Introduction

Silage maize (Zea mays L.), a globally significant forage crop, plays a pivotal role in the sustainable development of the livestock industry due to its direct influence on biomass production and nutritional quality1. In China alone, the planting area for silage maize has surpassed 2.33 million ha2, with an annual output exceeding 280 million tonnes3. Unlike grain maize, silage maize prioritizes whole-plant biomass maximization, with stalks contributing 45%-52% of the total dry matter, a critical determinant of forage feeding value4,5. Morphological stalk traits, including stalk width6, height7, and mechanical strength8, are shown to directly influence plant spatial competition strategies. For instance, thicker basal internodes enhance bending resistance, while shorter internodes optimize canopy light distribution.

Recent agricultural intensification has underscored the urgency of enhancing silage maize yield potential through high-density planting, a mainstream cultivation strategy aimed at increasing population biomass and storage capacity9. Within optimal density ranges, it has been demonstrated that maize yield exhibits a linear positive correlation with planting density. An increase in density can enhance maize population storage capacity and population biomass. However, excessive density compromises ventilation and light penetration, restricting individual plant growth and altering stalk morphology, such as increased plant height10, longer internodes11, elevated centres of gravity, reduced stalk diameter12. These ‘tall and thin’ adaptations, while temporarily alleviating population shading, significantly weaken stalk mechanical strength, heightening the potential of silage maize toppling13. Enhancing stalk strength is crucial for mitigating the risk of collapse. Rind penetration strength and bending strength serve as reliable proxies for stalk stability, with direct correlations to stalk resistance to lodging. A study demonstrated that stalk strength traits are highly significant and positively correlated with overall plant biomass14. Additionally, anatomical features, including epidermal thickness at the stalk base and its proportion relative to the total stalk cross-sectional area, the number of vascular bundles, and the proportion of thick-walled tissues in the stalk, have been confirmed to be strongly associated with stalk resistance to stunting, based on analyses of anatomical structure and histochemistry15,16. However, most of these studies have focused on single varieties or general analyses, lacking comparisons of the specific responses of different varieties in terms of stalk morphology and lodging resistance under high-density conditions. Moreover, planting density is closely linked to the quality traits of silage maize. Generally, as yield increases, crop nutritional value tends to decrease, and densification has been shown to exacerbate this reduction. Thus, investigating the impacts of high-density planting on silage maize quality traits is imperative.

Although high-density planting is an effective strategy for increasing yield, the comprehensive influencing mechanism of this method on stalk morphology traits, quality traits, and resistance to lodging has not been fully clarified. Moreover, there is a lack of systematic research on the response differences of different varieties in high-density environments. Xinjiang, situated in northwestern China, boasts a vast territory and abundant light resources. The cultivation of silage maize in this region can effectively utilize grassland resources, extend the forage supply period, and enhance both the yield and quality of feed, which are factors that provide unique conditions for achieving high maize yield in China17. Based on this, this study focuses on the Xinjiang region, conducted to investigate the effects of high-density environment on stalk-related traits and yield across different silage maize varieties, clarify the contribution rates of various stalk traits to yield, and analyze the stalk structural traits of dense-tolerant varieties. The aim is to provide a theoretical basis for improving the lodging resistance of silage maize stalks, thereby facilitating the rational increase of planting density.

Results

Analysis of variation in morphological traits of silage maize plants under high density

With increasing planting density, both maize plant height and ear height exhibited a downward trend (Fig.1-A), whereas the ear coefficient increased. Comparative analysis across varieties revealed that the ear height of M3 showed heightened sensitivity to density elevation. As illustrated in Fig.1-B, leaf area decreased progressively with increasing planting density. When comparing the upper ear leaf area, ear leaf area, and lower ear leaf area individually, the upper ear leaf area of M1 and M3 showed significant reductions under elevated density. Additionally, the ear leaf area decreased significantly when density increased from D2 to D3. Notably, the lower ear leaf area of M3 was found to be significantly larger than that of M1 and M2 across density treatments. Furthermore, the leaf width of all tested varieties tended to decrease with increasing planting density (Fig.1-C). With respect to stalk characteristics, the internode cross-sectional area at the basal 3rd internode of M1 was found to be significantly larger than that of M2 and M3, regardless of density treatment.

Fig. 1
figure 1

Effect of high-density planting on the morphology and mechanical traits of silage maize. D1, D2 and D3: Planting densities of 150,000 plants \(\hbox {ha}^{-1}\), 180,000 plants \(\hbox {ha}^{-1}\) and 210,000 plants \(\hbox {ha}^{-1}\), respectively; M1, M2 and M3: SheyuD1616, Dajingjiu 23 and Xianyu 1225, respectively; Horizontal coordinates (0, 10, 16, 20, 26, and 43): Growth stages corresponding to V6 (6-leaf stage), V9 (9-leaf stage), V12 (12-leaf stage), V15 (15-leaf stage), VT (tasseling stage), and R2 (blister stage), respectively. (A) Effect of high density on plant height, ear height and ear ratio. (B) Effect of high density on upper ear leaf area, ear leaf area, and lower ear leaf area. (C) Effect of basal 3rd internode on maximum diameter, minimal diameter, and stalk cross-sectional area. (D) Rind Penetration of basal internodes. (E) Bending strength of basal internodes. Different lowercase letters represent significant differences between different silage maize varieties at different planting densities at the 0.05 level. ns represents no significant level. *p<0.05, ** p<0.001.

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Effect of high density on the resistance of silage maize stalks to lodging

  • Changes in basal internode rind penetration strength

    Stalk rind penetration strength decreased significantly with increasing planting density. This parameter exhibited dynamic variation across growth stages, with values ranging from V12 to V15 stages, and peaking between VT and R1 (Fig. 1-D). Comparative analysis of silage maize varieties revealed that under D3 density at VT, the rind penetration strength of M3 was 16.42% lower than that of M1 and 20.11% lower than that of M2. Similarly, at D2 density, M3’s puncture resistance was 20.2% and 21.71% lower than that of M1 and M2, respectively. At D1 density, M3 showed reductions of 11.15% and 23.62% compared to M1 and M2, respectively.

  • Changes in basal internode bending strength

Bending strength increased during the reproductive period, peaking at the VT stage (Fig.1-E), but decreased with rising planting density. Varietal comparisons revealed that M3 exhibited significant differences in bending strength between D1, D2, and D3 as the reproductive period progressed. Specifically, at VT under D3 density, M3’s bending strength was 27.98% and 13.39% lower than that of M1 and M2, respectively.

Effect of high-density treatment on stalk microstructure

  • Changes in the mechanical organisation of stalk epidermis

Cross-sectional analysis revealed that the stalk is a heterogeneous structure composed mainly of stalk sclerotial tissue and a pith cavity (Fig. 2-A). The thickness of stalk sclerotial tissue was determined by measuring the width of the darkly stained bands formed by peripheral vascular bundles, which include small vascular bundles, epidermal mechanical tissues, and some of the thin-walled cells. At the VT stage, the mechanical tissue thickness diminished with increasing planting density (Fig. 2-B). M1 and M2 showed highly significant differences between D1 and D3, while M3 displayed no significant variation across all three densities. Similarly, the number of epidermal mechanical tissue layers decreased with increasing planting density (Fig. 2-C). M3 exhibited the fewest layers at D3 density, and across all three densities, its epidermal mechanical tissue layers were consistently fewer than those of M1 and M2.

  • Changes in the number of vascular bundles in the stalks

Fig. 2
figure 2

Mechanical organization differences in silage maize under high planting density. D1, D2 and D3: Planting densities of 150,000 plants \(\hbox {ha}^{-1}\), D1, D2 and D3: Planting densities of 150,000 plants \(\hbox {ha}^{-1}\), and 210,000 plants \(\hbox {ha}^{-1}\), respectively; M1, M2 and M3: SheyuD1616, Dajingjiu 23 and Xianyu 1225, respectively. (A) The microstructure of different silage maize varieties at different densities of \(5*10^{-5}\hbox {m}\). (B) Effect of high density of the mechanical tissue thickness. (C) Effect of high density of the mechanical tissue layers. (D) Vascular bundles number variations.

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At the VT stage, the total number of large and small vascular bundles in the stalk decreased with increasing planting density (Fig. 2-D). Notably, across all three density treatments, M3 maintained a higher count of both big and small vascular bundles compared to M1 and M2.

Effect of high density on yield and quality of silage maize

  • Analysis of yield variation of silage maize

Increasing planting density resulted in a concurrent increase in both fresh forage yield and the hay forage yield across all silage maize varieties (Fig. 3-A). Highly significant differences in fresh and hay forage yield were observed among M1, M2, and M3 at all three planting density levels. M2 consistently outperformed the other varieties, with a peak yield of 135.81 t \(\hbox {ha}^{^{-1}}\) (tonnes per hectare), for fresh forage and 31.97 t \(\hbox {ha}^{^{-1}}\) for hay, respectively.

  • Mean square and significance analysis of silage maize quality traits

Fig. 3
figure 3

Mechanical organization and yield-quality relationships in silage maize under high density. D1, D2 and D3: 150,000 plants \(\hbox {ha}^{-1}\), 180,000 plants \(\hbox {ha}^{-1}\) and 210,000 plants \(\hbox {ha}^{-1}\), respectively; M1, M2 and M3: Sheyu D1616, Dajingjiu 23 and Xianyu 1225, respectively. VTRPS: rind peneyration strength during tasseling stage, VTBS: bending strength during tasseling stage. (A) The yield of silage maize after harvest across different densities. (B) Density-dependent changes in the silage quality traits. (C) Correlation between stem microstructure characteristics and the rind penetration strength and bending strength during the VT stage. (D) Correlation between the yield and quality traits of silage maize and the rind penetration strength and bending strength at the VT stage. Different lowercase letters indicate significant levels of difference, ns indicates no significant difference, * p<0.05, ** p<0.001.

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Both planting density and silage maize variety significantly influenced key quality indicators, including acid detergent fiber, neutral detergent fiber, crude protein, dry matter, and starch content. Varietal performance varied across densities: M3 exhibited advantages in terms of acid and neutral detergent fibers under certain densities, while M2 showed outstanding starch trait across treatments (Fig. 3-B).

  • Correlation analysis of stalk, yield, and quality traits in maize silage

Correlation analyses revealed significant associations between stalk mechanical properties, microstructural traits, yield, and quality (Fig. 3-C). Notably, the number of big and small vascular bundles showed highly significant positive correlations with basal 3rd internode cross-sectional area, stalk diameter, and mechanical tissue thickness. Rind penetration strength and bending strength were shown to be strongly positively correlated with each other. Correlation analyses were conducted to evaluate relationships between silage maize stalk mechanical properties and key agronomic traits (Fig. 3-D). The results revealed that there was a highly significant positive correlation between fresh forage yield and hay forage yield, indicating that varieties with higher fresh biomass also produced greater dry matter yields. Moreover, both yields exhibited positive correlations with rind penetration strength and bending strength, suggesting that stronger stalks support higher biomass accumulation. Ring penetration strength and bending strength showed negative correlations with the acid detergent fiber and the neutral detergent fiber.

  • Analysis of yield components related to stalk traits in high-density silage maize

To elucidate the impact of stalk-related traits on yield under high-density conditions, multiple regression analysis was performed to quantify contributions of stalk-related traits, including variety, density, and yield (Table 1). \(\hbox {R}^{2}\) is a statistical measure used to assess the goodness of fit of a regression equation. It represents the proportion of the variation in the dependent variable that can be explained by the independent variable. The closer \(\hbox {R}^{2}\) is to 1, the better the regression equation fits the experiment. The results show that \(\hbox {R}^{2}\) = 0.928, that are large vascular bundle number, small vascular bundle number, mechanical tissue layer count, variety, and density collectively explained 92.8% of yield variation, demonstrating good fitting. The significance of the multiple regression model reached a highly significant level, indicating that the yield composition model of stalk-related traits was established in this study. The regression equation (y = - 0.065 x1 - 0.083 x2 + 4.807 x3 + 38.266), accurately reflected yield responses to different densities. As shown in Table 2, among the stalk-related traits evaluated under high-density conditions, only the number of big vascular bundles, the number of small vascular bundles, mechanical tissues layers, variety, and the density among the stalk related traits exhibited statistical significance or high significance in the significance test of the regression model. These factors collectively demonstrated a strong influence on yield in this experiment.

Table 1 Yield constitutive parameters and contribution of each trait to stalk-related traits in silage maize. *p<0.05, ** p<0.001.
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As shown in Table 2, the regression models for yield prediction differed significantly among silage maize varieties, reflecting varietal-specific contributions of stalk-related traits: M2 ( y = 0.463 x6 - 0.061 x7 + 1.675 x9 - 0.17 x10 - 5.059 x3 - 14.862 x5 + 85.19 ) and M3 ( y = 1.408 x6 - 0.043 x7 + 16.051 x9 - 0.762 x1 - 0.147 x1 - 0.13 x2 - 0.197 x11 + 21.21 ) introduced more significant traits into the yield model than M1 ( y = -4.094 x8 - 0.08 x2 + 2.557 x3 - 6.646 x5 + 114. 414 ). Density, basal 3rd internode maximum diameter, basal 3rd internode minimal diameter, and mechanical tissues layers exerted pronounced effects on the yield of under different silage maize varieties, and these traits had a greater effect on the yield of the silage maize varieties at different densities.

Table 2 Model parameters of yield components and contribution of stalk-related traits in silage maize varieties. *p<0.05, ** p<0.001.
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Discussion

Analysis of yield components related to stalk traits in high-density silage maize

Planting density stands as a pivotal factor influencing the growth patterns and yield dynamics of silage maize18, Among the various morphological attributes, plant height and stalk width serve as critical indicators reflecting the crop’s growth and development status, and to a certain extent, they can be indicative of the crop’s nutritional status19,20. In our experiment setup, distinct phased variations in plant height were observed in response to different planting densities. Specifically, under D1 density, both plant height and spike height were notably elevated. However, as the density surpassed thresholds D2 and D3, a significant reduction in the plant height was recorded, accompanied by a marked decrease in the stalk diameter. These findings corroborate the results reported by Yan et al.21, Liu et al.22, Zhang et al.23. This is also consistent with the findings of Tang et al.24, where it was observed that for the same variety of maize, plant height and ear height increase with increasing density, while stalk diameter decreases with increasing density. This “threshold effect” is closely related to the intensification of light competition and the change in resource allocation strategies. Below the threshold density, the light competition among plants is relatively weak, the group transmittance is higher, the photosynthetically active radiation is sufficient, the resource allocation is balanced, and the plants can simultaneously focus on both vertical growth and stalk thickening. Through optimizing the basal internode thickness, they enhance the mechanical support capacity, thereby forming better morphological traits25. However, when the density exceeds the threshold, the group canopy rapidly closes, the light intensity significantly decreases and the ventilation conditions are impaired. The plants initiate the “shading avoidance response” to compete for the limited light, and the resource allocation tilts towards vertical growth to prioritize the elongation of stalk nodes, resulting in a reduction in stalk diameter - this “tall and thin” morphological change is the adaptive adjustment of the plants to the high-density environment, but it will weaken the mechanical strength of the stalks26. This is consistent with the conclusion from previous studies in the Longdong Loess Plateau region that “plant density is negatively correlated with stem width”. The core reason for this lies in the fact that under high density, the efficiency of photosynthesis decreases, thereby limiting the accumulation of substances in the stalks. This trend can be primarily attributed to the reduced light intensity and impaired ventilation among plants at higher densities, which subsequently diminishes crop photosynthesis efficiency. Leaf length and width are equally pivotal factors affecting maize growth and development27, Our experiment demonstrated that the leaf area at the ear level of three silage maize varieties progressively diminished with increasing planting density, aligning with the observations made by Gou et al.28. This outcome underscores the notion that high-density planting substantially curtails the leaf area at the ear, likely due to the maize canopy closing rapidly under high-density treatment and the limited photosynthetically active radiation. These factors hinder the growth and development of maize, culminating in a reduced leaf area29. In summary, our study reveals a “threshold effect” in the influence of planting density on the morphological characteristics of silage maize. High-density planting exerts a pronounced impact on these morphological traits. However, it is feasible to enhance the morphological attributes of silage maize by judiciously increasing the planting density and maintaining it within a reasonable threshold, thereby increasing maize yield.

Effect of high density on yield and quality of silage maize

Density is recognized as a pivotal factor for achieving a high yield of silage maize30. Dry matter accumulation forms the cornerstone of maize yield, making increasing planting density a straightforward approach to achieve high yield. Jiang et al.31 concluded that the biological yield of silage maize initially increased and then declined with escalating density, highlighting that excessively high planting density could actually diminish the biological yield of maize. In our experiment, as silage maize planting density increased, both fresh forage yield and hay forage yield of silage maize initially rose and then fell, aligning with the findings of Qi et al.32. Notably, rind penetration strength and bending strength exhibited a significant positive correlation with yield. This is attributed to the robust stalks of silage maize, which maintain good uprightness in crowded planting space, effectively mitigating yield losses due to stalk lodging. This ensures a more rational spatial distribution between plants, optimizing ventilation and light penetration conditions, thereby enhancing individual plant yield and contributing to overall yield improvements13,33. Specifically, the hay forage yield and fresh forage yield of Sheyu D1616 and Dajingjiu 23 were higher at a density of 180,000 plants \(\hbox {ha}^{-1}\), with rind penetration strength and bending strength also relatively elevated at the tasseling stage compared to that of Siengyu 1225. Consequently, the density condition of 180,000 plants \(\hbox {ha}^{-1}\) was deemed more conducive to achieving high yield compared with the densities of 150,000 plants \(\hbox {ha}^{-1}\) and 210,000 plants \(\hbox {ha}^{-1}\).

The inherent traits of different varieties lead to varied responses to density. Yield and quality are two critical indicators of silage maize cultivation, and a reasonable planting density can significantly enhance silage maize quality. Silage maize quality was found to be positively correlated with crude protein and crude fat content, while negatively correlated with neutral detergent fibre and acid detergent fibre content34. Furthermore, Feng et al.35 demonstrated that the acid detergent fibre, neutral detergent fibre, and crude fat content in the upper part of maize increased with density, whereas crude protein content decreased. The forage nutritive value of silage maize planted at high density was lower than that at medium planting density36. The effect of planting density on crude protein and starch content varied among varieties, with significant differences observed and an overall negative correlation with planting density37. In our experiment, the impact of planting density on quality traits of silage maize was significant. The silage-specific variety Dajingjiu 26 maintained relatively low NDF levels at high density, which is related to its genetic predisposition for low lignification degree of the stalks. In contrast, the common grain variety Sheyu D1616 exhibited a significant increase in NDF under high-density conditions. This response may stalk from its thinner stalks and weaker lodging resistance, which prioritize resource allocation to grain development over stalk quality. The dual-purpose variety Xianyu 1225 displayed “intermediate” characteristics: at medium and low densities, its starch content approached that of Sheyu D1616 (grain advantage), while its biomass resembled Dajingjiu 26. Overall, silage maize quality declined with increasing planting density, aligning with findings from those reported by Tang et al.38 and Wang et al.36 , but diverging slightly from the findings of Lu et al.39. These discrepancies may arise from the varietal differences and distinct planting patterns. Thus, selecting an appropriate planting density is critical to balancing biological yield and forage nutritional value. Correlation analysis revealed that the rind penetration strength and bending strength were negatively correlated with acid-washed and neutral-washed fibres, respectively, but positively correlated with starch content. Notably, starch, the primary product of photosynthesis in silage maize, serves as the key metabolic energy source in this species2. These findings suggest that the enhancement of stalk quality traits may be achieved through the improvement of stalk strength by enhancing stalk-related correlation traits in a high-density environment.

To achieve the coordinated optimization of the yield and quality of silage maize under high-density conditions, the planting strategy should be adjusted according to the traits of the variety. The silage-specific variety Daqingjiu26 can maintain the phenotypic trait of low neutral detergent fiber content even under high-density conditions. At a planting density of around 180,000 plants \(\hbox {ha}^{-1}\), it can still ensure the biological yield while maintaining its advantages in feed quality. Although the common grain variety Sheyu D1616 shows a significant increase in starch content under high-density conditions, its stalk resistance to lodging is weak. Therefore, the planting density should be controlled within the range of 150,000-180,000 plants \(\hbox {ha}^{-1}\). Through the optimization of density gradients, the risk of lodging can be reduced and the yield, starch quality, and yield quality can be simultaneously improved. The grain-feed dual-purpose variety Xianyu 1225 can adjust the planting density according to different harvest targets of biomass and quality traits. At the same time, with the assistance of precise field management measures, the negative effects of high-density stress on quality traits can be alleviated, thereby strengthening the balance mechanism between yield and quality.

Response to high density and its contribution to yield components in traits related to resistance to inversion

Maize stalks serve as the primary locus of plant stress, with their strength directly influencing the crop’s resistance to stunting40. A notable correlation exists between maize stalk resistance to lodging and several stalk traits, including stalk tension, rind penetration strength, and stalk width41. Research has demonstrated that planting density significantly affects stalk traits, with stalk mechanical strength notably decreasing as planting density increases42. Basal internode traits have been identified as crucial factors influencing maize stalk lodging43,44, with the basal 3rd internode showing the strongest correlation with this phenomenon. Our experiment revealed a gradual decline in stalk rind penetration strength and bending strength with increasing planting density, corroborating the findings of Qi et al.32 and Gu et al.45. Additionally, Yao et al.46 investigated the response of the maize basal 3rd internode under dense planting conditions, demonstrating that stalk width and internal structure, such as vascular bundle density, play pivotal roles in stalk lodging resistance. Our experiment also found that stalk bending strength was positively correlated with the number of big and small vascular bundles, as well as stalk cross-sectional area. As planting density increased, the number of big and small vascular bundles, mechanical tissue layers, and mechanical tissue thickness decreased across three silage maize varieties, showing a highly significant positive correlation. This finding aligns with the results of Tian et al.47, suggesting that the stalk mechanical tissue layers, mechanical tissue thickness, vascular bundle number, and vascular bundle area are critical parameters for assessing stalk lodging performance in the improved selection of silage maize varieties.

Enhancing lodging resistance is a vital strategy for increasing maize yield. A comprehensive analysis of the contribution of stalk-related traits to the yield composition of silage maize, and an elucidation of their impact on yield, are essential for selecting and breeding silage maize varieties that are both dense-tolerant and high-yielding48. Our analysis of the yield composition of different silage maize varieties aimed to identify the factors that most significantly contribute to the average yield of these varieties. The study revealed that the number of big vascular bundles, as well as the number and density of small vascular bundles, were the primary contributors to the yield composition model. From a biological perspective, vascular bundles serve as the main channels for material transportation within the plant, and their quantity and structure directly affect the efficiency of transporting photosynthetic products, water, and nutrients. Big vascular bundles and small vascular bundles have a synergistic function in their functions. Big vascular bundles mainly undertake the long-distance transportation of a large amount of water and minerals, while small vascular bundles play an important role in the short-distance distribution and local supply of photosynthetic products49. A greater number of vascular bundles can increase the number of transportation channels and enhance the transportation rate, ensuring that photosynthetic products can be rapidly transported from the leaves to storage organs such as seeds, and at the same time ensuring that the water and nutrients absorbed by the roots are promptly transported to all growth parts of the plant, providing sufficient material support for the growth and development of the plant and the formation of yield50. In addition, the distribution and structure of vascular bundles are also related to the mechanical strength of the stalk. Adequate number of vascular bundles can enhance the supporting capacity of the stalk and reduce the risk of lodging, thereby indirectly ensuring the yield51. Additionally, rind penetration strength and bending strength were significantly positively correlated with silage maize yield. Previous studies52,53, various maize plant traits, including plant height, ear height, stalk width, basal internode diameter, and rind penetration strength, influence the plant’s resistance to lodging. Furthermore, stalk mechanical traits have been demonstrated to exhibit a strong correlation with lodging resistance and yield43,54. The results of this experiment align with previous studies, reinforcing the observation that under high-density planting conditions, stalk-related traits, such as the number of big and small vascular bundles, rind penetration strength, and bending strength, indirectly influence the key determinants of maize yield. Consequently, breeding strategies for high-density, high-yield maize varieties should prioritize the improvement of these traits. Specifically, enhancing stalk bark rind penetration strength, bending strength, and the number of vascular bundles directly contributes to stalk development, thereby promoting higher silage maize yields. The yield composition analysis, focusing on key maize stalk-related traits, was meticulously integrated into a linear regression model. These traits encompassed the maximum diameter of the 3rd basal internode, the minimum diameter of the 3rd internode at the base, and the number of mechanical tissue layers. Analysis shows that these traits contribute to the yield at different densities within the stalk. These findings suggest that under high-density conditions, enhancing stalk thickness and stalk wall diameter contributes indirectly to yield improvement.

The density tolerance and high-yield potential of maize are determined by the synergistic effects of various factors55. Previous studies have demonstrated that crop varieties exhibit enhanced lodging resistance when characterized by smaller ratios of plant height, ear height, base internode length, and stalk length, coupled with greater maximum stalk width and stalk rind penetration strength. Notably, lodging resistance has been found to show a significant positive correlation with yield56,57. In the present study, Dajingjiu23 achieved the highest fresh and hay forage yields, reaching 135.81 135.81 t \(\hbox {ha}^{-1}\) and 31.97 t \(\hbox {ha}^{-1}\) at 180,000 plants \(\hbox {ha}^{-1}\), respectively. These yields were significantly higher than those of Sheyu D1616 and Xianyu 1225. DajingJiu23 also displayed superior rind penetration strength, bending strength, and greater mechanical tissue thickness, all of which are advantageous for dense planting. Regression analysis revealed that the yield composition model of Da jingjiu23 incorporated a significantly greater number of impactful stalk traits compared to Sheyu D1616 and Xianyu 1225. However, there were also several stalk traits that were not conducive to dense planting. Therefore, in the breeding of high-yield, high-density varieties, emphasis should be placed on optimizing key density-tolerant traits. Breeding efforts should be tailored to specific needs, with a focus on balancing yield-related traits to an optimal state that promotes high productivity, rather than pursuing density tolerance in a blind manner.

Conclusion

As planting density increased, the three silage maize varieties exhibited significant differences in their performance and phenotypic variations. Both hay yield and fresh feed yield peaked at a density of 180,000 \(\hbox {ha}^{-1}\) plants. This density represents the optimal planting density for this specific variety and environmental conditions in this experiment. Correlation analysis revealed that rind penetration strength and bending strength were positively correlated with yield and starch content, suggesting that these mechanical properties may mitigate lodging risk and thereby indirectly influence yield formation. Multiple regression analysis indicated that the number of big and small vascular bundles, as well as the number of mechanical tissue layers, each contributed more than 5% to yield. Notably, the average contribution rates of the 3rd internode width and the number of mechanical tissue layers exceeded 15%, highlighting that under high-density conditions, stalk width and mechanical tissue exert a more prominent impact on yield. In conclusion, under the specific varietal and environmental conditions of this experiment, a planting density of 180,000 plants \(\hbox {ha}^{-1}\) is conducive to enhancing forage yield. Furthermore, the coordinated improvement of stalk lodging resistance at this density may serve as a critical safeguard for stable yield. These findings provide a theoretical basis for optimizing high-density cultivation of silage maize in the study area. Future research could encompass multi-environment field trials to validate the generalizability of this optimal planting density across diverse ecological contexts. Concurrently, genetic dissection of traits associated with yield stability should be pursued to elucidate the underlying genetic mechanisms, thereby furnishing a more robust theoretical framework for the breeding of high-yielding, density-tolerant maize varieties.

Methods

Experimental materials

The experiment was sowed at the South Xinjiang Industry-Education Integration Modern Agriculture Training Base of Tarim University, with sowing carried out on 21 April 2024, and the harvest achieved on August 2 2024. A randomized block design was adopted, incorporating three planting densities of D1 (150,000 plants \(\hbox {ha}^{-1}\)), D2 (180,000 plants \(\hbox {ha}^{-1}\)) and D3 (210,000 plants \(\hbox {ha}^{-1}\)). The test materials included M1 (SheYu D1616) is common grain variety, M2 (Dajingjiu 23) is specialized silage maize variety, and M3 (Xianyu 1225) is dual-purpose grain and forage varieties (Three varieties were sourced from the comprehensive variety screening work carried out by the previous research team. For 50 maize varieties intended for silage use, a planting experiment was conducted under a density of 105,000 plants \(\hbox {ha}^{-1}\). Through systematic measurement and analysis of various indicators such as yield, agronomic trait and nutritional quality, three maize varieties with overall excellent performance were selected from the numerous varieties. The yield and quality trait data of M1, M2, and M3 are detailed in Supplementary Table S1 and Supplementary Table S2). The test site was divided into 3 zones (representing 3 replicates), resulting in a total of 9 treatment combinations. Each treatment was replicated in three plots, with each plot covering an area of \(20\hbox { m}^{^{2}}\). Planting followed a wide-narrow row arrangement (Fig. 4-A), with two seeds sown per hole according to the planting density, at a depth of 4 to 5cm. For a single-year, single-location test.

Fig. 4
figure 4

(A) The silage maize cropping patterns. (B) Meteorological conditions in the Alar Reclamation Area in 2024. D1, D2 and D3: 150,000 plants \(\hbox {ha}^{-1}\), 180,000 plants \(\hbox {ha}^{-1}\) and 210,000 plants \(\hbox {ha}^{-1}\), respectively.

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Irrigation, fertilization and climate in the test area

The test site was characterized by a sandy loam soil texture. An assessment of the base fertility within the 0-20 cm soil layer revealed specific nutrient concentrations: soil organic matter was measured at 0.43 mg/g, while quick-acting potassium, nitrogen, and phosphorus levels were 126.78 mg/kg, 83.99 mg/kg and 2.19 mol/g, respectively. Irrigation at the site was implemented using a system of two rows of under-membrane drippers, designed to match the water needs of the maize. The preceding bcrop grown in this field was cotton. Detailed information regarding fertilization and irrigation regimes is presented in Table 3. In terms of agronomic management, fertilizer application, cultivation techniques, and pest control practices in the field adhered to standard field conditions. Geographically, the test site is located in the Alar Reclamation Area of Xinjiang, and its maximum temperature from April to September 2024 mainly occurred in July-August, and precipitation was mainly concentrated in May-August (Fig. 4-B).

Table 3 Fertilization and irrigation conditions. V6 (6-leaf stage), V9 (9-leaf stage), V12 (12-leaf stage), V15 (15-leaf stage), VT (tasseling stage), and R2 (blister stage), R4 (dough stage).
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Experimental methods and measurement indicators

In this study, the phenotypic characteristics of silage maize were systematically evaluated under field conditions, adhering to “the Specification and Data Standards for Maize Material Description”58. At the time of harvest, the following parameters were measured: plant height (cm)59, ear height (cm)60, stalk width (mm)61, leaf length (cm)61 and leaf width (cm)61.

Stalk Rind penetration strength (\(\hbox {N mm}^{^{-2}}\))52: To evaluate stalk strength, three plants were randomly selected from a small area at the five distinct growth stages: 9-leaf stage (V9), 12-leaf stage (V12), 15-leaf stage (V15), tasseling stage (VT), and blister stage (R2), and the tip of a certain cross-sectional area (e.g., \(1\hbox {mm}^{^{2}}\)) was inserted into the short-axis surface in the middle of the internode with the Stalk Strength Determining Instrument YYD-1B (Zhejiang Top Instrument Co., Ltd., Hangzhou, China), and the maximum value of penetration force recorded was taken as the rind penetration strength.

Stalk Bending strength (\(\hbox {N cm}^{^{-2}}\))52: Bending strength was assessed by applying the three-point bending method. Two points, located 5cm from the midpoint of the basal 3rd internode, were selected at 9-leaf (V9), 12-leaf (V12), 15-leaf (V15), tasseling stage (VT), and blister stage (R2) stages, and were fixed in the grooves of the supports at 10cm intervals. The maximum mechanical force required to break the basal 3rd internode was measured using the Stalk Strength Tester, that is, the bending strength of the stalk.

Determination of anatomical structure of basal 3rd internode: To investigate the internal anatomy of the stalk, three representative plants were selected at the tasseling stage. The middle portion of the basal 3rd internode above the ground was excised and fixed with FAA 7/15 fixative (95% anhydrous ethanol 150 mL + 5% formalin 100 mL + glycerol 50 mL)32. The fixed samples were then stained into a Plantago ovata red staining solution for 2h, followed by a gentle rinse in tap water, to remove excess dyes. The sections were subsequently dehydrated through a series of alcohol solutions (50%, 70%, 80%) for 3-8s, stained with plant solid green staining solution for 6-20s, and further dehydrated in anhydrous ethanol. After clearing in clean xylene for 5min, the sections were sealed with neutral gum. The prepared slides were examined using the MShot Image Analysis System microscope camera system and photographed. The number of large and small vascular bundles in the cross-sectional area of the 3-noded culm was counted for different varieties. Additionally, the number of cell layers and the thickness of the epidermal mechanical tissue of the culm were measured using ImageJ software. Sclerotial thickness was defined as the thickness of the peripheral group of vascular bundles, which are densely concentrated at the broad side of the culm. The diameter of the sclerotia was measured as the width of the darker-stained bands formed by these dense peripheral vascular bundles on the broad side of the culm. The number of big and small vascular bundles of the stalk was also quantified62.

Silage maize yield: To accurately determine the silage maize yield, all plants within the plot were meticulously mowed flush at the optimal harvesting period for silage maize. To ensure precision and eliminate edge effects, the side rows and the two ends of 40 cm were excluded from the harvest. The fresh weights of the harvested plants were then recorded, and the plot’s fresh grass yield was calculated and converted into fresh grass yield per hectare. In parallel, to assess the dry matter accumulation and yield, 10 representative plants of silage maize were randomly selected from each plot and transported to the laboratory, where they were killed in an oven at \(105^{\circ }\text {C}\) for 30min and dried at \(80^{\circ }\text {C}\) to a constant weight, and the dry-to-fresh ratio and hay yield were calculated63.

Silage maize quality: all the dried silage maize was crushed and passed through a 1mm sieve, and three grass samples were randomly selected from the well-mixed grass samples, each sample was about 0.5g, and all the nutritional indexes were determined in parallel. Crude protein (CP) content was determined by Kjeldahl method64; Neutral detergent fibre (NDF) and Acid detergent fiber (ADF) content was determined by Van Soest method64, and starch (ST) content was determined according to GB/T20194-2018. Starch) content65, and Dry matter (DM) content was determined by high performance liquid chromatography (HPLC)66.

Data analysis and methods

Descriptive statistics67,68 were conducted using Microsoft Excel 2021 software, encompassing maximum, minimum, and mean values of the data.

The traits under investigation were subjected to grouped ANOVA using GraphPad Prism version 9 software69, Following the ANOVA, histograms and line graphs were plotted to visually illustrate the distribution patterns.

R.4.33 was utilised for the execution of correlation analyses pertaining to stem-related traits, yield-related traits and quality-related traits70.

Regression analyses were conducted using IBM SPSS Statistics 27.0 to assess the contribution of stalk-related traits to silage maize hay yield through multiple regression models71,72. The selection of specific stalk-related traits (e.g., number of big and small vascular bundles, rind penetration strength, bending strength) as predictors in the regression models was based on two considerations: First, theoretical and prior research support—vascular bundles are critical for material transport in plants, and their quantity directly affects the efficiency of photosynthate translocation, which has been confirmed to be closely related to maize biomass accumulation15,16; stalk mechanical traits such as rind penetration strength and bending strength are core indicators of lodging resistance, and their association with yield stability under high-density conditions has been well-documented8,14. Second, results from preliminary correlation analyses in this study showed that these traits had significant correlations with hay yield (P<0.05), indicating strong intrinsic associations with yield and thus providing a statistical basis for their selection as predictors. To mitigate potential bias arising from differences in trait magnitudes, the data were standardized and converted into dimensionless data. The specific calculations for the regression analysis were formulated as follows:

$$\begin{aligned}Yn=A1\times x1+A2\times x2+A3\times x3+\cdots +An\times x_{\textrm{n}}\end{aligned}$$

yn is the standardised value of hay yield; x1, x2, x3... xn are the standardised values for each trait, and A1, A2, A3...An are the regression coefficients for each trait.

$$\begin{aligned} C_{1}=|A_{1}|/(|A_{1}|+|A_{2}|+|A_{3}|+\cdots +|A_{n}|) \end{aligned}$$

C1 is the contribution of trait x1 to yield. Also, the significance of the regression analysis was verified by F-test.