Genome editing: an introduction to CRISPR/Cas9

Damiano Martignago

Dr Damiano Martignago, Rothamsted Research

This week’s blog post was written by Dr Damiano Martignago, a genome editing specialist at Rothamsted Research.

 

Genome editing technologies comprise a diverse set of molecular tools that allow the targeted modification of a DNA sequence within a genome. Unlike “traditional” breeding, genome editing does not rely on random DNA recombination; instead it allows the precise targeting of specific DNA sequences of interest. Genome editing approaches induce a double strand break (DSB) of the DNA molecule at specific sites, activating the cell’s DNA repair system. This process could be either error-prone, thus used by scientists to deactivate “undesired” genes, or error-free, enabling target DNA sequences to be “re-written” or the insertion of DNA fragments in a specific genomic position.

The most promising among the genome editing technologies, CRISPR/Cas9, was chosen as Science’s 2015 Breakthrough of the Year. Cas9 is an enzyme able to target a specific position of a genome thanks to a small RNA molecule called guide RNA (gRNA). gRNAs are easy to design and can be delivered to cells along with the gene encoding Cas9, or as a pre-assembled Cas9-gRNA protein-RNA complex. Once inside the cell, Cas9 cuts the target DNA sequence homologous to the gRNAs, producing DSBs.

CRISPR/Cas9

The guide RNA (sgRNA) directs Cas9 to a specific region of the genome, where it induces a double-strand break in the DNA. On the left, the break is repaired by non-homologous-end joining, which can result in insertion/deletion (indel) mutations. On the right, the homologous-directed recombination pathway creates precise changes using a supplied template DNA. Credit: Ran et al. (2013). Nature Protocols.

 

Genome editing in crops

Together with the increased data availability on crop genomes, genome editing techniques such as CRISPR are allowing scientists to carry out ambitious research on crop plants directly, building on the knowledge obtained during decades of investigation in model plants.

The concept of CRISPR was first tested in crops by generating cultivars that are resistant to herbicides, as this is an easy trait to screen for and identify. One of the first genome-edited crops, a herbicide-resistant oilseed rape produced by Cibus, has already been grown and harvested in the USA in 2015.

Wheat powdery mildew

Researchers used CRISPR to engineer a wheat variety resistant to powdery mildew (shown here), a major disease of this crop. Image credit: NY State IPM Program. Used under license: CC BY 2.0.

 

Using CRISPR, scientists from the Chinese Academy of Sciences produced a wheat variety resistant to powdery mildew, one of the major diseases in wheat. Similarly, another Chinese research group exploited CRISPR to produce a rice line with enhanced rice blast resistance that will help to reduce the amount of fungicides used in rice farming. CRISPR/Cas9 has also been already applied to maize, tomato, potato, orange, lettuce, soybean and other legumes.

Genome editing could also revolutionize the management of viral plant disease. The CRISPR/Cas9 system was originally discovered in bacteria, where it provided them with molecular immunity against viruses, but it can also be moved into plants. Scientists can transform plants to produce the Cas9 and gRNAs that target viral DNA, reducing virus accumulation; alternatively, they can suppress those plant genes that are hijacked by the virus to mediate its own diffusion in the plants. Since most plants are defenseless against viruses and there are no chemical controls available for plant viruses, the main method to stop the spread of these diseases is still the destruction of the infected plant. For the first time in history, scientists have an effective weapon to fight back against plant viruses.

Cassava brown streak disease

The cassava brown streak disease virus can destroy cassava crops, threatening the food security of the 300 million people who rely on this crop in Africa. Image credit: Katie Tomlinson (for more on this topic, read her blog here).

 

Genome editing will be particularly useful in the genetic improvement of many crops that are propagated mainly by vegetative reproduction, and so very difficult to improve by traditional breeding methods involving crossing (e.g. cassava, banana, grape, potato). For example, using TALENs, scientists from Cellectis edited a potato line to minimize the accumulation of reducing sugars that may be converted into acrylamide (a possible carcinogen) during cooking.

 

Concerns about off-targets

One of the hypothesized risks of using CRISPR/Cas9 is the potential targeting of undesired DNA regions, called off-targets. It is possible to limit the potential for off-targets by designing very specific gRNAs, and all of the work published so far either did not detect any off-targets or, if detected, they occurred at a very low frequency. The number of off-target mutations produced by CRISPR/Cas9 is therefore minimal, especially if compared with the widely accepted random mutagenesis of crops used in plant breeding since the 1950s.

 

GM or not-GM

Genome editing is interesting from a regulatory point of view too. After obtaining the desired heritable mutation using CRISPR/Cas9, it is possible to remove the CRISPR/Cas9 integrated vectors from the genome using simple genetic segregation, leaving no trace of the genome modification other than the mutation itself. This means that some countries (including the USA, Canada, and Argentina) consider the products of genome editing on a case-by-case basis, ruling that a crop is non-GM when it contains gene combinations that could have been obtained through crossing or random mutation. Many other countries are yet to issue an official statement on CRISPR, however.

Recently, scientists showed that is possible to edit the genome of plants without adding any foreign DNA and without the need for bacteria- or virus-mediated plant transformation. Instead, a pre-assembled Cas9-gRNA ribonucleoprotein (RNP) is delivered to plant cells in vitro, which can edit the desired region of the genome before being rapidly degraded by the plant endogenous proteases and nucleases. This non-GM approach can also reduce the potential of off-target editing, because of the minimal time that the RNP is present inside the cell before being degraded. RNP-based genome editing has been already applied to tobacco plants, rice, and lettuce, as well as very recently to maize.

In conclusion, genome editing techniques, and CRISPR/Cas9 in particular, offers scientists and plant breeders a flexible and relatively easy approach to accelerate breeding practices in a wide variety of crop species, providing another tool that we can use to improve food security in the future.
For more on CRISPR, check out this recent TED Talk from Ellen Jorgensen:


About the author

Dr Damiano Martignago is a plant molecular biologist who graduated from Padua University, Italy, with a degree in Food Biotechnology in 2009. He obtained his PhD in Biology at Roma Tre University in 2014. His experience with CRISPR/Cas9 began in the lab of Prof. Fabio Fornara (University of Milan), where he used CRISPR/Cas9 to target photoperiod genes of interest in rice and generate mutants that were not previously available. He recently moved to Rothamsted Research, UK, where he works as Genome Editing Specialist, transferring CRISPR/Cas9 technology to hexaploid bread wheat with the aim of improving the efficiency of genome editing in this crop. He is actively involved with AIRIcerca (International Association of Italian Scientists), disseminating and promoting scientific news.

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Battening down the hatches: priming plant defense

This week’s blog post was written by Dr. Mike Roberts (Lancaster University, UK).

Dr. Mike Roberts

Dr. Mike Roberts, Lancaster University, UK, Mike with an experiment to test the effects of parental herbivory on defense priming in the next generation of Arabidopsis plants. (Photo credit: Lancaster University)

It is widely accepted that achieving agricultural sustainability means reducing our reliance on synthetic agrochemicals. One major group of agrochemicals is pesticides, which include the insecticides and fungicides that protect crop plants against pests and diseases. Pests and diseases aren’t going to go away, so reducing pesticide usage means that alternative crop protection approaches are needed. EU Directive 2009/128/EC (Sustainable Use of Pesticides) recommends the use of integrated pest and disease management (IPM) – the combined use of multiple approaches that together provide sufficient protection.

Our contribution to this challenge has been to identify ways in which we might enhance a plant’s own natural defense mechanisms. Plants have a wide array of structural and chemical defenses that they can employ to fight off enemies. Many of these are inducible, meaning that they are only activated in response to attack, which allows plants to balance the costs and benefits of defense. For crop plants, these costs can often translate into reduced yields. Spraying with compounds that switch on inducible defenses, such as the plant hormones jasmonic acid (JA) and salicylic acid (SA), can make plants more resistant, but this approach also risks unwanted growth reductions.

The JA team

(Left to right) Jane Taylor, Mike Roberts and Nigel Paul, who led the research on defense priming using seed treatments, in the glasshouse at the Lancaster Environment Center. (Photo credit: Lancaster University)

Fortunately, evolution has produced another way of regulating inducible defenses that we can take advantage of: the phenomenon we refer to as ‘priming’. When we ourselves get infected with something like a virus, our immune system generates antibodies to quickly fight it off, but it also produces memory cells that can respond to the same infection many months, or even years, in the future, with a more rapid and effective immune response. This is the basis of the familiar concept of vaccination. While plants don’t make antibodies, they are nevertheless able to alter future patterns of defense activation in response to previous infection by disease or feeding by herbivorous insects; thus, priming results in a faster and stronger activation of future inducible defense responses.

 

Benefits of plant defense priming

Transgenerational immune priming enhances disease resistance in Arabidopsis. All the plants in the photograph were inoculated by spraying the whole tray with a suspension of Pseudomonas syringae bacteria. The plants on the right side of the photo are from seed collected from parent plants that were infected with the same P. syringae bacteria, whilst those on the left come from healthy parents. (Photo credit: Belinda Ameyaw)

 

If we can find ways to prime defenses in crop plants, we might be able to improve pest and disease resistance with minimal impacts on yield. One way to do this is through seed treatments. We found that treating seeds with the defense hormone JA provides long-term enhanced resistance against herbivory and some fungal diseases, without affecting growth and development. We were able to patent this discovery, and the approach has since been successfully commercialized. The same approach can also be used to prime defenses against other forms of biotic and abiotic stress.

Seed treatment

Tomato seeds being treated with jasmonic acid solution. (Photo credit: Lancaster University)

How and why seed treatments provide long-term defense priming can be explained by the phenomenon of transgenerational immune priming, which my lab has also been investigating. After our success with the seed treatment, we wondered, “What if seeds were exposed to hormones like JA during the course of their development on the parent plant?” We tested this by infecting plants with bacteria or exposing them to herbivores, and then examined defense in their offspring. Remarkably, we saw that priming responses established in the parent were passed on to the offspring; something we refer to as transgenerational immune priming.

The evidence we have at present suggests that the mechanism for this heritable stress memory is epigenetic, meaning the genes that control priming are chemically tagged to alter their activity. These epigenetic modifications don’t involve changes in the DNA sequence and are reversible, allowing rapid, flexible responses to environmental stress. Understanding the nature of these epigenetic changes may provide another way to exploit priming for crop protection. Introducing the right epigenetic marks onto genes in elite crop varieties may enable the priming of defense without altering their genetic make-up. Given the difficulty of introducing new chemical and biological methods of crop protection, which require time-consuming and costly regulatory approval before they can be brought to market, this could prove an especially attractive option in the future.

Does Australia hold the key to food security?

This article is reposted from the Devex blog with kind permission from the author, Lisa Cornish.

CIAT research

Plant samples in the genebank at the International Center for Tropical Agriculture’s Genetic Resources Unit, at the institution’s headquarters in Colombia. Credit: Neil Palmer / CIAT. Used under license: CC BY-SA 2.0.

It was too dry in the Australian region of Wimmera to produce crops last summer. This year, floods are set to wipe out yields again. Like a number of other regions across the planet, climate change is starting to be felt.

“It’s like this every year somewhere,” said Sally Norton, head of the Australian Grains Genebank, which stores diverse genetic material for plant breeding and research.

For Norton and many of her colleagues in agricultural genetics, the picture is increasingly clear: The variety of crops used today are not able to withstand the changing conditions and changes expected in the future.

Australia’s biodiversity may offer some help, according to discussions at the recent International Genebank Managers Annual General Meeting held in Horsham, Victoria. The gathering, which brings together 11 countries, focused on how to better conserve seeds, build databases to manage collections, boost capacity across the world and fill gaps in genebanks.

Researchers are particularly interested in crop wilds, “the ancestors of our domesticated crops,” Marie Haga, executive director of the The Crop Trust, explained to Devex. Australia is one of the richest sources of these seeds. “It’s like the wolf being the ancestor to our domesticated dogs. Crop wild relatives have traits that we have lost in the domestication process — they might need less water, might live in unfriendly conditions, may be resistant to pests and diseases.”

As climate change continues to batter agricultural yields, crop wild relatives could provide resilience. The seeds give breeders and farmers new options of plant varieties with traits to withstand a variety of conditions based on the harsh climates they are found — drought, fire, flood, poor soil, high salinity.

For Haga, crop wild relatives are a solution for food security. “The challenge is that many of the varieties widely used in modern agriculture are very vulnerable, because we have been breeding on the same line and they are adapted to very specific environment,” Haga said. Varieties that flourish today, she said, could wither as the climate fluctuates.

“Utilization of the natural diversity of crops is key to the future,” she said. “The climate is rapidly changing and we need to feed a growing population with more nutritious food. It is very hard to see how we can do this unless we go back to the building blocks of agriculture.”

Norton agreed: “Crop wild relatives have an amazing adaptability to changing conditions,” she told Devex. “When we talk about food security, we are talking about getting varieties in farm paddocks that have greater resilience to extreme conditions. It may not be the highest yield, but you are going to get something from this crop.”

Why have they been overlooked?

Crop wild relatives have so far been underutilized in the research and breeding process of crops.

“We have this fabulous natural diversity out there including 125,000 varieties of wheat and 200,000 varieties of rice.” Haga said. “We have not at all unlocked the potential of these crops.”

One reason is a dearth of research. “Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives,” a 10-year project led by Haga to ensure long-term conservation of crop wild relatives, conducted a global survey of distribution and conservation and found that of 1,076 known wild relatives for 81 crops, more than 95 percent are insufficiently represented in genebanks and 29 percent are completely missing. They are missing purely due to the fact that they have yet to be collected.

“Genebank managers are generally open to include crop wild relatives in their collections.” Haga said. “It’s just quite simply that not enough work has been done in this area and the full potential is yet to be realized,” she said.

At the moment, seeds are being collected in 25 countries around the world as part of the crop wild relative project, but it is Australia that has been identified as one of the richest sources for crop wild relatives in the world. Because of the continent’s low population density and vast, undisturbed natural environment, a wide variety of species have been conserved, said Norton.

Australia holds significant diversity of wild relatives of rice, sorghum, pigeon pea, banana, sweet potato and eggplant currently missing from global collections, according to research by the Australian Seed Bank Partnership. Forty species have been prioritized for collection with high hopes that they will enable crops to withstand the harsh environmental conditions in which Australian species are found.

There are still many areas of Australia yet to be surveyed, and the full extent of its agricultural riches may yet to be tapped.

Australian researchers will play an important role in pre-breeding local species of wild relatives to improve their use in breeding programs. Crop wild relatives have historically been used in a variety of crops including synthetic wheat, but Australian native wild relatives have been harder to include in the breeding process.

“In the next 10 to 15 years it would be surprising if there is not something coming out that hasn’t got a component of Australian native wild relative in it,” Norton said who is currently involved in the collection of Australian crop wild relatives.

Collection of crop wild relatives is time sensitive

There is an urgency to collect crop wild relatives. Not only are wild species needed now to support changing environmental conditions affecting crops and farming, urbanization is putting crop wild relatives at risk of disappearing.

“We need to collect these sooner rather than later,” Norton told Devex. “Urbanization has a big impact on any native environment, let alone crop wild relatives. We know what species on our target list are more threatened than others — urbanization, flooding and fire are all risks to their security. We certainly have a priority list of species to collect and we need to make sure we target the ones that are under threat first.”

Once the varieties are conserved, breeders and farmers will need to be convinced to start using crop wild relatives. Many are already on board. “Most breeders understand these wild relatives have great potential,” Haga said.

Still, wild relatives can be difficult to work with and produce a lower yield. Haga expects there to be some reluctance, though limited.

“The understanding of the need is increasing and we feel very confident that this material will be used and some of them may be the game changer we are looking for,” she said.

The plans for crop wild relatives

Haga’s 10-year project on crop wild relatives is halfway complete. They are nearing the end of the collection phase and entering the pre-breeding process, before they are able to breed and deliver new species to farmers.

Australian support for the program includes an agreement for additional amount of $5 million. That comes on top of previous support of $21.2 million to the Crop Diversity Endowment Fund, which supports crop diversity globally and with a focus on the Indo-Pacific. Brazil, Chile, Germany, Japan, New Zealand, Norway, Switzerland and the United States are among other supporters of the endowment fund that hopes to reach $850 million. In Australia, further resources are still required to fund and support better seed collection at home.

Globally, plans for crop wild relatives includes raising greater awareness of their potential and importance.

“We have a big job to do to create awareness of the important of crop diversity generally and crop wild relatives specifically,” Haga said. “We have been speaking for years about biodiversity in birds and fish and a range of other animals, but we have talked very little about conserving the diversity of crops. I will fight for all types of diversity, but especially plants.”


 

This article is reposted from the Devex blog with kind permission from the author, Lisa Cornish.

Temperate matters in agriculture

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Most of the world’s food is produced in temperate zones. The Global Food Security program’s Evangelia Kougioumoutzi reports on the TempAg network.

Agricultural production in temperate regions is highly productive with a significant proportion of global output originating from temperate (i.e. non-tropical) countries – 21% of global meat production and 20% of global cereal production [link opens PDF] originate from Europe alone. This proportion is very likely to increase in light of climate change.

Temperature zones

Little fluffy clouds: temperate zones are well suited to agricultural production. Image credit: connect11/Thinkstock

TempAg is an international research collaboration network that was established to increase the impact of agricultural research and inform policy making in the world’s temperate regions. Its work does not solely focus on research, but also provides insights into current thinking through mapping existing scientific findings and outstanding knowledge gaps. In this way, the network aspires to become a platform for the alignment of national agricultural research and food partnership programs (such as Global Food Security) that will enable the development of more effective agricultural policies with a long-term vision.

Since its official inauguration in Paris in April 2015, TempAg has been leading a series of on-going workstreams around:

  • Boosting resilience of agricultural production systems at multiple scales and levels
  • Optimising land management for ecosystem services and food production
  • Improving sustainability of food productivity in the farms & enterprise level

You can read more about these themes on the TempAg website: http://tempag.net/themes/.

Future foresights

After 18 months of existence, TempAg held a foresight workshop in London on 5–7 October to determine its future priorities.

Forty delegates took part in the workshop, coming from the 14 different countries in the temperate region, and from academia, policy, industry, and professionals at the science–policy interface. Through a series of presentations and interactive sessions, participants were invited to consider what the current and future challenges are in temperate agriculture, taking into account the needs of policy makers and industry in helping them to improve sustainable agriculture practices.

 

Temperate zones

Temperate zones cover much of the world’s major food-growing areas. Image from Wikipedia/CIA-Factbook

 

To tackle sustainability in temperate agriculture, there is a need to better manage risks and stresses (both biotic and abiotic), as well as finding ways to manage the restoration of natural capital, ecosystem services, and soils. During the workshop, it was noted that utilizing the diversity within different agricultural systems, via identifying the best practice and using the appropriate technological mix, may be a way forward in making production systems more sustainable.

Participants stressed the importance of taking a holistic view of the sustainability agenda within agriculture, without just focusing on environmental aspects. This means also taking into consideration socioeconomic factors, such as making food value chains (like turning milk into cheese), more equitable by identifying who gets the equity from the food commodities’ prices, or identifying what the optimum legal framework for sharing data might be.

The group also considered sustainable agriculture issues from a policy and industry needs angle. It was interesting to see that dealing with shocks (environmental, socioeconomic, and technological) featured highly in this discussion as well. It was suggested that increasing resilience to these shocks could be facilitated via the widespread diffusion of existing technologies. Engaging with farmers during this time would be necessary to identify technology uptake barriers.

Forward moves

Future-proofing agricultural resilience and enhancing the capacity to respond to shocks was deemed an urgent priority, so the development of a comprehensive map identifying the multiple shocks that could impact on farm resilience in temperate zones might be a future workstream for TempAg. Work in this area could help develop models to assess the flexibility within agricultural production systems.

 

What we eat is largely based on the types of food we produce. Therefore, healthy diets are intrinsically linked with our production systems. Another area of interest for TempAg could be to explore what the nutritional value of crops should be for better health, and what a nutritional diet will look like for sustainable temperate agriculture. Developing frameworks in this area could further inform future farming practices in temperate areas.

Since TempAg’s initiation, two major global policy agendas have been adopted by the international community: the Sustainable Development Goals and the Paris COP21 agreement. Identifying what types of data and scientific evidence policy makers will need to achieve the agriculture-relevant targets was another area where TempAg could focus its activity moving forward.

Finally, delegates highlighted areas of work that could help to build more effective policies with a longer-term vision. These included developing economic tools for valuing natural capital and ecosystem services, as well as integrated assessment tools to monitor the performance and impact (environmental cost) of existing policies.

This article is cross-posted with the Global Food Security blog.


About Evangelia Kougioumoutzi

Evangelia is International Coordinator & Programme Manager for the Global Food Security program (GFS). Before joining GFS, Evangelia worked as an Innovation Manager for GFS partners BBSRC. She holds a PhD in plant development and genetics from the University of Oxford.

 

Professor Stefan Jansson on what makes a GMO, and the Scandinavian Plant Physiology Society

This week we speak to Professor Stefan Jansson, Umeå University, Sweden, who is the President of one of the Global Plant Council member organizations, the Scandinavian Plant Physiology Society (SPPS). He tells us more about his fascinating work, his prominent role in the GM debate, and his thoughts on the work of the SPPS and GPC, both now and in the future.

Stefan_Jansson

Could you tell us a little about your areas of research interest?

I have worked on (too) many things within plant science, but now I am focused on two subjects: “How do trees know that it is autumn?”, and “How can spruce needles stay green in the winter?” We use several approaches to answer these questions, including genetics, genomics, bioinformatics, biochemistry and biophysics.

 

Your ground-breaking work on CRISPR led to you being awarded the Forest Biotechnologist of the Year award by the Institute of Forest Biosciences. Could you tell us more about this work, and the role you have played in the GM debate?

In our work on photosynthetic light harvesting, we have generated and/or analyzed different lines lacking an important regulatory protein; PsbS. PsbS mutants resulting from treatment with radiation or chemical mutagens can be grown anywhere without restriction, but those that are genetically modified by the insertion of disrupting ‘T-DNA’ are, in reality, forbidden to be grown. For years, I, and many other scientists, have pointed out that it does not make sense for plants with the same properties to be treated so differently by legislators. In science we treat such plants as equivalents; when we publish our results we could be required to confirm that the correct gene was investigated by using an additional T-DNA gene knock-out line or an RNAi plant (RNA interference, where inserted RNA blocks the production of a particular protein), but in the legislation they and the ‘traditionally mutated’ plants are opposites.

This has been the situation for many years, but it has been impossible to change. To challenge this, we set up an experiment using a targeted gene-editing approach called CRISPR/Cas9 to make a deletion in the PsbS gene, which resulted in a plant with a non-functional PsbS gene but no residual T-DNA. We asked the Swedish competent authority if this would be treated as a GM plant or not, arguing that it is impossible to know if it is a ‘traditional’ deletion mutant or a gene-edited mutant. In the end, the authority said that, according to their interpretation of the law, this cannot be treated as a GMO.

If this interpretation is also used in other countries, plant breeders will have access to gene-editing techniques to aid them in their work to generate new varieties, which would otherwise not be a possibility. The reason we did this was to provide the authorities with a concrete case, and one which was not linked to a company or commercial crop but rather something that everyone would realize could only be important for basic science. Therefore most of the arguments that are used against GMOs could not be used, and this should be a step forward in the debate.

 

Check out Stefan’s fantastic TEDxUmeå talk to hear more on the GM debate:

spps_logoYou are the President of the Scandinavian Plant Physiology Society, one of the Global Plant Council member organizations. Could you briefly outline the work of the SPPS?

We support plant scientists – not only plant physiologists – in the Nordic countries, organize meetings, publish a journal (Physiologia Plantarum), etc.

 

What are the most important benefits that SPPS members receive?

This is an issue that we discuss a lot in the society right now. Only a limited fraction of Nordic plant scientists are members – obviously are the benefits not large enough – and this is something that we intend to change in the coming years. We think, for example, that we need to be a better platform for networking between researchers and research centers, and have a lot of ideas that we would like to implement.

 

How does the GPC benefit the SPPS?

Although there are country- and region-specific issues important for plant scientists, the biggest issues are global. The arguments why we need plant science are basically the same whether you are a plant scientist in Umeå or Ouagadougou, therefore we all benefit from a global plant organization.

 

What do you see as important roles for the future of the GPC, both for SPPS and the wider community?

This is quite clear to me: we will contribute to saving the planet.

 

What advice would you give to early career researchers in plant science?

Your curiosity is your biggest asset, so take good care of it.

 

Is there anything else you’d like to add?

The challenge for the GPC is clearly to get enough resources to be able to fulfil its very worthwhile ambitions. GPC has made a good start: the vision is clear and the roadmap is there, which are two prerequisites, but additional resources are needed to employ people to realize these ambitions, build upon current successes, and perform the important activities. It is easy to say that if we all contribute with a small fraction of our time that would be sufficient, but we all have may other obligations and commitments, and a few dedicated people are needed in all organizations.

Underutilized crops and insects replace fishmeal in aquaculture feed

Farmed fish are often fed with fishmeal, produced from the dried tissues of caught marine fish. In 2012, a total of 16.3 million metric tons of fish were caught to produce fishmeal and fish oil, 73% of which was used in aquaculture. This practice is unsustainable, and as the global human population is expected to rise to 9 over billion by 2050, capture fisheries will not be able to satisfy the demand for fish protein.

Barramundi

Barramundi fish

In recent decades there has been extensive research into ingredients to replace fishmeal, but this has focused mainly on sources of plant carbohydrate and protein such as maize and soy, which also serve as human foods. While these crops are now used in some commercial aquaculture feeds, they are not suitable for many species and have had less than optimal results. In addition, many countries do not grow these mainstream crops and are left in the undesirable position of having to import fishmeal alternatives, which can be cost prohibitive, and increase carbon emissions.

An alternative to fishmeal

Insect based feed

Insect based fish feed

The Crops for the Future (CFF) team in Malaysia is working with the University of Nottingham, UK, to investigate insect-based aquaculture feed as a replacement to fishmeal use in fisheries. Both organizations recognize that current rates of wild fish depletion are unsustainable and will not meet future demand for fishmeal under a ‘business as usual’ scenario. With support from the Newton-Ungku Omar Fund Institutional Linkages Programme, they have shown that the quality of insect larvae as an aquafeed ingredient is affected by the substrate on which the insects feed.

The CFF ‘FishPLUS’ program has revealed that black soldier fly (BSF; Hermetia illucens) larvae fed with underutilized crops can be used to produce insectmeal and replace up to 50% of fishmeal in formulated aquaculture. These crops are not used for human food and can be grown on marginal land close to areas of aquaculture production in tropical climates, increasing the sustainability of the process.

Producing insectmeal with underutilized crops

Ground Sesbiana

Ground Sesbania is used to feed the black soldier fly larvae

Over a year, the researchers worked with a private sector supplier to develop laboratory-scale BSF breeding pods in which different substrate combinations of underutilized crops could be trialed. BSF feeding trials were conducted using five separate or combined underutilized crops as substrate, i.e. Sesbania (Sesbania sp.); 90% Sesbania with 10% Moringa (Moringa oleifera); Bambara groundnut (Vigna subterranea) leaf; Bambara groundnut flour; and Moringa leaf.

The best results were obtained by feeding the larvae on Sesbiana, a nitrogen-fixing legume that grows well in marginal tropical landscapes and is not a human food crop. Overall, nutrient analyses indicated that the amino acid profile for insectmeal is encouraging and closely resembles fishmeal.

Successful feeding trials

Black soldier fly larvae

Black soldier fly larvae

Fish feeding trials using the BSF insectmeal were undertaken in Malaysia at the CFF Field Research Centre. The trial fish, barramundi, accepted a formulated feed with up to 50% replacement of fishmeal with Sesbania-fed BSF insectmeal. The feed conversion ratio, mortality rate and biomass growth rate were all comparable to control trials with commercial fishmeal aquaculture feed. Back in the UK, complementary antinutritional studies at the University of Nottingham contributed essential information to guide the development of an optimal aquaculture feed formulation in the future.

Waste not, want not

Amaranth alternative fertilizer

Amaranth growing with either commercial fertilizer (right) or FishPLUS substrate compost (left)

This project also embraces the use of undigested material from the insect feeding as compost for crops like okra and amaranth. For example, 10kg of Sesbania leaves produces 1kg of BSF pre-pupae and 9kg of undigested waste material. When used as a soil conditioner in our agronomy trial, this waste material improve the crop growth at a comparable level to commercial fertilizer. This could be used by terrestrial crop farmers to reduce their fertilizer bill.

The findings of this project are of importance to world food security. As leaders in this field of research, the UK and Malaysian partners are well placed to leverage these preliminary results and explore scalability and options for commercialization of benefit to both economies.


CFF is the world’s first and only organization dedicated to research on underutilized crops. Professor M.S. Swaminathan, World Food Prize Laureate and Father of the Asian Green Revolution, described CFF as `the need of the hour.’

You can see more about the FishPLUS project from Crops for the Future in the video below:



This article was written by FishPLUS Team, for Crops for the Future.

Newton-IUCAP workshop

Newton-IUCAP workshop

University_of_Nottingham CFFlogo

This work is supported by:

Funders links

Choosing your growth media for plant science

Considering its weedy nature, Arabidopsis thaliana is a fussy little plant. This can be a pain – even tiny environmental fluctuations can have significant impacts on the physiology and development that many of us are investigating.

As silly as it sounds, my labmates and I have spent many months debating the best compost media to use when growing Arabidopsis for research. It began when our trusted compost supplier changed the formula of its peat-based compost, which stressed our plants and turned them a lovely shade of purple! The conversation has continued to develop as we learn about the different media used in other laboratories.

A new paper from Drake et al. at my university (University of Bristol, UK) has added a new depth to the debate, so I thought I’d bring it all to your attention and perhaps receive some other suggestions to consider!

 

Peat-based vs non-peat compost

Arabidopsis growth media

Arabidopsis growth on peat-based and peat-free growth media. Drake et al., 2016.

The experiment, led by Dr Antony Dodd, was designed to test whether peat-based composts could be replaced by alternatives in Arabidopsis research, in an attempt to reduce plant science’s use of unsustainable peat extraction. The researchers grew two ecotypes of Arabidopsis (Col-0 and Ler) on both autoclaved and non-autoclaved composts, including peat-based compost and some formed of coir, composted bark, wood-fiber, and a domestic compost.

In terms of reducing peat use, Arabidopsis unfortunately grew best on the peat-based growing media, although some vegetative traits were comparable in some peat-free composts.

 

Autoclaving compost

This study caught my eye for another reason, however. We always sterilize our compost before growing Arabidopsis to reduce its contamination by fungi and insect pests; however, after learning that manganese toxicity can become a problem, we no longer autoclave it. As you can see in Boyd’s 1971 paper, manganese is converted to a more bioavailable form during the autoclave process, which can be toxic to plants.

Interestingly, Drake et al.’s research revealed no differences in Arabidopsis growth on autoclaved vs. non-autoclaved media, but I expect that in other environmental conditions the elevated manganese availability could become a problem. They did find that the autoclaved soil actually had more issues with mildew and algae, possibly because the natural microbiota had been killed and the compost was therefore easier to colonize.

 

Insecticide treatment

One of the biggest issues our lab has with non-autoclaved soil is the presence of small insects, which can predate our precious plants. A potential alternative to autoclaving is to treat the media with insecticide, such as imidacloprid, a neonicotinoid. However, many labs have stopped using these pesticides; in 2010, Ford et al. showed that several neonicotinoids, including imidacloprid, induce salicylate-associated plant defense responses associated with enhanced stress tolerance, while in 2012, Cheng et al. found 225 genes were differentially expressed in rice plants treated with imidacloprid. In experiments designed to measure precise physiological responses, I’m not convinced that it’s a good idea to use these pesticides!

 

Potential alternatives

To avoid using autoclaves and insecticides, you could consider baking compost overnight at 60°C (140°F) to try and kill fungal spores and insects, freezing the media, and/or using biocontrols to tackle insect pests, such as nematodes or mites.

In the peat vs. non-peat debate, it looks as though peat-based media are still the frontrunners in terms of compost, but hydroponic systems are becoming more popular as a way of tightly controlling nutrient regimes and manipulating whole plants more easily. Check out this video from Associate Professor Matthew Gilliham (University of Adelaide, Australia) to learn more about the technique:

If you have any other suggestions, please leave a comment and share your methods and ideas!

Brexit and agriculture

Professor Wyn Grant

Professor Wyn Grant

In June 2016, the UK Government will hold a public referendum for the people to decide whether or not Britain should exit the European Union. This contentious issue, popularly known as “Brexit”, has even divided the governing political party, with key parliamentary figures standing on either side of the debate.

There are many complex political issues for the UK to consider ahead of this referendum. One of these issues is: “what would be the consequences for UK agriculture if Britain were to leave the EU?” Professor Wyn Grant, a member of the Farmer–Scientist Network in the UK, tells us about a new report asking this very question.

Brexit and agriculture

by Wyn Grant

The Farmer–Scientist Network was set up by the Yorkshire Agricultural Society (UK) to facilitate practical cooperation between farmers and academics on the challenges facing agriculture. The Network felt there was a need to produce an assessment of the possible consequences of Brexit for agriculture. A working party was established, made up of leading experts on the EU’s Common Agricultural Policy and farmer members. I chaired this working party, and we produced what we hope is a comprehensive report, available here: http://yas.co.uk/charitable-activities/farmer-scientist-network/brexit.

Brexit ReportIn producing the Brexit report, one of our objectives was to provide information that farmers and others concerned with agriculture could use to question politicians during the referendum campaign. We also felt that agriculture and food had not been given sufficient attention during the negotiations and subsequent discussions. Should Brexit occur, our report draws attention to the issues that would have to be considered in exit negotiations.

The ins and outs of leaving

When evaluating the implications of Brexit for agriculture, we expected there would be complexities and uncertainties, but these were, in fact, greater than we anticipated. One reason for this is that, although the Lisbon Treaty on which the EU is founded makes provision for Member States to leave the EU under ‘Article 50’, none have ever done so before. It is difficult to know in advance how Britain’s exit would proceed, but it would almost certainly be necessary to use the entire two-year negotiating window provided for in the Treaty. Another complication is that the UK Government has not undertaken any formal contingency planning for exit, so it is difficult to know what a future domestic agricultural policy would look like.

In the event of Brexit taking place, the Farmer–Scientist Network feels that an optimal arrangement for the UK would be to establish a free trade area with the rest of the EU, with tariff-free access for UK farm products to the internal market. However, we don’t think the EU would want to give too generous a deal for fear of encouraging other member states to think about the benefits of exit.

Subsidies

Currently, two ‘pillars’ of financial subsidy are awarded to stakeholders in EU agriculture. We believe that the existing ‘Pillar 1’ subsidies that are given to EU farmers would be vulnerable after Brexit. This is an important issue, as for many farmers these subsidies make the difference between making a profit and running at a loss. Supporters of Brexit argue that the savings made from contributions to the EU budget would more than allow for subsidies to continue to be paid at the existing level. However, this overlooks the fact that the UK Treasury has for a long time targeted these subsidies as “market distorting”, and in the current climate of austerity in the UK, they could be at risk of being phased out as a means to reduce public expenditure.

We did, however, think that the ‘Pillar 2’ subsidies directed at agri-environmental and rural development objectives would be continued in some form. This is in part because they are embedded in contracts that continue beyond 2020, and because they have a coalition of domestic support from outside the industry from environmental and conservation lobbies.

Regulation

Some farmers resent what they see as excessive regulation emanating from Brussels. However, we think it is unlikely that many of these controls would be dropped or relaxed following Brexit. There are good reasons for regulations covering such areas as water pollution, pesticide use and animal welfare that have nothing to do with membership of the EU. Domestic support for such regulations would continue from environmental, conservation, public health, animal welfare and consumer organisations.

Some farmers hope that plant protection products that have been banned under EU regulations could be used after Brexit. However, there would still be domestic pressure to regulate these products and manufacturers might be unwilling to produce them just for the UK market.

Negotiation and trade

The UK at present negotiates in the World Trade Organisation (WTO) as a part of an EU bloc which provides additional leverage against powerful countries such as the United States. The agreements that the EU has with ‘third’ countries (those outside of Europe) would have to be renegotiated on a single country basis. Supporters of Brexit are confident that this task could be completed within two years. However, given that the UK has relied on the negotiating resources of the European Commission, it does not have many international trade diplomats and the process could take considerably longer.

Migrant labour

The horticulture industry in the UK is substantially dependent on migrant labour from elsewhere in the EU. This could not easily be replaced with domestic labour. It would be necessary to try and negotiate a new version of the Seasonal Agricultural Workers Scheme (SAWS) – a scheme (redundant since 2013) that was established to allow migrant workers from certain countries outside of the EU to work in UK agriculture – to ensure that the sector would have the labour it needs to function.

Conclusions

Being part of a larger political community gives British farmers some political cover from countries where farming makes up a large share of GDP or has strong cultural roots. The Farmer–Scientist Network concluded that it was difficult to see Brexit as beneficial to UK agriculture. However, we also emphasised that there are broader considerations about UK membership that needed to be weighed in any voting decision.

 

The Secrets of Seagrass

Zosteramarina

Zostera marina. Public domain, via Wikimedia Commons.

It’s the ancient story of plant evolution: photosynthetic algae moved to damp places on land, eventually evolving more complex architecture, and spreading across almost all terrestrial habitats. To cope with the drier conditions, plants developed roots to absorb water, and vascular tissue to transport it; a waxy cuticle coating their surfaces to prevent evaporation; and microscopic pores called stomata that open to allow carbon dioxide to diffuse in for photosynthesis but close to prevent excessive water loss.

How, then, does eelgrass (Zostera marina) fit in to this tale? It’s a monocot descended from the flowering plants, but it has turned its back on dry land and returned to the sea; a rare feat that only appears to have happened on three occasions. The recent sequencing of the eelgrass genome has revealed several interesting insights into the dramatic genetic changes that have allowed it to adapt to what lead author Professor Jeanine Olsen described as, “arguably the most extreme adaptation a terrestrial (and even a freshwater) species can undergo.”

Sayonara to stomata

If you live in the sea, conserving water isn’t your main concern. Eelgrass was known to lack stomata, but genetic comparisons to other species, including its freshwater relative Spirodela polyrhiza, revealed the first surprise of the study: eelgrass has lost not only its stomata but also the genes involved in their development and patterning. “The genes have just gone, so there’s no way back to land for seagrass,” said Olsen.

A difference in defense

When angiosperms are attacked by herbivores or pathogens, their defense response typically involves the release of volatile secondary metabolites through their stomata. How can eelgrass release these compounds without stomata? The answer is: it doesn’t. The genome study found that eelgrass is missing crucial genes involved in making ethylene (an important hormone release in times of stress), as well as those responsible for producing non-metabolic terpenoids, which act to repel pests.

Selective pressures of the marine environment differ greatly from those of terrestrial habitats, so different pathways may be involved. Second, eelgrass has a wide repertoire of pathogen resistance genes, which suggests that it is exposed to a very different set of pathogens that may not respond to typical immune responses. Third, volatile secondary metabolites are often involved in attracting pollinators; this is not believed to be necessary in eelgrass, where submarine pollination occurs using the water itself.

Zostera marina. Public domain, CC0 1.0.

Zostera marina – National Museum of Nature and Science, Tokyo. Public domain, CC0 1.0, via WikiMedia Commons.

Changing the cell wall

Eelgrass is subject to extremely salty conditions, and it’s had to adapt to osmotic stress. Unlike typical plant cell walls, eelgrass has engineered its cell wall matrix to retain water in the cell wall, even during low tide. This involves depositing sulfated polysaccharides and low methylated pectins in the cell wall matrix, but until its genome was sequenced no-one knew exactly how. It turns out that eelgrass has rearranged its metabolic pathways: “They have re-engineered themselves,” Olsen explains.

Living with a lack of light

Some species of Zostera can grow in water 50m deep, where light levels are reduced and shifted into a narrow wavelength range; ultraviolet (UV), red and far-red light have particularly low penetration after the first 1–2m of seawater. In a classic eelgrass ‘use it or lose it’ response, it has lost the UVR8 gene, which is responsible for sensing and responding to UV damage, as well as the phytochromes associated with red and far-red receptors. It does, however, retain the photosynthetic machinery, including photosystems I and II.

Unravelling angiosperm evolution

The recent eelgrass publication has revealed how this plant has either lost or adapted typical angiosperm traits to suit its needs, by ditching its stomata, volatile secondary metabolites and certain light sensing genes, or by altering the structure and function of the cell wall. It also developed adaptations that enable gas exchange, help pollen stick to submerged stigmas, and promote nutrient uptake.

Could these adaptations be useful in crop breeding? While a lack of defense compounds would probably be a step backwards, it would be extremely useful to understand how eelgrass copes with biotic stresses without them. Removing light receptors would also be problematic, but could eelgrass help us to develop crops that can grow in shaded conditions, perhaps in intercropping systems? What can we learn from eelgrass’ nutrient uptake and salt-tolerant adaptations?

Now that we have seen some of the secrets of eelgrass, how can we best make use of them?

 

Read the paper: The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea (Open Access)

Read the editorial: Genomics: From sea to sea (paywall)

Read the press release: Genome of the flowering plant that returned to the sea