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

Indian subsidies cost Queensland sugar industry $1 billion

Indian Government price and export subsidies have cost the Queensland sugar industry an estimated $1 billion.

The subsidies, which are currently under investigation by the World Trade Organisation, have contributed to substantially lower global sugar prices, according to a new report by Green Pool Commodity Specialists.

Peak industry organisations the Australian Sugar Milling Council (ASMC) and CANEGROWERS say the damaging impact confirms the need for continued pressure from the Australian Government and industry against India’s subsidies.

The ASMC commissioned report found Indian Government sugarcane price regulation was causing large production surpluses and India’s subsidised exports had contributed to substantially lower global sugar prices.

The report concludes that Queensland cane growers and sugar millers had incurred an estimated $1 billion revenue hit between 2017-18 and 2020-21 – or almost $5 million every week.

ASMC policy, economics and trade director David Rynne said Australia, Brazil and Guatemala made a formal complaint about the Indian Government’s subsidies to the WTO in February 2019.

A WTO report on the complaint may be handed down during 2021.

“Over the past four years, these subsidies have hurt Queensland growers, millers and sugar industry employees, their families and communities,” Mr Rynne said.

“The Green Pool report concluded that India’s current sugar production of more than 33 million tonnes exceeded its domestic sugar consumption of about 26mt.

“This excess subsidised production means export subsidies are likely to be an ongoing feature for many years if it is not reined in.”

ASMC and CANEGROWERS have praised the Australian Government for its advocacy on behalf of the sugar industry, urging continued support for growers, millers, dependent businesses and regional communities.

CANEGROWERS chairman Paul Schembri said the Australian Government had strongly backed the industry through the WTO case, but with concerning reports the Indian Government may be considering another package of export subsidies, it was clear that maintaining the collective effort of our diplomatic representatives to the Indian Government will be crucial.

“We are hopeful the WTO will find against the Indian subsidies because the economic pain for Australian growers, millers and sugar communities could be profound and prolonged if they continue,” Mr Schembri said.

“We will be sharing the findings of this report with the Australian and Queensland governments, and our local parliamentary representatives.

“We want to work with them to ensure progress on the path to a fairer global market for sugar.”

The Green Pool report found thatsince 2017-18 India’s sugar export subsidy program had become virtually institutionalised.

“India is now a structural exporter of sugar, with subsidies on exported sugar of up to one-third of an Indian mill’s cost of production of raw sugar,” the report reads.

“Besides export subsidies and regulated cane prices, India’s government also fixes domestic sugar prices well above global prices and provides soft loans to milling companies to build ethanol distilling operations to utilise sugar and processing by-products.

“The decision of India’s government to financially support significant levels of exports (initially compulsory) has had a real and perverse impact on the global market.”

ASMC is commissioning further analysis to determine the wider impact of India’s subsidies on Queensland’s regions and the state’s economy.

First 2021 sugar shipment

QSL has dispatched its first shipment of sugar from the current harvest.

The cargo of 42,000 tonnes of raw sugar from the Burdekin region was shipped on the Pacific Integrity out of Townsville earlier this month, bound for a refinery customer in South Korea.

QSL General Manager Marketing Mark Hampson said South Korea was one of Australia’s largest raw sugar markets, and had been one of QSL’s key customers for nearly 50 years.

“While we’ve had other shipments this year, those previous cargos delivered sugar produced last season,” Mr Hampson said.

“Queensland’s sugar is in high demand, so this first 2021-Season shipment marks the start of very a busy shipping program which will stretch through until mid-next year, with exports already planned for refining customers in South Korea, Indonesia, Japan and the USA.”

For further information contact Cathy Kelly on 0409 285 074.

Wilmar Sugar applications open soon for their 2022 apprenticeship round

Wilmar Sugar has announced the opening of applications for their 2022 apprenticeship round this Saturday July 10, calling on the next generation of apprentices to get their resumes ready.

With a variety of jobs on offer, applicants will have the opportunity to choose from apprenticeships in diesel fitting, electrical, fitting and turning, and fabricating trades, to be placed across any one of Wilmar’s eight sugar mills and bioethanol distillery.

Katelyn Smith, a first-year fitting and turning apprentice stationed at the Victoria Mill near Ingham, said she changed to an apprenticeship after six years of following a different career path and was glad she made the decision.

“I started as a fiber chemist at Victoria Mill. Through that, I spoke to some tradesmen and fitters, and I got to see the type of work they were doing,” Ms Smith said.

“I really like Wilmar’s safety values, so knowing you will come home safe every day.”

Wilmar Sugar apprentices.
 Wilmar Sugar apprentices.

First year fitting and turning apprentice, George Burns, said his apprenticeship gave him the opportunity to attain skills he had always wanted to learn.

“I grew up on a farm and used to pull machinery apart and put it back together, just never correctly, so this field will help me understand how it goes back together,” Mr Burns said.

“Most of the work in the mills is done in-house. You do it and you learn from that hands-on experience.”
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Wilmar have said the applications will remain open for three weeks, closing on Sunday August 1.

Training superintendent, Daniel Shipard said they are very proud of their apprenticeship program, and the level of interest they have received in the past.

“We currently employ about 130 apprentices across various trades,” Mr Shipard said.

“The four main trades that we focus on are fabricating, fitting and turning, electrical and diesel fitting.

Mr Shipard said the application is to be completed online and its fairly straightforward.

New investments to delivery exciting sugarcane research and development outcomes

Sugar Research Australia has announced the investment in multiple new research projects that will drive productivity, profitability and sustainability in the Australian sugarcane industry.

These new investments have targeted specific areas and issues that require solutions for growers and millers, with the new investments having been selected following a rigorous assessment process over the last few months.

SRA CEO Roslyn Baker said the new investments were aligned to the five Research Missions within SRA’s new Strategic Plan and involved extensive collaboration with investment and delivery partners.

“A key element of SRA’s new strategy is that SRA will collaborate more strongly than ever before – and we can see that clearly through these new project investments,” she said.

New and recent project investments include:

Project nameLead delivery agenciesSummaryInvestment agencies
E-network for rail-based cane transport systemsAdvisian Pty Ltd (lead); The University of NSW; Mobility Thinking Pty LtdThe development of a monitoring and communication system, operating across the sugar cane rolling stock and rail network, will seek to increase efficiency, improve safety, and reduce costs.SRA
Environmental DNA Technologies and Predictive Modelling for Rapid Detection and Identification of Sugarcane Priority Pests and DiseasesEnviro DNA Pty Ltd; SRAThis project builds on pest surveillance and in-field testing techniques to improve the ability of those undertaking Northern Australian sugar pest surveillance activities to identify pest incursion threats quickly and accurately.SRA and DAF Queensland
Transformational crop protection – Innovative RNAi biopesticides for management of sugarcane root feeding pestsUniversity of Queensland; SRAThis project aims to deliver a proof-of-concept demonstration for the BioClay/BenPol platform targeting root feeding pests of sugarcane including cane grubs and soldier flies.SRA and DAF Queensland
Sugar Industry Diversification Opportunities Investigation SupportProcom Consultants Pty LtdThis project will investigate the potential application of two sugarcane biorefinery opportunities (hydrogen production from bagasse and compostable bioplastic production from cane juice) and identify any investment barriers to their application.SRA and DAF Queensland
A Common Approach to Sector-Level GHG Accounting for Australian AgricultureCSIROThis project will develop a common approach for greenhouse gas accounting that is shared by agricultural commodity sectors, via a collective action process.SRA + 9 other RDCS; DAWE, West Australia DPIRD & CRSPI (via Agriculture Innovation Australia)
Environmental Risk Assessment & Life Cycle Assessment of the Raw Sugar ManufacturingIntegrity Ag & Environment Pty LtdLife Cycle Analysis (LCA) is a standardised method for quantifying the environmental and potentially human health impacts of the inputs, outputs, and emissions across all relevant phases of a product life cycle. The purpose of this LCA project is to help the industry better understand, benchmark, and improve its environmental contribution to human health, environment, ecosystem quality and resource use, including waste management.SRA and DAF Queensland
Beyond Imidacloprid – Chemical and Biorational Alternatives for Managing CanegrubsSRAWith the only canegrub control  compound (imidacloprid) at the risk of being withdrawn from the market, this project will (if successful) generate efficacy and runoff data that is required to support the registration of alternative compounds for the canegrub control.SRA and DAF Queensland
Engineering bacterial enzyme secretion for cellulose utilisationQueensland University of TechnologyThis project aims to develop microbial technology to reduce the cost of extracting fermentable sugars, which could lead to the creation of a bacterium that can directly extract sugars from lignocellulosic biomass and convert them into biochemicals.SRA
Maximising cane recovery through the development of a harvesting decision-support toolSRA andDAF QueenslandThis project will provide an online platform that helps growers and contractors look at the potential gains associated with improved harvesting practices, and costs, and then examine the costs of harvesting.SRA and DAF Queensland

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.