Post-harvest evaluation of high β-carotene maize (Zea mays L.) derived through marker-assisted introgression

Introduction

Maize (Zea mays L.) is a vital global cereal, providing up to 61% of caloric intake in some developing nations, yet typical varieties are severely deficient in PVA carotenoids, containing only 0.15 to 1.5 µg/g. This deficiency is a primary driver of childhood blindness and increased mortality rates globally. The carotenoid biosynthetic pathway in maize is regulated by the crtRB1 gene, which encodes β-carotene hydroxylase 1; the favorable allele 1 (543 bp) is known to reduce the conversion of β-carotene into non-PVA compounds, thereby increasing its accumulation in the endosperm (Yan et al., 2010 and Vinodhana et al., 2025) [13, 11] . For biofortification to be effective, these crops must not only possess high PVA content but also maintain nutritional stability during post-harvest processing (Garrett et [4,1,2,3,14] and show high bioavailability for human absorption.

Materials and Methods

Genetic Material and Hybridization: Eleven elite inbred lines (females) were crossed with two high beta-carotene UMI testers in a Line × Tester design at the Agricultural Research Station, Bhavanisagar.

Marker-Assisted Screening:

Foreground selection for the crtRB1 3'TE gene was conducted using specific primers (65F, 62R, 66R) to identify the favourable 543 bp allele.

Extraction and HPLC Quantification of Beta-Carotene

Seeds harvested from the hybrids and their corresponding parental lines were evaluated for β-carotene content. To minimize carotenoid degradation, seeds were harvested at approximately 14% moisture content and stored at 22-26 °C until analysis. β-Carotene extraction and quantification were performed according to the HarvestPlus protocol described by Rodriguez et al. (2004) [9] , with appropriate precautions to prevent photooxidative losses. Seed samples were finely ground in ice-cold acetone, and all subsequent extraction procedures were conducted under yellow light (Quackenbush, 1963; Weber, 1987) [8,12] . The carotenoid extracts were concentrated under reduced pressure using a rotary evaporator at 45 °C and reconstituted in methanol to a final volume of 2 mL. β-Carotene was separated and quantified using high-performance liquid chromatography (HPLC) equipped with a C18G 120A column (250 × 4.6 mm) and a photodiode array detector set at 450 nm. The mobile phase consisted of acetonitrile, methanol, and ethyl acetate (80:10:10, v/v/v) and was delivered at a flow rate of 1 mL min⁻¹. A calibration curve was generated using an authentic β-carotene standard (M/s. Sigma Aldrich, India) prepared in methanol at concentrations of 0.1, 1, 10, 50, and 100 μg/g β-Carotene was identified by matching its characteristic absorption spectrum and chromatographic retention time with those of the authentic standard. Quantification was subsequently performed using the corresponding standard calibration curve.

Bioavailability and Post-Processing Retention

Provitamin A bio-accessibility was determined via an in vitro stimulated gastrointestinal digestion model according to the procedure given by Garett (1999) [4] and Lakshminarayana et al., (2009) [5] with slight modification in both beta carotene introgressed and normal inbred lines. All analyses were performed in triplicate for raw maize samples, in vitro digested samples, and processed food products. Digestive stability (bio-availability) was determined by calculating the ratio of β-carotene recovered after the oral and intestinal phases of in vitro digestion to the initial β-carotene content present in the untreated maize samples. Post-processing retention was evaluated by cooking biofortified raw maize flour into traditional food products: popcorn, chapati, roti, and noodles, tracking terminal beta-carotene survival. Data analysis was carried out using STATA statistical software (StataCorp version 14.2). Descriptive statistics, including the mean and standard deviation (SD), were calculated from triplicate measurements to evaluate βcarotenoid retention in processed maize samples. The effects of processing on β-carotene levels were assessed using oneway analysis of variance (ANOVA), followed by Tukey's post hoc multiple comparison test. Statistical significance was defined as p < 0.05.

Results and Discussion

Beta-Carotene Accumulation, Bio-accessibility and Post-Processing Retention

The nutritional enhancement objective was conclusively met. HPLC quantification revealed that standard elite lines possessed negligible beta-carotene i.e., N 42 at 0.07 µg/g and N 104 at 0.10 µg/g, representing severe baseline deficiency (Table 1). The donors UMI 1200-7-26-1-6-1 and UMI 1230-2-70-9-6-1 naturally accumulated 8.40 µg/g and 6.44 µg/g, respectively. Estimation of β-carotene in hybrids revealed that among the 22 hybrids, the hybrid N 14 × UMI 1200-7-26-1-6-1 showed high β-carotene content followed by the hybrids N 5 × UMI 1200-7-26-1-6-1, N 90 × UMI 1200-7-26-1-6-1 and N 14 × UMI 1230-2-70-9-6-1. The top-performing agronomic hybrid N 14 × UMI 1200-7-26-1-6-1 synergistically accumulated a high beta-carotene content of 6.27 µg/g, making it the paramount dual-purpose cross.

In vitro-stimulated gastrointestinal digestion of the biofortified hybrid cross (UMI 1200β × UMI 1230β) showed an initial beta-carotene content of 7.41 µg/g, which decreased to 5.15 µg/g post-digestion (Table 2). This represents a remarkably high bio accessibility recovery rate of 69.51%, significantly superior to that of standard, nonfortified maize (49.38%) (Figure 1). Because carotenoids are structurally sensitive to isomerization and oxidation through heat and light, postprocessing retention was evaluated. Thermal food processing of biofortified raw flour initially (6.38 µg/g) led to varying degrees of degradation depending on the cooking method. In case of processed food products as expected the raw maize flour has a carotenoid content of (6.38±0.171 µg/g) which was higher when compared to the concentration (0.25-2.5 µg/g) generally reported for typical yellow maize varieties (Nuss and Tanumihardjo, 2010) [6] but still lower than the current breeding target for biofortified maize as set by HarvestPlus (17 µg/g of Provitamin A). The retention of β-carotene in the individual processed products roti, chapati and the noodles and popcorns revealed that, the exposure of β-carotene for high temperature and for prolonged time will reduce the retention of β-carotene and among these food products popcorn retained 57.68% (3.68 µg/g), chapati displayed the absolute lowest retention at just 27.90% (1.78 µg/g) and in noodles a moderate retention of 45.77% (2.92 µg/g) was observed (Table 3), since these were considerably exposed to light and temperature for long time when compared to roti which exhibited the highest retention at 67.87% (4.33 µg/g). As supported by Ortiz-Monasterio et al. (2007), β-carotene is affected by light, storage, and other environmental factors. These results indicate that additional research is required to improve the food processing methods to increase the retention of carotenoid concentration in maize hybrids, to reach the target set by HarvestPlus. This, in turn, reduces PVA deficiency and malnutrition. Crucially, the bio accessibility and postharvest fate of betacarotene determine the real-world success of this intervention. The structural integrity of carotenoids renders them deeply susceptible to thermal breakdown (Sowa et al., 2017) [10] . The processing data clearly show that severe dry heat (e.g., chapati, popcorn) destroys the vast majority of provitamin A. However, the preparation of roti has nearly 68% retention and a 63.47% digestive bio-accessibility, indicating that biofortified maize provides a highly effective, metabolizable dietary matrix for delivering provitamin A directly to vulnerable demographics.

Conclusion

The study concludes that integrating MAB for the crtRB1 allele is a highly efficient method for developing elite biofortified maize hybrids. The hybrid N 14 × UMI 1200-7-26-1-6-1 was identified as the paramount genotype, combining high yield potential with a beta-carotene content of 6.27 µg/g. Its superior digestive bio-accessibility (69.51%) and high retention during roti preparation demonstrate that biofortified maize is a ready-to-deploy tool to systematically alleviate Vitamin A deficiency and "hidden hunger" globally.

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