First Breeding of Sugar Cane Using CRISPR/Cas9

Sugarcane is one of the most productive plants on Earth, providing 80 percent of the sugar and 30 percent of the bioethanol produced worldwide. Its size and efficient use of water and light give it tremendous potential for the production of renewable value-added bioproducts and biofuels.

But the highly complex sugarcane genome poses challenges for conventional breeding, requiring more than a decade of trials for the development of an improved cultivar.

Two recently published innovations by University of Florida researchers at the Department of Energy’s Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) demonstrated the first successful precision breeding of sugarcane by using CRISPR/Cas9 genome editing — a far more targeted and efficient way to develop new varieties.

CRISPR/Cas9 allows scientists to introduce precision changes in almost any gene and, depending on the selected approach, to turn the gene off or replace it with a superior version. The latter is technically more challenging and has rarely been reported for crops so far.

In the first report, researchers demonstrated the ability to turn off variable numbers of copies of the magnesium chelatase gene, a key enzyme for chlorophyll biosynthesis in sugarcane, producing rapidly identifiable plants with light green to yellow leaves. Light green plants did not show growth reduction and may require less nitrogen fertilizer to produce the same amount of biomass. That study, published in Frontiers in Genome Editing, was led by CABBI researchers Fredy Altpeter, Professor of Agronomy at the University of Florida’s Institute of Food and Agricultural Sciences (IFAS), and Ayman Eid, a Postdoctoral Research Associate in Altpeter’s lab.

The second study, also published in Frontiers in Genome Editing, achieved efficient and reproducible gene targeting in sugarcane, demonstrating the precise substitution of multiple copies of the target gene with a superior version, conferring herbicide resistance. Scientists co-introduced a repair template together with the gene-editing tool to direct the plant’s own DNA repair process so that one or two of the thousands of building blocks of the gene, called nucleotides, were precisely replaced in the targeted location. The result was that the gene product was still fully functional and could no longer be inhibited by the herbicide. That study was led by Altpeter and former CABBI Postdoc Mehmet Tufan Oz.

Altpeter’s lab, part of CABBI’s groundbreaking project to develop new oil-rich sugarcane varieties, has pioneered research with sugarcane genome editing using the TALEN gene-editing system. But the two recent publications are the first to successfully demonstrate CRISPR gene-editing in sugarcane as well as gene targeting for precision nucleotide substitution in sugarcane using any genome-editing tool.

“Now we have very effective tools to modify sugarcane into a crop with higher productivity or improved sustainability,” Altpeter said. “It’s important since sugarcane is the ideal crop to fuel the emerging bioeconomy.”

Sugarcane is a hybrid of two kinds of parent plants, so it has multiple sets of chromosomes rather than just two, as with humans or “diploid” plants. That creates genetic redundancy — with many sets of genes doing the same job — which may contribute to the plant’s productivity: If one set breaks, there’s a backup. But it makes sugarcane extremely difficult to modify. Crop scientists have to target all the genes and copies that govern a particular trait in order to make improvements.

With conventional breeding, two types of sugarcane are cross-bred to reshuffle the genetic information present in each parent in the hope of enhancing a desirable trait such as disease resistance. The problem is that genes are transferred from the parents to offspring in blocks, and desirable traits are often linked with deleterious genetic material. This means scientists often have to do multiple rounds of backcrossing and screen thousands of plants to restore the elite background, or underlying plant characteristics, in addition to improving one trait they’re attempting to modify. The process is more time-consuming and costly in plants with complex genomes like sugarcane.

Precise gene-editing technologies such as CRISPR-Cas9 offer a much more targeted path to crop improvement because it avoids the reshuffling of genetic information and simply changes inferior gene versions into superior ones. Given the sugarcane genome’s complexity, Altpeter and his team focused initially on genes that control noticeable traits — leaf color and herbicide resistance — so they could determine if the edits worked.

Beyond providing an easily identifiable phenotype, the targeted genes may prove useful in future research. Changing the chlorophyll content of sugarcane has the potential to increase canopy level photosynthesis or reduce the requirement for nitrogen fertilizer, based on previous plant modeling. Sugarcane is a tall, dense plant, with the top leaves getting lots of sun and shading lower foliage. If the upper leaves have less chlorophyll, sunlight can penetrate deeper into the plant, increasing its biomass with the same amount of light and less fertilizer. Herbicide resistance is not only an agronomically desirable trait to facilitate weed management; it will also facilitate future gene-editing efforts by enabling suppression of non-edited plant cells.

At CABBI, Altpeter and his team are already applying the results to develop improved sugarcane lines. Sugarcane has many different gene targets that can translate into more biomass or the production of lipids or specialty fatty acids — all of which would advance CABBI’s goals to produce fuels and other products from plants to replace petroleum. Because the crop is already harvested and processed for sugar extraction, the basic infrastructure to process its raw material into a product on a shelf is essentially in place.

“Adding value streams is relatively inexpensive compared to other crop alternatives,” Altpeter said.

Reference: Oz MT, Altpeter A, Karan R, Merotto A, Altpeter F. CRISPR/Cas9-mediated multi-allelic gene targeting in sugarcane confers herbicide tolerance. Front Genome Ed. 2021;3. doi: 10.3389/fgeed.2021.673566

Student engineers sweetening the deal on clean energy

Chemical engineering students from the University of Queensland (UQ) have helped investigate how sugarcane could be used as a clean energy source to create hydrogen.

Professor Damien Batstone of UQ’s Faculty of Engineering, Architecture and Information 

Technology, said bagasse, or sugarcane pulp, and other agricultural residues were an abundant resource that could generate ‘green’ or carbon-negative hydrogen at scale.

Biomass from crushed sugarcane stalks and leaves could also potentially produce hydrogen for under $3 per kilogram, one third of the cost of current options, he added.

Last year, 150 students in 36 teams were tasked with designing a process to produce hydrogen gas from bagasse with either thermal gasification or hydrothermal gasification as their process.

Generating pure hydrogen

Caitlin Welsh, a chemical and materials engineer who has a Bachelor of Engineering (Hons) from UQ, and her team, were responsible for designing a thermal gasification process for an input of 2000 t/day of bagasse. 

“I was allocated the pre-treatment node where I was required to design units to heat and dry the bagasse in preparation for gasification,” she said.  

“Part of my role was to ensure the bagasse was pre-treated to ensure highest possible efficiency. I needed to do this while ensuring the pre-treatment node did not counter the energy savings in the gasifier. 

“So, I applied energy integration in my design by utilising steam produced downstream in my pre-treatment for heating of bagasse.”

Welsh, who is currently working at Visy Board as a graduate engineer, said that, with thermal gasification, there will always be by-products such as ash, tar, carbon dioxide and carbon monoxide gas.

“The hydrogen can, however, be extracted and the by-products captured in the downstream process,” she said.

Eva I Iong Lam, who also has a Bachelor of Engineering (Hons) from UQ, and her team were involved in the cutting-edge hydrothermal gasification process.

“It is similar to that of thermal gasification, except it involves wet biomass,” she said.

“Not only does it save energy in the drying of bagasse, but it also allows for lower operating temperatures with the possibilities to utilise a variety of biomasses as feedstocks.”

Chemical engineering students from the University of Queensland (UQ) have helped investigate how sugarcane could be used as a clean energy source to create hydrogen.
Eva I Iong Lam and Caitlin Welsh helped investigate the use of sugarcane to produce hydrogen.

A graduate chemical engineer who now works at Engeny Water Management, I Iong Lam said the slurry was fed into the reactor, then produced a supercritical fluid where its heat energy was integrated with other processing units through a series of heat exchangers.

“Hydrogen is produced more readily under supercritical water conditions in this process,” she said. 

“The gases were then separated by chemisorption using Methyldiethanolamine (MDEA) solvent.” 

The models she created were also able to recover the majority of water present in the gas streams using flash tanks, so it could be repurposed within the plant.

“A key challenge was the limited availability of materials that could withstand high temperatures and pressures,” I Iong Lam said.

“I chose stainless steel 316 as the construction material for a flash tank receiving supercritical fluids as it has excellent resistance to corrosion, thermal and pressure stress.”

A future biomass technology

While gasification has been widely applied to coal processing, it has not been applied to hydrogen production from biomass at large scale, Batstone said.

“This offers an alternative pathway with potential for higher profits for canegrowers, and for sugarcane to be used in ethanol and plastic production, while fully utilising the biomass residues,” he said.

Welsh hopes that her career will include further involvement in sustainable engineering solutions, while I Iong Lam wants to continue to focus on water consulting.

“You get clean water by just turning on a tap, we never really spend a moment to appreciate the processes and people behind it,” she said. “Now, I am hoping to make a positive difference to the community I live in.”.

To develop the work done by the students, a future project by UQ will involve growers, sugar companies and likely end-users and include governments investing in a hydrogen economy.

Energycane produces more biodiesel than soybean at a lower cost

Bioenergy from crops is a sustainable alternative to fossil fuels. New crops such as energycane can produce several times more fuel per acre than soybeans. Yet, challenges remain in processing the crops to extract fuel efficiently.

Four new studies from the University of Illinois explore chemical-free pretreatment methods, development of high-throughput phenotyping methods, and commercial-scale techno-economic feasibility of producing fuel from energycane in various scenarios.

The studies are part of the ROGUE (Renewable Oil Generated with Ultra-productive Energycane) project at U of I. ROGUE focuses on bioengineering accumulation of triacylglycerides (TAGs) in the leaves and stems of energycane, enabling the production of much more industrial vegetable oil per acre than previously possible.

“The productivity of these non-food crops is very high per unit of land. Soybean is the traditional crop used for biodiesel, but we can get higher yield, more oil, and subsequently more biofuel from lipid-producing energycane,” says Vijay Singh, Founder professor in the Department of Agricultural and Biological Engineering (ABE) at U of I and co-author on all four papers.

Biofuel production from crops involves breaking down the cellulosic material and extracting the oil in a series of steps, explains study co-author Deepak Kumar, assistant professor in the Chemical Engineering Department at State University of New York College of Environmental Science and Forestry (SUNY-ESF) and adjunct research scientist at the Carl R. Woese Institute for Genomic Biology at U of I.

“The first step is to extract the juice. That leaves bagasse, a lignocellulosic material you can process to produce sugars and subsequently ferment to bioethanol,” Kumar says.

“One of the critical things in processing any lignocellulosic biomass is a pretreatment step. You need to break the recalcitrant structure of the material, so enzymes can access the cellulose,” he adds. “Because energycane is a relatively new crop, there are very few studies on the pretreatment and breakdown of this bagasse to produce sugars, and to convert those sugars into biofuels.”

The pretreatment process also yields some unwanted compounds, which inhibit enzymes that convert the sugar into biofuels. The U of I researchers investigated the best pretreatment methods to maximize the breakdown while minimizing the production of inhibitors. Typically, the pretreatment process uses chemicals such as sulfuric acid to break down the biomass at high temperature and pressure.

“We use a chemical-free method, which makes it more environmentally friendly,” Kumar explains. “Furthermore, harsh chemicals may alter the oil structure or quality in the biomass.”

The researchers tested their method using nine different combinations of temperature and time intervals. They were able to achieve more than 90% cellulose conversion at the optimal conditions, which is equivalent to results from chemical pretreatment methods.

The second study built on those results to further investigate the relationship between temperature, inhibitor production, and sugar recovery.

“We pretreated the lignocellulosic biomass over a range of different temperatures to optimize the condition for minimal inhibitor generation without affecting the sugar recovery. Then we added cryogenic grinding to the process,” says Shraddha Maitra, postdoctoral research associate in ABE and lead author on the study.

“In cryogenic grinding, you treat the bagasse with liquid nitrogen, which makes it very brittle, so upon grinding the biomass fractures easily to release the sugars. This further increased sugar recovery, mainly xylose, by about 10% compared to other refining processes,” Maitra explains.

Other industries use similar methods, for example for spices and essential oils, where it is important to preserve the qualities of the product. But applying them to biofuel production is new.

In a third study, Maitra and her co-authors investigated time-domain nuclear magnetic resonance (NMR) technology to determine the stability and recovery of lipids by monitoring changes in total, bound, and free lipids after various physical and chemical feedstock preprocessing procedures.

The research team’s fourth study investigated the commercial-scale techno-economic feasibility of engineered energycane-based biorefinery. They used computer modeling to simulate the production process under two different scenarios to determine capital investment, production costs, and output compared with soybean-based biodiesel.

“Although the capital investment is higher compared to soybean biodiesel, production costs are lower (66 to 90 cents per liter) than for soybean (91 cents per liter). For the first scenario, processing energycane had overall slightly lower profitability than soybean biodiesel, but yields five times as much biodiesel per unit of land,” says Kumar, the lead author on the study.

“Energycane is attractive in its ability to grow across a much wider geography of the U.S. south east than sugarcane. This is a region with much underutilized land, yet capable of rain-fed agriculture,” says ROGUE Director Steve Long, Ikenberry Endowed Chair of Plant Biology and Crop Sciences at the University of Illinois.

“As a perennial, energycane is suitable for land that might be damaged by annual crop cultivation. Our research shows the potential to produce a remarkable 7.5 barrels of diesel per acre of land annually. Together with co-products, this would be considerably more profitable than most current land use, while having the potential to contribute greatly to the national U.S. goal of achieving net zero greenhouse gas emissions by 2050. This proves how valuable it is to build on the successes already achieved in bioengineering energycane to accumulate oils that are easily converted into biodiesel and biojet,” Long states.


Story Source:

Materials provided by University of Illinois College of Agricultural, Consumer and Environmental Sciences. Original written by Marianne Stein. Note: Content may be edited for style and length.

A new era of research, development and adoption (RD&A) begins for the Australian sugarcane industry

Today (Thursday July 1) marks ‘day one’ of an exciting era of sugarcane research, development and adoption (RD&A) investment for the Australian sugarcane industry, with Sugar Research Australia (SRA) officially commencing a new five-year Strategic Plan.

SRA CEO Roslyn Baker said that the Strategic Plan 2021-2026 had been developed with extensive consultation and feedback over the last 18 months and had culminated in a new plan built on five specific pillars of value.

These pillars are: strong foundations, a high-performing research portfolio, translation expertise, world-class sugarcane varieties, and commercial benefits and rewards.

“By focusing on these five strategic pillars, we have created a new direction for SRA that puts the company in the best possible position to deliver on our new vision,” she said.

SRA’s new vision is to be: A trusted partner, shaping the future prosperity of the Australian sugarcane industry and regional communities through innovation and ingenuity.

“This plan represents the biggest transformation of SRA since the company began operation in 2013,” Ms Baker said.

“Our plan is a growth strategy for research and development for the sugar industry. We are focused on delivering immediate value by providing valuable industry services, while also ensuring long-term sustainability and outcomes through re-invigorating the research investment portfolio and focusing on potential commercial opportunities.”

SRA’s new strategy will:

  • Support a portfolio approach to investment in RD&A to ensure a balance of investments that address both the current-day productivity and sustainability constraints for the industry, while anticipating the future opportunities and challenges ahead.
  • Leverage SRA’s internal research capability and regional footprint to increase the awareness and use of research knowledge to improve regional productivity and sustainability, facilitate regional collaboration and partnerships, and boost co-investment opportunities.
  • Continue to evolve and modernise our world-class sugarcane variety development program to meet the current and future needs of the industry.
  • Strategically invest in innovative crop protection that uses new science and technology so that the Australian sugarcane industry can lead the world, and exceed community expectations, in protecting our precious natural environment.

Ms Baker said she was grateful for the industry support and guidance for the development of the plan, as well as strong support from the SRA staff and Board.

“We have reshaped SRA to deliver the best bang for the buck when it comes to the investment that our government and industry stakeholders make in SRA,” she said. “We have also aligned our new plan strongly with the innovation agendas being targeted by the Australian and Queensland Governments.

“We look forward to talking with all of SRA’s partners and investors in coming weeks and months about our new strategic direction and how we can all work together to achieve the best possible outcomes for the industry.”

Joint media release: Sweet victory for sugarcane growers

A unique pilot project involving more than 100 canegrowers in North Queensland has delivered a solution to reduce the amount of nitrates that end up in the Great Barrier Reef by at least 15 per cent.

The successful trial funded by the Australian Government’s National Environmental Science Program (NESP) saw scientists and canegrowers working together across the Russell and Mulgrave river catchments south of Cairns to install hi-tech telemetry and water quality sensors close to their properties.

For the first time, farmers were able to access year-round and real time measurements of nitrate amounts in their creeks and rivers using a specially developed mobile phone app.

This showed that if landholders can hold back the first flush of Wet Season rain using the existing farm drainage network and let it settle for a few days, it can significantly reduce runoff into the reef.

Minister for the Environment Sussan Ley said giving growers access to real time data had helped to build trust and provide confidence when making on-farm decisions that support the environment.

“This is an example of farmers and scientists working closely together to improve ecosystem health, ensuring farmers are fully informed and can have faith in the data they are receiving,” Minister Ley said.

“Protecting our waterways means cleaner rivers, larger fish stocks, resilient marine life, healthier coral and stronger coastal tourism.

“To do this we need to work closely with growers and maintain their trust. The project has partnered with local farmers to design the program and carry out the research.

“Being able to accurately track and reduce dissolved inorganic nitrogen flowing onto the reef, and work closely with communities, is key to meeting our water quality targets under the Reef 2050 Plan.”

Special Envoy for the Great Barrier Reef and Federal Member for Leichhardt Warren Entsch said the pilot showed how farmers were part of the solution to help the reef thrive.

“Projects like these are building relationships and goodwill between farmers and scientists, allowing landholders to be closely involved in planning discussions and scientific trials.

“Growers have been able to have a say about methods that are acceptable to improve water quality without having financial and practical repercussions on their farms and families.

“This study represents an inspiring commitment by canegrowers to do what they can to help the reef, and how positive these efforts continue to be for the environment, industry and local communities.”

North Queensland-based Senator for Queensland, Susan McDonald, who recently took part in a Senate Inquiry into farming effects on Great Barrier Reef waters, said the Morrison Government was leading the way in showing collaboration as the best way to achieve positive outcomes for all stakeholders.

“One of the glaring revelations in the Inquiry was the lack of on-farm engagement undertaken by the Queensland Labor Government when they drafted their anti-farming Reef regulations,” she said.

“This federal program is giving farmers the tools and information they need to make sound decisions on how they manage their land, but more importantly it shows them the results of their efforts.

“Farmers are the first to say that caring for the environment is a top priority, but they should be encouraged and helped by governments, not dictated to with harsh laws and endless paperwork.”

The project was funded by the Australian Government’s NESP Tropical Water Quality Hub through the Cairns-based Reef and Rainforest Research Centre (RRRC) and delivered by scientists from CSIRO and James Cook University.

SRA to build industry knowledge on interactions between on-farm practice and water quality in the Central Region

Sugarcane growers in the Mackay and Plane Creek areas of Central Queensland will have the opportunity to gain a clearer understanding of the complex relationship between farming systems and water quality, through a new project that is about to commence.

This new project is led by Sugar Research Australia (SRA) and is an addition to SRA’s work on the “Cane to Creek” series of projects, bringing this work to multiple sub-catchments in the Central Region.

Regional Coordinator for the Central Region, Phil Ross, said that the Mackay-Whitsunday Cane to Creek project would work closely with productivity services organisations, Farmacist, and growers on adaptive learning through demonstration sites.

“Through Cane to Creek in several other regions, SRA has worked hand-in-glove with growers to look at locally-specific issues that are relevant to them,” Mr Ross said.

“Growers are continually changing and improving practices to improve productivity, profitability, and sustainability. The industry also operates in a very complex system, which means we are always looking for new and practical information to help implement new practice.

“Through this project, we will be working with growers to better understand these various factors in their own local conditions.

“This will lead to increased adoption of improved practices that meet the goals of improving productivity, profitability and sustainability, including nutrient and pesticide management strategies that contribute to achieving the dissolved inorganic nitrogen and pesticide load reduction targets for the region.”

Mr Ross said that along with the work already underway in other regions, this project will provide a platform for growers, researchers and advisors to agree on and test potential solutions to better match nitrogen and herbicide application to their specific requirements.

The Mackay-Whitsunday Cane to Creek project is funded by the partnership between the Australian Government’s Reef Trust and the Great Barrier Reef Foundation (GBRF), with support from SRA.

It will run over the next three years and will leverage decision support tools such as the SRA SIX EASY STEPS Toolbox and the forthcoming DAF Queensland Pesticide Selection Tool. In-field surveys of liquid imidacloprid applicators and sampling for imidacloprid in runoff water will also contribute to our understanding of this key chemical control for cane grubs.

“We will be working with growers on crop nutrition, pesticide stewardship and water quality science, as well as breaking down the barriers to bring science and industry to the same table.”