Introduction

Sugarcane (Saccharum spp. hybrids) cultivation has expanded to over 110 countries, covering an area of 25.98 million ha (Singh and Gupta 2019) and contributing to nearly 80% of global sugar production (Venail et al. 2022), highlighting its significant economic importance. Due to substantial genetic heterogeneity in the seed population, sugarcane is typically propagated from vegetative cuttings (Franklin et al. 2006). Sugarcane stalks usually possess a single axillary bud at each node (Burner and Legendre 1998). In commercial sugarcane cultivation, stem sections (setts) or entire topped stems are commonly used for planting (Tew and Cobill 2008). Consequently, it's essential to plant an adequate amount of seed cane to ensure a satisfactory number of millable stalks and achieve higher cane yields in a field. Currently, commercial sugarcane fields plant approximately 112,500 to 125,000 buds per hectare, with the seed fee representing about 15–20% of the planting cost (Lu et al. 2020). Thus, finding ways to minimize both the number of cane setts and the planting cost is crucial for maximizing profits in the sugarcane business, presenting a challenge to the sugarcane industry.

In previous studies on the innovative utilization of hybrids from Saccharum robustum at the Yunnan Sugarcane Research Institute (YSRI), the YZ 07-86 sugarcane genotype has been identified as a natural mutant genotype with dual-axillary buds in the nodes (Lu et al. 2020). Remarkably, both buds in a node can sprout normally and develop into stems. Additionally, the millable stalks and cane yield of YZ 07-86 were not significantly different compared to the single-axillary budded genotype (YZ 07-87) when planting an equal number of buds, but the required cane setts were halved under the dual-axillary budded genotype (Lu et al. 2020). As stated by Sharma (1955), the multiple-bud trait was not inherited in sugarcane except in some cases where they reported triple-buds in a clone persisting even after 4 years of vegetative propagation. Burner and Legendre (1998) also reported that with in vitro propagation, the multiple-bud characteristic of CP 68-413 (a sugarcane variety) was comparatively stable and sexually transferred. Lu et al. (2020) observed that the dual-axillary bud trait of YZ 07-86 has remained stable in field trials, whether in new plantings or ratoon crops. Furthermore, they conducted crosses involving the YZ 07-86 mutant genotype with 27 different sugarcane parents. From these crosses, they found that hybrid offspring clones resulting from 11 combinations exhibited dual-axillary buds, thus demonstrating the heritable nature of the dual-axillary bud trait in the YZ 07-86 genotype. Therefore, it is noteworthy that the dual-axillary bud trait in the YZ 07-86 genotype is both stable and heritable (Lu et al. 2020). Therefore, if the dual-axillary bud trait can be utilized in breeding, new double-bud varieties can be developed, and the amount of seed cane can be significantly reduced while ensuring sufficient bud quantity, thereby reducing the costs associated with planting and enhancing the profitability of the sugarcane business.

To better develop and utilize the excellent characteristics of dual-bud mutants, the key scientific problem to be addressed is clarifying the physiological mechanism of dual-bud formation and development. Several authors (McSteen 2009; Chen et al. 2020) have reported that the formation and development of axillary buds are regulated by shoot-branching-related genes through interactions with plant hormones. Gibberellins (GAs), ethylene, cytokinins (CKs), abscisic acid (ABA), and auxin are collectively regarded as the classical five plant hormones, with indole-3-acetic acid (IAA) identified as the primary auxin in plants (Kende and Zeevaart 1997). Importantly, Chen et al. (2020) suggested that the pathways of IAA and ABA are involved in controlling axillary bud formation in the dual-axillary budded sugarcane genotype. Although there are approximately 136 known GAs (Hedden 2020), only a few are present in bioactive forms (Binenbaum et al. 2018), with GA3 (gibberellin 3) and GA4 (gibberellin 4) included in the bioactive group (Davière and Achard 2013). Additionally, CKs are considered the primary class of hormones involved in shoot development (Wybouw and De Rybel 2019), with isopentenyl adenosine (iPA), zeatin riboside (ZR), and dihydrozeatin (DHZR) considered the active forms of CKs (Kende and Zeevaart 1997; Ongaro and Leyser 2008).

As plant hormones play a direct role in plant physiological development, investigating the major hormones present in both genotypes and their dynamic changes at the bud differentiation stage is crucial for understanding their involvement in dual-axillary bud formation. To achieve this, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze key endogenous hormones, including IAA, ABA, GA3, GA4, iPA, ZR, and DHZR, in both the dual-axillary budded YZ 07-86 genotype and its single-axillary budded sister line, YZ 07-87. The objective was to elucidate the physiological mechanism behind dual-axillary bud formation in YZ 07-86.

Materials and Methods

The experiment was conducted at the first research base of the Sugarcane Research Institute of the Yunnan Academy of Agricultural Sciences, located in Kaiyuan, China. Assessment of the plant endogenous hormones (GA3, GA4, IAA, ABA, iPA, ZR, and DHZR) and their dynamic changes in the YZ 07-86 and YZ 07-87 genotypes was performed using shoot apical meristem (SAM) tissue samples.

Experimental Design and Sampling Procedure

A systematic field trial was established to collect shoot apical meristem (SAM) tissue samples. The selected field was divided into six plots, each with an area of 20 m2 (with 1 m spacing between adjacent plots). Within each plot, five furrows, each 4 m long, were prepared for planting test materials. The two sugarcane genotypes (YZ 07-86 and YZ 07-87) were randomly assigned to the plots following a completely randomized design, with three replicated plots for each genotype. Seed cane consisting of 9-month-old plants was used, with an equal number of buds (192 buds) planted in each plot using cane setts (stem pieces) containing three nodes. Standard cultural practices were followed to manage the field trial.

Sampling commenced 106 days after planting (DAP) and continued at intervals of 5 days up to 201 DAP. Data collection was not possible at 186 DAP, resulting in a total of 19 sampling time points: specifically, at 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 191, 196, and 201 DAP. At each sampling time, two randomly selected stalks were harvested from each plot, immediately placed in water to immerse the cut stem surface, and then transported to the nearby laboratory. Sampling was consistently conducted between 8 a.m. and 9 a.m., following the same procedure for each sampling time. In the laboratory, the leaves and leaf sheaths were removed from the stalks, and the SAM tissues of the plants were separated. The collected tissues were individually wrapped in aluminum foil, frozen in liquid nitrogen for 30 min, and temporarily stored in a refrigerator at − 20 °C (Zhang et al. 2019). Hormone extraction was performed immediately after completing the tissue sample collection process.

Sample Preparation and Extraction of Endogenous Hormones

The plant hormone test kits purchased from the Plant Chemical Control Center of the China Agricultural University were utilized, following the instructions provided with the kits for sample preparation and extraction of endogenous hormones (Zhang et al. 2019; 2020a). A precise 1 g portion of shoot apical meristem tissue sample was ground into homogenates using a mortar and pestle placed on an ice bath, with a total of 6 mL of extracting solution (80% methanol containing 1 mmol/L 2-tert-butyl-4-methylphenol). The resulting mixture was transferred into a 10 mL centrifuge tube and stored at 4 °C for 4 h to extract the hormones. After extraction, the mixture was centrifuged at 3500 RPM for 8 min, and the supernatant was collected. Subsequently, 1 mL of extracting solution was added to the precipitate for re-extracting the hormones. This mixture was stored at 4 °C for 1 h, followed by centrifugation under the same conditions, and the supernatant was collected. The supernatants from the first and second extractions were combined. The resulting supernatant was filtered using a Sep-Pak C-18 extraction column (Waters, WAT043395, USA). Methanol from the extraction was removed using a nitrogen blow-drying device (ZGDCY-24S, ZiGui, China), and the solution was diluted by adding 2 mL of sample diluent (consisting of 1 mL/L tween-20, 1 g/L gelatin, and PBS—phosphate buffer solution: 8 g/L NaCl, 0.2 g/L KH2PH4, 2.96 g/L Na2HPO4·12H2O, pH = 7.5). All stock solutions were stored at -70 °C for further use.

Determination of Endogenous Hormone Contents and Data Analysis

The levels of endogenous hormone contents were determined using enzyme-linked immunosorbent assays (ELISA) (He et al. 2009; Zhang et al. 2019, 2020a). The corresponding hormone test kits were employed according to the kit instructions for determining hormone contents in samples. Standard samples were mixed with the sample diluent, and eight concentrations (including 0 ng/mL) were prepared for each hormone sequentially by a factor of 2. The maximum concentration for the standard curves of IAA and ABA was 100 ng/mL, while for ZR, iPA, and DHZR, it was 20 ng/mL, and for GAs, it was 10 ng/mL. Test samples were diluted using the sample diluent as necessary to achieve accurate estimates within the standard concentration range.

For the determination of ZR, a series of standard samples were added to the first two rows of a 96-microwell plate (antigen coated and blocked), while test samples were placed in the remaining wells. Each sample was duplicated, and 50 μL of sample was added to each well. Subsequently, 50 μL of antibody solution (rabbits' antisera) (5 mL sample diluent, 2.5 μL of antibodies) was added to each well and incubated at 37 °C for 30 min for competition. Following washing of the plate (using an ImmunoWashTM 1575 microplate washer, BIO-RAD) with washing liquid (1000 mL PBS, 1 mL Tween-20), 100 μL of enzyme-labeled secondary antibody solution (RaMIgG-HRP) (10 mL sample diluent, 10 μL of antibodies) was added to each well and incubated for 30 min at 37 °C. After another round of washing, 100 μL of substrate solution (containing 20 mg O-phenylenediamine (OPD), 10 mL substrate buffer, 4 μL 30% H202) was added to the wells for coloration. The reaction was terminated by adding 50 μL of 2 mol/L sulfuric acid to each well when the color was appropriate (the standard samples exhibited a color gradient, with the maximum concentration well showing a light color). The absorbance was measured at 490 nm using a microplate reader (BIO-RAD iMarkTM, USA). Standard curves were prepared, and the level of hormone content was calculated using a logit curve (Zhang et al. 2019).

The horizontal coordinate of the logit curve was represented by the natural logarithm of each concentration (ng/mL) of the standard hormone sample, while the vertical coordinate was expressed by the logit value of the colorimetric value of each concentration. The logit value was calculated using the formula: Logit (B/B0) = ln [(B/B0)/(1-B/B0)] = ln [B/(B0-B)], where B0 represents the colorimetric value of the 0 ng/mL well and B represents the colorimetric value of other concentrations. By plotting the logit value against the natural logarithm of the hormone concentration of the sample, the concentration of the hormone (in ng/mL) could be determined from the graph. Subsequently, the content of hormone in the sample (in ng/g. FW = nanograms per gram of fresh weight) was calculated. This procedure was followed for determining the concentration of other hormones as per the kit instructions.

The average value of the duplicate measurements for each sample was considered as the hormone content in the sample. For statistical analysis, three replicates were performed by utilizing the average hormone content of the two plants collected from each replicate in the field trial. In addition to the individual hormone contents, various hormone content ratios, such as ZR/IAA, iPA/IAA, DHZR/IAA, ZR/ABA, iPA/ABA, DHZR/ABA, ZR/GA3, iPA/GA3, DHZR/GA3, ZR/GA4, iPA/GA4, and DHZR/GA4, were also calculated. The data underwent statistical analysis to calculate the standard error. Mean values, standard error, analysis of variance, and LSD (least significant difference) were computed using SPSS 25.0 statistical software. The mean values were compared using F statistics at significance levels of P < 0.05 and P < 0.01. Graphs were generated using Microsoft Excel 2016.

Results and Discussion

The present study revealed that the mean contents of DHZR, GA4, GA3, iPA, and IAA did not show significant differences between YZ 07-86 and YZ 07-87, while mean ABA and ZR contents exhibited significant differences (Table 1). Additionally, DHZR content was significantly higher (P < 0.05) in YZ 07-86 compared to YZ 07-87 at 116 DAP (M = 28.3, SE =  ± 3.8), 141 DAP (M = 82.6, SE =  ± 4.3), and 156 DAP (M = 53.3, SE =  ± 7.8); however, no specific pattern for significant differences was observed throughout the evaluation period. Furthermore, GA4 content differed significantly (P < 0.05) between genotypes only at 131 DAP and 176 DAP. Specifically, significantly higher GA4 content was recorded at 131 DAP in YZ 07-86 (M = 12.2, SE =  ± 2.2), while significantly lower GA4 content was observed at 176 DAP in YZ 07-86 (M = 2.4, SE =  ± 0.4). Similarly, iPA content was significantly higher (P < 0.01) in the YZ 07-86 genotype at 141 DAP (M = 27.7, SE =  ± 0.9) and 156 DAP (M = 26.9, SE =  ± 3.4); however, it was significantly lower (P < 0.01) than that of YZ 07-87 (M = 8.1, SE =  ± 0.2) at 166 DAP.

Table 1 Mean hormone contents in the YZ 07-86 and YZ 07-87 sugarcane genotypes
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In the group of cytokinins (CKs), DHZR and iPA are considered minor cytokinins (Tantikanjana et al. 2001). Although CKs are known to play roles in bud regulation (Rameau et al. 2015) and shoot development (Wybouw and De Rybel 2019), this experiment found non-significant mean contents (P > 0.05) of both minor CKs between genotypes. This finding aligns with Tantikanjana et al. (2001), who observed similar results in mutant Arabidopsis with extensive proliferative shoot branching compared to their wild-type counterparts. Additionally, Bredmose et al. (2005) noted a decrease in iPA and DHZR in axillary bud tissues of rose cuttings with the initiation of axillary bud growth. Although hormone contents were not measured in the axillary buds in the current study, the mean contents of these hormones in shoot apical meristem (SAM) tissues did not decrease, even with the development of additional buds in the nodes of YZ 07-86.

Furthermore, although gibberellins play diverse roles in the plant life cycle (Achard and Genschik 2009) by directly affecting cell division and expansion through the integration of internal and external stimuli (Martins et al. 2018), this experiment did not reveal a significant difference in mean GA4 contents between dual-axillary or single-axillary bud genotypes, despite GA4 belonging to the bioactive group of gibberellins.

The IAA contents of both genotypes followed a similar pattern during the evaluation period (Fig. 1). The mean IAA content did not exhibit significant differences (P > 0.05) between genotypes at any sampling point during the evaluation period, with IAA content showing an increasing trend in both genotypes during the early sampling points. In the YZ 07-86 genotype, the IAA content (ng/g FW) reached its maximum (M = 643.6, SE =  ± 88.5) at 136 DAP, while in the YZ 07-87 genotype, it reached its maximum (M = 549.0, SE =  ± 111.7) at 126 DAP (Fig. 1). Auxin is primarily synthesized in the shoot apex of young leaves (Ljung et al. 2001), transported basipetally (Ongaro and Leyser 2008), and plays a role in the axillary bud formation process (McSteen and Leyser 2005). Chen et al. (2020) suggested that axillary bud development in the dual-axillary bud genotype occurs under the influence of the IAA pathway of genetic regulation. However, our results revealed statistically similar (P > 0.05) mean IAA contents during the evaluation period, as well as at each sampling point, which contradicts the findings of Chen et al. (2020).

Fig. 1
figure 1

Changes in IAA content in the two sugarcane genotypes over the evaluation period

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It is well established that GA3 is a crucial type of gibberellin widely employed in regulating plant growth and development (Rizza and Jones 2019). Although the mean contents of GA3 did not show significant differences between the two genotypes (Table 1), an erratic pattern of GA3 contents was observed between the two genotypes at each sampling point up to 141 DAP (Fig. 2). During this period, YZ 07-87 exhibited significantly higher GA3 contents at 106, 121, and 136 DAP compared to YZ 07-86. According to Vasantha et al. (2010), this period corresponds to the formative stage of sugarcane development, which spans from 60 to 150 days of crop age. However, no significant difference in GA3 content between the two genotypes was observed at any sampling point after 136 DAP (Fig. 2). Previous studies (Liu et al. 2011, 2020) suggest that the effects of exogenous GA3 on axillary bud formation and lateral bud growth vary across plant species. Nevertheless, based on the current results, it can be hypothesized that GA3 has only limited and pronounced effects in both sugarcane genotypes during the formative stage.

Fig. 2
figure 2

Changes in GA3 content in the two sugarcane genotypes over the evaluation period

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Davies (2010) described plant hormones as naturally occurring organic substances that affect physiological functions such as growth, differentiation, and development at low doses. Although not directly involved in the formation of dual-axillary buds, auxin is recognized as an important plant hormone for axillary meristem initiation (McSteen 2009); cytokinins (CKs) play a significant role in the initiation of axillary meristem development (Zhang et al. 2020b) and branching in plants, including the regulation of meristem size (Shani et al. 2006); and gibberellins (GAs) are considered important hormones for regulating various developmental processes in plants (Liu et al. 2020), including the regulation of axillary meristem outgrowth (Zhang et al. 2020b). However, the current experiment has not revealed a significant difference in mean contents of DHZR, iPA, GA3, GA4, and IAA between dual-axillary and single-axillary bud genotypes during the evaluation period. Therefore, it can be hypothesized that these hormones per se may not contribute to dual-axillary bud formation in YZ 07-86. This hypothesis is supported by Rameau et al. (2015), who reported the hormonal involvement and importance of sugars in bud regulation in plants.

In the assay of ABA, significant differences were observed only at 111, 156, 171, and 196 DAP. At all other sampling points, the differences were not statistically significant (P > 0.05) (Fig. 3). The ABA contents of YZ 07-86 were significantly lower than that of YZ 07-87 at 111 and 171 DAP. Additionally, the ABA contents of YZ 07-86 were significantly higher (P < 0.05) than the control genotype at 156 and 196 DAP. Consequently, the mean ABA content of YZ 07-86 was significantly lower than that of YZ 07-87 (Table 1).

Fig. 3
figure 3

Changes in ABA content in the two sugarcane genotypes over the evaluation period

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ABA is recognized as an important plant hormone involved in various stages of the plant life cycle, including dormancy, seed development, and plant responses to environmental stresses (Seo and Koshiba 2002). Plants exhibit tolerance to diverse stress conditions under elevated ABA levels (Kende and Zeevaart 1997), with Zeevaart and Creelman (1988) reporting an increase in ABA biosynthesis when plant cells lose turgor. Verslues (2016) also noted that reduced turgor pressure is essential for ABA accumulation. In this study, both YZ 07-86 and YZ 07-87 were subjected to identical conditions, with sufficient irrigation provided, thereby excluding the effects of water stress on ABA contents.

Although influenced by sugars, ABA plays a role in bud regulation (Rameau et al. 2015); exogenous ABA has been found to slightly inhibit the growth of tiller buds in rice, and higher ABA levels have been associated with decreased wheat tillers (Liu et al. 2011). Similar results were reported by Mader et al. (2003), who observed a close correlation between ABA and the inhibition of bud growth. It can thus be suggested that the lower level of ABA in YZ 07-86 contributes to promoting bud development, thereby resulting in dual-axillary buds, while the higher ABA content in YZ 07-87 could maintain a normal single-bud state.

The assay of ZR garnered significant attention because of the marked difference observed in ZR contents between the YZ 07-86 mutant and the YZ 07-87 control genotypes. Our results revealed that mean ZR contents were significantly higher in YZ 07-86 compared to the YZ 07-87 genotype (Table 1). Figure 4 illustrates the comparison of ZR contents in both genotypes at each sampling point.

Fig. 4
figure 4

Changes in ZR content in the two sugarcane genotypes over the evaluation period

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Interestingly, the ZR contents of YZ 07-86 were consistently higher than those of YZ 07-87 at all sampling points (Fig. 4). Significant differences (P < 0.05) in ZR contents in YZ 07-86 were observed from 146 DAP onward. However, ZR contents did not significantly differ at 151, 171, 176, and 196 DAP. From the beginning to about the midpoint (from 106 to 121 DAP) of the formative stage, the ZR content of both genotypes gradually decreased, with no specific pattern observed thereafter, except for the consistently higher ZR contents in YZ 07-86.

Bredmose et al. (2005) cited that phytohormones, produced in root and shoot meristems, coordinate morphological processes of plants via phytohormone gradients, while Wolters and Jürgens (2009) reported that hormones interact through synergistic or antagonistic crosstalk, influencing each other's biosynthesis or response. In addition, Vanstraelen and Benkova (2012) reported that interconnected hormonal pathways collectively influence individual hormone actions and overlap in function across various developmental processes. Then, the final developmental outcome is determined by a sophisticated network where one hormone's influence is modulated by the interactions of other hormonal pathways (Vanstraelen and Benkova, 2012). Understanding these hormonal interactions and their dynamic balance is also crucial for interpreting the underlying mechanisms driving bud development in plants. Therefore, in addition to the absolute content of plant hormones, different content ratios of endogenous hormones were also calculated (Table 2). In the present study, the mean content ratios iPA/IAA, DHZR/IAA, iPA/ABA, DHZR/ABA, iPA/GA3, DHZR/GA3, iPA/GA4, and DHZR/GA4 did not significantly differ between the YZ 07-86 and YZ 07-87 genotypes.

Table 2 Changes in different hormone ratios in the two genotypes during the evaluation period
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However, in YZ 07-86, the mean content ratios of ZR/IAA, ZR/ABA, ZR/GA3, and ZR/GA4 were significantly higher compared to YZ 07-87. The major contributing factor for the significantly higher mean content ratios ZR/IAA, ZR/GA3, and ZR/GA4 in YZ 07-86 would be the significantly higher level of ZR in this genotype. This conclusion is supported by the observation that other ratios, except for ZR/ABA, which do not involve ZR, were not significantly different (P > 0.05) between the two genotypes. However, for the significantly higher ZR/ABA content ratio, both ZR and ABA would be contributing factors since both hormone contents were significantly different between genotypes, with ZR being higher and ABA being lower in YZ 07-86.

The significantly different hormone content ratios of ZR/IAA, ZR/GA3, and ZR/GA4 at different sampling points did not follow a specific pattern during the evaluation period, although significantly different (P < 0.05) content ratios were observed at certain sampling points. Specifically, the content ratios of ZR/ABA were not significantly different at any sampling point up to 121 DAP. However, from 126 DAP onward, they were significantly higher in YZ 07-86 at multiple sampling points (126, 131, 141, 146, 151, 161, 171, 176, 181, and 201 DAP).

Yao and Finlayson (2015) elucidated the intricate relationship between cytokinins and ABA, proposing that cytokinins may regulate both the levels and response of ABA. They emphasized that ABA suppresses cytokinin accumulation in wheat (Triticum aestivum) roots and shoots while stimulating cytokinin oxidase activity, ultimately leading to reduced cytokinin levels. In our study, we observed significantly higher levels of ZR (a cytokinin) and significantly lower levels of ABA in the YZ 07-86 genotype, resulting in a higher ZR/ABA ratio. This finding aligns with Yao and Finlayson's (2015) proposition that lower levels of ABA create an environment conducive to ZR accumulation in YZ 07-86. Given that ZR is recognized as a bud growth stimulator (Tantikanjana et al. 2001), it can be inferred that this condition promotes the formation of double buds in the YZ 07-86 genotype.

Our results reveal that although the average levels of GA3 or GA4 were not significantly different between genotypes, the ratios of ZR/GA3 and ZR/GA4 were notably higher in the YZ 07-86 genotype compared to YZ 07-87. In their study on the woody plant Jatropha curcas, Ni et al. (2015) observed that gibberellin and cytokinin (CK) synergistically promote lateral bud outgrowth and influence the expression of key transcription factors, BRANCHED1 and BRANCHED2, which regulate bud dormancy. They also found that gibberellin is necessary for CK-mediated axillary bud outgrowth. Consequently, it can be inferred that the effects of ZR in the YZ 07-86 genotype are not hindered by gibberellins, as both genotypes exhibited statistically similar gibberellin contents.

The current study revealed a significantly higher ZR content in the dual-axillary bud genotype, resulting in comparable IAA levels (P > 0.05) in both genotypes. The relationship between auxin and CKs in relation to the development of shoot meristems is reported as antagonistic (Hussain et al. 2021); auxin inhibits bud growth (Tantikanjana et al. 2001) while regulating the biosynthesis of CKs (Ongaro and Leyser 2008). Mader et al. (2003) observed a dominant zeatin riboside (ZR) content in the axillary buds of chickpea (Cicer arietinum) at the onset of lateral bud growth, despite a decrease in auxin content. However, our results revealed that the content ratio of ZR/IAA was also significantly higher in the YZ 07-86 genotype under similar endogenous IAA contents (P > 0.05) between the two genotypes. Therefore, it can be suggested that the elevated ZR content in the mutant genotype happened independently of the IAA content, providing a clue that ZR may have contributed to the dual-axillary bud formation of YZ 07-86. Tantikanjana et al. (2001) explained that the pattern of proliferative shoot branching in zeatin-elevated sps mutant Arabidopsis plants did not result from decreased auxin levels because the free IAA level in mutant plants was higher than that in wild-type plants in their experiment. Therefore, it can be further suggested that dual-axillary bud formation in YZ 07-86 may not occur due to decreased IAA levels but rather as a consequence of elevated ZR content.

It is widely acknowledged that cytokinins (CKs) play a crucial role in shoot branching (Ongaro and Leyser 2008), directly stimulating bud growth (Tantikanjana et al. 2001), and determining shoot cell fate across various cell types, thus making them a key class of hormones involved in shoot development (Wybouw and De Rybel 2019). Tanimoto and Harada (1981) provided evidence for CKs' involvement in bud formation by suggesting the promotion of floral bud formation in in vitro-cultured Torenia fournieri by zeatin, a type of CK (Werner et al. 2001). They proposed that this might occur due to CKs stimulating shoot formation from vegetative bud primordia.

Groot et al. (1995) observed an increase in endogenous zeatin riboside (ZR) levels in the ls mutant of tomato, known for its inability to produce axillary buds in most leaf axils. This led to the formation of bulbous structures in initially empty leaf axils, from which adventitious shoots could arise, suggesting a role for cytokinins in inducing tissue proliferation in these regions. Furthermore, Chen et al. (1997) reported considerable increases in free cytokinins, such as zeatin and zeatin riboside, promoting flower bud development in Euphoria longana. Additionally, Tantikanjana et al. (2001) documented higher levels of zeatin in Arabidopsis sps mutant plants exhibiting massive shoot proliferation due to increased bud initials. Moreover, they reported an increase in bud initiation under the application of exogenous cytokinins and in transgenic plants overproducing cytokinins.

Rameau et al. (2015) demonstrated the involvement of cytokinins (CKs) in bud regulation, while Liu et al. (2020) specifically reported the regulation of axillary bud outgrowth in garlic (Allium sativum L.) by ZR, influenced by sugar levels. Additionally, studies have shown a positive association between CKs, particularly ZR, and axillary bud growth in other plant species. For instance, miniature roses exhibit enhanced axillary bud growth in response to CKs (Bredmose et al. 2005), while ZR predominates in axillary buds during the onset of lateral bud growth in chickpea (Cicer arietinum) (Mader et al. 2003). These findings collectively support the view that elevated ZR levels promote dual-axillary bud formation, as observed in YZ 07-86. While these findings support the concept that elevated ZR levels lead to dual-axillary bud formation in YZ 07-86, they also present conflicting evidence. For example, CKs inhibit flower bud formation in tobacco (Tanimoto and Harada 1981), and despite increased endogenous ZR levels, the ls mutant of tomato fails to restore axillary meristem formation (Groot et al. 1995). These contradictions challenge our suggestion regarding the role of ZR.

Lu et al. (2020) provided a detailed description of the occurrence of dual-axillary buds in YZ 07-86, noting that this phenomenon predominantly occurred above the 5th internode from the bottom. They observed that more than 70% of buds doubled between the 15th and 20th internodes, with a progressive decrease thereafter (10% double buds between the 25th and 29th internodes, and no double buds in the top internodes) toward the stem tip. However, dual-axillary bud formation was not uniform across all nodes; some nodes exhibited only a single axillary bud despite the initiation of dual-axillary bud formation. Furthermore, Lu et al. (2020) reported that both buds in a node germinate well and develop into stems. However, no information is available related to bud dormancy in YZ 07-86 or in YZ 07-87.

In the present study, hormone contents were assessed in shoot apical meristem (SAM) tissues, revealing no significant difference in zeatin riboside (ZR) content between genotypes from 106 to 141 days after planting (DAP). However, after 141 DAP, significantly higher ZR contents were consistently recorded in YZ 07-86. Notably, there was no significant elevation in ZR content in YZ 07-86 at certain sampling points after 146 DAP. The ratios of ZR to abscisic acid (ABA) content displayed a similar pattern to ZR content in YZ 07-86, with no significant differences during early sampling points (up to 121 DAP) but significantly higher ratios observed during later sampling points (from 126 DAP onward). However, similar to ZR content, the ZR/ABA content ratio did not exhibit significant differences at certain sampling points.

The observed variations in ZR content and ZR/ABA ratio appear to correlate with the pattern of dual bud formation in YZ 07-86. Therefore, as a hypothesis, it can be suggested that dual buds may not develop in the first few nodes due to the absence of elevated ZR levels, with dual bud formation commencing with the elevated ZR contents characteristic of YZ 07-86. Additionally, the variation in ZR content might be responsible for the changes in bud formation along the stalk of the YZ 07-86 genotype. Furthermore, it can be hypothesized that the higher ZR/ABA ratio may create a more conducive environment for dual-axillary bud formation in YZ 07-86.

Cytokinins act as long-distance signaling molecules synthesized in both roots and shoots (Ongaro and Leyser 2008), coordinating various aspects of plant development and serving as regulatory signals at both local and distant levels (Ko et al. 2014). The CKs play a crucial role in promoting cell differentiation (Yonova 2010). While CKs of the trans-zeatin type are primarily produced in the root vasculature, they are transported to the shoot, where they regulate shoot growth (Ko et al. 2014). However, studies suggest that trans-zeatin type CKs are not essential for normal shoot growth, as they are also synthesized in aerial parts of the plant (Kiba et al. 2013; Kieber and Schaller 2014). Moreover, the influence of environmental factors, such as nitrogen status, significantly impacts plant growth and development mediated by CKs. Nitrogen availability triggers an increase in xylem CK translocation, not only in roots but also in leaves, with CK synthesis being nitrogen-dependent (Hirose et al. 2008). Consequently, variations in ZR content over time can be expected, as observed in this study, and analyzing ZR contents in both stalks and nodal regions would be helpful for further elucidating the effects of ZR on dual bud formation in sugarcane.

Conclusions

In summary, we investigated the dynamic changes in endogenous hormone levels in the double-bud mutant YZ 07-86 sugarcane genotype compared to the control genotype YZ 07-87, aiming to understand the physiological mechanism underlying dual-axillary bud formation in YZ 07-86. Our findings suggest that DHZR, iPA, GA4, and IAA may not directly contribute to the double-axillary bud phenomenon observed in YZ 07-86. Noteworthy differences between YZ 07-86 and YZ 07-87 were identified, particularly the significantly higher level of ZR and the significantly lower level of ABA in YZ 07-86. We hypothesize that the lower ABA level in YZ 07-86 might foster the development of dual-axillary buds, while elevated ABA in YZ 07-87 maintained a single-bud state. Moreover, our analysis suggests that elevated ZR content and higher ZR/ABA content ratios could be pivotal factors in dual-axillary buds formation in YZ 07-86. However, further investigation is warranted to validate the physiological mechanism underlying dual-axillary bud formation in sugarcane. Analyzing hormone contents in the axillary buds and nodal regions of both genotypes could provide deeper insights. Furthermore, future research should focus on validating the effect of exogenous ZR on bud development in sugarcane of different ages, as well as exploring related gene mining. These avenues promise to enhance our understanding of the intricate processes governing bud development in sugarcane, potentially offering valuable insights for agricultural practices and crop improvement strategies.