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Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia.

ABSTRACT: The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.

SUBMITTER: Koehorst-van Putten HJ

PROVIDER: S-EPMC3264866 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia.

Koehorst-van Putten H J J HJ Sudarmonowati E E Herman M M Pereira-Bertram I J IJ Wolters A M A AM Meima H H de Vetten N N Raemakers C J J M CJ Visser R G F RG

Transgenic research 20110405 1

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of gene ...[more]

PMID: 21465166

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Project description:Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.

Project description:BackgroundStarch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed "resistant starch" (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss.ResultsIn this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants.ConclusionsThis is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Project description:Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.

Dataset Information

Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia.

Publications

Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia.

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