STTR Phase II: Novel Analysis Tools for Production of Higher Indican Yielding Plants for Bio-based Indigo

This Small Business Technology Transfer (STTR) Phase II project will develop a new tool for the analysis of in situ indican precursor in indigo plants, which when combined with genomic analysis and genetic linkage mapping in selectively bred indigo crops, will lead to high indican yielding breeding parental lines and ultimately competitively price natural indigo dye. Additionally, characterization of genetic markers will accelerate further crop improvement by understanding and harnessing the genes crucial to indican synthesis and other aspects of significance to overall indigo yield. These advancements will benefit customers, denim mills, by leading to a more reliable, lower cost plant-derived indigo supply. The success of this multiphase STTR project will enable a cost-competitive, cleaner, and more sustainable denim dyeing process, while greatly expanding the market for domestically produced natural indigo. Commercialization of a more consistent and higher yielding US-grown indigo plant that produces high purity indigo powder can replace the current standard of synthetic, imported indigo powder. While in demand by the marketplace today, plant-derived indigo is currently only used in premium denim products due to the high cost per pound resulting from low yields per plant per acre. The direct result of this research will be to open new market segments and expand existing market penetration for US-grown and manufactured biobased indigo for the textile industry, an addressable market of $1.86B. The methods and technology developed through this project have a direct path to the commercial marketplace and the industry is ready to support biobased textile dyes such as plant-derived indigo. During this project, reference genome resources will be constructed for indigo feedstock crops, F1 mapping populations will be constructed for P. tinctoria, I. tinctoria, and I. suffruticosa varieties, and design of the handheld rapid assay device will be validated through laboratory analysis. The reference genome for I. suffruticosa will be built using Pacific Biosciences SMRT sequencing and assembly. In addition to this reference genome, whole-genome resequencing will be performed on available I. suffruticosa and I. tinctoria varieties. Nucleotide variation and variant effect prediction will be made between the Indigofera-based indigo varieties. This variation will be used to develop markers to evaluate intervarietal crosses. Controlled greenhouse crosses of three species will be made to create F1 mapping populations for use in constructing a genetic map and linkage mapping, in combination with the resources developed. Intraspecies varieties exhibiting distinct phenotypic traits of commercial interest or notable dye yield differences will be selected for crossing to generate the F1 populations. Results from laboratory-based fluorometry equipment will be evaluated for efficacy and then compared against extractive indigo dye analysis from the leaf biomass. A final prototype will be constructed based on these findings and validated through use in the laboratory and in the field. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.