Environmental impact of phytic acid in Maize (Zea mays. L) genotypes for the identification of stable inbreds for low phytic acid

Abstract

Phytic acid is a ubiquitous compound that chelates the micronutrients in food and hinder their absorption. Hence, breeding for low phytate content for producing stable low phytic acid (lpa) hybrids is essential. Phytic acid content in maize grains has been found to vary across environments and its stable expression has yet to be explored. In a view of this, forty inbreds were screened with two checks viz., CO-6 and CO-H(M)-8 across three locations. Twenty morphological and three quality traits were observed to identify the stable lines for low phytic acid with higher free inorganic phosphorous and starch. Among all the lines, UMI-467, LPA-2-285, LPA-2-395 and UMI-447 recorded a stable performance in both AMMI and GGE biplot analysis for low phytic acid (2.52–3.32 mg/g). These lines also had a higher free inorganic phosphorous, ensuring its bioavailability (1.78–1.88 mg/g). There were perturbations in yield, starch and seed characteristics of the stable low phytic acid lines due to their lower phytic acid concentrations. This stated the role of phytic acid in plant physiology and established the constraints to be faced in breeding for low phytic acid in maize. Among the lpa lines, LPA-2-285 (57.83%) and UMI-447 (55.78%) had the highest average starch content. The lowest stable phytic acid content was observed in UMI-467 (2.52 mg/g) and this line had severe reductions in yield parameters. Considering the seed and yield characteristics, LPA-2-285, LPA-2-395 and UMI-447 performed better than UMI-467. Although these four stable lines were poor in their adaptability among all the genotypes, they could be utilised as promising stable donors to facilitate the development of stable lpa hybrids.

Electronic supplementary material

The online version of this article (10.1007/s12298-020-00818-x) contains supplementary material, which is available to authorized users.

Introduction

The current century thrives on increased food production with enhanced nutritional values to combat the prevailing nutritional deficiencies. More than 50% of the world’s calorie intake is provided by the cereals with lower bioavailability of micronutrients. Maize being the third important cereal crop after rice and wheat, is utilized by around 63% of the world as food and poultry feed (Shah et al. 2016). It is an essential source of carbohydrate, protein, iron, vitamin B and minerals such as phosphorous, magnesium, manganese, zinc, copper, iron, selenium and calcium, but its nutritional bioavailability is affected by the presence of an anti-nutritional factor known as phytic acid.

Phytic acid (Myoinositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is a ubiquitous compound of the myoinositol-6 phosphate pathway. In plants, 85% of the total phosphorous is present in the form of phytic acid and this is degraded by phytase during germination to restore the phosphorous required for the plant development. This phytic acid also plays a key role in signalling, responses to environmental stimuli and acts as a major component in sugar signalling and chromatin modification (Latrasse et al. 2013). Considering its physiological pH, it is a polyanion with six to eight negative charges and acts as a strong chelator of positively charged mineral cations (Raboy 2000). When consumed, this dietary phytic acid binds to seed derived minerals and endogenous minerals in the gut. Due to the lack of phytase in monogastric animals, the undigested phytic acid-mineral complex is excreted as salts (Raboy 2000). Thus, phytic acid is considered as an anti-nutritional factor, affecting the mineral bioavailability in food (Zhou and Erdman 1995). This issue has been critical in the poultry sector as 60% of the poultry feed includes maize. In poultry, to enhance the body weight gain and feed conversion ratio, fungal phytase has been supplemented with maize to enhance the absorption of these minerals. This supplementation, on the other hand, increased the cost of feed with poor efficiency in micronutrient absorption (Lelis et al. 2012). Hence, it was found that utilization of natural lpa (low phytic acid) mutant lines in breeding programs could help in the enrichment of nutrient absorption in humans and other non-ruminants (Raboy 2000).

Phytic acid is produced from the branching myoinositol 6-phosphate pathway involving several components of starch and polysaccharides like raffinose and fructose, that responds to diverse environmental stimuli (Sparvoli and Cominelli 2015). This suggests the possibilities of its variation across environments. Further, the studies on phytic acid conducted previously for screening low phytic acid lines in maize exhibited variation in phytic acid content across locations (Jacob 2016 unpublished). The literatures on stability of phytate in maize are limited and the research programs to identify stable lpa lines are in progress.

In other cereals like wheat, phytic acid is reported to vary across environments due to its crossover interactions towards temperature and water stress conditions (Brankovick et al. 2015). There are also studies reporting the influence of environment on phytic acid in crops such as sorghum and flaxseed. They also identified potential varietal lines for stable low phytic acid content in sorghum and flaxseed (Kayode et al. 2006; OOmah et al. 1996).

Hence, stable expression of phytic acid content in maize across environments has to be ensured (Sparvoli and Cominelli 2015) for its effective utilization in hybrid breeding programs. Further, it is expected that breeding for reduced phytate levels might compromise seed germination and yield, as there are negative correlations between grain yield and low grain phytate (Lorenz et al. 2007). Thus, this study was undertaken to identify the stable inbreds for low phytic acid to reinforce the development of hybrids with stable low phytic acid in maize.

Materials and methods

Forty inbreds with two checks CO-6 and CO-H(M)-8 were raised in randomized block design across three locations (E1: Coimbatore, E2: Vagarai and E3: Bhavanisagar) with two replications for studying the environmental influence of phytic acid in maize (Table 1). Observations on twenty morphological characters viz., days to 50% tasselling, days to 50% silking, anthesis silking interval, tassel length, number of tassel branches, cob placement height, plant height, number of kernels per row, number of rows per kernel, cob length, cob girth, cob weight, shelling per cent, shank weight single plant yield, hundred seed weight, seed length, seed girth, seed thickness, seedling vigour index (Abdul-Baki and Anderson 1973) and three biochemical traits viz; phytic acid, free inorganic phosphorous and starch were recorded. The estimation of phytic acid was done by the Davies and Reid method (1979), free inorganic phosphorous was by HIP assay (Raboy 2000) and finally starch was done by the method put forward by Clegg 1956 with three biological replicates.

Table 1.

List of inbreds in the study

Code	Entries	Code	Entries
G1	DMR-QPM-01-06-2	G22	IMR-269
G2	DMR-QPM-03–05-1	G23	IMR-294
G3	DMR-QPM-03-09-01	G24	UMI-1099
G4	DMR-QPM-03-72	G25	UMI-467
G5	DMR-QPM-04-05	G26	UMI-447
G6	DMR-QPM-06-12	G27	UMI-300-1
G7	DMR-QPM-06-20	G28	UMI-158
G8	DMR-QPM-06-20-1	G29	UMI-113
G9	DMR-QPM-08-04	G30	IMR-326
G10	DMR-QPM-08-07	G31	IMR-314
G11	DMR-QPM-09-07	G32	IMR-271
G12	DMR-QPM-09-13-1	G33	IMR-19
G13	DMR-QPM-09-15	G34	IMR-225
G14	DMR-QPM-10-04	G35	IMR-255
G15	DMR-QPM-10-06-2	G36	IMR-118
G16	DMR-QPM-10-11	G37	IMR-29
G17	DMR-QPM-11-04-2	G38	IMR-20
G18	DMR-QPM-11-17	G39	LPA-2-285
G19	DMR-QPM-215	G40	LPA-2-395
G20	IMR-335	G41	CO-6
G21	IMR-353	G42	CO-H(M)-8

*Set of inbreds, checks and their codes used in the study. The bold entries depicts the checks

Phytic acid estimation (Davies and Reid 1979)

Phytic acid was analysed from the maize ground samples. 0.5 g of the ground samples were subjected to shaking with 10 ml of 0.5 M HNO₃ and the samples were filtered with a whatman No.1 paper. Following this, 0.2 ml of the filtrate plus 0.2 ml of freshly prepared ferrous ammonium sulphate (2.16 mg/ml) in eppendorf tubes were kept for incubation in a waterbath for 20 min. After cooling of these tubes, 1 ml of isoamyl alcohol and 20 µl of ammonium thiocyanate (5 g/50 ml) were added and shaken well. The tubes were then centrifuged at 3000 rpm for 10 min at 4°C and the colour developed was read at 460 nm. The content was estimated from the standard graph by a series of standards prepared from sodium phytate (0.5 mg/ml).

Free inorganic phosphorous (HIP assay) (Raboy 2000)

0.1 g of the powdered maize samples were soaked in 0.4 M HCl and was incubated overnight at 4°C overnight. The next day, the samples were mixed and an aliquot of 100 µl were taken in eppendorf tubes. To this, 900 µl of freshly prepared Chen’s reagent (1 part of 6 N H₂SO₄:1 part of 2.5% ammonium molybdate: 1 part of 10% ascorbic acid: 2 parts of H₂O is mixed) was added. The blue color phosphomolybdate complex developed was then read at 660 nm. Series of standards from KH₂PO₄ were prepared to obtain a linear graph from which the content was calculated.

Starch estimation (Clegg 1956)

From maize ground samples, 2 g was taken and homogenized with 80% ethanol. They were then centrifuged at 12,000 rpm for 15 min. The centrifuge of the samples in 80% ethanol was continued until the supernatant developed colour on treatment with anthrone. After obtaining a colourless supernatant, the residue was retained and dried in a hot water bath. After drying, 5 ml of H₂O and 6 ml of 52% perchloric acid were added. These tubes were allowed for extraction at 0°C for 20 min and their supernatants were collected. Again the extraction with perchloric acid and H₂O was repeated and the supernatant obtained was pooled with the previous one in a volumetric flask. The volume of the pooled extracts was made upto 100 ml with H₂O and finally, 0.1 ml of the extract was taken and to this, 4 ml of anthrone (200 mg of anthrone in distilled ethanol made upto 100 ml with ice cold 95% sulphuric acid) was added and kept in boiling water bath for 8 min and the color developed was read at 630 nm. Standard series were prepared from glucose.

Statistical analysis

The analysis of variance was calculated using the model:

$$Y_{\mathit{ij}}=\mu +G_i+E_j+G{E}_{\mathit{ij}}$$

where Y_ij is the corresponding variable of the ith genotype in jth environment (location), μ is the total mean, G_iis the main effect of ith genotype, E_j is the main effect of jth environment, and GE_ij is the effect of genotype × environment interaction.

Additive main-effect and Multiplicative interaction (AMMI) model was used to analyse the stability of the genotypes by analysing its principal component scores and this model can be presented with the following formula:

$$Y_{\mathit{ger}}=\mu +{\alpha }_g+{\beta }_e+\sum _{n=1}^N{\lambda }_n{\gamma }_{\mathit{gn}}{\delta }_{\mathit{en}}+{\rho }_{\mathit{ge}}+{\mathcal{E}}_{\mathit{ger}}$$

where $\mu$ is the grand mean estimated by $\overrightarrow{y}$, genotype deviation from the grand mean ${\alpha }_g$ by $\overline{y}$_g − $\overline{y}$, and the environment deviation ${\beta }_e$ by $\overline{y}$_e − $\overline{y}$.

The GGE biplot software for capturing the stable genotypes (Yan et al.2001) generate graphs showing (1) “what-won-where” pattern, (2) ranking of cultivars on the basis of yield and stability, (3) location vectors, and (4) comparison of locations to ideal location (Yan et al. 2007). The data was analysed by PBTools 1.4 package from IRRI.

Results and discussion

The analysis of variance revealed significant G × E interaction (Table 2 in the Supplementary file) indicating the influence of environments on the genotypes for phytic acid content (Kayode et al. 2006; Liu et al. 2007). Among the forty inbreds studied, ten exhibited a favourable reduced phytic acid content (< 10 mg/g). Out of these four low phytic acid inbreds, viz, UMI-467, LPA-2-285, LPA-2-395 and UMI-447 exhibited a lower interaction towards the environment. These genotypes expressed a lower phytic acid content in all the three locations and the coincidence of the curves for their phytic acid content in corresponding locations in the response plot clearly shows the stable performance for their low phytic acid (Fig. 1) among all the other genotypes (Table 3, 4, 5 and 6 in supplementary file). The genotypes with high phytic acid content was found to vary across environments and were observed to be high yielding (Fig. 2). This further coincides with the correlation of phytic acid with yield as reported by Raboy et al. 2000. The elite high phytate inbreds and checks CO-6, CO-H(M)-8 were superior in their yield levels with varying higher phytic acid content across locations (Table 7 in supplementary file). This is in accordance with the findings of Kayode et al. 2006 stating that none of the higher phytic acid lines were stable for their phytic acid content which has to be further explored. Hence, this elucidates the importance of phytic acid × environment interaction for understanding the factors enduring the adaptability and stability of the low phytic acid lines across locations (Brankovick et al. 2015).

Fig. 1.

Response Plot of Genotypes. *Graphical Variation of phytic acid across environments, E1 & E3: green lines, E2: Red

Fig. 2.

Adaptation of Genotypes for Phytic acid. *Levels of phytic acid in the genotypes screened

Stability for phytic acid by AMMI-biplot

The AMMI biplot captured 95.70% of the G + GE variation for phytic acid content with two major principal components across the locations. The coordinate point of the two principal components for the response factor lies in the origin of the AMMI biplot and the lines placed near this origin are considered to be stable in their performance. Among all the ten low phytic acid lines, UMI-467, LPA-2-395, LPA-2-285 and UMI-447 were found to be placed near the origin and they had the lowest stable phytic acid content across the environments (Fig. 3). Following them, the lines UMI-113, UMI-300-1, UMI 158 and UMI-1099 were moderately stable (Table 7 in supplementary file) in the AMMI biplot and they were lying nearer to the circle of origin (Aslam and Pavlu 2007). These moderately stable lines showed a variation in their phytic acid content in environment 2.

Fig. 3.

AMMI Biplot for PA (Phytic acid). *Biplot of PC 1 and PC 2 in AMMI indicating the expressivity and stability of the genotypes

The environment 2 had the highest interaction (Fig. 4) with genotypes having a long spooked arrow for the phytic acid content towards the negative quadrant (Aslam and Pavlu 2007). This environment falls under the rainfed zone of Tamil Nadu and is found to be highly interactive towards the performance of the lines raised in it due to its prevailing extreme condition (Zhai et al. 2016). This confirms the effect of strong genotype × environment interaction for phytic acid due to the water stress and temperature as reported by Brankovick et al. (2015). Therefore, this states the importance of stable lpa maize lines in cultivation for providing high nutrimental feed to the poultry sector across the country.

Fig. 4.

Environmental Interactions in AMMI biplot. *Extent of Environmental influences towards the genotypes

Stability by GGE biplot for phytic acid

The GGE biplot is considered to be superior than AMMI biplot and these plots also had similar interpretation for stability (Yan et al. 2001). The GGE biplot had captured a greater G + GE variation of 97.90% in the PC plot. The genotypes were ranked in the GGE biplot based on their overall mean and PC (principal component) scores. The PC1in the GGE plot portrays the adaptability and the expression of the line for the trait and PC2 exhibits its phenotypic stability (Mitrovick et al. 2012). A higher PC1 is favourably preferred for positive traits like yield and negative traits like phytic acid directs a selection towards the lines with lower PC1 and PC2 nearing zero (Balestre et al. 2009). The inbreds, UMI-467 (2.52 mg/g), LPA-2-395 (2.73 mg/g), LPA-2-285 (2.83 mg/g) and UMI-447 (3.47 mg/g) had the stable and lowest phytic acid content with lower PC1 and PC 2 scores nearing zero (Fig. 5). The lines UMI-113, UMI-158, UMI-300-1 and UMI-1099 were moderately stable and were placed on the circle nearer to the origin. These lines also had a moderate phytic acid content ranging from 5.91–8.60 mg/g (Table 7 in supplementary file). Considering the performance of the high phytate lines, they were not stable for their phytic acid and were placed far away from the origin. This shows the differential response of cultivar × location (low versus high phytic acid lines) which may be genetically determined (Kayode et al. 2006). The major components that confer stability of phytic acid in these stable lpa lines have to be further explored.

Fig. 5.

Genotype view in GGE Biplot. *Placement of genotypes in the GGE biplot, the red lines indicates the level of expression for phytic acid in both extremes (high and low)

The main advantage of GGE biplot is the grouping of similar environments in What-Won Where plot (Yan et al. 2007). This plot grouped environment 1 and environment 3 as a mega environment (Fig. 6) due to the similarity in their interactions. Thus, any one among these environments could be omitted in the further multi-locational analysis (Balestre et al. 2009).

Fig. 6.

Classification of Environments. *Classification of environments in different sectors, Environments within a sector are a mega-environment and extreme genotypes are placed in the peaks of the green lines

From what-won where plot, it could also be found that the genotype IMR 335 had higher extreme performances for phytic acid in environment 1 and environment 3. The lines IMR 353 and DMR-QPM-09-07 were observed to exert their higher extreme expression for phytic acid in environment 2. Considering all the three environments, the stable lines UMI-467, LPA-2-285, LPA-2-395 and UMI-447 performed with extreme lower values for phytic acid content and were placed on the extreme left quadrant (Fig. 6). This presented their unique expression for phytic acid and enhances the opportunities to explore its molecular mechanism. The ideal favourable environment in GGE biplot was observed to be environment 1 and environment 3 (Fig. 7). This suggested that all the genotypes favourably performed in both these locations in the positive direction for most of the traits. Therefore, these genotypes could be recommended to these environments for analysing their favourable performances.

Fig. 7.

GGE biplot for Environments. *Identification of ideal genotype and environment, the environment that passes through the average environmental axis has a low interaction towards the genotypes

In this study, both AMMI and GGE biplot were informative and it was found that AMMI was more informative for classifying the genotypes and GGE biplot was advantageous in implicating the role of environment on the genotypes (Yan et al. 2007; Gauch et al. 2008). From both AMMI and GGE biplot, the line UMI-467 followed by LPA-2-285, LPA-2-395 and UMI-447 were stable for their low phytic acid content with higher free inorganic phosphorous and these lines could be employed as potential pre-breeding materials in low phytic acid programs.

Effect of phytic acid with other key components

Across the three locations, phytic acid had significant interactions with the other key traits like free inorganic phosphorous, single plant yield, starch, hundred seed weight and seedling vigour index.

Phytic acid has the critical issue of chelating cationic minerals like phosphorous in food. Earlier reports on phytic acid has confirmed the negative association of phytic acid with free inorganic phosphorous (Raboy 2001), as this phytic acid serves as a storage complex for phosphorous in maize (Raboy et al. 2000). Hence, the free inorganic phosphorous in these lpa inbreds were analysed to ensure its bioavailability in food and this was eventually observed to be high in the stable lpa lines. The free inorganic phosphorous ranged from 1.78 (UMI-447) -1.87 (UMI-467) mg/g in the stable lpa lines (Table 8 in supplementary file). On the other hand, the checks CO-6 and CO-H(M)-8 with higher phytic acid exhibited the minimum levels of free inorganic phosphorous in them (Fig. 8). This portrays the importance of lowering the phytic acid in maize feeds to ensure the maximum absorption of phosphorous by animals feeding on it. Among all the low phytic acid lines, UMI-467 (2.52 mg/g) and LPA-2-395 (2.73 mg/g) had the most stable low phytic acid with higher free inorganic phosphorous (1.87 and 1.85 mg/g). This exhibits their utility as potential donors for developing stable low phytic acid lines with higher free inorganic phosphorous (Fig. 8).

Fig. 8.

Performance of stable lpa lines with the checks for Biochemical traits and Seedling vigour. PA Phytic acid, FIP free inorganic phosphorous, SVI seedling vigour index

Several multi-environmental studies illustrate the importance of stability and adaptability of genotypes across locations. Although these two terms seem to be similar, adaptability is the acclimatization of the genotype to a particular environment with higher yield and stability is the uniform performance of the genotypes across locations (Balestre et al.2009). Considering this, the adaptability of the stable low phytic acid lines were poor in all the environments (Fig. 9). Among all the inbreds, the checks CO-6 and CO-H(M)-8 had higher phytic acid content with greater adaptability in all the three locations (Fig. 9). Other than the checks, DMR-QPM-04-05, DMR-QPM-11-17, DMR-QPM-01-06-2 had a stable performance for yield with higher phytic acid ranges (Table 3, 4, 5 and 6 in supplementary file).

Fig. 9.

Yield levels of stable lpa lines with checks. PA Phytic acid and SPY Single Plant yield

Whereas, the yield of the lpa lines were much poor and unstable (Fig. 9) when compared to other lines including the checks CO-6 and CO-H(M)-8. It ranged from 28.16 g (UMI 467) to 45.90 g (LPA-2-285) (Fig. 9). The most stable lpa line, UMI-467 had the lowest yield of 28.16 g due to a poor seed set (Bregitzer and Raboy 2006). This clearly states that perturbations in the production of phytic acid would significantly reduce the yield levels and hence careful selection of the parental sources is essential (Donahue et al. 2010). Among all the low phytic acid lines, the highest moderate stable yield was observed in LPA-2-285 and LPA-2-395 (Fig. 9). Therefore, these lines could be used in low phytate programs without compromising the seed yield traits (Table 9 in supplementary file).

Considering the yield parameters, starch is also an essential component in maize. The myoinositol that involves in the production of phytic acid also acts as a transporter of the Uridine di-phosphate glucose in starch synthesis (Raboy 2001). This propels the importance of observing the starch accumulation in lpa inbreds. Supporting this report in this study, all the lines with a higher phytic acid and yield had exhibited a higher starch content across locations (Table 3, 4, 5, 6 and 11 in supplementary file). Whereas, the low phytic acid lines were examined with a lower starch content (Lorenz et al. 2008). The highest starch content among the stable low phytate lines was in UMI 447 (55.77%) and the lowest starch was found in UMI 467 (51.38%) (Fig. 10). This suggests the reason beneath the reduction in the yield levels of lpa lines as starch is the major dry weight factor in cereals (Figs. 8, 9).

Fig. 10.

Performance of seed sphericity and seed weight in Stable lines. PA Phytic acid, SPY single plant yield, SL seed length, ST seed thickness, SG seed girth 100 swt: hundred seed weight

Following the yield levels, perturbations in phytic acid and starch content has also affected the seed traits namely, seed spherecity (seed length × seed girth × seed thickness) and seed weight. The lpa lines had a lower hundred seed weight and smaller seed spherecity among the rest of the inbreds. The lowest seed length and hundred seed weight was observed in UMI-467 and UMI-447 (Fig. 10). The line LPA-2-395 had a higher seed length (0.90 cm) with the lowest seed girth (0.26 cm) among the lpa lines. These values project the small, obovate and shrivelled nature of seeds in the low phytic acid lines and is similar with the findings of Lorenz et al. 2007 (Table 10 and 12 in supplementary file).

Comparing all the yield attributing characters, it could be understood that the phytic acid is the major storage component of all the minerals required for the plant’s germination and vigour. This exudes the importance of observing the seedling vigour of the stable lpa inbreds due to its abnormal seed characteristics and it was found that their germinability were found to be reduced. The line, UMI-467 (597.76) followed by UMI-447 (1206.80) had the lowest seedling vigour index among all the inbreds (Figs. 8, 9) (Naidoo et al. 2012). These adverse effects with poor germination in lpa lines have also been encountered as a major negative pleiotropic effect in other crops like barley (Raboy et al. 2015). This states the physiological role of phytic acid in plant development. Although the stable lpa lines had poor seedling vigour index, LPA-2-285 (1511.06) and LPA-2-395 (1272.62) had a better germinability among them and hence, these lines (Fig. 8) as parents in lpa hybrid development programs could resolve the constraints in seed production.

The predicted BLUP values and genetic progress of the stable lines

The genetic gain or progress is the breeding value gained after a selection cycle for a particular trait. Several studies prioritising yield have reported the accumulation of favourable alleles for the trait after each selection. This enhancement in the breeding value is estimated by the Best Linear Unbiased Prediction value (BLUP). The BLUP values of the identified stable lines for their genetic gain for low phytic acid were estimated (Table 13 in supplementary file). The predicted values indicate a slight increased variation of phytic acid in UMI-447 and LPA-2-395 (Fig. 11). There is not much variation predicted for UMI-467 and LPA-2-285 in the upcoming generations. This necessitates the further studies to evaluate their stability in crossing programs to reveal their underlying genetic and molecular mechanisms.

Fig. 11.

a BLUP values for Genetic Progress of Stable lpa lines, b Actual performance of stable lpa observed in the experiment. BLUP values for successive generation vs the actual performance of the stable lines

Conclusion

The data presented indicates that the maize lines differ in its phytate content and their stability is genetically as well as environmentally controlled. The biplots indicated the maximum G × E variation for phytic acid and also exhibited the drastic reduction of yield in lpa lines. This elucidates the importance of phytic acid in seed health. The findings suggest the possibility of developing maize hybrids with low phytic acid for its utilization as a more bioavailable diet both for humans and non-ruminants. Though variation of phytic acid in response to the environments differs within the genotypes by environmental interaction, the genetical reason behind their stability could further be explored. These identified stable lines viz., UMI-467, LPA-2-395, LPA-2-285 and UMI-447 for phytic acid with a similar interaction across the environments could be employed in breeding maize for low phytic acid in future.

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Conflict of interest

All authors declare that they have no conflict of interest.