Maize is an excellent nutritional source and is consumed as a staple food in different parts of the world, including India. Developing a maize genotype with a combination of higher lysine and tryptophan, along with β-carotene, can help alleviate the problem of protein-energy malnutrition (PEM) and vitamin A deficiency (VAD). This study is aimed at improving lysine and tryptophan content by transferring opaque-2 (o2) gene from donor HKI163 to β-carotene-rich inbred lines viz., UMI1200β⁺ and UMI1230β⁺. For this purpose, F₁, BC₁F₁, BC₂F₁, BC₂F₂, and BC₂F₃ plants were developed using an o2 line HKI163 and two β-carotene-rich inbred lines, UMI1200β⁺ and UMI1230β⁺, as the parents. Foreground selection using the associated marker umc1066 for the o2 gene and the marker crtRB1 3'TE for the crtRB1 gene was used to select the target genes. A total of 236 simple sequence repeat (SSR) markers distributed evenly across the maize genome were employed for the background selection. To fix the crtRB1 allele in the BC₁F₁ stage, individual plants homozygous at the crtRB1 locus and heterozygous at the o2 locus were selected and used for backcrossing to produce BC₂F₁ plants. Furthermore, the selected heterozygous BC₂F₁ plants from both crosses were selfed to obtain the BC₂F₂ plants, which were then selected for the target gene and selfed to generate the BC₂F₃ lines. From each cross, five improved lines with homozygous marker alleles for the crtRB1 and o2 genes with a recurrent parent genome (RPG) recovery ranging from 86.75 to 91.21% in UMI1200β⁺×HKI163 and 80.00 to 90.08% in UMI1230β⁺×HKI163 were identified. The improved lines had good agronomic performance and possessed high lysine (0.294-0.332%), tryptophan (0.073-0.081%), and β-carotene (6.12-7.38 µg/g) content. These improved lines can be used as genetic resources for maize improvement.

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