Genetics to boost sugarcane production

Scientists in Brazil are taking steps towards genetically modifying sugar cane so it produces more sucrose naturally, looking to eventually boost the productivity and economic benefits of the tropical grass.

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.
sucrose accumulation.jpg

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.”

Taken from a newsletter by FAPESP, a SciDev.Net donor, edited by our Latin America and the Caribbean desk

 

This article was originally published on SciDev.Net. Read the original article.

Striga hermonthica – a beautiful but devastating plant…

This week’s post was written by Caroline Wood, a PhD candidate at the University of Sheffield.

When it comes to crop diseases, insects, viruses, and fungi may get the media limelight but in certain regions it is actually other plants which are a farmer’s greatest enemy. In sub-Saharan Africa, one weed in particular – Striga hermonthica – is an almost unstoppable scourge and one of the main limiting factors for food security.

Striga is a parasitic plant; it attaches to and feeds off a host plant. For most of us, parasitic plants are simply harmless curiosities. Over 4,000 plants are known to have adopted a parasitic mode of life, including the seasonal favorite mistletoe (a stem parasite of conifers) and Rafflesia arnoldii, nicknamed the “corpse flower” for its huge, smelly blooms. Although the latter produces the world’s largest flower, it has no true roots – only thread-like structures that infect tropical vines.

When parasitic plants infect food crops, they can turn very nasty indeed. Striga hermonthica is particularly notorious because it infects almost every cereal crop, including rice, maize, and sorghum. Striga is a hemiparasite, meaning that it mainly withdraws water from the host (parasitic plants can also be holoparasites, which withdraw both water and carbon sugars from the host). However, Striga also causes a severe stunting effect on the host crop (see Figure 1), reducing their  yield to practically nothing. Little wonder then, that the common name for Striga is ‘witchweed’.

Striga-infected sorghum

Figure 1: Striga-infected sorghum. Note the withered, shrunken appearance of the infected plants. Image credit: Joel Ransom.

 

Several features of the Striga lifecycle make it especially difficult to control. The seeds can remain dormant for decades and only germinate in response to signals produced by the host root (called strigolactones) (Figure 2). Once farmland becomes infested with Striga seed, it becomes virtually useless for crop production. Germination and attachment takes place underground, so the farmer can’t tell if the land is infected until the parasite sends up shoots (with ironically beautiful purple flowers). Some chemical treatments can be effective but these remain too expensive for the subsistence farmers who are mostly affected by the weed. Many resort to simply pulling the shoots out as they appear; a time-consuming and labor-intensive process. It is estimated that Striga spp. cause crop losses of around US $10 billion each year [1].

Certain crop cultivars and their wild relatives show natural resistance to Striga. Here at the University of Sheffield, our lab group (headed by Professor Julie Scholes) is working to identify resistance genes in rice and maize, with the eventual aim of breeding these into high-yielding cultivars. To do this, we grow the host plants in rhizotrons (root observation chambers) which allow us to observe the process of Striga attachment and infection (see Figure 3). Already this has been successful in identifying rice cultivars that have broad-spectrum resistance to Striga, and which are now being used by farmers across Africa.

 

Life cycle of Striga

Figure 2: Life cycle of Striga spp. A single plant produces up to 100,000 seeds, which can remain viable in the soil for 20 years. Following a warm, moist conditioning phase, parasite seeds become responsive to chemical cues produced by the roots of suitable hosts, which cause them to germinate and attach to the host root. The parasite then develops a haustorium: an absorptive organ which penetrates the root and connects to the xylem vessels in the host’s vascular system. This fuels the development of the Striga shoots, which eventually emerge above ground and flower. Figure from [2].

 

But many fundamental aspects of the infection process remain almost a complete mystery, particularly how the parasite overcomes the host’s intrinsic defense systems. It is possible that Striga deliberately triggers certain host signaling pathways; a strategy used by other root pathogens such as the fungus Fusarium oxysporum. This is the focus of my project: to identify the key defense pathways that determine the level of host resistance to Striga. It would be very difficult to investigate this in crop plants, which typically have incredibly large genomes, so my model organism is Arabidopsis thaliana, the workhorse of the plant science world, whose genome has been fully sequenced and mapped. Arabidopsis cannot be infected by Striga hermonthica but it is susceptible to the related species, Striga gesnerioides, which normally infects cowpea.  I am currently working through a range of different Arabidopsis mutants, each affected in a certain defense pathway, to test whether these have an altered resistance to the parasite.  Once I have an idea of which plant defense hormones may be involved (such as salicylic acid or jasmonic acid), I plant to test the expression of candidate genes to decipher what is happening at the molecular level.

Striga-infected Arabidopsis

Figure 3: One of my Arabidopsis plants growing in a rhizotron. Preconditioned Striga seeds were applied to the roots three weeks ago with a paintbrush. Those that successfully attached and infected the host have now developed into haustoria. The number of haustoria indicates the level of resistance in the host. Image credit: Caroline Wood.

 

It’s early days yet, but I am excited by the prospect of shedding light on how these devastating weeds are so effective in breaking into their hosts. Ultimately this could lead to new ways of ‘priming’ host plants so that they are armed and ready when Striga attacks. It’s an ambitious challenge, and one that will certainly keep me going for the remaining two years of my PhD!

 

You can follow my journey by reading my blog and keeping up with me on Twitter (@sciencedestiny).

 

References:

[1] Westwood, J. H. et al. (2010). The evolution of parasitism in plants. Trends in Plant Science, 15(4): 227-235.

[2] Scholes, J. D. and Press, M. C. (2008). Striga infestation of cereal crops – an unsolved problem in resource limited agriculture. Current Opinion in Plant Biology, 11(2): 180-186.

Just add water: Could resurrection plants help feed the world?

This week we spoke to Professor Henk Hilhorst (Wageningen University and Research) about his research on desiccation tolerance in seeds and plants.

 

Could you begin by telling us a little about your research?

I am a plant physiologist specializing in seed biology. I have a long research record on various aspects of seeds, including the mechanisms and regulation of germination and dormancy, desiccation tolerance, as well as issues in seed technology. Being six years from retirement now, I decided to extend my desiccation tolerance studies from seeds to resurrection plants, which display vegetative desiccation tolerance. I strongly believe that unveiling of the mechanism of vegetative desiccation tolerance may help us create crops that are truly tolerant to severe drought, rather than (temporarily) resistant.

 

How did you become interested in this field of study, and how has your career progressed?

As with many things in life, it was coincidence. I majored in plant biochemistry and applied for a PhD position in seed biology. After obtaining the degree I was offered a tenure track position in seed physiology by the Laboratory of Plant Physiology at Wageningen University, where I still work as a faculty member. My career has progressed nicely and I am an authority in the field of seed science, editor-in-chief of the journal Seed Science Research, and will become the President of the International Society for Seed Science in September of this year.

I see my current work on vegetative desiccation tolerance as a highlight in my professional life. I have always been more interested in the desiccation tolerance of seeds until about five years ago, when my current collaborator Prof Jill Farrant of the University of Cape Town, South-Africa, made me enthusiastic about these wonderful resurrection plants. We started to work together and published our first study recently in Nature Plants.

Read the paper here ($): A footprint of desiccation tolerance in the genome of Xerophyta viscosa.


 

In your recent paper, you sequenced the genome of the resurrection plant, Xerophyta viscosa, which can survive with less than a 5% relative water content. How is it possible for a plant to lose so much of its water and still survive?

These plants have a lot of characteristics that we’ve seen in seeds. They display protective desiccation tolerance mechanisms in their leaves, including anti-oxidants, protective proteins, and even dismantle their photosynthetic machinery during periods of drought. Even the cell wall structure and composition of resurrection plants resemble those of seeds. We are currently working on a paper describing the striking similarities between seeds and resurrection plants.

 

What was the most interesting discovery you made upon sequencing the genome of the resurrection plant?

First, the similarities between resurrection plants and seeds listed above were also apparent at the molecular level. For example, previous work suggested that the “ABI3 regulon”, consisting of about 100 genes regulated by the transcription factor ABI3, is specific to seeds, but we found that it is almost completely present (and active) in the leaves of Xerophyta viscosa too!

Secondly, we found “islands” or clusters of genes specific for desiccation tolerance that aren’t found in other species. Many of these regulate secondary metabolite pathways.

 

How challenging was it to sequence the genome of this plant? How did you overcome any difficulties?

It was very challenging. First, the species is an octoploid, meaning it has eight copies of each chromosome. This meant that we had to sequence its genome at very high coverage and employing the most advanced sequencing facilities, e.g. PacBio. Getting funding for this complex analysis was another challenge. We then took almost a year to assemble the genome and annotate it at the desired quality.

 

Xerophyta viscosa

Xerophyta viscosa before and after the rains. Image credit: Prof. Henk Hilhorst.

 

You identified some of the most important genes involved in desiccation tolerance. Is it possible to translate this work into other species, such as crops that may be threatened by drought as the climate changes?

That will be our ultimate goal. It’s important to remember that desiccation-sensitive plants, including all our major crops, produce seeds that are desiccation tolerant. This implies that the information for desiccation tolerance is present in the genomes of these crops but that it is only turned on in the seeds. We are trying to determine how this is localized, in order to find a method to turn on the desiccation tolerance mechanism in vegetative parts of the (crop) plant too. In parallel we are expressing some of the key transcription factors from Xerophyta viscosa in some important crops to see how this affects them.

 

Are there any other interesting aspects of Xerophyta viscosa biology?

Contrary to plants that wilt and ultimately die because of (severe) drought, leaves of resurrection species do not show such stress-related senescence. This is related to the engagement of active anti-senescence genes during the drying of the leaves of resurrection species. We are currently investigating these senescence-related mechanisms too.

 

Rose of Jericho (Anastatica hierochuntica)

The rose of Jericho (Anastatica hierochuntica) is another resurrection plant. Image credit: FloraTrek. Used under license: CC BY-SA 3.0.

 

Do you expect to find that different types of desiccation-tolerant plants use the same subset of genes to survive drought, or could they have developed other pathways to resilience?

We expect that the core mechanism is very similar among the resurrection species but that each species may have adapted to its specific environment.

Funding permitting, we will sequence the genomes of at least another ten resurrection species to further clarify the various evolutionary pathways to desiccation tolerance and, importantly, to discriminate between species-specific and desiccation tolerance-specific genes.

 

What advice do you have for early career researchers?

Stick to what you believe in, even if you have to (temporarily) be involved in research that you appreciate less, e.g., because of better funding opportunities.

 


Read Henk’s recent paper in Nature Plants here ($): A footprint of desiccation tolerance in the genome of Xerophyta viscosa.

Roots of a second green revolution

This week we spoke to Professor Jonathan Lynch, Penn State University, whose research on root traits has deepened our understanding of how plants adapt to drought and low soil fertility.

 

 

Could you begin by giving us a brief introduction to your research?

We are trying to understand how plants adapt to drought and low soil fertility. This is important because all plants in terrestrial ecosystems experience suboptimal water and nutrient availability, so in rich nations we maintain crop yields with irrigation and fertilizer, which is not sustainable in the long term. Furthermore, climate change is further degrading soil fertility and increasing plant stress. This topic is therefore both a central question in plant evolution and a key challenge for our civilization. We need to develop better ways to sustain so many people on this planet, and a big part of that will be developing more resilient, efficient crop plants.

 

Drought is devastating for crops

Drought and low soil fertility are devastating for crops. Image credit: CIAT. Used under license: CC BY-SA 2.0.

 

What got you interested in this field, and how has your career developed over time?

When I was 9 years old I became aware of a famine in Africa related to crop failure and resolved to do something about it. I studied soils and plant nutrition as an undergraduate, and in graduate school worked on plant adaptation to low phosphorus and salinity stress, moving to a research position at the CIAT headquarters in Colombia. Later I moved to Penn State, where I have maintained this focus, working to understand the stress tolerance of staple crops, and collaborating with crop breeders in the USA, Europe, Africa, Asia, and Latin America.

 

Your recent publications feature a variety of different crop plants. Could you talk about how you select a species to study?

We work with species that are important for food security, that grow in our field environments, and that I think are cool. We have devoted most of our efforts to the common bean – globally the most important food legume – and maize, which is the most important global crop. These species are often grown together in Africa and Latin America, and part of our work has been geared to understanding how maize/bean and maize/bean/squash polycultures perform under stress. These are fascinating, beautiful plants with huge cultural importance in human history. They are also supported by talented, cooperative research communities. One nice feature of working with food security crops is that their research communities share common goals of achieving impact to improve human welfare.

 

Common bean (Phaseolus vulgaris)

The common bean (Phaseolus vulgaris) is an important staple in many parts of the world. Image credit: Ervins Strauhmanis. Used under license: CC BY 2.0.

 

Many researchers use Arabidopsis thaliana for plant research, but are crops better suited for root research than the delicate roots of Arabidopsis? Are crop plants more or less difficult to work with in your research than Arabidopsis?

The best research system is entirely a function of your goals and questions. We have worked with Arabidopsis for some questions. Since we work with processes at multiple scales, including crop stands, whole organisms, organs, tissues, and cells, it has been useful to work with large plants such as maize, which are large enough to easily measure and to work with in the field. The most interesting stress adaptations for crop breeding are those that differ among genotypes of the same species, and at that level of organization there is a lot of biology that is specific to that species, that cannot readily be generalized from model organisms with very different life strategies. There has been considerable attention to model genomes and much less attention to model phenomes.

 

You have developed methodologies for the high-throughput phenotyping of crop plants. What does this technique involve and what challenges did you have to overcome to succeed?

We have developed multiple phenotyping approaches – too many to summarize readily here. Our overall approach is simply to develop a tool that helps us achieve our goals. For example, we have developed tools to quantify the root architecture of thousands of plants in the field, to measure anatomical phenotypes of thousands of samples from field-grown roots, to help us determine which root phenotypes might affect soil resource capture, etc. Working with geneticists and breeders, we are constantly asked to measure something meaningful on thousands of plants in a field, in many fields, every season. ARPA-E (the US Advanced Research Projects Agency for Energy) has recently funded us to develop phenotyping tools for root depth in the field, but this is the first time we have been funded to develop phenotyping tools – generally we just come up with things to help us do our work, which fortunately have been useful for other researchers as well.

 

Could you talk about some of the computational models you have developed for investigating plant growth and development?

The biological interactions between plants and their environment are so complex, we need computational (in silico) tools to help us evaluate them. Increasingly, in silico tools can integrate information across multiple scales, from gene expression to crop stands. These tools also allow us to evaluate things that are difficult to measure, such as phenotypes that do not yet exist, or future climates. In silico biology will be an essential tool in 21st Century biology, which will have access to huge amounts of data at multiple scales that can be used to try to understand incredibly complex systems, such as the human brain or roots interacting with living soil. Our main in silico tool is SimRoot, developed over the past 25 years to understand how root phenotypes affect soil resource capture.

Check out a SimRoot model below:


 

You have been working on breeding plants that have improved yield in soils with low fertility. What have you achieved in this work?

In collaboration with crop breeders and colleagues in various nations we have developed improved common bean lines with better yield under drought and low soil fertility that are being deployed in Africa and Latin America, improved soybean lines with better yield in soils with low phosphorus being deployed in Africa and Asia, and are now working with maize breeders in Africa to develop lines with better yield under drought and low nitrogen stress. Many crop breeders are using our methods for root phenotyping to target root phenotypes in their selection regimes in multiple crops.

 

What piece of advice do you have for early career researchers?

You are at the forefront of an unprecedented challenge we face as a species – how to sustain 10 billion people in a degrading environment. Plant biologists are an essential part of the effort to reshape how we live on this planet. Do not doubt the importance of your efforts. Do not lose sight of the very real human impact of your scientific choices. Do not be deterred by the gamesmanship and ‘primate politics’ of science. You can make a difference. We need you.

Creole maize reveals adaptation secrets

By Lucina Melesio

[MEXICO CITY] An international team of scientists identified a hundred genes that influence adaptation to the latitude, altitude, growing season and flowering time of nearly 4,500 native maize varieties in Mexico and in almost all Latin American and Caribbean countries.

Creole — or native — varieties of maize are derived from improvements made over thousands of years by local farmers, and contain genes that help them adapt to different environments.

“We are now using this analysis to find other genes that are of vital importance to breeders, such as those resistant to extreme heat, frost or drought — environmental conditions associated with climate change and that could affect maize production.”

Sarah Hearne, CIMMYT

“Latin American breeders will be able to use these results to identify native varieties that could contribute to improved adaptation”, Edward Buckler, a Cornell University researcher and co-author of the study published in Nature Genetics (February 6), told SciDev.Net.

The information on the genetic markers described in the study will be available online, said Sarah Hearne, a researcher at the International Maize and Wheat Improvement Center (CIMMYT) and co-author of the study. “Meanwhile, any breeder can contact us to request information”, she said.

“We are now using this analysis to find other genes that are of vital importance to breeders, such as those resistant to extreme heat, frost or drought — environmental conditions associated with climate change and that could affect maize production”, Hearne said.

Maize ears from CIMMYT’s collection, showing a wide variety of colors and shapes. CIMMYT’s germplasm bank contains about 28,000 unique samples of cultivated maize and its wild relatives, teosinte and Tripsacum. These include about 26,000 samples of farmer landraces—traditional, locally-adapted varieties that are rich in diversity. The bank both conserves this diversity and makes it available as a resource for breeding.
Photo credit: Xochiquetzal Fonseca/CIMMYT.

Studying native maize varieties is extremely difficult because of their genetic variation. Although domesticated, they are wilder than commercial varieties.

For this study, the researchers cultivated hybrid creole varieties in various environments in Latin America and identified regions of the genome that control growth rates. They looked into where the varieties came from and what genetic features contributed to their growth in that environment.

 In comments to SciDev.Net, James Holland, a researcher at North Carolina State University, Jeffrey Ross-Ibarra, a researcher at the University of California Davis, and Rodomiro Ortiz, a researcher at the Swedish University of Agricultural Sciences — who did not participate in the study — commended the magnitude of the study and the original method developed by the researchers to access the rich set of genetic information about native maize varieties.

Hearne added that the research team has initiated a “pre-breeding” programme with a small group of breeders in Mexico. As part of that programme, CIMMYT delivers to breeders materials from its germplasm bank of Creole maize; it also provides molecular information the breeders can use to generate new varieties.

This piece was produced by SciDev.Net’s Latin America and Carribean edition.

This article was originally published on SciDev.Net. Read the original article.

Global Plant Council stress resilience commentaries published in Food and Energy Security

In October 2015, researchers from around the world came together in Iguassu Falls, Brazil, for the Stress Resilience Symposium, organized by the Global Plant Council and the Society for Experimental Biology (SEB), to discuss the current research efforts in developing plants resistant to the changing climate. (See our blog by GPC’s Lisa Martin for more on this meeting!)

Building on the success of the meeting, the Global Plant Council team and attendees compiled a set of papers to provide a powerful call to action for stress resilience scientists around the world to come together to tackle some of the biggest challenges we will face in the future. These four papers were published in the Open Access journal Food and Energy Security alongside an editorial about the Global Plant Council.

In the editorial, the Global Plant Council team (Lisa Martin, Sarah Jose, and Ruth Bastow) introduce readers to the Global Plant Council mission, and describe the Stress Resilience initiative, the meeting, and introduce the papers that came from it.

In the first of the commentaries, Matthew Gilliham (University of Adelaide), Scott Chapman (CSIRO), Lisa Martin, Sarah Jose, and Ruth Bastow discuss ‘The case for evidence-based policy to support stress-resilient cropping systems‘, commenting on the important relationships between research and policy and how each must influence the other.

Global Plant Council President Bill Davies (Lancaster University) and CIMMYT‘s Jean-Marcel Ribaut outline the ways in which research can be translated into locally adapted agricultural best practices in their article, ‘Stress resilience in crop plants: strategic thinking to address local food production problems‘.

In the next paper, ‘Harnessing diversity from ecosystems to crops to genes‘, Vicky Buchanan-Wollaston (University of Warwick), Zoe Wilson (University of Nottingham), François Tardieu (INRA), Jim Beynon (University of Warwick), and Katherine Denby (University of York) describe the challenges that must be overcome to promote effective and efficient international research collaboration to develop new solutions and stress resilience plants to enhance food security in the future.

University of Queensland‘s Andrew Borrell and CIMMYT‘s Matthew Reynolds discuss how best to bring together researchers from different disciplines, highlighting great examples of this in their paper, ‘Integrating islands of knowledge for greater synergy and efficiency in crop research‘.

In all of these papers, the authors suggest practical short- and long-term action steps and highlight ways in which the Global Plant Council could help to bring researchers together to coordinate these changes most effectively.

Read the papers in Food and Energy Security here.

Synthetic biology in chloroplasts

Dr Anil Day, University of Manchester

Dr Anil Day, University of Manchester

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.

 

Anil Day examines transformed plants

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.

Plastids in plants

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.

 

Edited chloroplasts

A tobacco plant containing leaf areas with edited (pale green) and normal (darker green) chloroplasts. Credit: Dr. Anil Day.

Last year you reported that chloroplasts degrade in mature sperm cells just prior to fertilization. Could you elaborate on how this might be utilized in future crop breeding?

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 PrimavesiHuixia 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.

 

Selection of transformed plants

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.

Registration open for GPC/SEB New Breeding Technologies Workshop!

New Breeding Technologies in the Plant Sciences – Applications and Implications in Genome Editing

Gothenburg, Sweden, 7-8th July 2017

REGISTRATION FOR THIS MEETING IS NOW OPEN!

Organised by: Dr Ruth Bastow (Global Plant Council), Dr Geraint Parry (GARNet), Professor Stefan Jansson (Umeå University, Sweden) and Professor Barry Pogson (Australian National University, Australia).

Targeted genome engineering has been described as a “game-changing technology” for fields as diverse as human genetics and plant biotechnology. Novel techniques such as CRISPR-Cas9, Science’s 2015 Breakthrough of the Year, are revolutionizing scientific research, allowing the targeted and precise editing of genomes in ways that were not previously possible.

Used alongside other tools and strategies, gene-editing technologies have the potential to help combat food and nutritional insecurity and assist in the transition to more sustainable food production systems. The application and use of these technologies is therefore a hot topic for a wide range of stakeholders including scientists, funders, regulators, policy makers and the public. Despite its potential, there are a number of challenges in the adoption and uptake of genome editing, which we propose to highlight during this SEB satellite meeting.

One of the challenges that scientists face in applying technologies such as CRISPR-Cas9 to their research is the technique itself. Although the theoretical framework for using these techniques is easy to follow, the reality is often not so simple. This meeting will therefore explain the principles of applying CRISPR-Cas9 from experts who have successfully used this system in a variety of plant species. We will explore the challenges they encountered as well as some of the solutions and systems they adopted to achieve stably transformed gene-edited plants.

The second challenge for these transformative technologies is how regulatory bodies will treat and asses them. In many countries gene editing technologies do not fit within current policies and guidelines regarding the genetic modification and breeding of plants, as it possible to generate phenotypic variation that is indistinguishable from that generated by traditional breeding methods. Dealing with the ambiguities that techniques such as CRISPR-Cas9 have generated will be critical for the uptake and future use of new breeding technologies. This workshop will therefore outline the current regulatory environment in Europe surrounding gene editing, as well as the approaches being taken in other countries, and will discuss the potential implications and impacts of the use of genome engineering for crop improvement.

Overall this meeting will be of great interest to plant and crop scientists who are invested in the future of gene editing both on a practical and regulatory level. We will provide a forum for debate around the broader policy issues whilst include opportunities for in-depth discussion regarding the techniques required to make this technology work in your own research.

This meeting is being held as a satellite event to the Society for Experimental Biology’s Annual Main Meeting, which takes place in Gothenburg, Sweden, from the 3–6th July 2017.