Houston, Texas, USA : Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing.
In a new study published in Science Advances, the researchers demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)–mediated targeted mutagenesis of two genes involved in amylose biosynthesis, protein targeting to starch (ptst1) or granule bound starch synthase (gbss), can reduce or eliminate amylose content in root starch.
Cassava (Manihot esculenta Crantz) is a multipurpose crop cultivated for food and as an industrial feedstock. Successful domestication of the species that originated from South America has promoted cassava to among the top five most important sources of carbohydrate globally. The starch-rich storage roots are a staple food in tropical and subtropical countries where the plant is relatively drought-tolerant and suitable for growth in marginal environments. The roots are also an important commodity and processed by a multibillion-dollar industry for use in manufacturing paper, beverages, biodegradable materials, animal feed, pharmaceuticals, and biofuels. In western countries, the gluten-free property of cassava is further popularizing the crop as an alternative food source to help alleviate symptoms of celiac disease and gluten intolerance. This broadening interest in cassava led to a 60% increase in global harvest between 2000 and 2012. However, with climate change and a world population projected to exceed 9 billion by 2050, there is a continual need to produce and distribute elite crop varieties, including cassava, to improve yields and adaptation.
Cassava is an exceptional candidate for genetic engineering, but implementation of new plant breeding technologies (NPBT), including the genome editing CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated protein 9) system, remains a challenge. Similar to many other crops, cassava is recalcitrant toward genetic transformation and in vitro regeneration and exhibits poor fertility in some breeding lines and farmer-preferred varieties . Asynchronous maturation of the monoecious flowers means progeny are highly heterozygous and make conventional breeding programs for the introgression of traits very time-consuming. Moreover, flowering in a glasshouse environment seldom occurs, and sexual reproduction yields few seeds. Consequently, there is a necessity for accelerated flowering and improved breeding cycles in cassava to fully capitalize on genome editing technology for agriculture.
A NPBT combining genome editing to efficiently introduce homozygous mutations with accelerated flowering will allow the production of a segregated progeny for rapid crop improvement even in glasshouse environments.
Here, the researchers describe such a NPBT for cassava and demonstrate its use for engineering starch biosynthesis, modifying the quality of starch in storage roots.
The importance of starch in food and nutrition, as well as for industrial processing, has motivated the diversification of crops with desired starch traits
Integration of the Arabidopsis Flowering locus T gene in the genome-editing cassette allowed the researchers to accelerate flowering—an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes.
“Cassava plants harboring the transgene cassettes (pCas9-sgGBSS-FT or pCas9-sgPTST-FT) required for genome editing of GBSS or PTST1 and accelerated flowering were successfully produced using stable transformation of embryogenic calli. We confirmed in vivo expression of the transgenes, as well as localization of Cas9 fused to enhanced green fluorescent protein (eGFP) in the nuclei of protoplasts (in a transient assay) and stably transformed, regenerating somatic embryos.
A cleavage assay provided evidence that the designed ribonucleoprotein complexes encoded for in the cassettes were capable of DNA cleavage in vitro. Nucleotide insertions/deletions (indels) resulting from Cas9-mediated cleavage and nonhomologous end joining (NHEJ) were also successfully identified following sequencing of plant DNA” the researchers wrote.
This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.
Video : A new plant breeding technique to improve cassava cultivation: a proof-of-concept study for improved starch. This material relates to a paper that appeared in the Sep. 5th 2018, issue of Science Advances. The paper, by S.E. Bull at ETH Zurich in Zurich, Switzerland, and colleagues was titled, ” Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch.”