Global cereal demand for human consumption and livestock feed is rapidly increasing due to continuous population growth (Alexandratos and Bruinsma, 2012). Among cereals, maize demand is the highest, followed by wheat and rice, which together account for 94% of all cereal consumption (Ranum et al., 2014). The increased maize demand will have a great impact on the low and middle-income countries such as those in sub-Saharan Africa (SSA) and south Asia with an undernourishment prevalence of 22.8% and 14.7%, respectively (Abegaz, 2018). Besides food insufficiency, several millions of people in these low and middle-income regions suffer from micronutrient deficiencies such as Zn and vitamin A, as well as essential amino acids such as lysine and tryptophan (Nair, 2020; Nkhata et al., 2020). One of the reasons for this is overreliance on maize-based foods with limited supplementary foods, which exposes a high proportion of the population to the risk of micronutrient and protein deficiency (Prasanna et al., 2001; Nuss and Tanumihardjo, 2011). Vitamin A and Zn deficiency negatively affects the physical well-being, eyesight and cognitive development of people and can lead to birth defects and child mortality (Menkir, 2008; Rautiainen et al., 2016). Tryptophan is a precursor amino acid for niacin (vitamin B3) biosynthesis, and therefore its deficiency causes pellagra (Gupta et al., 2015; Kipsang et al., 2019). General lack of dietary protein in weaned infants adversely affects their overall well-being, and symptoms include peripheral oedema, diarrhea and severe wasting, collectively known as “kwashiorkor” (Goredema-Matongera et al., 2021).

To meet both nutritional and high maize demand, several studies have suggested the need to develop multi-nutrient biofortified maize hybrids with high yield potential (Ray et al., 2013). This breeding initiative could have huge benefits to maize-based low-income societies such as in SSA. Breeding for high yielding biofortified hybrid maize is a cost-effective and sustainable strategy to increase both maize productivity and nutritional quality. Hybrid maize is developed by crossing two or more parents to exploit maximum heterosis. Whilst hybrids generally have high yield potential, developing stable biofortified hybrids with outstanding performance under both stress and non-stress environments is a key priority in SSA (Miranda et al., 2008). Most of the cropping systems in SSA are characterized by various abiotic stress factors that threaten food security and humanity in this region and these include low soil nitrogen (Makumbi et al., 2011), and combined heat and drought stress (Cairns et al., 2013; Meseka et al., 2018). The incidence of drought and heat stress and the continuous decline in soil fertility and water holding capacity is expected to increase due to global climate change (Ertiro et al., 2017). Projections are that by 2030, 40% of the arable land will be unsuitable for the maize varieties grown currently (Meseka et al., 2018). The simultaneous occurrence of heat and drought stress in the tropical lowlands is likely to increase and has greater adverse effects on agronomic traits than when each stress occurs separately (Cairns et al., 2013; Bhardwaj et al., 2021). In addition, the intensified crop production in SSA, monocultural practices and growing crops in marginal areas, have contributed significantly to the rapid decline of soil fertility in SSA (Makumbi et al., 2011). Therefore, strong adaptation measures such as developing multiple stress tolerant maize are required to enable small-holder farmers to cope with these production constraints (Salami et al., 2010). Breeding for nutritionally superior hybrids that can withstand multiple stress conditions is an attractive strategy to mitigate both food insecurity and several nutritional challenges experienced in SSA simultaneously (Chakraborti et al., 2009; Hindu et al., 2018).

While concerted efforts are being made towards developing biofortified maize hybrids, breeding for Zn-enhanced hybrid cultivars is lagging behind (Garcia-Oliveira et al., 2018). The International Maize and Wheat Improvement Centre (CIMMYT) and its partners have initiated breeding programs for Zn-enhancement in maize (Goredema-Matongera et al., 2021). Wide genetic variation for Zn exists in normal maize (Garcia-Oliveira et al., 2018), QPM (Chakraborti et al., 2009; Hindu et al., 2018) and provitamin A maize (Hoisington, 2002). Improving maize kernel Zn content in maize already biofortified for provitamin A and QPM increases its overall nutritional composition (Maziya-Dixon et al., 2000; Prasanna et al., 2020). In this way, millions of lives could be saved from various macro- and micro-nutrient deficiencies and this concurs with the agenda for the United Nation’s Sustainable Development Goals for 2030 to end hunger in all its forms (Gil et al., 2019).

To develop highly productive multi-nutrient maize hybrids, breeders should know the GCA and SCA of inbred lines, since they both indicate the breeding value of inbred lines in hybrid combinations (Karim et al., 2018). GCA has been defined by Sprague and Tatum (1942) as the average performance of the inbred line based on its performance in crosses with other inbred lines. SCA refers to the performance of an inbred line in a specific cross. Combining ability is a powerful tool for identifying the best performing inbred lines for use in different cross combinations either to exploit heterosis or accumulate fixable genes (Uddin et al., 2008). This reveals the mode of gene action for a particular trait. GCA is associated with additive gene effects, whereas SCA is associated with non-additive effects including dominance and epistatic gene effects (Aguiar et al., 2003). Several studies evaluated the combining ability of maize inbred lines grown under managed drought stress (Bänziger et al., 2006; Derera et al., 2007), drought and low N stress (Makumbi et al., 2011; Beyene et al., 2013), optimum conditions (Ertiro et al., 2017) and heat stress (Archana et al., 2018). Despite all this, studies on combining ability for GY for biofortified maize inbred lines and testcross performance under stress and non-stress conditions are still limited. Such studies are useful to breeders pursuing breeding for quality traits using exotic nutrient donors that introgress desired nutritional traits into local germplasm pools. Therefore, the objective of this study was to estimate the GCA and SCA of introduced Zn donors in testcrosses involving normal, provitamin A and QPM testers, under WW, managed low N and HMDS conditions.

The different management conditions used in this study showed differences in the way Zn-enhanced genotypes responded to these growing conditions. Compared to the OPT conditions, GY was reduced by 60%, 86%, and 10% under HMDS, LN and WW conditions, respectively. While a very small reduction was observed under WW conditions, GY was significantly reduced under LN, followed by HMDS. The negative effect of LN and HMDS conditions on GY observed in the current study was in agreement with several previous studies (Betrán et al., 2003; Makumbi et al., 2011; Beyene et al., 2013; Ertiro et al., 2017). The average days to mid-anthesis were lowest (65.5 days) under HMDS and highest under LN (71 days). Although the number of days to mid-anthesis for OPT and WW did not vary much, HMDS and LN stress induced earlier and later flowering, respectively, compared to non-stress conditions. A similar trend was reported by Ortiz-Covarrubias et al. (2019) using provitamin A hybrids. This could have been attributed to the combined adverse effects of heat and drought stress conditions compared to heat or drought separately. Meseka et al. (2018) reported that the effects of combined heat and drought stress on normal maize are much greater than the effect of each stress separately. Therefore, early flowering observed under HMDS conditions was probably a sign of efficient utilization of the limited resources such as moisture to enable completion of the reproductive cycle (Bänziger and Lafitte, 1997). Under excessive stress conditions, most plants signal stress-induced response mechanisms, such as accumulation of high levels of abscisic acid (ABA), a hormone that regulates growth, development, dormancy, floral induction and senescence (Bruce et al., 2002; Ali et al., 2020). Therefore, high levels of ABA in plants under severe HMDS stress could signal early flowering as stress adaptive mechanisms as compared to well-watered conditions.

In addition, LN and HMDS increased ASI by 188% and 169%, respectively. WW conditions had a significant impact on ASI. Findings from previous studies of non-biofortified maize (Menkir et al., 2006; Ertiro et al., 2017), also reported an increase of ASI by 144% and 149% due to excessive moisture stress compared to optimum conditions. Therefore, a wider ASI observed in the current study under HMDS stress could be attributed to high severity of the combined stresses. High ASI was also observed under LN stress conditions. The GY reduction observed under both stress management conditions could be partly due to increased ASI, a secondary trait, which has high negative correlation with GY under stress conditions (Westgate, 1997; Ertiro et al., 2017; Meseka et al., 2018). Both LN and drought stress at flowering favors the development of male inflorescence but inhibits ear and silk development (Maiti et al., 1996; Badr et al., 2020). However, HMDS negatively affects tassel quality, pollen production and viability compared to drought alone and this ultimately negatively impacts the quality of pollen produced as well as reduction in period of pollen shedding (Cairns et al., 2012; Meseka et al., 2018). All these factors result in incomplete or nil fertilization, which ultimately increases kernel abortion, decreases kernel development, leading to fewer ears per plant causing a reduction in GY (Jacobs and Pearson, 1991; Beck et al., 1996; Magorokosho et al., 2003). ASI has been widely used for indirect selection of genotypes with high GY potential under LN, drought and HMDS conditions, by selecting genotypes with improved synchrony between the female and male flowering (Bänziger et al., 2000; Meseka et al., 2018). In addition to ASI, both LN and HMDS reduced PH and EPP compared to OPT and WW conditions. PH was reduced by 16% and 14% under LN and HMDS respectively, but did not differ much for plants grown under WW and OPT conditions. Furthermore, WW, LN and HMDS reduced EPP by 0.07%, 50% and 30%, respectively. The reduction of both PH and EPP under stress conditions observed in this study concurs with previous studies conducted on normal maize (Badu-Apraku et al., 2011; Badu-Apraku et al., 2012; Talabi et al., 2017).

The average GY of the top 20 Zn-enhanced experimental hybrids was higher than the average of all the checks under all the management conditions. In addition, slightly lower GY of the experimental hybrids than the best check demonstrates significant yield improvement of the biofortified hybrids developed by CIMMYT. In the past, biofortified maize varieties have been associated with low yield potential, and this has hindered full adoption of such nutrient-dense cultivars by farmers (Bänziger and Long, 2000; Nkhata et al., 2020). When ranking the GY performance of all the 84 hybrid entries, checks (E84) only occupied the first position under OPT conditions ( Supplementary Table 1 ). This indicates that Zn-biofortified experimental hybrids performed better under WW, HMDS and LN. It is encouraging that among the commercial checks, provitamin A hybrids occupied the first position for GY performance under both stress conditions ( Supplementary Table 1 ). In addition, the fourth best check hybrid under OPT conditions was E80, a QPM commercial hybrid. Although the normal checks dominated in the overall best performing check hybrids, provitamin A and QPM biofortified check hybrids also showed outstanding performance under all management conditions. These findings confirm the great progress made so far by CIMMYT to improve GY performance of tropically adapted biofortified varieties. The knowledge of the correlation between environments or traits between management levels is critical for breeders in examining similarities between environments for GY performance and secondary traits that could be useful for indirect selection under stress conditions. In this regard, the positive correlation of GY and secondary traits such as PH and EPP observed under all management conditions agrees with previous studies on normal maize reported by Badu-Apraku et al. (2011) and Talabi et al. (2017). In addition to that, GY was negatively correlated with ASI under both stress conditions, and this further confirms the effective use of ASI for indirect selection of high yielding genotypes under stress conditions reported in previous studies of normal maize (Bolaños and Edmeades, 1993; Bänziger and Lafitte, 1997; Ortiz-Covarrubias et al., 2019). Furthermore, GY was positively correlated with AD under OPT conditions and this indicates that given adequate moisture, late maturing genotypes yield more as a result of prolonged grain filling period (Edmeades et al., 1989). Clustering of environments with similar growing conditions as observed in this study was expected. Therefore, maize breeders for quality traits should develop nutrient-dense cultivars for specific environments, since the cultivars respond to these environments differently.

The most widely used breeding strategy is to select for Zn-enhanced genotypes with high yield potential under OPT conditions and then evaluate for stable performance of the selections under different stress conditions (Myers, 1985; Magorokosho et al., 2003). In this regard, L9/T7 and L10/T7 were the best Zn-enhanced experimental hybrids that combined high GY performance and stability under stress and non-stress environments. The hybrids were constituted from inbred lines with Zn and normal genetic backgrounds. These hybrids could have combined nitrogen and water-use efficiency, as well as considerable tolerance to heat stress, possibly due to heterosis or additive effects when both parents contributed favorable alleles (Makumbi et al., 2011; Ertiro et al., 2017). For instance, all the parents of L9/T7 contributed positive GCA effects for GY under LN and HMDS, but only the tester had positive GCA under non-stress conditions. Conversely, all the parents of L10/T7 showed positive GCA under OPT conditions, with the tester contributing positive GCA under stress conditions. This indicates that superior GY performance of Zn-enhanced hybrids were due to fixation of favorable genes in the respective parental inbred lines. CIMMYT and IITA usually select inbred lines for hybrid development based on desirable traits, inclusive of those indicative of stress-tolerance such as ASI, EPP, tassel blast, chlorophyll content, leaf firing and senescence (Alam et al., 2017; Meseka et al., 2018). In addition to L10/T7 and L9/T7, hybrids L2/T4, L4/T4, and L3/T5 were identified as good Zn-enhanced hybrids in a provitamin A background. Similarly, L8/T6 and L11/T3 combined high GY and desirable secondary traits across all management conditions. Favorable alleles could have been contributed by the respective parents. High GY performance of this multi-nutrient maize across management conditions demonstrates the possibility to develop multiple-stress tolerant multi-nutrient maize genotypes for commercialization in SSA.

In breeding for a particular trait, knowledge of the associated gene action is important in optimizing the breeding strategy. In the present study, the presence of both additive and non-additive gene effects was observed, confirming the complex genetic nature of GY. Dominance variances were more important for GY than secondary traits, except for PH under OPT and WW conditions. Under OPT conditions, equal proportional contribution was observed for EPP. Conversely, additive variance was significant for GY and other traits under LN and HMDS except for EPP under LN. The predominant nature of non-additive gene effects in controlling GY and secondary traits under non-stress conditions observed in the current study were also reported in previous studies but focusing on normal maize (Mhike et al., 2011; Fahad et al., 2018). Under LN and HMDS, preponderance of additive gene action was observed for GY and most secondary traits. These results are in agreement with previously studies (Derera et al., 2007; Musila et al., 2010; Ertiro et al., 2017). Despite this, knowledge on the genetic factors governing GY and secondary traits under HMDS is still limited. The presence of both additive and dominance gene action for the traits measured indicate the presence of greater genetic diversity among the parental inbred lines used to make crosses (Fahad et al., 2018). Traits controlled by additive gene effects can be effectively improved by standard selection during inbred line development and also through recurrent selection. Some Zn-enhanced hybrids showed outstanding performance under both LN and HMDS, possibly due to fixation and contribution of multiple-stress tolerant alleles from their parents. Despite the contribution of additive effects under stress conditions, dominant gene effects were important for most traits under OPT and WW conditions. This implies that the best breeding strategy would be to exploit both GCA for the parents and SCA effects for the resulting Zn-enhanced or multi-nutrient hybrids under both stress and non-stress conditions (Chiuta and Mutengwa, 2020).

The GCA effects of both lines (Zn donors) and testers were significant across all the management levels. However, the GCA effects of line x site or tester x site were only significant under OPT and HMDS conditions. This shows the need to select lines or testers that are well adapted to specific growing conditions. Comparing the lines and the testers, results show that the testers from the normal, provitamin A, and QPM genetic backgrounds were more adapted to the Zimbabwean growing conditions than the newly introduced Zn donors. Since the testers are currently used in different breeding programs at CIMMYT, their wide adaptation to both stress and adverse growing conditions reflects emphasis and breeding efforts made in breeding for multiple stress tolerance. The inbred lines with high GCA for GY and secondary traits could be used (i) in recurrent selection schemes to improve the frequency of desirable alleles for a trait in a population, (ii) as testers to evaluate newly developed or introduced Zn-enhanced inbred lines, and (iii) as parents for Zn-enhanced synthetic varieties (Makumbi et al., 2011; Mutimaamba et al., 2020). Therefore, lines with high GCA effects for GY and other important traits were identified as L6, L7 and L11 and could be useful as testers for newly developed Zn-enhanced inbred lines under stress and non-stress environments. The results of the present study demonstrate that GY potential of the Zn-enhanced normal, provitamin A and QPM hybrids can be comparable to the normal counterparts. Hence, the possibility of developing multi-nutrient stacked hybrids with high yield potential. In addition, the current study also demonstrated the possibility of identifying Zn-enhanced lines and testers from the normal, provitamin A, and QPM nutritional groups that can be useful to make multi-nutrient hybrids adapted to stress and non-stress conditions. However, further studies should focus on understanding the physiology and genetics involved in controlling the accumulation of Zn in maize kernels, as this is critical in designing an effective breeding strategy for hybrids stacked with nutrients including grain Zn. Further studies should also focus on developing Zn-enhanced inbred lines that are well adapted to a wide range of environments.

Commercialization of the identified multi-nutrient hybrids could contribute significantly to both production and productivity of multi-nutrient biofortified maize in SSA. Such hybrids include L10/T7 and L9/T7 (Zn + normal), L2/T4, L4/T4, L3/T5 (Zn + provitamin A), L8/T6 and L11/T3 (Zn + QPM). Although no single Zn donor with high GCA effects for GY and other traits were identified across all management levels, some inbred lines such as L6 and L7 combined positive GCA effects for GY and most secondary traits under optimum and managed LN conditions. Similarly, L8 and L9 were identified for good GCA effects for GY under stress conditions. This indicates that Zn donors were adapted to specific environments and thus selection should be based on specific adaptation. Such Zn-enhanced lines could be used as testers or to form synthetics in biofortification programs. However, the locally developed testers were less affected by environmental changes. This shows evidence of the progress made by CIMMYT in breeding for multiple stress tolerance in maize to withstand harsh growing conditions in SSA. However, lines that showed consistency in contributing for low nitrogen and drought tolerance could be further evaluated for nitrogen and water use efficiency. High yielding zinc-enhanced hybrids with specific combinations were identified and could be commercialized in Zimbabwe. Our results demonstrates that some biofortified maize hybrids can be comparable to normal maize in terms of grain yield potential as opposed to previous studies reporting strong dilution effects, grain yield versus nutritional quality.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Conceptualization, NM and ML. Investigation, NM. Resources, TN and ML. Writing–original draft preparation, NM. Writing–review and editing, TN, CK, AB, and ML. Data analysis, CK and NM. Supervision, ML, TN, and AB. Funding acquisition, TN and ML. All authors contributed to the article and approved the submitted version.