Breeding Tomato Hybrids Rich in the Colorless Carotenoids Phytoene and Phytofluene with Resistance to Broomrape

Abstract Phytoene and phytofluene (P&P) are colorless carotenoids with emerging nutritional and health benefits, yet they accumulate at low levels in most edible crops. Tomato represents the principal dietary source of these compounds, but conventional cultivars contain only trace amounts. Here, we describe the development of tomato hybrids highly enriched in P&P through targeted breeding of carotenoid biosynthesis. Two recessive alleles of the ζ-carotene isomerase gene (Z-iso), designated ZETA (z), were identified in the processing cultivar M82. Loss of Z-ISO activity blocked downstream carotenoid synthesis, leading to massive accumulation of phytoene, phytofluene, and ζ-carotene in fruits and flowers. Despite the metabolic block, ZETA fruits displayed elevated total carotenoid content, associated with transcriptional upregulation of the phytoene synthase gene Psy1 and the formation of enlarged plastoglobuli. Stacking ZETA with the HIGH-PIGMENT1 (hp1) mutation and the dominant GOLDEN2-LIKE (GLK2) allele produced hybrids containing over 330 μg.g−1 fresh weight (FW) of total carotenoids, including 255 μg.g−1 FW P&P, levels unprecedented in any crop. However, ZETA roots showed severe deficiencies in abscisic acid (ABA) and strigolactones, associated with reduced yield. Reciprocal grafting demonstrated that wild-type rootstocks restored plant vigor and doubled the yield of P&P lines. Because ZETA roots produce lower levels of strigolactone, the mutation also conferred partial resistance to the parasitic weed Phelipanche aegyptiaca. Complete resistance was achieved by grafting z/hp1 scions onto SHOOT-BRANCHING1 (sb1) rootstock. Notably, extracts from P&P-rich tomato fruit have recently received approval from the European Food Safety Authority, underscoring the regulatory readiness and commercial relevance of this biofortification strategy.