Experts’ interest in utilizing gene editing for the breeding crops has seen revolutionary growth. Meanwhile, people’s awareness for food safety has also been increasing.
According to a study, participants who had expert knowledge of molecular biology perceived emerging technologies to offer the lowest risk and highest benefits or value for food application, while lay public showed the highest risk and lowest benefit.
A research team recently developed new methods that will make it significantly faster to produce gene-edited plants. They hope to alleviate a long-standing bottleneck in gene editing and, in the process, make it easier and faster to develop and test new crop varieties with two new approaches.
At July’s New Breeding Technologies workshop held in Gothenburg, Sweden, Dr. Staffan Eklöf, Swedish Board of Agriculture, gave us an insight into their analysis of European Union (EU) regulations, which led to their interpretation that some gene-edited plants are not regulated as genetically modified organisms. We speak to him here on the blog to share the story with you.
Could you begin with a brief explanation of your job, and the role of the Competent Authority for GM Plants / Swedish Board of Agriculture?
I am an administrative officer at the Swedish Board of Agriculture (SBA). The SBA is the Swedish Competent authority for most GM plants and ensures that EU regulations and national laws regarding these plants are followed. This includes issuing permits.
You reached a key decision on the regulation of some types of CRISPR-Cas9 gene-edited plants. Before we get to that, could you start by explaining what led your team to start working on this issue?
It started when we received questions from two universities about whether they needed to apply for permission to undertake field trials with some plant lines modified using CRISPR/Cas9. The underlying question was whether these plants are included in the gene technology directive or not. According to the Swedish service obligation for authorities, the SBA had to deliver an answer, and thus had to interpret the directive on this point.
Could you give a brief overview of Sweden’s analysis of the current EU regulations that led to your interpretation that some CRISPR-Cas9 gene-edited plants are not covered by this legislation?
The following simplification describes our interpretation pretty well; if there is foreign DNA in the plants in question, they are regulated. If not, they are not regulated.
Our interpretation touches on issues such as what is a mutation and what is a hybrid nucleic acid. The first issue is currently under analysis in the European Court of Justice. Other ongoing initiatives in the EU may also change the interpretations we made in the future, as the directive is common for all member states in the EU.
CRISPR-Cas9 is a powerful tool that can result in plants with no trace of transgenic material, so it is impossible to tell whether a particular mutation is natural. How did this influence your interpretation?
We based our interpretation on the legal text. The fact that one cannot tell if a plant without foreign DNA is the progeny of a plant that carried foreign DNA or the result of natural mutation strengthened the position that foreign DNA in previous generations should not be an issue. It is the plant in question that should be the matter for analysis.
Does your interpretation apply to all plants generated using CRISPR-Cas9, or a subset of them?
It applies to a subset of these gene-edited plants. CRISPR/Cas9 is a tool that can be used in many different ways. Plants carrying foreign DNA are still regulated, according to our interpretation.
What does your interpretation mean for researchers working on CRISPR-Cas9, or farmers who would like to grow gene-edited crops in Sweden?
It is important to note that, with this interpretation, we don’t remove the responsibility of Swedish users to assess whether or not their specific plants are included in the EU directive. We can only tell them how we interpret the directive and what we request from the users in Sweden. Eventually I think there will be EU-wide guidelines on this matter. I should add that our interpretation is also limited to the types of CRISPR-modified plants described in the letters from the two universities.
We are currently waiting for the EU to declare whether CRISPR-Cas9 gene-edited plants will be regulated in Europe. Have policymakers in other European countries been in contact with you regarding Sweden’s decision process?
Yes, there is a clear interest; for example, Finland handled a very similar case. Other European colleagues have also shown an interest.
What message would you like plant scientists to take away from this interview? If you could help them to better understand one aspect of policymaking, what would it be?
Our interpretation is just an interpretation and as such, it is limited and can change as a result of what happens; for example, what does not require permission today may do tomorrow. Bear this in mind when planning your research and if you are unsure, it is better to ask. Moreover, even if the SBA (or your country’s equivalent) can’t request any information about the cultivation of plants that are not regulated, it is good to keep us informed.
I think it is vital that legislation meets reality for any subject. It is therefore good that pioneers drive us to deal with difficult questions.
Every morning I slice a banana onto my breakfast cereal.
And I am not alone.
Every person in the UK eats, on average, 100 bananas per year.
Bananas are rich in fiber, vitamins, and minerals like potassium and magnesium. Their high carbohydrate and potassium content makes them a favorite snack for professional sports players; the sugar provides energy and the potassium protects the players from muscle fatigue. Every year, around 5000 kg of bananas are consumed by tennis players at Wimbledon.
But bananas are not only delicious snacks and handy energy kicks. For around 100 million people in Sub-Saharan Africa, bananas are staple crops vital for food security. Staple crops are those foods that constitute the dominant portion of a standard diet and supply the major daily calorie intake. In the UK, the staple crop is wheat. We eat wheat-based products for breakfast (toast, cereals), lunch (sandwich), and dinner (pasta, pizza, beer).
In Uganda, bananas are staple crops. Every Ugandan eats 240 kg bananas per year. That is around 7–8 bananas per day. Ugandans do not only eat the sweet dessert banana that we know; in the East African countries such as Kenya, Burundi, Rwanda, and Uganda, the East African Highland banana, called Matooke, is the preferred banana for cooking. Highland bananas are large and starchy, and are harvested green. They can be cooked, fried, boiled, or even brewed into beer, so have very similar uses wheat in the UK.
In West Africa and many Middle and South American countries, another cooking banana, the plantain, is cooked and fried as a staple crop.
In terms of production, the sweet dessert banana we buy in supermarkets is still the most popular. This banana variety is called Cavendish and makes up 47% of the world’s banana production, followed by Highland bananas (24%) and plantains (17%). Last year, I visited Uganda and I managed to combine the top three banana cultivars in one dish: cooked and mashed Matooke, a fried plantain and a local sweet dessert banana!
Three types of banana in a single dish in Uganda.
Another important banana cultivar is the sweet dessert banana cultivar Gros Michel, which constitutes 12% of the global production. Gros Michel used to be the most popular banana cultivar worldwide until an epidemic of Fusarium wilt disease devastated the banana export plantations in the so-called “banana republics” in Middle America (Panama, Honduras, Guatemala, Costa Rica) in the 1950s.
Fusarium wilt disease is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (FOC). The fungus infects the roots of the banana plants and grows up through the water-conducting, vascular system of the plant. Eventually, this blocks the water transport of the plant and the banana plants start wilting before they can set fruits.
Fusarium Wilt symptoms
The Fusarium wilt epidemic in Middle America marked the rise of the Cavendish, the only cultivar that could be grown on soils infested with FOC. The fact that they are also the highest yielding banana cultivar quickly made Cavendish the most popular banana variety, both for export and for local consumption.
Currently, Fusarium wilt is once again the biggest threat to worldwide banana production. In the 1990s, a new race of Fusarium wilt – called Tropical Race 4 (TR4) – occurred in Cavendish plantations in Indonesia and Malaysia. Since then, TR4 has spread to the neighboring countries (Taiwan, the Philippines, China, and Australia), but also to distant locations such as Pakistan, Oman, Jordan, and Mozambique.
Current presence of Fusarium wilt Tropical Race 4. Affected countries are colored in red.
In Mozambique, the losses incurred by TR4 amounted to USD 7.5 million within just two years. Other countries suffer even more; TR4 causes annual economic losses of around USD 14 million in Malaysia, USD 121 million in Indonesia, and in Taiwan the annual losses amount to a whopping USD 253 million.
TR4 is not only diminishing harvests. It also raises the price of production, because producers have to implement expensive preventative measures and treatments of affected plantations. These preventive measures and treatments are part of the discussion at The World Banana Forum (WBF). The WBF is a permanent platform for all stakeholders of the banana supply chain, and is housed by the United Nation’s Food and Agricultural Organization (FAO). In December 2013, the WBF created a special taskforce to deal with the threat posed by TR4.
Despite its massive impact on banana production, we know very little about the pathogen that is causing Fusarium wilt disease. We don’t know how it spreads, why the new TR4 is so aggressive, or how we can stop it.
Fusarium Wilt symptoms in the discolored banana corm.
Breeding bananas is incredibly tedious, because edible cultivars are sterile and do not produce seeds. I am therefore exploring other ways to engineer resistance in banana against Fusarium wilt. As a scientist in the 2Blades group at The Sainsbury Laboratory, I am investigating how we can transfer resistance genes from other crop species into banana and, more recently, I have been investigating bacteria that are able to inhibit the growth and sporulation of F. oxysporum. These biologicals would be a fast and cost-effective way of preventing or even curing Fusarium wilt disease.
[RIO DE JANEIRO] A genetically modified (GM) cane variety that can kill the sugarcane borer (Diatraea saccharalis) has been approved in Brazil, to the delight of some scientists and the dismay of others, who say it may threaten Brazilian biodiversity.
Brazil is the second country, after Indonesia, to approve the commercial cultivation of GM sugarcane. The approval was announced by the Brazilian National Biosafety Technical Commission (CTNBio) on June 8.
Sugarcane borer is one of the main pests of the sugarcane fields of South-Central Brazil, causing losses of approximately US$1.5 billion per year.
“Breeding programmes could not produce plants resistant to this pest, and the existing chemical controls are both not effective and severely damaging to the environment,” says Adriana Hemerly, a professor at the Federal University of Rio de Janeiro, in an interview with SciDev.Net.
“Studies conducted outside Brazil prove that protein from genetically modified organisms harms non-target insects, soil fauna and microorganisms.”
“Therefore, the [GM variety] is a biotechnological tool that helps solve a problem that other technologies could not, and its commercial application will certainly have a positive impact on the productivity of sugarcane in the country.”
Jesus Aparecido Ferro, a member of CTNBio and professor at the Paulista Júlio de Mesquita Filho State University, believes the move followed a thorough debate that began in December 2015 — that was when the Canavieira Technology Center (Sugarcane Research Center) asked for approval to commercially cultivate the GM sugarcane variety.
“The data does not provide evidence that the cane variety has a potential to harm the environment or human or animal health,” Ferro told SciDev.Net.
To develop the variety, scientists inserted the gene for a toxin [Cry] from the bacterium Bacillus thuringiensis (Bt) into the sugarcane genome, so it could produce its own insecticide against some insects’ larvae.
This is a technology that “has been in use for 20 years and is very safe”, says Aníbal Eugênio Vercesi, another member of the CTNBio, and a professor at the State University of Campinas.
But Valério De Patta Pillar, also a member of the CTNBio and a professor at the Federal University of Rio Grande do Sul, points to deficiencies in environmental risk assessment studies for the GM variety — and the absence of assessments of how consuming it might affect humans and animals.
According to Pillar, there is a lack of data about the frequency with which it breeds with wild varieties. Data is also missing on issues such as the techniques used to create the GM variety and the effects of its widespread use.
Rogério Magalhães, an environmental analyst at Brazil’s Ministry of the Environment, also expressed concern about the approval of the commercial transgenic cane.
“I understand that studies related to the impacts that genetically modified sugarcane might have on Brazilian biodiversity were not done by the company that owns the technology,” said Magalhães in an interview with SciDev.Net. This is very important because Brazil’s climate, species, and soils differ from locations where studies might have taken place, he explained.
Among the risks that Magalhães identified is contamination of the GM variety’s wild relatives. “The wild relative, when contaminated with transgenic sugarcane, will have a competitive advantage over other uncontaminated individuals, as it will exhibit resistance to insect-plague that others will not have,” he explained.
Another risk that Magalhães warns about is damage to biodiversity. “Studies conducted outside Brazil prove that Cry protein from genetically modified organisms harms non-target insects, soil fauna and microorganisms.”
Magalhães added that some pests have already developed resistance to the Bt Cry protein, prompting farmers to apply agrochemicals that are harmful to the environment and human health.
A man stacks sugarcane at the Ver-o-Peso (Check the Weight) market in Belem.
Currently, it is common for producers to raise sucrose levels in sugar cane by applying artificial growth regulators or chemical ripeners. This inhibits flowering, which in turn prolongs harvest and milling periods.
One of these growth regulators, ethephon, is used to manage agricultural, horticultural and forestry crops around the world. It is widely used to manipulate and stimulate the maturation of sugarcane as it contains ethylene, which is released to the plant on spraying.
Ethylene, considered a ripening hormone in plants, contributes to increasing the storage of sucrose in sugar cane.
“Although we knew ethylene helps increase the amount of sugar in the cane, it was not clear how the synthesis and action of this hormone affected the maturation of the plant,” said Marcelo Menossi, professor at the University of Campinas (Unicamp) and coordinator of the project, which is supported by the Brazilian research foundation FAPESP.
To study how ethylene acts on sugarcane, the researchers sprayed ethephon and an ethylene inhibitor, aminoethoxyvinylglycine (AVG), on sugar cane before it began to mature.
After spraying both compounds, they quantified sucrose levels in tissue samples from the leaves and stem of the cane. They did this five days after application and again 32 days later, on harvest.
Those plants treated with the ethephon ripener had 60 per cent more sucrose in the upper and middle internodes at the time of harvest, while the plants treated with the AVG inhibitor had a sucrose content that was lower by 42 per cent.
The researchers were then able to identify genes that respond to the action of ethylene during ripening of the sugar cane. They also successfully identified the genes involved in regulating sucrose metabolism, as well as how the hormone acts on sucrose accumulation sites in the plant.
Based on the findings, the team has proposed a molecular model of how ethylene interacts with other hormones.
“Knowing which genes or ripeners make it possible for the plant to increase the accumulation of sucrose will allow us to make genetic improvements in sugarcane and develop varieties that over-express these genes, without the need to apply ethylene, for example,” explained Menossi.
This research could also help with spotting the most productive sugar cane, as some varieties that do not respond well to hormones, he added. “It will be possible to identify those [varieties] that best express these genes and facilitate the ripening action.”
This week we spoke to Dr. Anil Day, a synthetic biologist at the University of Manchester who has developed an impressive array of tools and techniques to transform chloroplast genomes.
Could you begin by giving our readers a brief overview of synthetic biology?
Synthetic biology involves the application of engineering principles to biological systems. One approach to understanding a biological system is to break it down into smaller parts, which can be used to design new properties. These redesigned pieces can be reassembled into a new system, tested experimentally, and refined in an iterative process. Synthetic biology projects that are underway in our lab include designing plastids such as chloroplasts with new metabolic functions, and in the longer term the design and assembly of synthetic chloroplast genomes.
Dr. Anil Day examines a cabinet of transformed plants. Credit: Dr. Anil Day.
Why do you use chloroplasts for synthetic biology systems?
Chloroplasts have a relatively small genome, coding for about 100 genes. Importantly, exogenous (foreign) genes coding for new functions can be precisely introduced into the chloroplast genome. All of the plastids within a plant contain the same genome so, once established, the user-designed reprogrammed plastids will be present throughout the plant. Chloroplasts can also produce very high levels of protein; researchers have achieved expression levels where over 70% of the total soluble protein in the leaves is the engineered protein. Expression in tomato fruit is also possible.
Multiple genes can be introduced into chloroplasts and expressed coordinately, allowing the metabolic engineering of more complex processes. The upper size limit for insertions is not known but is likely to be above the 50,000 nucleotide insertion achieved to date. Furthermore, chloroplasts and other plastids are important metabolic hubs and contain a wide variety of chemical substrates useful for metabolic engineering.
Plants have several types of plastids, including green photosynthetic chloroplasts, pigment-containing chromoplasts, and starch-containing amyloplasts. Credit: Dr. Anil Day.
Could you describe the current state of our ability to engineer chloroplasts?
Chloroplast engineering is routine in many labs around the globe. Although there are multiple chloroplasts in every cell, the process of converting all the chloroplasts to a single population of engineered genomes is not an issue. Most researchers use the tobacco plant because it is easily transformed, but other crops are amenable to transformation, including oilseed rape, soybean, tomato, and potato (cereals such as rice and wheat are more problematic). There has been progress with developing the inducible expression of exogenous genes in chloroplasts too.
What challenges/differences do you face when transforming chloroplast genomes when compared to the nuclear genome?
Typical genetic modification of the DNA in the nucleus is performed by introducing exogenous genes in T-DNA. T-DNA is transferred to the plant using the bacterium Agrobacterium tumefaciens, which is an efficient process, but the T-DNA integrates ‘randomly’ at many sites within chromosomes and different lines can have variable expression levels due to positional effects and gene silencing.
A. tumefaciens-mediated gene delivery systems do not work for chloroplast transformation. Most chloroplast transformation labs introduce genes into plastids by blasting cells with gold or tungsten particles coated with DNA. Because chloroplast genomes are present in multiple copies per cell, the process of converting all resident chloroplasts to the transgenic genome requires a continued period of selection. This means that the isolation of chloroplast transformants can take slightly longer than nuclear transformation. In our lab, we speed up this process by using restoration of photosynthesis to select chloroplasts with exogenous genes. Once plants with a uniform population of transgenic plastid genomes have been isolated, the transgenes are stable and inherited through the maternal line.
For the novice, I would say nuclear transformation using A. tumefaciens is easier to accomplish than chloroplast transformation.
A tobacco plant containing leaf areas with edited (pale green) and normal (darker green) chloroplasts. Credit: Dr. Anil Day.
Chloroplasts are inherited from the female parent in wheat. This is useful because it restricts the pollen-mediated spread of chloroplast-localized transgenes into the environment. Previously, no-one had studied the mechanism of maternal chloroplast inheritance in wheat using modern cell biology tools. With our collaborators Lucia Primavesi, Huixia Wu, and Huw Jones at Rothamsted Research, we developed an efficient method to observe small non-green plastids in wheat pollen in real time. We found that the plastids were destroyed during the maturation of sperm cells, which explained the absence of paternal plastids in the offspring.
This discovery has applications in crop breeding. Anther culture is a powerful technique where new homozygous plants can be produced by doubling the chromosome numbers of haploid plants regenerated from pollen. This technique has been challenging in cereals, as chloroplast degradation in pollen leads to a high percentage of albino plants (in some cases 100% albinos). Understanding how to prevent the destruction of plastids in pollen sperm cells will improve this technique in cereals, which could speed up crop breeding in the future.
Transformed plantlets are selected by their ability to survive on a herbicide-containing agar plate, and can then be grown up into mature plants. Credit: Dr. Anil Day.
What sorts of processes have you successfully transformed into chloroplasts, and what kinds of results have you achieved?
We have expressed a variety of exogenous genes in chloroplasts, from those conferring resistance to herbicides to vaccine epitopes and pharmaceutical proteins:
Plants expressing the bar gene in chloroplasts were resistant to the herbicide glufosinate (also known as phosphinothricin).
A chloroplast-expressed viral epitope was used to identify samples of human blood infected with the hepatitis C virus.
Human transforming growth factor 3 (hTGFβ3), a potential wound healing drug, accumulated to high concentrations in chloroplasts, and could be processed to a pure active form resembling clinical grade hTGFβ3.
In collaboration with Ray Dixon, Cheng Qi, and Mandy Dowson-Day at the John Innes Centre, we investigated the feasibility of introducing nitrogen-fixing genes into chloroplasts. This work was initiated in a unicellular green alga with the bacterial nifH gene.
What is the cutting edge of chloroplast transformation research?
Chloroplast genes are important for plant growth and development but they are difficult to improve by conventional breeding methods. We recently developed a method to edit plastid genomes, which allows beneficial single point mutations to be introduced into chloroplast genes. This is important because the resulting plants have an identical genome to the original cultivar apart the single base substitution, potentially leading to a new class of biotech crop.