Genetic Evaluation of Heterosis and Combining Ability of Quality Protein Maize (Zea mays L.) Hybrids under Terminal Heat Stress Conditions

The development of quality protein maize (QPM) hybrids tolerant to terminal stress environments has been an essential strategy for reducing the associated loss in grain yield. A study by Badu-Apraku et al., (2019) employed line × tester analysis in QPM breeding, highlighting its efficacy in selecting QPM hybrids. While QPM varieties resistant to disease, drought and environmental stresses have been developed conventionally, the adoption of improved QPM genotypes in farmers’ field is restricted mainly due to lack of proper team effort among maize breeders, farmers, agricultural extension workers and other relevant stakeholders (Tandzi et al., 2017). In this study attempt was made to develop elite hybrids/segregants of quality protein maize with heat stress tolerance (Edreira et al., 2014).
       
Combining ability evaluation is a crucial factor in determining the value of inbred lines to get best hybrid combinations (Rajesh et al., 2014). General combining ability (GCA) effects are useful for managing existing diversity, identifying superior parental genotypes with desirable characteristics and establishing linkages between breeding materials. Specific combining ability (SCA) effects, on the other hand, are useful for evaluating recombination frequencies in order to identify potential single crosses between populations or inbred generations. These combined insights provided by GCA and SCA are essential for effective hybrid breeding programs and the development of superior inbred (Thakur et al., 2023); (Prasad et al., 2017). Genetic enhancement in tropical maize under terminal heat stress can also be achieved through secondary traits that exhibit strong association with yield such as membrane stability index, tassel blast, leaf firing, root lodging and biochemical parameters like chlorophyll content, catalase and peroxidase Geetha et al., (2019). Based on the above findings, a research program was conducted to estimate the nature and magnitude of gene action and variation components under heat stress condition. The goal was to identify potential inbred lines and cross combinations by evaluating their general and specific combining ability, respectively along with the heterotic effects.

Nine parental QPM lines were subjected to a crossing programme during summer (Mid February to Mid June, 2022) to generate 18 F₁s in a line × tester mating design. Materials used for the present investigation comprises of 18 F_1s, parental lines (six lines and three testers) and one standard check (Table 1). The 28 genotypes (18 F_1’s + 9 Parents + 1 Check) were evaluated at the post graduate research farm, genetics and plant breeding department, School of MSSSoA, Centurion University of Technology and Management, Odisha in a randomized block design (RBD) with three replications during summer 2023. Days to 50% tasseling and days to 50% silking were recorded by counting the number of days from sowing to floral emergence in 50% plants per plot. Five randomly qualified plants were chosen in each replication for recording plant height (cm), ear height (cm), ear length (cm), ear girth (cm), number of kernels row^-1, number of kernels row cob^-1, 100 grain weight (g), Grain yield plant^-1 (g), canopy temperature (!), chlorophyll content (%), membrane stability index (%), estimation of protein content (%), estimation of oil content (%), catalase, peroxidase, leaf firing, tassel blast and root lodging. Canopy temperature at midday was measured using an infrared thermometer. Chlorophyll content was assessed using SPAD meter on leaf samples from different parts of the plant. Membrane stability index (MSI) was recorded using electrolyte leakage method, with MSI calculated from electrical conductivity measurements. Catalase activity was determined through an enzymatic assay by monitoring the decrease in absorbance at 240 nm and peroxidase activity was measured by the increase in absorbance at 470 nm in a spectrophotometer.
 

       
Based on the mean values, line × tester analysis and standard heterosis assessment for yield and its related traits was performed as per the method suggested by Kempthorne (1957). Heterosis was calculated according to the method suggested by Shull (1908). Heterosis expressed as increase or decrease of F₁ hybrid value over the best commercial check HQPM-1 (standard heterosis) for each character was recorded as per the formulas suggested by Hayes et al., (1955). Analysis was done using Windostat version 9.2 and graphs were plotted using Minitab v16.0 software.

Analysis of variance
 
ANOVA for yield and associated traits showed significant variation among parents and crosses (line × tester) for all the traits except chlorophyll content and canopy temperature (Table 2a and 2b). Lines were significant for all traits except cob height, chlorophyll content and canopy temperature. Testers were significant for days to 50% tasseling, days to 50% silking, plant height, cob height, cob length, number of kernel rows cob^-1, number of kernels row^-1, membrane stability index and peroxidase. Line × tester interaction was significant for most traits except cob height, cob length, chlorophyll content, canopy temperature and oil percentage. The partitioning of variances among the hybrids into components namely, lines and crosses showed significant differences for all the traits except cob height and chlorophyll content (Table 2a and 2b) Subba et al., (2022). Genotypes exhibited a predominance of non-additive genetic effects with σ²GCA/σ²SCA ratios less than one and the degree of dominance (σ²A/σ²D)^1/2 greater than 1 for all traits (Table 3). These results suggest further exploitation of non-additive gene effects for traits through heterosis breeding. In our study, traits such as protein content (71.5%) and oil content (85.55%) exhibited high narrow sense heritability. Therefore, hybridization is the desirable choice for utilizing the presumed heterosis in selective crosses. Sanghera et al., (2013) reported high specific heritability for yield related traits.
 

 

 

 
General combining ability (GCA) effects
 
The estimates of GCA effects revealed significant differences among the parents suggesting general combining ability for all the traits in preferred direction (Table 4a and 4b). Among the parents, the lines CML149 and CML143 showed highly significant positive GCA effects for grain yield per plant indicating the presence of additive gene action. While line CML149 and tester CML330 exhibited desirable significant GCA effects for 100 grain weight, CML149 for number of kernels row^-1, CML143 for number of kernel rows cob^-1, CML138 for cob girth. CML138 and CML332 recorded negative and significant GCA effects indicating that they are good general combiners for obtaining hybrids with reduced plant height. The parents CML149, CML334, CML143 and CML145 recorded positive significant GCA effects for protein content, lines CML 149 and CML334 recorded positive significant GCA effects for oil percentage, lines CML149, CML334, CML332 and CML143 recorded positive significant GCA effects for membrane stability index. Parents CML138, CML332 and CML330 recorded positive significant GCA effects for catalase activity. Line CML167 recorded positive significant GCA effects for peroxidase activity (Table 4b). Similarly, for days to 50% tasseling negative GCA effects were recorded by CML149, CML334, CML143, CML167 and CML330 and for days to 50% silking CML149, CML334 and CML167 and CML330 exhibit negative significant GCA effects (Table 4a). Two lines viz., CML334 and CML332 recorded negatively significant GCA effects for anthesis silking interval. It is evident that the line CML149 was adjudged as the best combiner for grain yield per plant, number of kernel row-1, protein content, oil content and membrane stability index and thus can be utilized as potential parent due to high per se performance with significant GCA effects for the respective traits. Among the nine parents CML149 and CML143 were chosen as the best general combiner for grain yield per plant. Hence these two lines can be utilized in the hybridization programme Barh et al., (2015). Good general combiners for earliness were reported by Ahmed et al., (2017); Elmyhun et al., (2020); Bharti et al., (2020); Raihan et al., (2023). Similar results were also reported earlier in maize for grain yield per plant Matin et al., (2016).
 

 

 
Specific combining ability (SCA) effects
 
The SCA is a consequence of dominance and epistasis (Sprague and Tatum, 1942). Estimates of specific combining ability for different crosses is presented in Table 4a and 4b. Out of 18 hybrids, nine hybrids, viz., CML149 × CML145, CML149 × CML330, CML138 × CML145, CML138 × CML193, CML334 × CML145, CML332 × CML193, CML143 × CML145, CML143 × CML330 and CML167 × CML330 showed significant and negative SCA for days to 50% tasseling and crosses CML149 × CML145, CML138 × CML193, CML334 × CML145, CML332 × CML193, CML143 × CML145 and CML143 × CML330 showed negative significant SCA effects for days to 50% silking (Table 5a). Negative significant SCA effects were exhibited by two hybrids viz., CML138 × CML193 and CML332 × CML145 for plant height. The hybrid CML167 × CML145 had positive SCA effect for number kernel rows cob^-1. Two hybrids namely, CML149 × CML330 and CML143 × CML193 had positive SCA effect for grain yield per plant and five hybrids had positive SCA effect for 100-grain weight in CML149 × CML330, CML332 × CML145, CML143 × CML145, CML167 × CML193 and CML167 × CML330 (Table 5a). CML149 × CML330 showed positive SCA effect for protein content. Seven hybrids namely, CML149 × CML330, CML138 × CML193, CML334 × CML145, CML332 × CML330, CML143 × CML145, CML167 × CML145 and CML167 × CML193 showed positive significant SCA effects for membrane stability index. CML332 × CML193 and CML143 × CML145 showed positive significant SCA effects for catalase activity.  Six hybrids viz., CML149 × CML145, CML138 × CML145, CML334 × CML193, CML332 × CML193, CML143 × CML145 and CML167 × CML330 showed positive significant SCA effects for peroxidase activity. Based on SCA effect (Table 5a), hybrids, CML149 × CML330 and CML143 × CML193 were identified as promising specific combiners for grain yield per plant, indicating their potential in heterosis breeding for increased grain yield (Table 8) Barh et al., (2015); Lane et al., (2015). Besides yield, CML149 × CML330 possessed significant negative SCA effect for days to 50% tasseling and significant positive SCA effects for 100 grain weight, protein content and membrane stability index.  In the present study it was observed that the grain yield was predominantly controlled by non-additive gene action (dominance and epistasis). Two hybrids, CML138 × CML193 and CML332 × CML145 showed negative SCA effects for plant height and were considered appropriate for development medium tall, non-lodging hybrids. Bharti et al., (2020) reported elite specific combiners for earliness and grain yield. Similar results were reported by Raihan et al., (2023) for grain yield; Ahmed et al., (2017) for grain yield, 100 grain weight and early flowering.
 

 

 
Standard heterosis
 
The per cent of standard heterosis expressed by different F₁ hybrids over the commercial check varieties HQPM-1 for yield and yield contributing characters are presented in (Table 6a and 6b). The extent of heterosis in F₁ hybrids varied within the spectrum of characters and among the crosses. Negative heterosis is considered desirable for days to 50% tasseling and days to 50% silking in the development of hybrids with earliness whereas, for plant height in developing medium tall, non-lodging hybrids.
 

 

       
The magnitude of heterosis depends on the degree of non-additive gene action and broad genetic diversity among parents. All characters studied in eighteen hybrids were compared with standard check HQPM-1. Eleven hybrids showed negative significant standard heterosis for days to 50% tassel emergence ranging from -4.15% to 8.29% and the hybrid CML332 × CML193 exhibited maximum negative significant standard heterosis. Seven hybrids registered negative significant standard heterosis for days to 50% silking ranging from -3.55% to 8.63% and the hybrid CML334 × CML145 displayed maximum negative significant standard heterosis for days to 50% silking (Table 6a). The cross CML138 × CML193 recorded most negative significant standard heterosis and can be used to produce short stature hybrids for lodging resistance. Out of three hybrids registered positive significant standard heterosis for cob length, the cross CML149 × CML145 displayed maximum positive significant standard heterosis (47.37%). For number of kernels row-1 cross CML149 × CML330 displayed most positive significant standard heterosis. For grain yield per plant standard heterosis value ranged from -29.82% to 33.31% and the cross CML149 × CML330 showed most positive significant standard heterosis (Table 7). For 100 grain weight the standard heterosis of hybrids ranged from -36.96% to 6.52% and hybrid CML149 × CML330 exhibited maximum positive significant standard heterosis (6.52%). Standard heterosis for protein content ranged from -39.71% to 25.54% and hybrid CML149 × CML330 showed maximum positive significant standard heterosis (Table 8).
 

 

       
Twelve hybrids registered positive significant standard heterosis for peroxidase activity ranging from -72.73% to 349.35% and hybrid CML167 × CML193 recorded most significant standard heterosis. Similar findings were reported by Mohammad et al., (2017) for protein content along with grain yield per plant in QPM maize. Aswin et al., (2020); Singh et al., (2022) also reported similar results in grain yield per plant.

The evaluation of parents based on per se performance and general combining ability (GCA) effects revealed that lines CML149, CML143 and tester CML330 were selected as potential contributors for future breeding programs. The importance of considering both mean performance and GCA effects in parent selection was emphasized. Crosses involving good GCA parents namely, CML149 × CML330 and CML143 × CML193 where shown to have high SCA effects for grain yield, terminal heat stress tolerance and high mean performance. These crosses could be exploited for development of high performing QPM hybrids due to non additive gene action for yield and its associated traits. Hybrids CML149 × CML330 and CML143 × CML193 exhibited significant per se performance, favourable SCA effects and standard heterosis for grain yield and other important traits making them promising candidates for further evaluation and utilization in breeding programs.
       
The assessment of hybrids based on per se performance, specific combining ability (SCA) effects and standard heterosis provided a comprehensive understanding of their potential. None of the crosses, exhibiting tolerance to terminal heat stress, displayed symptoms of leaf firing, tassel blast, root lodging and yield reduction. Crosses exhibiting high SCA were derived from parents that are generally good combiners for grain yield per plant. Such crosses deliver good segregants provided additive genetic system in a good combiner and epistatic effects in the crosses play in same direction for maximal character expression under consideration. Ample scope exists for QPM hybrid development under high temperature conditions and the need to expedite by following appropriate breeding procedures.

The authors express their gratitude to M. S. Swaminathan School of Agriculture for providing financial grant and technical assistant. Additionally the first authors show gratitude towards the Centurion University of Technology and Management for providing the fellowship to pursue doctoral research.

The authors declare that there is no conflict of interest regarding the publication of this paper.