Integrating Cytochrome P450-Mediated Herbicide Tolerance into Anthocyanin-Rich Maize Through Conventional Breeding

Abstract

Meeting the rising demand for staple grains now requires cultivars that combine high yield, enhanced nutritional value, and strong chemical resilience. Blue-kernel landraces from central Mexico are rich in anthocyanins yet remain highly susceptible to post-emergence herbicides, whereas modern hybrids detoxify these compounds through cytochrome P450 (CYP450) enzymes. We crossed the anthocyanin-rich variety Polimaize with a CYP450-tolerant hybrid and evaluated the two parents and their F₁ segregants (designated “White” and “Yellow”) under greenhouse applications of mesotrione (75 g a.i. ha⁻¹), nicosulfuron (30 g a.i. ha⁻¹), and their mixture. Across 160 plants, the hybrid retained 95% of control dry matter and showed ≤7% foliar injury under all treatments, whereas Polimaize lost 28% biomass and exhibited 36% injury after nicosulfuron. The Yellow class matched hybrid performance while maintaining a blue pericarp and a β-carotene-rich endosperm, demonstrating that nutritional and agronomic traits can be stacked. The White class displayed heterosis-driven compensatory growth, exceeding its untreated biomass by 60% with nicosulfuron and by 82% with the mixture despite transient bleaching. Chlorophyll and carotenoid fluorescence revealed rapid, zeaxanthin-linked photoprotection in all tolerant genotypes, consistent with accelerated CYP450-mediated detoxification. These findings show that broad-spectrum herbicide tolerance can be introgressed into pigment-rich germplasm through conventional breeding, providing a non-transgenic path to herbicide-ready, anthocyanin-rich maize. The strategy preserves local biodiversity while delivering cultivars suited to intensive, weed-competitive agriculture and offers a template for integrating metabolic resilience into other native crops.

1. Introduction

Feeding a global population that is projected to approach ten billion within the next three decades requires agriculture to produce larger harvests while also improving the nutritional profile of staple foods and reducing environmental impacts [1,2]. The dominant strategy of the Green Revolution (replacing heterogeneous landraces with high-input hybrids) delivered remarkable yield gains but simultaneously narrowed the genetic base of major crops. This contraction threatens long-term resilience and weakens the cultural ties between farming communities and the germplasm they have stewarded for millennia [3].

Nowhere is this tension clearer than in Mexico, the center of origin and diversification of maize (Zea mays L.). Molecular, archeological, and ethnobotanical evidence identifies the state of Michoacán as a focal point in the domestication and subsequent evolution of maize, common bean (Phaseolus vulgaris L.), squash (Cucurbita spp.), and Physalis spp. [4,5]. Recent landscape-genomic surveys confirm that this region still harbors extensive allelic diversity [6], a resource now leveraged by the Polimaize initiative, which introduces agronomically valuable traits from local landraces (chiefly anthocyanin pigmentation) into elite backgrounds through conventional breeding [7,8].

Anthocyanins deposited in the pericarp give Michoacán maize its deep blue-to-purple color. Beyond their culinary appeal, these flavonoids scavenge reactive oxygen species, improve endothelial function, lower blood pressure, and are associated with reduced cognitive decline [9,10,11]. In plants, anthocyanins also contribute to photoprotection and stress signaling, roles partly orchestrated by cytochrome P450 (CYP) mono-oxygenases such as CYP93B, CYP93E, and CYP93G, which channel phenylpropanoid precursors into flavonoid branches of the pathway. Cytochrome P450 mono-oxygenases (CYPs) constitute a superfamily of ≈300 genes in maize, grouped into ten major clans. Among them, subfamilies CYP81, CYP72, and CYP709 have repeatedly been implicated in the oxidative detoxification of sulfonylurea and triketone herbicides. In particular, CYP81A6 and CYP81A9 catalyze hydroxylation steps that render nicosulfuron and mesotrione biologically inactive, thereby conferring field-level tolerance [11,12,13]. These combined agronomic and nutraceutical benefits explain the growing market demand for colored maize.

A major barrier to wider adoption of blue landraces is their pronounced sensitivity to standard post-emergence herbicides. Mesotrione, a 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) inhibitor, induces transient bleaching, whereas nicosulfuron, an acetolactate-synthase (ALS) inhibitor, causes chlorosis, necrosis, and yield loss at recommended doses; Polimaize shows both symptoms, in contrast to modern white hybrids that remain virtually uninjured. Tolerance in those hybrids derives from rapid induction of a small subset of CYP450 genes (about one percent of the maize genome) that oxidize mesotrione, nicosulfuron, and related xenobiotics, thereby accelerating conjugation and vacuolar sequestration [12,13,14]. Isoforms such as CYP81A9 and CYP81A6, responsible for phase I herbicide detoxification through hydroxylation reactions, achieve peak transcript levels within twenty-four hours of exposure; this surge precedes visible plant recovery and demonstrates their direct contribution to herbicide tolerance [15].

Embedding such metabolic tolerance without transgenes is feasible. Conventional selection in sweet corn populations produced lines able to withstand four-fold mesotrione rates, and heterozygous hybrids often displayed greater detoxification capacity than either parent, suggesting additive or synergistic CYP activity [16,17,18,19]. Accurate phenotyping is essential for similar progress in colored maize. Chlorophyll and carotenoid fluorescence provide early, nondestructive indicators of HPPD inhibition (mesotrione typically elevates zeaxanthin fluorescence and depresses β-carotene and lutein signals) whereas ALS inhibitors perturb chlorophyll-a fluorescence through branched-chain amino-acid depletion [20,21,22]. Combined applications can stimulate de novo anthocyanin synthesis, detectable as red-edge fluorescence shifts [23]. When these optical markers are integrated with dry-matter reduction indices and absolute growth rates, they offer a comprehensive view of physiological resilience [24].

Against this backdrop, we asked whether CYP450-mediated herbicide tolerance can be combined with anthocyanin-rich germplasm without sacrificing either trait. By crossing Polimaize with a proven tolerant hybrid and examining parental lines and segregating progeny under mesotrione, nicosulfuron, and their mixture, we quantified foliar damage, biomass dynamics, and multi-waveband fluorescence to elucidate the biochemical and physiological bases of tolerance. Demonstrating compatibility between metabolic detoxification and high anthocyanin content would provide a blueprint for breeding maize that meets the intertwined goals of agronomic performance, nutritional enhancement, and conservation of Mexico’s living crop heritage.

2. Materials and Methods

2.1. Biological Material

Two parental genotypes were used: the anthocyanin-rich variety Polimaize, developed at the Instituto Politécnico Nacional through recurrent selection among Michoacán landraces and CIMMYT CML lines [8], and the white-kernel hybrid Cimarrón^® released by Asgrow Mexico (hereafter Commercial Hybrid). Polimaize bears a blue pericarp with high anthocyanin content and a yellow endosperm that accumulates carotenoids, whereas the Commercial Hybrid adapted to temperate, sub-humid regions (mean 25 °C; 600–1000 mm annual rainfall) has white pericarp and endosperm and has shown field tolerance to mesotrione and nicosulfuron. Controlled crosses were carried out in a glasshouse (28 ± 3 °C, 60 ± 5% RH, 14 h photoperiod). Polimaize served as the pollen donor and the Commercial Hybrid as the female parent. F₁ ears were air-dried at 25 °C for ten days, then kernels were sorted by pericarp and endosperm color. Two segregant classes were retained: blue-pericarp/white-endosperm (White) and blue-pericarp/yellow-endosperm (Yellow). All four genetic groups (Polimaize, Commercial Hybrid, White, and Yellow) were evaluated concurrently. The experiments were conducted from June to September 2024 in naturally ventilated, passively temperature-controlled greenhouses (19–31 °C; 45–65% RH) at the Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR), Michoacán Unit, Instituto Politécnico Nacional (19°59′58″ N, 102°42′22.6″ W; 1980 m a.s.l.).

2.2. Plant Establishment and Herbicide Treatments

Surface-sterilized seeds (2% NaOCl, 5 min) were rinsed and germinated for 48 h on moist filter paper at 25 °C. Seedlings with a 30 mm radicle were transplanted into 8 L pots (25 cm Ø × 20 cm h). They were planted with a single maize seedling (one plant pot⁻¹) to prevent intraspecific competition and to ensure that the pot constituted the experimental unit, filled with a 2:1:1 (v/v/v) mix of peat moss, perlite, and vermiculite (pH 6.0; EC 0.8 dS m⁻¹). A total of 160 pots were planted (10 per genotype × treatment combination). Soil water potential was kept near field capacity (≈−10 kPa) by drip irrigation. When plants reached 20 ± 1 cm (V3–V4 stage), they were sprayed with a CO₂-pressurized backpack sprayer (R & D Sprayers, Opelousas, LA, USA, 250 kPa, XR11002 nozzle, 200 L ha⁻¹). Three post-emergence herbicide regimes were evaluated using Syngenta formulations registered in Mexico. Mesotrione, supplied as Callisto^® 480 SC (480 g a.i. L⁻¹) was applied at 0.156 L ha⁻¹, delivering 75 g a.i. ha⁻¹; nicosulfuron, formulated as Sanson^® 4 SC (40 g a.i. L⁻¹), was applied at 0.75 L ha⁻¹, providing 30 g a.i. ha⁻¹; and a tank mix containing both actives at these respective rates completed the set of treatments. Untreated controls received distilled water plus 0.1% (v/v) of the non-ionic surfactant Trend^® 90. All applications were conducted at 09:00 h under ambient conditions of 26 ± 2 °C and 55 ± 4% relative humidity. The chosen doses lie within the label ranges for post-emergence maize treatments and reflect common agronomic practice in the study region.

2.3. Foliar Phenotyping

Leaf injury was quantified fifteen days after treatment (15 DAT). High-resolution images of the two youngest fully expanded leaves were taken with a Canon EOS 80D (Canon Inc., Tokyo, Japan) under diffuse light and analyzed in ImageJ v1.54p to calculate the proportion of tissue exhibiting bleaching or necrosis relative to total leaf area. For pigment fluorescence, four 2 cm² disks were excised from the third expanded leaf, rinsed, blotted dry, and placed adaxial side up in a Cytation™ 5 multimode reader. Chlorophyll fluorescence was recorded across nine excitation wavelengths between 400 and 650 nm; fluorescence of luteolin (Ex 260 nm), quercetin (Ex 340 nm) and β-carotene (Ex 520 nm) was also measured. Raw values were normalized to the corresponding untreated control of each genotype. Relative fluorescence (RF) was calculated as the ratio of the signal recorded in herbicide-treated tissue to the basal fluorescence of the corresponding control, thereby normalizing variability arising from leaf anatomy and intrinsic pigment content. An RF > 1 denotes increased emission, indicative of compensatory pigment accumulation, whereas an RF < 1 points to photobleaching or pigment degradation.

2.4. Dry-Matter Reduction Index and Absolute Growth Rate

Above-ground biomass was harvested 2 h before herbicide application (0 DAT) and again at 15 DAT, oven-dried at 80 °C for 72 h, and weighed. The dry-matter reduction index (DMRI) was calculated as the ratio of dry mass in treated plants to that in controls; values < 1 indicate biomass loss, whereas values > 1 indicate compensatory growth. The absolute growth rate (AGR) for the 15-day interval was calculated following Hunt [25]:

AGR = (DW₂ − DW₁)/(t₂ − t₁)

(1)

Initial dry weight (DW₁) was obtained from a parallel destructive sampling of ten plants per genotype harvested immediately before herbicide application (0 DAT); samples were oven-dried at 80 °C for 72 h, weighed, and the mean value was used in AGR calculations. Where DW₁ and DW₂ are dry weights at 0 and 15 DAT, and t₁ and t₂ the corresponding days after sowing.

2.5. Experimental Design and Statistical Analysis

The experiment followed a completely randomized factorial design with two factors: genotype (Polimaize, White, Yellow, Commercial Hybrid) and herbicide treatment (control, mesotrione, nicosulfuron, mesotrione + nicosulfuron). Each of the 16 genotype-by-treatment combinations was replicated ten times (one pot = one replicate). Response variables were expressed as percentages relative to their respective control to minimize baseline heterogeneity. Data were checked for normality (Shapiro–Wilk) and homoscedasticity (Levene); where necessary, log₁₀ or square-root transformations were applied. Analyses of variance were conducted at α = 0.05, and mean separation used Tukey’s HSD test (α = 0.05) in R v4.3.2.

3. Results

3.1. Dry-Matter Retention and Visual Injury

Two-way ANOVA showed significant main effects of genotype, herbicide, and their interaction on the dry-matter reduction index (DMRI) and foliar damage (FD) (p < 0.05). After mesotrione application (75 g a.i. ha⁻¹), all lines retained at least 75% of control biomass (DMRI ≥ 0.75;

Table 1. Dry-matter reduction index (DMRI) and visual injury (FD) of four maize genotypes 15 days after post-emergence herbicide treatments.

	Mesotrione		Nicosulfuron		Meso + Nico
Genotype	DMRI	FD (%)	DMRI	FD (%)	DMRI	FD (%)
Parents
Polimaize (blue landrace)	0.75 ± 0.11 a	0.30 ± 0.10 ab	0.72 ± 0.18 b	0.36 ± 0.09 a	0.77 ± 0.17 b	0.45 ± 0.08 a
Cimarrón® (white hybrid)	0.96 ± 0.09 a	0.07 ± 0.08 b	0.98 ± 0.16 ab	0.03 ± 0.07 b	0.95 ± 0.15 b	0.04 ± 0.07 b
F1 segregants
Yellow (blue/yellow)	0.83 ± 0.10 a	0.26 ± 0.09 ab	0.97 ± 0.17 ab	0.42 ± 0.08 a	0.87 ± 0.15 b	0.10 ± 0.07 b
White (blue/white)	0.89 ± 0.12 a	0.48 ± 0.09 a	1.60 ± 0.19 a	0.44 ± 0.09 a	1.82 ± 0.18 a	0.10 ± 0.08 b
p-value (ANOVA)	NS	**	***	***	***	*
HSD (α = 0.05)	0.41	0.37	0.68	0.33	0.62	0.30

DMRI = dry-matter reduction index (DWtreated/DWcontrol); FD = percentage of leaf area with bleaching or necrosis. Values are means ± standard error (n = 10 pots, one plant pot⁻¹). Different superscript letters within a column indicate significant differences by Tukey’s HSD test (α = 0.05). Significance codes for genotype × treatment ANOVA: NS = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001. Genotype classes: Polimaize = blue-kernel landrace; Cimarrón^® = cytochrome-P450-tolerant white hybrid; Yellow and White = F₁ segregants differing in endosperm color.

). Visual symptoms, however, diverged sharply: the White segregant displayed 48% bleaching, the Commercial Hybrid only 7%, and Polimaize and the Yellow segregant intermediate values of 30% and 26%, respectively. At 3 DAT, bleaching in susceptible leaves first appeared as a translucent ivory halo along the distal margins; by 7 DAT, this halo had expanded into porcelain-white bands covering the distal two-thirds of Polimaize blades, whereas tolerant genotypes remained fully green except for scattered straw-colored pin-points (<0.5 mm) on tertiary veins. By 15 DAT, susceptible leaves exhibited cinnamon-brown mosaics of coalescing necrotic speckles (≈0.8 mm), while the Hybrid and Yellow segregant preserved >90% green area and the White segregant produced additional leaf tissue despite persistent whitening. Thus, mesotrione injury was largely cosmetic and genotype dependent.

Nicosulfuron (30 g a.i. ha⁻¹) produced a sharper contrast. Polimaize lost 28% dry matter, and its FD reached 36%, confirming sensitivity. Early nicosulfuron damage manifested as diffuse interveinal chlorosis at 3 DAT, progressing to complete bleaching with marginal necrosis by 10 DAT in Polimaize; in contrast, tolerant genotypes showed only faint midrib halos and maintained turgid, fully green canopies throughout the assessment period. Biomass in the Commercial Hybrid and Yellow segregant remained unchanged (DMRI ≈ 1.0), and FD stayed below 5%. The White segregant behaved atypically: despite 44% bleaching, it accumulated 60% more biomass than its control (DMRI = 1.60). The mesotrione + nicosulfuron mixture reinforced the pattern: Polimaize fell to DMRI = 0.77 with 55% damage; Yellow showed a moderate decrease (DMRI = 0.87); the Hybrid again was virtually unaffected (DMRI = 0.95; FD = 4%); and White over-compensated further (DMRI = 1.82) without additional injury.

3.2. Absolute Growth Rate

Absolute growth rate (AGR) mirrored the biomass trends (

Table 2. Absolute growth rate (AGR) of four maize genotypes 15 days after post-emergence herbicide treatments (greenhouse, n = 10).

	Parents		F1 Segregants
Treatment	Polimaize (Blue Landrace)	Cimarrón® (White Hybrid)	Yellow (Blue/Yellow)	White (Blue/White)
Control	7.1 ± 0.95 a	4.5 ± 0.90 a	4.9 ± 1.60 a	0.5 ± 1.20 b
Mesotrione	2.6 ± 0.85 ab	3.8 ± 0.88 a	2.3 ± 1.50 a	0.1 ± 1.10 b
Nicosulfuron	2.4 ± 1.05 b	4.7 ± 1.02 a	4.0 ± 1.70 a	5.7 ± 1.90 ab
Meso + Nico	3.0 ± 1.00 ab	3.9 ± 0.94 a	2.7 ± 1.55 a	7.6 ± 2.10 a
p-value (ANOVA)	*	NS	*	NS
HSD (α = 0.05)	4.6	4.9	8.6	6.5

Different letters within columns indicate significant differences (Tukey, p ≤ 0.05). The data are presented as the means ± standard error. n = 10. AGR = (DW₂ − DW₁)/(t₂ − t₁), where DW = dry weight and t = time (days). Values are means ± standard error (n = 10 pots, one plant pot⁻¹). Superscript letters within a column indicate significant differences among treatments for each genotype (Tukey’s HSD, α = 0.05). ANOVA significance codes: NS = p > 0.05; * = p ≤ 0.05. Genotype classes: Polimaize = blue-kernel landrace; Cimarrón^® = cytochrome-P450-tolerant white hybrid; Yellow and White = F₁ segregants differing in endosperm color.

). Under control conditions, Polimaize grew fastest (7.1 mg plant⁻¹ day⁻¹), followed by Yellow (4.9 mg day⁻¹) and the Commercial Hybrid (4.5 mg day⁻¹); White expanded only 0.5 mg day⁻¹. Mesotrione reduced Polimaize AGR to 2.6 mg day⁻¹ but left the other genotypes unaffected. Nicosulfuron imposed the steepest penalty on Polimaize (−66%) yet propelled White to 5.7 mg day⁻¹—a tenfold rise. The mixture maintained this polarity: Polimaize stabilized at 3.0 mg day⁻¹, whereas White climbed to 7.6 mg day⁻¹, exceeding Polimaize’s untreated rate; Yellow and the Hybrid held their reference values.

3.3. Chlorophyll Fluorescence

Spectral data confirmed photosynthetic recovery after herbicide stress. Mesotrione increased total chlorophyll fluorescence (400–650 nm) in Polimaize (+22%), Yellow (+34%), and the Hybrid (+29%) but pushed it below unity in White, consistent with its high FD (Figure 1A). After nicosulfuron, all genotypes converged near the control value, and the mixture restored fluorescence in White to the tolerant range (Figure 1B,C), indicating that photosystems rebound even under dual herbicide pressure.

3.4. Secondary-Pigment Fluorescence

Ratios for luteolin and β-carotene remained near 1.0 across all treatments, indicating that xanthophyll cycling was scarcely disturbed. Quercetin fluorescence rose slightly after nicosulfuron and the mixture in every genotype, though not significantly. Under mesotrione alone, Polimaize and the Hybrid displayed the strongest quercetin signals (Figure 2), suggesting faster mobilization of antioxidant flavonols in backgrounds equipped with a full CYP450 detoxification complement.

4. Discussion

The dataset reveals a nuanced physiological landscape in which three defensive modes emerge. At one extreme, the Commercial Hybrid appears metabolically primed: we infer that CYP81A6, CYP81A9, and allied isoforms are present at appreciable basal levels and may be induced early enough to hydroxylate mesotrione and nicosulfuron before they disrupt carotenoid synthesis or branched-chain amino acid metabolism [15,26]. Fluorescence data support this view; under mesotrione, the hybrid’s chlorophyll emission rises by roughly 30%, a hallmark of photoprotective xanthophyll cycling, and its quercetin signal (an early marker of flavonoid biosynthesis) also ranks among the highest (Figure 2), consistent with CYP97-mediated antioxidant bursts in tolerant lines [12]. Although the phenotypic tolerance is congruent with early CYP induction, confirmation will require RT-qPCR or RNA-seq profiling in follow-up studies. Together, these traits create a physiological buffer that safeguards growth and photosystem integrity even under the tank mix, mirroring field scenarios in which growers routinely combine HPPD and ALS inhibitors for broad-spectrum weed control [17].

Polimaize sits at the opposite pole. Its capacity to tolerate mesotrione was expected because most maize backgrounds harbor at least one HPPD-detoxifying CYP allele. Crucially, its 25–30% foliar bleaching is purely cosmetic: no biomass is lost, confirming that oxidative injury is transient and that carotenoid biosynthesis rebounds once mesotrione is hydroxylated [13,22]. Yet the same genotype fails under nicosulfuron, losing roughly one-third of its dry matter even though chlorophyll fluorescence barely changes. ALS inhibition compromises protein synthesis and cell expansion long before photochemical signatures appear [27], explaining why necrosis in Polimaize does not track the modest fluorescence shift in Figure 1B. These results remind breeders that optical sensors alone cannot classify ALS tolerance; biomass- or growth-based metrics must accompany any high-throughput screen.

The two segregants illustrate how these antagonistic alleles recombine. Yellow inherits the hybrid’s tolerance while retaining the anthocyanin-rich pericarp and β-carotene endosperm of the landrace, aligning with the trend to stack quality and tolerance traits [28]. White, however, is more provocative. Heterozygous for the hybrid’s detoxification alleles, it shows deeper mesotrione bleaching and a transient 12% decline in chlorophyll but, after nicosulfuron, rebounds into an anabolic surge that raises dry matter 60–80% above the control. Such overcompensation recalls herbivore-induced reallocations mediated by flavonoid- and auxin-responsive CYPs [12,13]. Tolerant inbreds up-regulate more than 50 CYP genes within 24 h of ALS stress [29]; in a heterozygous background, expression waves may become desynchronized, creating a window in which detoxification is active while growth signaling exceeds parental baselines, consistent with White’s AGR of 7.6 mg plant⁻¹ day⁻¹ under the mix.

This compensatory phenotype is double-edged. It can buffer yield under herbicide drift, drought pulses, or mild hail (any stress that removes photosynthetic leaf area but leaves meristems alive) yet it increases phenotypic variance. Dissecting CYP81A6/A9 dosage and promoter haplotypes will clarify whether heterozygosity per se, or specific regulatory combinations, drives the response [19]. Marker-assisted selection could then stabilize desirable plasticity while discarding lines that channel resources into pigment repair rather than grain fill.

Ecological implications also warrant attention. Gene flow among landraces in the Trans-Mexican Volcanic Belt is common; introgression of broad-spectrum detoxification alleles into that mosaic could buffer ultra-sensitive blue varieties against drift or, conversely, blur distinctive phenotypes an issue that calls for socio-genomic monitoring [3].

Nutritionally, Yellow’s dual enrichment addresses pro-vitamin A and antioxidant deficiencies without the yield penalty that has hampered earlier biofortified cultivars. Multilocation trials will determine whether its hybrid-level performance holds under farm conditions.

The absence of synergistic injury in the tank mix challenges the notion that combining modes of action necessarily heightens crop stress. CYP81A9 metabolizes both mesotrione and sulfonylureas [12], and mesotrione can reduce nicosulfuron translocation by about 9% [30]. Even sensitive Polimaize suffers no additive biomass loss beyond that caused by nicosulfuron alone, suggesting that simultaneous spraying could save labor and fuel where tolerant alleles are fixed.

Overall, the Polimaize cross demonstrates that conventional breeding can reconcile high-value pigmentation, carotenoid enrichment, and modern herbicide tolerance. It reveals an unexpected heterosis window in which ALS stress becomes a growth stimulus, underscoring how little of the CYP450 network’s adaptive breadth has been tapped. Moving from pots to on-farm trials, from bulk fluorescence to tissue-specific transcriptomes, and from single crosses to multiparent populations will indicate whether this fusion of heritage and innovation can sustain Mexican blue maize in the twenty-first century—an achievement grounded not in transgenic shortcuts but in the evolutionary versatility of the CYP450 family.

5. Conclusions

A single conventional cross successfully combined the vivid pigmentation of a Michoacán landrace with the broad-spectrum herbicide tolerance typical of elite hybrids. All four genotypes handled mesotrione efficiently, confirming that cosmetic leaf bleaching is a poor proxy for functional susceptibility and that biomass- or growth-based metrics are indispensable for large-scale screening. Under nicosulfuron, three distinct responses emerged: a constitutively tolerant hybrid; a sensitive landrace; and two segregants—Yellow, which paired full tolerance with elevated carotenoid and anthocyanin content, and White, for which we hypothesize that a heterozygous CYP81A profile attenuated ALS stress, leading to an unexpected biomass surge. The absence of synergistic injury in the mesotrione + nicosulfuron mix suggests functional redundancy within the maize CYPome, although molecular confirmation of this redundancy remains to be demonstrated, and it indicates that simultaneous field application is agronomically sound once tolerant haplotypes are fixed. Overall, our findings demonstrate that nutritional enhancement and chemical resilience are compatible breeding objectives and that judicious use of native CYP450 (pending molecular validation of the candidate isoforms) diversity can yield cultivars meeting modern weed-management demands while preserving the cultural and nutritional heritage of Mexican blue maize. Validation across multiple environments, together with allele-dosage mapping and transcriptomic dissection, will be essential for translating these greenhouse insights into stable, farmer-ready varieties and for confirming the CYP-driven mechanisms proposed here.