration and grafting, will be employed in the project.
Most importantly, NEWCOTIANA goals include the improvement of the awareness and public understanding of NPBTs. Our aim is to create a two-way dialogue between science on the one hand and stakeholders, policymakers and the general public on the other, through provision of unbiased information and reliable data arising from the project, through innovative and highly participative channels. In this way, NEWCOTIANA will provide industry, policy-makers and consumers with experimental evidence and social communication channels to facilitate the decision-making process for the adoption of NPBTs.
During the course of the first 48 months of the project a number of important results have been achieved:
- A high quality N. benthamiana genome assembly has been produced that will allow researchers to better design their breeding strategies, select CRISPR targets, and will provide important clues on the genetic basis of N. benthamiana favourable biofactory traits (www.nbenth.com).
- Newcotiana has engaged in COVID-19 activities, such as the development of positive controls for SARS-CoV-2 PCR diagnostic assays. These controls have been used in the Norwich Testing Initiative, a diagnostics programme that provides regular testing of staff and students on the Norwich Research Park (UK).
- New tools for NPBTs in Nicotiana have been created, including more efficient CRISPR enzymes, alternative methods for gRNA delivery, optimised methods for gene targeting, or new intragenic elements.
- Significant advances have been made in the generation of Nicotiana biofactory lines: N. benthamiana lines with increased levels of zeaxanthin, a carotenoid that can slow macular degeneration, have been produced. These plants can also be used for the production of crocins, a potent antioxidant with pharmacological properties. As for tobacco, plants with altered glycosylation have been generated that will improve the functionality of recombinant glycoproteins. On the other hand, tobacco plants with low nicotine but high anatabine content, a compound that has shown anti-inflammatory properties, as well as lines enriched in squalene, a compound widely used as vaccine adjuvant, have also been produced. In addition, as a background trait for improved plant yield and biosafety, non-flowering tobacco lines have been generated. Finally, some of these traits have been combined to create non-flowering anatabine rich or non-flowering-squalene rich tobacco plants.
- New Nicotiana species have been produced by grafting-mediated horizontal genome transfer. In addition, data on the transcriptome and metabolome of 22 different Nicotiana species has been generated that will provide information on the natural variation available within the Nicotiana genus and will contribute to the discovery of new or missing genes.
- Constant interactions have been maintained with stakeholders, including policymakers, farmers’ associations and the industry, as well as the general public. More than 180 dissemination/communication activities have been undertaken, including conferences, workshops, scientific publications (zenodo.org/communities/newcotiana) exhibitions, videos (newcotiana.org/videos). Reports on stakeholder opinions and public perception on NPBTs have been generated. Webpage and social media (twitter @NewcotianaP) are being maintained.
- A high quality N. benthamiana genome assembly has been produced that will allow researchers to better design their breeding strategies, select CRISPR targets, and will provide important clues on the genetic basis of N. benthamiana favourable biofactory traits (www.nbenth.com).
- Newcotiana has engaged in COVID-19 activities, such as the development of positive controls for SARS-CoV-2 PCR diagnostic assays. These controls have been used in the Norwich Testing Initiative, a diagnostics programme that provides regular testing of staff and students on the Norwich Research Park (UK).
- New tools for NPBTs in Nicotiana have been created, including more efficient CRISPR enzymes, alternative methods for gRNA delivery, optimised methods for gene targeting, or new intragenic elements.
- Significant advances have been made in the generation of Nicotiana biofactory lines: N. benthamiana lines with increased levels of zeaxanthin, a carotenoid that can slow macular degeneration, have been produced. These plants can also be used for the production of crocins, a potent antioxidant with pharmacological properties. As for tobacco, plants with altered glycosylation have been generated that will improve the functionality of recombinant glycoproteins. On the other hand, tobacco plants with low nicotine but high anatabine content, a compound that has shown anti-inflammatory properties, as well as lines enriched in squalene, a compound widely used as vaccine adjuvant, have also been produced. In addition, as a background trait for improved plant yield and biosafety, non-flowering tobacco lines have been generated. Finally, some of these traits have been combined to create non-flowering anatabine rich or non-flowering-squalene rich tobacco plants.
- New Nicotiana species have been produced by grafting-mediated horizontal genome transfer. In addition, data on the transcriptome and metabolome of 22 different Nicotiana species has been generated that will provide information on the natural variation available within the Nicotiana genus and will contribute to the discovery of new or missing genes.
- Constant interactions have been maintained with stakeholders, including policymakers, farmers’ associations and the industry, as well as the general public. More than 180 dissemination/communication activities have been undertaken, including conferences, workshops, scientific publications (zenodo.org/communities/newcotiana) exhibitions, videos (newcotiana.org/videos). Reports on stakeholder opinions and public perception on NPBTs have been generated. Webpage and social media (twitter @NewcotianaP) are being maintained.
NEWCOTIANA has already advanced beyond the state-of-the-art in sequence-specific nuclease (SSN) technologies and platform optimization. Concerning SSNs technologies, we have developed alternative nucleases and more efficient gene editing/targeting methods. As for platform optimization, the new N. benthamiana genome assembly has set a crucial milestone in plant biofactories that will facilitate future breeding strategies; the expanded collection of regulatory elements represents a crucial step forward in the application of intragenesis in breeding; first prototype Nicotiana lines incorporating new valuable biofactory traits have been obtained and validated at laboratory scale and in the open field. In addition to the current advances, the following impacts are expected:
Scientific impact: the project will deepen in the understanding of gene function in Nicotiana species and the interactions between the metabolome and agronomic behaviour.
Technical impact: the prototype varieties generated will be validated at pilot scale resulting in the selection of a number of elite plant varieties. Elite lines should show improved production of functional recombinant proteins (with higher quality and/or yield) or added-value metabolites.
Social impact: In the EU, there are approximately 60.000 tobacco farmers and as many indirect jobs threatened by the decline in tobacco demand. NEWCOTIANA will bring the opportunity to maintain those jobs and to generate new ones, revitalizing rural areas where tobacco is a traditional crop as well as new others that will incorporate NEWCOTIANA. The scalability of the NEWCOTIANA biofactory will improve accessibility to biopharmaceuticals (vaccines, antibodies and others) whose availability is endangered due to high manufacturing costs and/or long production cycles. Furthermore, NEWCOTIANA aims to provide industry, policy-makers and consumers with the experimental evidence that will facilitate the decision-making process for the adoption of NPBTs.
Scientific impact: the project will deepen in the understanding of gene function in Nicotiana species and the interactions between the metabolome and agronomic behaviour.
Technical impact: the prototype varieties generated will be validated at pilot scale resulting in the selection of a number of elite plant varieties. Elite lines should show improved production of functional recombinant proteins (with higher quality and/or yield) or added-value metabolites.
Social impact: In the EU, there are approximately 60.000 tobacco farmers and as many indirect jobs threatened by the decline in tobacco demand. NEWCOTIANA will bring the opportunity to maintain those jobs and to generate new ones, revitalizing rural areas where tobacco is a traditional crop as well as new others that will incorporate NEWCOTIANA. The scalability of the NEWCOTIANA biofactory will improve accessibility to biopharmaceuticals (vaccines, antibodies and others) whose availability is endangered due to high manufacturing costs and/or long production cycles. Furthermore, NEWCOTIANA aims to provide industry, policy-makers and consumers with the experimental evidence that will facilitate the decision-making process for the adoption of NPBTs.