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Cottonseed remains a low-value by-product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ-cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild-type levels of gossypol and related terpenoids. Although it was a relatively small-scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild-type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%-8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever-increasing needs of humanity. [ABSTRACT FROM AUTHOR]

Global cottonseed production can potentially provide the protein requirements for half a billion people per year; however, it is woefully underutilized because of the presence of toxic gossypol within seed glands. Therefore, elimination of gossypol from cottonseed has been a long-standing goal of geneticists. Attempts were made to meet this objective by developing so-called ‘glandless cotton’ in the 1950s by conventional breeding techniques; however, the glandless varieties were commercially unviable because of the increased susceptibility of the plant to insect pests due to the systemic absence of glands that contain gossypol and other protective terpenoids. Thus, the promise of cottonseed in contributing to the food requirements of the burgeoning world population remained unfulfilled. We have successfully used RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with the expression of the δ-cadinene synthase gene during seed development. We demonstrate that it is possible to significantly reduce cottonseed-gossypol levels in a stable and heritable manner. Results from enzyme activity and molecular analyses on developing transgenic embryos were consistent with the observed phenotype in the mature seeds. Most relevant, the levels of gossypol and related terpenoids in the foliage and floral parts were not diminished, and thus their potential function in plant defense against insects and diseases remained untouched. These results illustrate that a targeted genetic modification, applied to an underutilized agricultural byproduct, provides a mechanism to open up a new source of nutrition for hundreds of millions of people. [ABSTRACT FROM AUTHOR]

The global output of cottonseed contains ∼10.8 trillion grams of protein that can meet the basic protein requirements of ∼590 million people at a rate of 50 g/day. However, gossypol, a toxic terpenoid present in seed glands, renders cottonseed unfit as food for human consumption or as feed for nonruminants. RNAi, under the control of a seed-specific promoter, was used to selectively silence δ-cadinene synthase gene to reduce gossypol levels in the seed by 97% without affecting the levels of gossypol and related terpenoids in rest of the plant where they are needed for defense against insects and diseases. The resulting Ultra-low Gossypol Cottonseed (ULGCS) is considered safe as food or as feed for more efficient (both, in terms of feed conversion ratio and protein conversion ratio) monogastric animals. Field trials conducted over multiple years in multiple states confirm the stability and heritability of the trait with no diminution of fiber/seed yield, quality or agronomic performance. A ULGCS event, TAM66274, was deregulated by USDA-APHIS in October 2018 and FDA concluded its food safety consultation in September 2019 and concurred with our determination of its safety. Global adoption of TAM66274, with more efficient and expanded usage of its protein, has the potential to significantly improve nutrition security and boost farmers' income without requiring additional inputs or acreage under cultivation, thus making cotton farming more sustainable. [ABSTRACT FROM AUTHOR]

Summary: In seeds and other parts of cultivated, tetraploid cotton (Gossypium hirsutum L.), multicellular groups of cells lysigenously form dark glands containing toxic terpenoids such as gossypol that defend the plant against pests and pathogens. Using RNA‐seq analysis of embryos from near‐isogenic glanded (Gl2Gl2Gl3Gl3) versus glandless (gl2gl2gl3gl3) plants, we identified 33 genes that expressed exclusively or at higher levels in embryos just prior to gland formation in glanded plants. Virus‐induced gene silencing against three gene pairs led to significant reductions in the number of glands in the leaves, and significantly lower levels of gossypol and related terpenoids. These genes encode transcription factors and have been designated the 'Cotton Gland Formation' (CGF) genes. No sequence differences were found between glanded and glandless cotton for CGF1 and CGF2 gene pairs. The glandless cotton has a transposon insertion within the coding sequence of the GoPGF (synonym CGF3) gene of the A subgenome and extensive mutations in the promoter of D subgenome homeolog. Overexpression of GoPGF (synonym CGF3) led to a dramatic increase in gossypol and related terpenoids in cultured cells, whereas CRISPR/Cas9 knockout of GoPGF (synonym CGF3) genes resulted in glandless phenotype. Taken collectively, the results show that the GoPGF (synonym CGF3) gene plays a critical role in the formation of glands in the cotton plant. Seed‐specific silencing of CGF genes, either individually or in combination, could eliminate glands, thus gossypol, from the cottonseed to render it safe as food or feed for monogastrics. [ABSTRACT FROM AUTHOR]

Summary Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability. [ABSTRACT FROM AUTHOR]