BluApple™: A Genetically Engineered Blue-Fleshed Apple via Dual Pathway Modulation

BluApple™ is a genetically engineered apple that exhibits stable blue to blue-purple flesh coloration through coordinated metabolic engineering of carotenoid and anthocyanin pathways. This technical report documents the full scientific basis, construct designs, sequence elements, and transformation strategies underlying the BluApple system.

The blue-flesh phenotype is achieved through a dual-pathway approach:

1. Carotenoid suppression:
   Disruption of key carotenoid biosynthesis genes (including PSY1, LCYB, ZDS, and PDS) using CRISPR-Cas or other gene-silencing methods to eliminate yellow background pigments.

2. Anthocyanin enhancement:
   Introduction of a multigene anthocyanin expression cassette encoding CHS, CHI, F3H, F3'5'H, DFR, ANS, and UFGT under a fruit-specific promoter, enabling high-level biosynthesis of delphinidin-based pigments in apple cortex tissue.

Optional modules for vacuolar pH modification and metal-ion cofactors are included to stabilize blue anthocyanin complexes, based on prior findings in ornamental and model species.

This report provides:

• A complete patent-style specification of the BluApple platform

• Detailed background on pigment biosynthesis in fruit crops

• Full construct designs for carotenoid suppression (Vector A) and anthocyanin gene stacking (Vector B)

• FASTA sequences for all pathway genes

• A WIPO ST.26-compliant sequence listing (XML)

• Transformation and regeneration methodology

• Phenotypic evaluation protocols

• A comprehensive scientific bibliography supporting each component of the design

The BluApple platform extends to other fleshy fruits (pear, cherry, peach, plum, nectarine) with compatible metabolic architectures.

This Zenodo deposit serves as a formal and citable record of the underlying scientific design, enabling archival reference, reproducibility, and future development.