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MAP  >  Grass Research
2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Plant materials, growth conditions, and stress treatment
Vector construction
Chlorophyll and anthocyanin content determination
Physiological analysis
RNA isolation and qRT-PCR analysis
Results
Screening and characterization of lncRNAs
Generation of lncRNA overexpression lines
Effect of lncRNA overexpression on heat tolerance in Arabidopsis
Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis
Effect of lncRNA overexpression on drought tolerance in Arabidopsis
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
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ARTICLE   Open Access    

Effects of different plant growth regulators on phenotypic variation and seed yield of Dactylis glomerata

  • 1.

    College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, Sichuan, China

  • 2.

    Seed Research Centre (SRC), Lincoln University, Lincoln, Christchurch 7647, New Zealand

More Information
  • Received: 12 August 2024
    Revised: 24 September 2024
    Accepted: 09 October 2024
    Published online: 29 October 2024
    Grass Research  4,  Article number: e022 (2024)  |  Cite this article
  • Plant growth regulators (PGRs) have been widely used in the production of forage seeds. In a two-year field experiment, different concentrations of CCC (500, 750, 1,000, 1,250 g·hm−2) and TE (100, 200, 300, 400 g·hm−2) were sprayed on Dactylis glomerata as well as a mixture of CCC and TE (500 + 50, 500 + 150, 1,000 + 50, 1,000 + 150 g·hm−2). By studying the effects of different PGRs and different concentrations on the characters and seed yield of Dactylis glomerata, it was found that the plant height was significantly reduced by spraying two plant growth regulators alone, and the stem diameter of increased by spraying a mixture of CCC and TE. Different PGRs had different effects on the functional leaves of Dactylis glomerata under different concentrations. Spraying of two PGRs in a certain range of concentrations could significantly improve the seed yield, but when the concentration was too high, the seed yield was reduced. The seed yield of Dactylis glomerata could be significantly increased by spraying a mixture of CCC and TE in the range of the test concentrations. Among them, spraying a mixture of CCC and TE at a concentration of 500 + 150 g·hm−2 had the best effect on increasing seed yield, which increased the average yield by 47.91% in two years. Additionally, the experiment also found that spraying PGRs mainly increased the yield of Dactylis glomerata by increasing the number of fertile tillers·m−2, spikes per fertile tillers, and seed setting rate.

  • Introduction

    Plants are continuously subjected to unpredictable environmental conditions and encounter a multitude of stressors throughout their growth and development, posing a significant challenge to global crop production and food security[1]. Heat and drought are undoubtedly the two most important stresses that have a huge impact on crops. Both elicit a wide array of biochemical, molecular, and physiological alterations and responses, impacting diverse cellular processes and ultimately influencing crop yield and quality[2].

    A primary physiological consequence of both stresses is the diminished photosynthetic capacity, partially resulting from the degradation of chlorophyll due to leaf senescence under stress conditions. Chlorophyll accumulation was diminished in numerous plants subjected to drought or heat stress conditions[3,4]. Various environmental stresses prompt excessive generation of reactive oxygen species (ROS), initiating oxidative damage that compromises lipids, and proteins, and poses a serious threat to cellular functions[2]. To mitigate oxidative stress and minimize damage, plants have developed various protective mechanisms to neutralize ROS. Several antioxidant enzymes, such as SOD, POD, and CAT, are integral to cellular antioxidative defense mechanisms. Additionally, antioxidants such as anthocyanins and proline serve as crucial ROS scavengers[5,6]. The elevation in temperature typically induces the transient synthesis of heat shock proteins (Hsps), which function as molecular chaperones in protecting proteins from denaturation and aggregation, with their activity primarily regulated at the transcriptional level by heat shock factors (Hsfs)[7]. The significance of Hsps and Hsfs in all organisms, including plants, has been assessed in various stress conditions that could disrupt cellular homeostasis and result in protein dysfunction[7]. Drought stress can also trigger the transcription of a suite of marker genes, including RD29A, RD29B, NCED3, AREB1, Rab18, etc., which assist plants in mitigating cellular damage during dehydration and bolstering their resilience to stress[8−10].

    Previous research efforts focusing on the regulatory control of stress-related genes have largely centered around protein-coding genes. In recent years, non-protein-coding transcripts have emerged as important regulatory factors in gene expression. Among them, long non-coding RNAs (lncRNAs) lncRNAs have been identified as implicated in various abiotic stresses[11,12]. LncRNAs are a class of non-coding RNAs (ncRNAs) exceeding 200 nucleotides in length. They possess minimal or no protein-coding potential[13]. In plants, lncRNAs are specifically transcribed by RNA polymerases Pol IV, Pol V, Pol II, and Pol III[14,15]. LncRNAs exhibit low abundance and display strong tissue and cellular expression specificity relative to mRNAs. Moreover, sequence conservation of lncRNAs is was very poor across different plant species[13,16,17]. The widespread adoption of high-throughput RNA sequencing technology has revealed lncRNAs as potential regulators of plant development and environmental responses. In cucumber, RNA-seq analysis has predicted 2,085 lncRNAs to be heat-responsive, with some potentially acting as competitive endogenous RNAs (ceRNAs) to execute their functions[18]. In radish, a strand-specific RNA-seq (ssRNA-seq) technique identified 169 lncRNAs that were differentially expressed following heat treatment[19]. In Arabidopsis, asHSFB2a, the natural antisense transcript of HSFB2a was massively induced upon heat stress and exhibited a counteracted expression trend relative to HSFB2a. Overexpression of asHSFB2a entirely suppressed the expression of HSFB2a and impacted the plant's response to heat stress[20]. For drought stress resistance, 244 lncRNAs were predicted in tomatoes to be drought responsive probably by interacting with miRNAs and mRNAs[21]. Under drought stress and rehydration, 477 and 706 lncRNAs were differentially expressed in drought-tolerant Brassica napus Q2 compared to drought-sensitive B. napus, respectively[22]. In foxtail millet and maize, 19 and 644 lncRNAs, respectively, were identified as drought-responsive[23,24]. Despite the identification of numerous lncRNAs by high-throughput sequencing, which suggests their potential involvement in various abiotic stress processes, only a minority have been experimentally validated for function.

    In our previous study, we characterized 1,229 differentially expressed (DE) lncRNAs in Chinese cabbage as heat-responsive, and subsequent bioinformatics analysis reduced this number to 81, which are more likely associated with heat resistance[25]. lnc000283 and lnc012465 were selected from among them for further functional investigation. The findings indicated that both lnc000283 and lnc012465 could be promptly induced by heat shock (HS). Overexpression of either lnc000283 or lnc012465 in Arabidopsis plants enhanced their capacity to tolerate heat stress. Additionally, both lnc000283 and lnc012465 conferred drought tolerance to transgenic Arabidopsis.

    Materials and methods

    Plant materials, growth conditions, and stress treatment

    The lncRNA sequences examined in this study were from Chiifu-401-42 Chinese cabbage and all Arabidopsis plants were of the Col-0 background. Transgenic plants expressing lnc000283 and lnc012465 were generated using the Agrobacterium tumefaciens-mediated floral dip method[26]. Single-copy and homozygous T3 plants were identified through genetic segregation on an agar medium supplemented with kanamycin. The T3 generation plants, or their homozygous progeny, were utilized in the experiments.

    For phenotypic assessment, Arabidopsis seeds were initially sown on filter paper moistened with ddH2O and placed in a 4 °C freezer for 2 d. Subsequently, they were evenly planted in nutrient-rich soil and transferred to a growth chamber operating a 16-h day/8-h night cycle, with day/night temperatures of 22 °C/18 °C and a light intensity of 250 μmol·m−2·s−1. After 10 d of growth, Arabidopsis plants with uniform growth were transferred to 50-hole plates. Arabidopsis plants grown in Petri dishes were firstly seed-sterilized and then sown on 1/2 MS medium supplemented with 10 g·L−1 sucrose. The seeds were then placed in a 4 °C refrigerator for 2 d in the dark before transferring them to a light incubator. The day/night duration was set to 16 h/8 h, the day/night temperature to 21 °C/18 °C, and the light intensity to 100 μmol·m−2·s−1.

    For heat treatment, 3-week-old seedlings were subjected to 38 °C for 4 d within a light incubator, subsequently transferred to their original growth conditions under the same light/dark cycles. For drought treatment, 3-week-old Arabidopsis seedlings were deprived of water for 10 d, followed by rehydration to facilitate a 2-d recovery period. Plants were photographed and surveyed both before and after treatment.

    Vector construction

    The lncRNA sequences (lnc000283 and lnc012465) were chemically synthesized based on RNA-seq data, with restriction sites for BamH1 and Kpn1 engineered upstream and downstream. The resultant lncRNA constructs were subcloned into the pCambia2301 binary vector, incorporating a cauliflower mosaic virus (CaMV) 35S promoter. The recombinant vectors were transformed into Escherichia coli TOP10 competent cells (Clontech), incubated at 37 °C overnight, after which single clones were selected for PCR verification, and the confirmed positive colonies were submitted for sequencing. Following verification, the correct plasmids were introduced into A. tumefaciens strain GV3101 using the freeze-thaw method and subsequently transformed into Arabidopsis wild-type (Col) plants.

    Chlorophyll and anthocyanin content determination

    To quantify the chlorophyll content, the aerial portions of wild-type and transgenic Arabidopsis plants, grown in Petri dishes were weighed, minced, and then subjected to boiling in 95% ethanol until fully decolorized. Aliquots of 200 μL from the extract were transferred to a 96-well plate and the absorbance at 663 nm and 645 nm was measured via spectrophotometry by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Three biological replicates were analyzed for WT and each transgenic line. Chlorophyll content was determined according to the formula of the Arnon method[27]: Chlorophyll a = (12.72A663 − 2.59A645) v/w, Chlorophyll b = (22.88A645 − 4.67A663) v/w, Total chlorophyll = (20.29A645 + 8.05A663) v/w.

    The quantification of anthocyanin was performed as follows: aerial parts of wild-type and transgenic Arabidopsis plants, cultivated in Petri dishes, were weighed and ground to powder in liquid nitrogen. Subsequently, the samples were incubated in 600 μL of acidified methanol (containing 1% HCl) at 70 °C for 1 h. Following this, 1 mL of chloroform was added, and the mixture was vigorously shaken to remove chlorophyll. The mixture was then centrifuged at 12,000 rpm for 5 min, after which the absorbance of the aqueous phase was determined at 535 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). Three biological replicates were analyzed for WT and each transgenic line. The relative anthocyanin content was calculated according to anthocyanin concentration and extraction solution volume. One anthocyanin unit is defined as an absorption unit at a wavelength of 535 nm in 1 mL of extract solution. In the end, the quantity was normalized to the fresh weight of each sample.

    Physiological analysis

    Three-week-old transgenic and WT A. thaliana plants, subjected to normal conditions or varying durations of heat or drought stress, were utilized for subsequent physiological assessments. All assays were performed in accordance with the method described by Chen & Zhang[28]. In brief, 0.1 g of fresh leaf tissue was homogenized in 500 μL of 100 mM PBS (pH 7.8) while chilled on ice. The homogenate was then centrifuged at 4 °C, and the resultant supernatant was employed for further analysis. For the determination of MDA content, 100 μL of the supernatant was combined with 500 μL of a 0.25% thiobarbituric acid (TBA) solution (which was prepared by dissolving 0.125 g of TBA in 5 mL of 1 mol·L−1 NaOH before being added to 45 mL of 10% TCA) and boiled for 15 min. Following a 5 min cooling period on ice, the absorbance was measured at 532 nm and 600 nm. The activity of POD was determined as follows: initially, 28 μL of 0.2% guaiacol and 19 μL of 30% H2O2 were sequentially added to 50 mL of 10mM PBS (pH 7.0), after thorough heating and mixing, 1 mL was transferred into a cuvette, then 50 μL of the supernatant was added to the cuvette and the absorbance at 470 nm was monitored every 15 s for 1 min. To determine the proline content, a reaction solution was prepared by mixing 3% sulfosalicylic acid, acetic acid, and 2.5% acidic ninhydrin in a ratio of 1:1:2, then 50 μL of the supernatant was added to 1 mL of the reaction solution, which was then subjected to a boiling water bath for 15 min (the solution turned red after the boiling water bath). Following cooling on ice, the absorbance at 520 nm was recorded. For the quantification of proline, an L-proline standard curve was prepared by dissolving 0, 5, 10, 15, 20, 25, and 30 μg of L-proline in 0.5 mL of ddH2O, followed by the addition of 1 mL of the reaction solution and measuring the absorbance at 520 nm. The proline content in the samples was then determined based on the L-proline standard curve.

    RNA isolation and qRT-PCR analysis

    Total RNA was isolated from the aerial parts of Arabidopsis using the TaKaRa MiniBEST Plant RNA Extraction Kit, followed by purification and reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). The cDNA product was diluted 10 times and real-time PCR was conducted in triplicate for each biological replicate using SYBR PCR Master Mix (Applied Biosystems) on the ABI 7500 system under the following conditions: 98 °C for 3 min, followed by 40 cycles of 98 °C for 2 s and 60 °C for 30 s. The relative expression levels of each gene were normalized against the transcript abundance of the endogenous control UBC30 (At5g56150) and calculated using the 2−ΔCᴛ method. The specific primers employed for qRT-PCR are detailed in Supplemental Table S1.

    Results

    Screening and characterization of lncRNAs

    In our prior investigation, dozens of lncRNAs associated with the heat stress response in Chinese cabbage were identified through informatics analysis. Two lncRNAs (lnc000283 and lnc012465) were chosen for genetic transformation in Arabidopsis to elucidate their functions comprehensively. Transcriptome data analysis indicated that the expression of lnc000283 and lnc012465 in Chinese cabbage were both induced by HS. To verify the accuracy, the expression patterns of lnc000283 and lnc012465 were confirmed through quantitative real-time PCR (qRT-PCR), and the results from qRT-PCR were consistent with those obtained from RNA-seq (Fig. 1a). The corresponding homologous genes in Arabidopsis were identified as CNT2088434 and CNT2088742, exhibiting sequence similarities of 88% and 87%, respectively (Supplemental Fig. S1). Subcellular localization predictions using the lnclocator database (www.csbio.sjtu.edu.cn/bioinf/lncLocator) suggested that both lncRNAs are localized within the nucleus (Supplemental Table S2). Bioinformatics analysis was conducted using the CPC tool (http://cpc.cbi.pku.edu.cn/) indicated that lnc000283 and lnc012465 are noncoding sequences, with coding probabilities of 0.0466805 and 0.0432148, respectively comparable to the well-characterized lncRNAs COLDAIR and Xist, but significantly lower than those of the protein-coding genes UBC10 and ACT2 (Fig. 1b).

    Figure 1.  Characteristics of lnc000283 and lnc012465. (a) Expression level of lnc000283 and lnc012465 in Chinese cabbage leaves treated at 38 °C at different time points, as determined by qRT-PCR and RNA-seq. CK is a representative plant before heating, and T1, T4, T8, and T12 denote plants that were subjected to 38 °C for 1, 4, 8, and 12 h, respectively. The expression levels were normalized to the expression level of Actin. (b) Analysis of coding potential for lnc000283 and lnc012465. The coding potential scores were calculated using the CPC program. UBC10 (At5g53300) and ACT2 (At3g18780) are positive controls that encode proteins. COLDAIR (HG975388) and Xist (L04961) serve as negative controls, exhibiting minimal protein-coding potential.
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    Generation of lncRNA overexpression lines

    To elucidate the role of lnc000283 and lnc012465 in response to abiotic stress, overexpression vectors were constructed for these lncRNAs, driven by the CaMV 35S promoter, and they were introduced into Arabidopsis thaliana (Col-0 ecotype). Through PCR identification and generational antibiotic screening, two homozygous positive lines for lnc012465 and lnc000283 were obtained. The relative expression levels of these lncRNAs were assessed using qRT-PCR (Fig. 2a). When plants were grown in 1/2 MS medium, with the consumption of nutrients, and reduction of water, the leaves of WT began to turn yellow, but the lnc000283 and lnc012465 overexpression lines developed a deep purple color of leaf veins (Fig. 2b). Examination of chlorophyll and anthocyanin contents in the plants revealed that both overexpression lines had higher levels of chlorophyll and anthocyanin compared to the WT, suggesting that the transgenic plants might possess enhanced resistance to nutritional or water stress (Fig. 2c, d).

    Figure 2.  Arabidopsis plants overexpressing lnc000283 and lnc012465 had higher anthocyanins and chlorophyll content. (a) The relative expression level of lnc000283 and lnc012465 in WT and different transgenic lines. UBC10 (At5g53300) was used as an internal control. Each value is mean ± sd (n = 3). (b) The phenotype of WT and Arabidopsis overexpressing lnc000283 or lnc012465 grown on 1/2 MS medium 50 d after sowing. The (c) anthocyanin and (d) chlorophyll content of WT and transgenic Arabidopsis overexpressing lnc000283 or lnc012465. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01).
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    Effect of lncRNA overexpression on heat tolerance in Arabidopsis

    Given that lnc000283 and lnc012465 were highly induced by heat, the thermotolerance of the overexpressing (OE) plants were compared to that of the wild type. Arabidopsis plants were initially exposed to a an HS treatment at 38 °C for 4 d, followed by recovery at room temperature. The death caused by HS was processive. Post-severe HS challenge for 4 d, OE plants initially appeared similar to WT, but upon recovery, their leaves started to fold or curl, followed by a transition to yellow, white, and eventually drying out (Fig. 3a). OE lnc000283 and OE lnc012465 plants exhibited enhanced thermotolerance compared to WT, with lnc012465 showing particularly strong tolerance (Fig. 3a; Supplemental Fig. S2a). After 5 d of recovery, leaf coloration indicated that transgenic plants maintained a significantly higher percentage of green leaves and a lower percentage of bleached leaves compared to WT (Fig. 3b; Supplemental Fig. S2b). Under non-heat-stress conditions, WT and OE plants possessed comparable water content. However, following heat stress, the fresh-to-dry weight ratio of OE lnc000283 and lnc012465 lines was significantly greater than that of WT (Fig. 3c; Supplemental Fig. S2c). Abiotic stresses frequently trigger the production of excessive reactive oxygen species (ROS), which are believed to cause lipid peroxidation of membrane lipids, leading to damage to macromolecules. Leaf MDA content is commonly used as an indicator of lipid peroxidation under stress conditions; therefore, the MDA content in both transgenic and WT plants was assessed. Figure 3d shows that the MDA content in WT plants progressively increased after heat treatment, whereas in the two lines overexpressing lnc012465, the MDA content increased only slightly and remained significantly lower than that in WT at all time points. In plants overexpressing lnc000283, the MDA content did not significantly differ from that of WT before heat stress. However, after 4 d of heat treatment, the MDA content was significantly lower compared to WT (Supplemental Fig. S2d). The results suggested that the expression of both lnc012465 and lnc000283 can mitigate injury caused by membrane lipid peroxidation under heat-stress conditions. Peroxidase (POD) is a crucial antioxidant enzyme involved in ROS scavenging. Figure 3e and Supplemental Fig. S2e demonstrate that POD activity increased in both transgenic and WT plants after heat treatment. However, the increase in WT plants was modest, whereas OE lnc000283 and OE lnc012465 plants exhibited consistently higher POD activity. As anticipated, proline levels were induced in response to stress in all studied plants (Fig. 3f; Supplemental Fig. S2f). However, under normal conditions and 2 d post-heat stress treatment, the proline content in OE lnc000283 and OE lnc012465 plants did not exhibit significant changes compared to WT (Fig. 3f; Supplemental Fig. S2f). Moreover, after 4 d of heat stress, the proline content in OE lnc012465 lines was significantly lower than in WT, and the OE lnc000283 transgenic line 12-6 also showed a marked decrease in proline content compared to WT (Fig. 3f; Supplemental Fig. S2f). The results indicated that the thermotolerance of plants overexpressing either lnc000283 or lnc012465 was independent of proline accumulation.

    Figure 3.  Overexpressing lnc012465 lines are more tolerant to heat stress. (a) Phenotypes of WT and OE lnc012465 plants were assessed before and after exposure to heat stress. The heat treatment was applied to 25-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh-to-dry weight ratio of Arabidopsis leaves was measured before and after 38 °C heat treatment. (d)−(f) depict the MDA content, POD activity, and proline content in Arabidopsis leaves at varying durations of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis

    To elucidate the molecular mechanisms by which lncRNAs enhance thermotolerance in Arabidopsis, the expression of the Hsf gene HsfA7a and three Hsps (Hsp25.3, Hsa32, and Hsp18.1-CI) in OE lnc000283, OE lnc012465, and WT Arabidopsis plants were investigated at various time points following heat treatment. As shown in Fig. 4 and Supplemental Fig. S3, both Hsf and Hsps exhibited a rapid response to heat stress with strong induction. Notably, the transcripts of HsfA7a and Hsp25.3 were significantly upregulated at 1 h after heat exposure, then experienced a sharp decrease. Hsa32 and Hsp18.1-CI were highly induced at 1 h and, unlike the other proteins, sustained high expression levels at 3 h (Fig. 4; Supplemental Fig. S3). At 1 h post-heat treatment, the transcript levels of Hsa32 and HsfA7a in OE lnc000283 did not significantly differ from those in WT. However, by 3 h, Hsa32 expression was roughly 50% of the WT level, while HsfA7a expression was approximately double that of WT (Supplemental Fig. S3). The overexpression of lnc000283 did not significantly affect the transcript level of Hsp25.3 at any of the tested time points. Notably, Hsp18.1-CI expression in both lines overexpressing lnc000283 was significantly induced at all three detection points post-heat treatment, reaching approximately 4-9-fold higher levels than in the WT (Supplemental Fig. S3). In Arabidopsis plants with elevated expression of lnc012465, the expression patterns of all Hsp and Hsf genes were similar to those in plants overexpressing lnc000283, with the notable exception of Hsa32. Unlike the WT, Hsa32 did not show a trend of down-regulation at 3 h post-heat treatment (Fig. 4). The findings suggest that the substantial induction of Hsp18.1-CI may play a role in enhancing the thermotolerance of Arabidopsis plants overexpressing lnc000283 and lnc012465.

    Figure 4.  The expression of HSF and HSP genes in lnc012465 overexpressing lines before and after different heat treatment times. Gene expression levels were quantified using RT-qPCR and normalized to UBC10 (At5g53300). Each value represents the mean ± standard deviation (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on drought tolerance in Arabidopsis

    Prior research has implicated a significant proportion of genes in conferring resistance to various abiotic stresses. To elucidate the functions of lnc000283 and lnc012465 more thoroughly, WT and transgenic plants were subjected to drought stress by depriving them of water for 9 d. It was noted that the majority of leaves in WT plants withered and dried, whereas the OE lnc000283 and OE lnc012465 plants exhibited reduced withering, with only a minority displaying dryness (Fig. 5a; Supplemental Fig. S4a). Eight days post-rewatering, a negligible fraction of WT seedlings exhibited recovery, whereas the overwhelming majority of transgenic plants regained vigorous growth (Fig. 5a; Supplemental Fig. S4a). The transgenic plants demonstrated a significantly higher survival rate compared to the WT plants. Following 9 d of water deficit treatment, less than 40% of the WT plants survived, whereas the OE 012465 lines 8-7 and 9-1 exhibited survival rates of 100% and 95%, respectively, and the OE 000283 lines 11-10 and 12-6 had survival rates of 87% each. (Fig. 5b; Supplemental Fig. S4b). Water loss serves as a critical metric for assessing plant drought tolerance, hence the fresh-to-dry weight ratio of excised leaves was assessed via desiccation analysis. Following 4 d of drought treatment, the fresh-to-dry weight ratio for WT plants was reduced to 43%, whereas for OE lnc000283 lines 11-10 and 12-6, it was reduced to 73% and 75%, respectively. For OE 012465 lines 8-7 and 9-1, the ratios were reduced to 67% and 62%, respectively (Fig. 5c; Supplemental Fig. S4c). The findings indicated that lnc000283 and lnc012465 endow the transgenic plants with drought tolerance.

    Figure 5.  Overexpressing lnc012465 lines are more tolerant to drought stress. (a) Phenotype of WT and OE lnc012465 plants before and after subjecting to drought stress. Drought treatment was carried out on 20-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh weight to dry weight ratio of Arabidopsis leaves before and after undergoing 38 °C heat treatment. (d)−(f) MDA content, POD activity, and proline content in Arabidopsis leaves under different times of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001)
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    MDA content in leaves is a standard biomarker for assessing the extent of drought stress-induced damage. Prior to drought stress exposure, MDA levels in WT and transgenic plants were comparable. However, following 7 and 9 d of water deficit, the MDA content in the transgenic plants was markedly reduced compared to the WT, suggesting a less severe degree of membrane lipid peroxidation in the transgenic plants (Fig. 5d; Supplemental Fig. S4d). Oxidative stress frequently coincides with drought stress, hence the activity of POD was assessed to evaluate the ROS scavenging ability. The findings indicated that as the duration of drought treatment increased, POD activity progressively rose. Before drought exposure, the POD activity in lines 11-10 and 12-6 of OE 000283 was 2.4-fold and 2.2-fold higher than that of the WT, respectively (refer to Supplemental Fig. S4e). Following drought treatment, the POD activity in the transgenic lines remained significantly elevated compared to the wild type, although the enhancement was less pronounced than before the treatment (Supplemental Fig. S4e). In the OE 012465 plants, the POD activity in lines 8-7 and 9-1 significantly surpassed that of the wild type, with the discrepancy being more pronounced during drought stress (Fig. 5e). The proline content in WT and OE 000283 plants exhibited no significant differences before and after 7 d of treatment. However, after 9 d of drought, the proline content in OE 000283 plants was significantly lower compared to that in the WT (Supplemental Fig. S4f). OE 000465 plants showed no significant difference from the wild type before and after drought treatment (Fig. 5f). The findings were consistent with those under heat stress, indicating that the enhanced stress resistance due to the overexpression of lnc000283 and lnc012465 in Arabidopsis is not reliant on proline accumulation.

    Following drought stress treatment, the expression levels of drought-related genes such as RD29A, RD29B, NCED3, AREB1, and Rab18 were significantly elevated in plants overexpressing lnc000283 and lnc012465 compared to WT plants. These findings suggest that lnc000283 and lnc012465 modulate Arabidopsis drought tolerance by regulating the expression of genes associated with the drought stress response (Fig. 6; Supplemental Fig. S5).

    Figure 6.  The expression of drought-responsive genes in lnc012465 overexpressing lines before and after different drought treatment time. Gene expression levels were determined by qRT-PCR normalized against UBC10 (At5g53300). Each value is mean ± sd (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Discussion

    The integrity of global food security is under threat due to the confluence of rapid population expansion and profound climatic shifts[29]. Amidst the shifting climatic landscape, heat and drought stress have emerged as primary limitations to crop yield and global food security. Understanding how plants detect stress cues and acclimate to challenging conditions is a pivotal biological inquiry. Moreover, enhancing plant resilience to stress is essential for maintaining agricultural productivity and fostering environmental sustainability[2]. Concurrently, the advancement of next-generation sequencing (NGS) technology has led to the identification of a substantial number of lncRNAs that participate in diverse stress responses, with functional analyses having been conducted on several of these molecules.[30] For instance, in the case of potatoes, the lncRNA StFLORE has been identified to modulate water loss through its interaction with the homologous gene StCDF1[31]. LncRNA TCONS_00021861 can activate the IAA biosynthetic pathway, thereby endowing rice with resistance to drought stress[32]. In wheat, the expression of TalnRNA27 and TalnRNA5 was upregulated in response to heat stress[33]. Our prior investigation identified a total of 81 lncRNAs in Chinese cabbage that engage in intricate interactions with their respective mRNA targets across various phases of heat treatment[25]. Two lncRNAs, lnc000283 and lnc012465, were chosen for subsequent functional analysis. Findings confirmed that these lncRNAs endow transgenic Arabidopsis plants with enhanced tolerance to both heat and drought, thereby offering novel resources for enhancing stress resistance through genetic engineering.

    Abiotic stresses frequently trigger the synthesis of anthocyanins, serving as natural antioxidants that mitigate oxidative damage by neutralizing surplus reactive oxygen species (ROS), thereby protecting plants from growth inhibition and cell death, allowing plants to adapt to abiotic stresses[34,35]. For instance, during chilling stress, the accumulation of anthocyanins within leaves can mitigate oxidative damage, thereby enhancing the photosynthetic rate[36]. Consequently, the level of abiotic stress tolerance can be inferred from the concentration of anthocyanins. The reduction of photosynthetic ability is one of the key physiological phenomena of stresses, which is partly due to the degradation of chlorophyll caused by leaf senescence during stress. The reduced accumulation of chlorophyll in the plants was seen in many plants when exposed to drought or heat stress conditions. The current investigation revealed that lncRNA-overexpressing plants cultivated in Petri dishes exhibited increased accumulation of both chlorophyll and anthocyanins in advanced growth phases, indicating that these transgenic plants, overexpressing lnc000283 and lnc012465, demonstrated enhanced stress tolerance and superior growth performance relative to WT (Fig. 2c, d).

    Upon exposure to heat stress, there is a marked induction of transcription for numerous genes that encode molecular chaperones in plants, with the vast majority of these genes contributing to the prevention of protein denaturation-related damage and the augmentation of thermotolerance[37−39]. The present investigation identified multiple heat-inducible genes in plants overexpressing lnc000283 and lnc012465, as well as in WT (Fig. 4; Supplemental Fig. S3). The findings indicated that of the four HSP or HSF genes examined, Hsp18.1-CI exhibited a significantly greater abundance in both OE lnc000283 and OE lnc012465 plants compared to the WT following heat treatment for several days. Hsp18.1-CI, formerly referred to as Hsp18.2 has been the subject of investigation since 1989.[40] Following the fusion of the 5' region of Hsp18.2 in frame with the uidA gene of Escherichia coli, the activity of GUS, serving as the driver gene was observed to increase upon exposure to HS[40]. The Arabidopsis hsfA2 mutant exhibited diminished thermotolerance after heat acclimation, with the transcript levels of Hsp18.1-CI being substantially reduced compared to those in wild-type plants following a 4-h recovery period[41]. The findings revealed that the upregulation of Hsp18.1-CI protein is a critical mechanism by which plants achieve enhanced protection against heat stress in adverse environmental conditions, thereby bolstering their thermotolerance.

    Plants cultivated in natural settings are often subjected to concurrent multiple abiotic stresses, which can exacerbate threats to their routine physiological functions, growth, and developmental processes[42,43]. Elucidating the molecular mechanisms underlying plant responses to abiotic stress is crucial for the development of new crop varieties with enhanced tolerance to multiple abiotic stresses. Previous research has indicated that the overexpression of certain protein-coding genes can endow plants with resistance to a variety of abiotic stresses. For instance, tomatoes with robust expression of ShCML44 demonstrated significantly enhanced tolerance to drought, cold, and salinity stresses[44]. Overexpression of PeCBF4a in poplar plants confers enhanced tolerance to a range of abiotic stresses[45]. With respect to lncRNAs, transgenic Arabidopsis plants that overexpress lncRNA-DRIR displayed marked increased tolerance to salt and drought stresses compared to the wild-type[46]. In the present study, both overexpression lines of lnc000283 and lnc012465 exhibited resistance to heat and drought stresses, thereby contributing to the enhancement of plant resilience against multiple stresses (Figs 3, 5; Supplemental Figs S2, S4).

    The number of genes implicated in plant drought resistance is regulated by both ABA-dependent and ABA-independent pathways[47,48]. It is well established that the expression of RD29A exhibits a high level of responsiveness to drought stress, operating through both ABA-dependent and ABA-independent mechanisms[49]. RD29B, AREB1, and RAB18 are governed by an ABA-dependent regulatory pathway[10,49,50]. NCED3 is involved in ABA biosynthesis[51]. In the present study, the transcript levels of RD29A, RD29B, NCED3, AREB1, and RAB18 were significantly elevated in OE lnc000283 and OE lnc012465 plants compared to those in the WT plants (Fig. 6; Supplemental Fig. S5). The findings indicated that the drought tolerance imparted by OE lnc000283 and OE lnc012465 plants is contingent upon an ABA-dependent mechanism.

    Prior research has indicated that certain long non-coding RNAs (lncRNAs) can assume analogous roles across diverse biological contexts. For example, the lncRNA bra-miR156HG has been shown to modulate leaf morphology and flowering time in both B. campestris and Arabidopsis[52]. Heterogeneous expression of MSL-lncRNAs in Arabidopsis has been associated with the promotion of maleness, and similarly, it is implicated in the sexual lability observed in female poplars[53]. In the present study, lnc000283 and lnc012465 were induced by heat in Chinese cabbage, and their heterologous expression was found to confer heat tolerance in Arabidopsis. Additionally, sequences homologous to lnc000283 and lnc012465 were identified in Arabidopsis (Supplemental Fig. S1). The data suggest that these sequences may share a comparable function to that of heat-inducible sequences, potentially accounting for the conservation of lnc000283 and lnc012465'os functionality across various species.

    Conclusions

    In conclusion, the functions of two heat-inducible lncRNAs, lnc000283 and lnc012465 have been elucidated. Transgenic Arabidopsis lines overexpressing these lncRNAs accumulated higher levels of anthocyanins and chlorophyll at a later stage of growth compared to the WT when grown on Petri dishes. Furthermore, under heat and drought stress conditions, these OE plants exhibited enhanced stress tolerance, with several genes related to the stress resistance pathway being significantly upregulated. Collectively, these findings offer novel insights for the development of new varieties with tolerance to multiple stresses.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and supervision: Li N, Song X; experiment performing: Wang Y, Sun S; manuscript preparation and revision: Wang Y, Feng X, Li N. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information files.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32172583), the Natural Science Foundation of Hebei (C2021209019), the Natural Science Foundation for Distinguished Young Scholars of Hebei (C2022209010), and the Basic Research Program of Tangshan (22130231H).

  • The authors declare that they have no conflict of interest.

  • Supplementary Table S1 Eigenvalue, contribution rate and cumulative contribution rate of main components of traits under CK.
    Supplementary Table S2 Component matrix among traits under CK.
    Supplementary Table S3 Eigenvalue, contribution rate and cumulative contribution rate of main components of traits under CCC.
    Supplementary Table S4 Component matrix among traits under CCC.
    Supplementary Table S5 Eigenvalue, contribution rate and cumulative contribution rate of main components of traits under TE.
    Supplementary Table S6 Component matrix among traits under TE.
    Supplementary Table S7 Eigenvalue, contribution rate and cumulative contribution rate of main components of traits under CCC+TE.
    Supplementary Table S8 Component matrix among traits under CCC+TE.
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  • Cite this article

    Zhang R, Zhang X, Rolston P, Yang Z, Feng G, et al. 2024. Effects of different plant growth regulators on phenotypic variation and seed yield of Dactylis glomerata. Grass Research 4: e022 doi: 10.48130/grares-0024-0021
    Zhang R, Zhang X, Rolston P, Yang Z, Feng G, et al. 2024. Effects of different plant growth regulators on phenotypic variation and seed yield of Dactylis glomerata. Grass Research 4: e022 doi: 10.48130/grares-0024-0021
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Effects of different plant growth regulators on phenotypic variation and seed yield of Dactylis glomerata

  • 1.

    College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, Sichuan, China

  • 2.

    Seed Research Centre (SRC), Lincoln University, Lincoln, Christchurch 7647, New Zealand

  • Received: 12 August 2024
  • Revised: 24 September 2024
  • Accepted: 09 October 2024
  • Published online: 29 October 2024
Grass Research  4,  Article number: e022  (2024)  |  Cite this article

Abstract: Plant growth regulators (PGRs) have been widely used in the production of forage seeds. In a two-year field experiment, different concentrations of CCC (500, 750, 1,000, 1,250 g·hm−2) and TE (100, 200, 300, 400 g·hm−2) were sprayed on Dactylis glomerata as well as a mixture of CCC and TE (500 + 50, 500 + 150, 1,000 + 50, 1,000 + 150 g·hm−2). By studying the effects of different PGRs and different concentrations on the characters and seed yield of Dactylis glomerata, it was found that the plant height was significantly reduced by spraying two plant growth regulators alone, and the stem diameter of increased by spraying a mixture of CCC and TE. Different PGRs had different effects on the functional leaves of Dactylis glomerata under different concentrations. Spraying of two PGRs in a certain range of concentrations could significantly improve the seed yield, but when the concentration was too high, the seed yield was reduced. The seed yield of Dactylis glomerata could be significantly increased by spraying a mixture of CCC and TE in the range of the test concentrations. Among them, spraying a mixture of CCC and TE at a concentration of 500 + 150 g·hm−2 had the best effect on increasing seed yield, which increased the average yield by 47.91% in two years. Additionally, the experiment also found that spraying PGRs mainly increased the yield of Dactylis glomerata by increasing the number of fertile tillers·m−2, spikes per fertile tillers, and seed setting rate.

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    Introduction

    • Orchardgrass (Dactylis glomerata L.) is an excellent cool season perennial grass that is widely cultivated around the world and it is the fourth most crucial forage grass[1−3]. Because of its strong adaptability, rich nutrition, large biomass, and soft grass quality, orchardgrass is widely used in grazing and for the production of hay or silage[4,5]. In addition, it also plays an important role in ecological remediation and grassland construction, such as stone desertification control and returning farmland to grass[6]. As a material basis for reproduction and application in improving degraded grassland and planting artificial grassland, seed is critical in grassland construction and economic development of animal husbandry[7]. However, lodging can seriously affect seed production when it encounters wind and rain during the filling stage due to the large biomass and softness of orchardgrass[8].

      Seed yield is an important characteristic of forage and turf grass, thus seed multiplication is economically relevant for novel grass cultivars to compete commercially[9]. In recent years, seed production had induced wide concern for the breeders and producers. The yield of forage seeds is compared with crop seeds, which is usually very low and variable, only 10%−20% of the above-ground matter harvested as seed[10]. Grass seed yield is a complex trait which is affected by genetic properties, cultivation management as well as environmental factors[11]. Cultivation management could effectively optimize seed yield by establishment techniques, the use of plant growth regulators, fertilization, harvesting, and seed cleaning[9].

      Plant growth regulators (PGRs) are synthetic compounds that have similar effects to natural plant hormones and are recognized as exogenous non-nutritive chemicals for target plants. As a growth blocker, Chlorocholine chloride (CCC) (also called Chlormequat chloride) is an excellent PGR to reduce reproductive stem length and lodging, make the leaves green and thicker, increase the chlorophyll content, and develop the root system. CCC blocks the biosynthesis of endogenous gibberellin (GA), thus reducing cell division in the internode region without affecting the division of apical meristem and delaying cell elongation and plant dwarfing[12,13]. CCC has been recommended to be applied to reduce lodging in seed production[14]. Stahli et al. also found that CCC could improve lodging resistance and increase Triticum aestivum grain yield[15,16]. Trinexapac-ethyl (TE) is also a plant growth regulator and has been widely adopted as a lodging control agent in forage and turf grass seed production[17]. It controls the growth of plants by reducing the content of GA, which is similar to CCC. TE also controls the height of plants when the crop is growing vigorously, thereby increasing seed yield[18]. TE-induced increases in seed yield were related to improvements in floret production and seed set[19]. The increase in seed yield had been reported in a lot of grass species by the use of chemical PGRs[9]. To date, the fertilization, cultivation mode, and application of PGRs have been proven to increase the number of inflorescence[20], the number of seeds per panicle[21], and the number of seeds[22]. Those seed production factors in orchardgrass directly or indirectly improve the yield of orchardgrass seed.

      Currently, there is a large demand for orchardgrass seeds. Besides the improvement of traditional cultivation and fertilization measures, it is also crucial to increase seed yield through the use of plant exogenous hormones. This study aimed to explore the effects of various plant growth regulators and their combinations on reproductive traits, seed yield components, and cell level of orchardgrass. Thus, the effects of different plant growth regulators on orchardgrass seed yield, traits, and microstructure cells, and the most suitable treatment method for the production of their seeds were studied and then incorporated into production to help increase the yield of orchardgrass seed.

    Materials and methods

      Plant material and experimental design

    • The plant material used in this experiment was a nationally-approved variety 'Dianbei' (Dactylis glomerata cv. 'Dianbei') selected by Sichuan Agricultural University, with good palatability, well-adapted, high nutritional value, and high yield. The experiment was carried out at the experimental site (E103°39', N30°33') of the modern agricultural base of Sichuan Agricultural University (Chengdu, China) for two years. The experimental site was in the subtropical humid monsoon climate zone, with an altitude of 514 m. The soil was purple loam soil with a pH of 6.2. The soil available phosphorus content was 1.81 mg·kg−1, available nitrogen content was 52 mg·kg−1, and available potassium content was 79 mg·kg−1. Before sowing, 20,000 kg·hm−2 of organic fertilizer was applied to the test site and topdressing was applied during tillering and jointing. After removal of weeds and debris for one week, 'Dianbei' was sown on September 15, 2016. The sowing density was equivalent to 20 kg·hm−2. By using drill seeding, 3 m × 5 m of the plot area was prepared, with row spacing at 50 cm. The protection lines were set at 3 m around the whole test site, whereas the spacing between plots was 1 m. Throughout the entire growth observation period, tilling and weed removal were carried out from time to time to prevent diseases and insect pests.

      Treatments

    • The trial was designed in a single-factor randomized plot with three replicates. The growth period of this experiment was based on the expression of Zadoks et al.[23]. Within the period of GS32 (jointing stage: two nodes), foliar sprays were applied to 'Dianbei' at different concentrations, followed by second spray after two weeks. Tap water acted as a control (CK) in this study.

      The concentrations of plant growth regulators were determined based on the results from previous studies[24,25], and the plot experiment which was conducted in the early stage of the experiment. The dosage of each plant growth regulator is listed in Table 1.

      Table 1.  Experiment application rate.

      Treatment Serial number Application rate in 2017 and 2018
      at the stage of GS32 (g·hm−2)
      Chlorocholine chloride (A)
      		A1 500
      A2 750
      A3 1,000
      A4 1,250
      Trinexapac-ethyl (B)
      		B1 100
      B2 200
      B3 300
      B4 400
      Chlorocholine chloride & Trinexapac-ethyl (C) C1 500 + 50
      C2 500 + 150
      C3 1,000 + 50
      C4 1,000 + 150

      Plant measurements

    • A few measurements were taken at the mature stage of orchardgrass, including plant height, internode number, the length and stem diameter of the second internode, the length and width of the flag leaf, and the length and width of the top second leaf. During the flowering period, a 1 m2 sample (excluding marginal rows) was randomly selected from each experimental plot to determine the number of fertile tillers of orchardgrass. The procedures were repeated four times for each plot. Twenty fertile tillers were randomly selected from each plot to determine the number of spikelets per fertile tiller, whereas 20 spikelets were randomly selected from each plot to determine the number of florets per spikelet. The theoretical seed yield (fertiletillersUnitarea×spikeletfertiletillers×floretsspikelet×averageseedweight) was calculated. Harvested seeds were weighed after drying, threshing, and cleaning. After that, the number of seeds with a caryopsis was counted, and the number of these seeds was divided by the number of flowers per spikelet to calculate the seed-setting rate. All the measuring indexes are shown in Table 2.

      Table 2.  Total measurement index.

      Morphological character Reproductive character
      Plant height, PH (cm) The number of fertile, NFT (tillers·m−2)
      Internode number, IN Spikes per fertile tillers, SFT
      Internode length, IL (cm) Florets per spikelet, FS
      Stem diameter, SD (mm) Seed setting rate, SSR (%)
      Flag leaf width, FLW (mm) 1000-seed weight, TSW (g)
      Flag leaf length, FLL (cm) Actual seed yield, ASY (kg·hm−2)
      Width of top second leaf,
      WTSL (mm)      		 Theoretical seed yield, TSY (kg·hm−2)
      Length of top second leaf,
      LTSL (cm)

      The histological analysis of vascular bundle cells was carried out by selecting the cross-section of the middle stem of the second segment of the stem base. After that, the plant cells were stained with Safranin O-Fast Green Stain, followed by observation of the sclerenchyma and xylem cells under the electron microscope at 400 x.

      Statistical analysis

    • All the experimental data were summarized using Microsoft Excel 2016. To investigate the effects of different PGRs on the seed yield and related traits of orchardgrass, One-Way ANOVA was used in SPSS Statistics 27 (SPSS Inc., New York, USA) to conduct a single factor variance analysis for each indicator. The level of significant difference was p < 0.05. LSD tests were used to compare the levels of each indicator for two years. The correlation heat map visualization (Pearson) and principal component analysis of orchardgrass were analyzed by Origin 2024 (Origin Lab Corp Inc., USA).

    Results

      Effect of plant growth regulators on phenotypic variation of Dactylis glomerata

    • After treatment with different concentrations of CCC and TE, the plant height of orchardgrass was significantly reduced, and the effect was more significant with the increased concentration (p < 0.05). The application of CCC with a dosage of 500 g·hm−2 inhibited the internode number, and the inhibitory effect decreased with the increase of CCC concentration, showing low promotion and high inhibition. The internode number was significantly reduced for treatment TE alone and a CCC + TE mixture compared with the control. After the application of CCC, internode length was inhibited, and with the increase in concentration, the internode length became shorter. When TE and CCC + TE were combined, a low concentration had a significantly promoting effect on internode length, and with the increase in concentration, the internode length of D. glomerata was first increased and then decreased. CCC significantly increased the stem diameter of orchardgrass at 750 g·hm−2, and the stem diameter was decreased by TE treatment at all concentrations. Under the mixed application of CCC + TE, with the concentration at 500 + 50 g·hm−2, the stem diameter was the largest. However, the stem diameter was reduced when the concentration was too high (Table 3).

      Table 3.  Effects of plant growth regulators on the morphology characters of Dactylis glomerata in 2017−2018.

      PGR No. Plant height (cm) Internode number Internode length (cm) Stem diameter (mm) Flag leaf width (mm) Flag leaf length (cm) Width of top
      second leaf (mm)      		 Length of top second leaf (cm)
      A CK 129.9a 4.58d 14.90a 5.18b 7.82b 31.42a 7.96a 39.34a
      A1 119.6b 4.22e 12.12b 4.75c 7.66c 29.20b 7.35c 36.72b
      A2 106.7c 4.81c 12.00bc 5.30a 7.84b 31.56a 7.68b 36.12bc
      A3 98.6d 5.25b 10.20c 4.92b 8.01a 27.65b 7.92a 33.40c
      A4 94.0d 5.52a 9.40c 4.69c 8.13a 28.66b 7.94a 34.29c
      B CK 129.9a 4.58a 14.90b 5.18a 7.82bc 31.42a 7.96d 39.34ab
      B1 119.8b 4.25bc 15.43a 4.98b 7.67c 31.02a 7.87d 40.00a
      B2 116.0c 4.30b 14.98b 4.68c 8.28a 30.03ab 8.61a 36.96c
      B3 111.6d 4.26bc 13.63c 5.15a 7.99b 27.99c 8.15c 36.23c
      B4 104.2e 3.91c 13.62c 4.87b 8.34a 29.31b 8.51b 35.70c
      C CK 129.9a 4.58a 14.90b 5.18b 7.82bc 31.42a 7.96b 39.34a
      C1 118.9b 4.06b 15.61a 5.39a 8.41a 30.33b 8.39a 37.85b
      C2 119.1b 4.07b 14.65b 4.79c 7.46d 25.93d 7.67c 34.72c
      C3 110.8c 3.71d 13.97bc 4.72d 7.78c 31.10a 7.87b 37.61b
      C4 114.0c 3.83c 13.60bc 4.79d 7.92b 28.13c 8.43a 35.15c
      Different letters in the same column indicate significant differences at the p < 0. 05 level.

      When high concentrations of CCC above 750 g·hm−2 and over 200 g·hm−2 of TE were applied, the width of the flag leaf was increased. Under a mixture of CCC + TE at 500 + 50 g·hm−2, the flag leaf width was the largest, which was 8.41 mm. On the contrary, except for the mixed application of CCC + TE at 500 + 50 and 1,000 + 150 g·hm−2, other concentrations inhibited the width of the flag leaf. The length of the flag leaf decreased with increasing concentrations of TE. However, the CCC alone and the mixed application of CCC + TE showed a trend of decreasing and then increasing with the increase in concentration. The variation of the width and length of the top second leaf was similar to the width and length of flag leaf (Table 3).

      In terms of the effects of two PGRs on the morphological characters of orchardgrass, the present study showed that the high concentration of CCC alone (1,250 g·hm−2) had the lowest plant height, which was 94.0 cm, whereas the shortest internode length was 9.40 cm. Application of TE alone had the greatest effect on the length and width of the top second leaf. At a TE concentration of 200 g·hm−2, the largest width of the top second leaf was 8.61 mm. On the other hand, the longest length of the second to top leaf was 40 cm when the concentration was 100 g·hm−2. Mixed application of CCC + TE had the greatest effects on internode number, stem diameter, and flag leaf width. When the concentration was 1,000 + 50 g·hm−2, the minimum internode number was 3.71. At a concentration of 500 + 50 g·hm−2, the maximum stem diameter and flag leaf width were 5.39 and 8.41 mm respectively among all treatments.

      Effects of plant growth regulators on seed yield and yield component of Dactylis glomerata

    • According to the average data from a previous study in 2017−2018, CCC significantly increased the number of fertile tillers·m−2, but when the concentration reached 1,250 g·hm−2, the number of fertile tillers·m−2 decreased. The mixed application of different concentrations of TE, CCC + TE significantly increased the number of fertile tillers·m−2. The number of spikelets increased significantly under certain concentration conditions of PGRs, except for the concentration above 1,000 + 150 g·hm−2 under the mixed application of CCC + TE and when the concentration of TE was over 200 g·hm−2. PGRs increased the number of florets and the seed setting rate. When the concentration of CCC was 750 g·hm−2 and the concentration of TE was 100 g·hm−2, the 1000-grain weight of D. glomerata seeds was significantly increased compared with the control. For CCC + TE mixed treatments, the concentration of 500 + 50 g·hm−2 and 1,000 + 50 g·hm−2 significantly increased the 1000-grain weight of seeds. When the concentration of CCC was lower than 1,250 g·hm−2, the actual seed yield and theoretical yield of D. glomerata were significantly increased (p < 0.05). The actual seed yield and the theoretical yield increased with different concentrations of TE, except for the TE concentration at 400 g·hm−2, which was lower than other concentrations in the actual seed yield. For the combined application of CCC + TE, the actual and theoretical seed yields of all treatments were significantly higher than the control (p < 0.05) (Table 4).

      Table 4.  Effects of PGRs on seed yield and yield characters of Dactylis glomerata L. in 2017−2018.

      PGR No. The number of fertile (tillers·m−2) Spikes per fertile tillers Florets per spikelet Seed setting rate (%) 1000-grain weight (g) Actual seed yield (kg·hm−2) Theoretical seed yield (kg·hm−2)
      A CK 116.50d 366.28a 4.01c 45d 0.62b 216c 1,057d
      A1 150.165b 365.57a 4.29a 53b 0.61b 253a 1,443b
      A2 167.25a 367.19a 4.11bc 55a 0.64a 248a 1,601a
      A3 132.75c 353.35b 4.13b 52bc 0.61b 222b 1,194c
      A4 89.42e 350.16b 4.15b 50c 0.63ab 186d 823e
      B CK 116.50d 366.44c 4.01c 45c 0.62b 216d 1,057e
      B1 141.67bc 449.22a 4.18b 52b 0.64a 278b 1,690b
      B2 163.59a 424.595b 4.27a 55a 0.62b 297a 1,820a
      B3 146.25b 355.58d 4.43a 53ab 0.62b 240c 1,412c
      B4 131.42c 359.17d 4.35a 45c 0.63b 209d 1,270d
      C CK 116.5e 366.44d 4.01b 45d 0.62b 216d 1,057e
      C1 135.17c 432.67c 4.06b 61b 0.66a 270bc 1,561c
      C2 147.34b 443.33b 4.42a 63ab 0.61b 320a 1,783b
      C3 159.42a 485.24a 4.12b 66a 0.65a 280b 2,068a
      C4 132.58d 362.69e 4.51a 53c 0.62b 256c 1,322d
      Different letters in the same column indicate significant differences at the p < 0.05 level.

      In terms of the effects of two PGRs on seed yield and yield component of orchardgrass, it was found that the high concentration of CCC alone was sprayed (1,250 g·hm−2), the lowest number of fertile tillers·m−2 was 89.42, while the lowest actual seed yield and theoretical yield were 186 kg·hm−2, and 823 kg·hm−2 respectively. The mixed application of CCC + TE had the greatest effects on actual seed yield at 500 + 150 g·hm−2, making the actual seed yield 320 kg·hm−2.

      Correlation between the morphological and reproductive traits of Dactylis glomerata

    • The correlation analysis showed that the correlations among the morphological traits were not consistent under the different PGRs. Among them, under the CK, the number of internodes, the width of the flag leaf, and the length of the top second leaf were significantly negatively correlated, the length of the internode and the length of the top second leaf were also negatively correlated (Fig. 1a). After the treatment of CCC, the plant height of orchardgrass was extremely negatively correlated with the number of internodes, the width of the flag leaf, and the width of the top second leaf, but significantly positively correlated with internode length and the length of the top second leaf. The actual seed yield was not significantly correlated with plant height, but positively correlated with internode length, stem diameter, and the length of the top second leaf (Fig. 1b). Under TE treatment, plant height was extremely positively correlated with internode length, internode number, and the length of the top second leaf, but significantly negatively correlated with the flag leaf width. Actual seed yield was extremely positively correlated with plant height, internode number, and length. The length of the top second leaf was positively correlated (Fig. 1c). Under the combination of CCC + TE, the actual seed yield was significantly negatively correlated with the width of the flag leaf, and extremely negatively correlated with the length of the top second leaf (Fig. 1d).

      Figure 1. 

      Correlation between the morphological traits of Dactylis glomerata. The correlation heat map visualization of (a) control, (b) CCC, (c) TE, and (d) CCC + TE are presented. ** significant correlation at the 0.01 level; * significant correlation at the level of 0.05.

      The correlation analysis of reproductive traits of orchardgrass showed that under control treatment, the correlation coefficient between the actual seed yield and the number of fertile tillers·m−2 was −0.69, showing a significant negative correlation (Fig. 2a), but under treatment of CCC, the correlation coefficient was 0.95, showing a significant positive correlation (Fig. 2b). Under treatment of TE, there was a significant positive correlation between the actual seed yield and the number of fertile tillers·m−2, spikes per fertile tiller, and seed setting rate (Fig. 2c). Under the mixed treatment of CCC and TE, the actual seed yield was positively significant correlated with the seed setting rate, while the theoretical seed yield was extremely positively correlated with the number of fertile tillers·m−2, spikes per fertile tillers, and seed setting rate (Fig. 2d).

      Figure 2. 

      Correlation between the reproductive traits of Dactylis glomerata. The correlation heat map visualization of (a) control, (b) CCC, (c) TE, and (d) CCC + TE are presented. ** significant correlation at the 0.01 level; * significant correlation at the level of 0.05.

      Principal component analysis of the traits

    • Principal component analysis of the investigated traits showed that under control treatment (Fig. 3a), among the 15 factors examined, the eigenvalues greater than 1 were the first two principal components, and the cumulative contribution rate reached 100%, representing most of the original data information. The contribution rate of the first principal component was 76.1%. The width of the flag leaf and internode number were the main positive contributions whereas the internode length was the main negative contribution with a factor coefficient of −0.31. On the other hand, the contribution rate of the second principal component was 24.0%, while the length of the top second leaf and actual seed yield were the main positive contributions. The actual seed yield had a major positive contribution, with a factor coefficient as high as 0.52 (Supplementary Tables S1 & S2).

      Figure 3. 

      Principal component analysis of morphological and reproductive traits of Dactylis glomerata under different PGRs of treatment, (a) CK, (b) CCC, (c) TE, and (d) CCC + TE. Arrows represent physiological and reproductive traits with various lengths based on the impact of each trait on the separation of treatments. The 15 traits parameters (PH Plant height, IN Internode number, IL Internode length, SD Stem diameter, FLW Flag leaf width, FLL Flag leaf length, WTSL Width of top second leaf, LTSL Length of top second leaf, NFT The number of fertile tillers, SFT Spikes per fertile tillers, FS Florets per spikelet, SSR Seed setting rate, TSW 1000-seed weight, ASY Actual seed yield, TSY Theoretical seed yield) are shown.

      When treated with CCC (Fig. 3b), among the 15 factors examined, those with eigenvalues greater than 1 were the first three principal components, and the cumulative contribution rate reached 92.1%. The contribution rate of the first principal component was 62.8%. Most of the traits were positive contributions, except for the internode number, flag leaf width, top second leaf width, and 1000-grain weight. The internode length and the actual seed yield had a major positive contribution, both with a factor coefficient of 0.32. The contribution ratio of the second principal component was 19.6%. All the traits were positive contributions, excluding plant height, length of the top second leaf, and florets per spikelet. Stem diameter had a major positive contribution, with a factor coefficient of 0.50. The third main component contributed 9.7%, and the 1000-grain weight was the main positive contributor, with a factor coefficient of 0.68 (Supplementary Tables S3 & S4).

      Under TE treatment (Fig. 3c), among the 15 factors examined, the eigenvalues greater than 1 were the first four principal components, and the cumulative contribution rate reached 94.0%. The contribution rate of the first principal component was 55.2%. All other traits were positive contributions, except for stem diameter, flag leaf width, top second leaf width, and florets per spikelet. The contribution rate of the second principal component was 18.9%, and the number of fertile tillers was the largest positive contributor to the component, with a factor coefficient of 0.48. The third main component contributes 13.5%, with the flag leaf width and the width of the top second leaf as the main positive contributions, both with a factor coefficient of 0.42. The fourth main component contribution rate was 6.4%, and the major positive contribution was the stem diameter, with a factor coefficient as high as 0.62 (Supplementary Tables S5 & S6).

      Under CCC + TE treatment (Fig. 3d), among the 15 factors examined, the eigenvalues greater than 1 were the first three principal components, and the cumulative contribution rate occupied 91.41%. The contribution rate of the first principal component was 37.7%. All the traits were positive contributions, except for plant height, internode number, width of the top second leaf, and florets per spikelet. The number of fertile tillers had a major positive contribution, with a factor coefficient of 0.40. The contribution ratio of the second principal component was 33.7%, with the flag leaf width as the main positive contribution and factor coefficient of 0.43. The third main component contributed 19.0%, and the plant height was the main positive contribution, with a factor coefficient as high as 0.54 (Supplementary Tables S7 & S8).

      Effects of PGRs at different concentrations on actual seed yield of Dactylis glomerata

    • In the two-year experiment, the actual seed yield was different under different treatments of orchardgrass. Low concentrations of CCC increased seed yield, with the highest yield increase rate at 17.3%, but when the concentration reached 1,250 g·hm−2, it reduced the yield of orchardgrass, which was decreased by 14.0%. TE increased seed yield at low concentrations but decreased seed yield at high concentrations. Within the range of tested concentrations, the mixture of CCC + TE increased the seed yield of orchardgrass by 47.9% when the concentration was 500 + 150 g·hm−2 (Table 5).

      Table 5.  Effect of PGRs of different concentrations on the actual seed yield of Dactylis glomerata in 2017−2018.

      PGR No. Actual seed yield (kg·hm−2) Increase production compared with the control (kg·hm−2) Yield increase rate (%)
      CK 216.3 − −
      A A1 253.7 37.3 17.26
      A2 248.4 32.1 14.85
      A3 221.7 5.4 2.49
      A4 186.1 −30.2 −13.97
      B B1 278.3 62.0 28.65
      B2 297.1 80.7 37.32
      B3 240.8 24.5 11.32
      B4 209.2 −7.1 −3.29
      C C1 269.5 53.1 24.56
      C2 320.0 103.7 47.91
      C3 280.2 63.9 29.53
      C4 255.7 39.4 18.22

      Effects of PGRs at different concentrations on the microstructure of vascular bundle cells of Dactylis glomerata

    • The different concentrations of PGRs affected the seed yield and the vascular bundle tissue cells of orchardgrass stems. Under CCC treatment, the number of sclerenchyma cells increased with the increased concentration but reached the maximum when the concentration was 1,000 g·hm−2. The change of xylem cell number was consistent with that of sclerenchyma cells, reaching the maximum at 1,000 g·hm−2. The number of xylem cell layers was three except for the control under CCC treatment. The number of sclerenchyma cells was first increased and then decreased with the increase in TE concentrations, reached the maximum at 200 g·hm−2, and compared with the control, the number of sclerenchyma cells increased under all TE concentrations. Similarly, the changes in xylem cell number and layer number were consistent with the changes in sclerenchyma cell number, which first increased and then decreased with the increase in concentration. Generally, all treatments had two layers, except for concentrations at 100 and 200 g·hm−2, which had three layers. When CCC + TE were applied in combination at a concentration of 1,000 + 150 g·hm−2, the number of sclerenchyma cells was the highest, with 40 sclerenchyma cells. In addition, the number and layer of xylem cells first increased and then decreased with the increase in concentration. Among them, the mixed treatment of CCC + TE with concentration at 500 + 150 g·hm−2 had the best effect, which was significantly higher than other treatments. There was no significant change in cell layer numbers of sclerenchyma under all treatment concentrations (Table 6).

      Table 6.  Effect of PGRs of different concentrations on the vascular bundle cells of Dactylis glomerata.

      PGR No. Sclerenchyma Xylem
      Cell
      population      		 Cellular
      layer      		 Cell
      population      		 Cellular
      layer
      A CK 27c 2a 8b 2b
      A1 32b 2a 13ab 3a
      A2 35a 2a 13ab 3a
      A3 36a 2a 14a 3a
      A4 35a 2a 14ab 3a
      B CK 27c 2a 8c 2b
      B1 38a 2a 12b 3a
      B2 38a 2a 14a 3a
      B3 36b 2a 14ab 2b
      B4 36b 2a 8c 2b
      C CK 27c 2a 8c 2c
      C1 26c 2a 11b 3b
      C2 37ab 2a 17a 4a
      C3 35b 2a 12b 3b
      C4 40a 2a 11b 3b
      Different letters in the same column indicate significant differences at the p < 0.05 level.

      The biopsy tissue showed that the stem vascular bundle of orchardgrass treated with CCC, TE, CCC + TE became larger than the untreated control (Fig. 4a−d). Sclerenchyma could thicken the cell walls. After being treated with CCC + TE, the xylem cells were not only more numerous but also larger (Fig. 4c). Based on the effects of these PGRs on vascular bundle cell populations in the stem of orchardgrass, and the effects of combined PGRs on various phenotypic traits of orchardgrass, the use of PGRs could increase the stem diameter, shorten the internode length and plant height, etc. by increasing the number of sclerenchyma cells and layers as well as the number of xylem cells and layers (Table 3, Fig. 4). Thus, the orchardgrass had better resistance to lodging during the harvest period and facilitated the transportation of assimilated substances into grains for storage. It might be due to the promotion of reproductive growth in orchardgrass at the expense of vegetative growth, thereby directly or indirectly increasing the seed yield.

      Figure 4. 

      Transverse section of vascular bundle of Dactylis glomerata. The microstructure of vascular bundle cells of (a) CCC, (b) TE, (c) CCC + TE, and (d) control are shown. All the images are at 400 x microscopic. The cells between the two black circles were sclerenchyma cells as shown in (a) and the arrows point to phloem and xylem in (b).

    Discussion

    • Seed yield is the primary commercial feature for grass species[26]. The chlorocholine chloride (CCC) and trinexapac-ethyl (TE) used in this study are plant growth inhibitors, which could improve the seed yield of orchardgrass by regulating vegetative growth and promoting reproductive growth[27]. In this study, different plant growth regulators were applied at the GS32 period to investigate the effects on the agronomic traits and seed yield of orchardgrass. It was found that within a certain concentration range, all the treatments (CCC, TE, and CCC + TE) reduced the plant height of orchardgrass, while the stem diameter was significantly increased. Meanwhile, the application of CCC and TE might reduce lodging by lowering plant height, while mixed application of CCC + TE might reduce lodging by increasing stem diameter. The effects of CCC and TE on the reduction of plant height of orchardgrass might be different. The results show that CCC may reduce the plant height by reducing the internode length, while TE reduced the plant height by reducing the internode number, thereby, reducing potential lodging and increasing seed yield. Chastain et al. showed that TE reduced the stem length, and the higher the concentration, the greater the reduction. The increases in tall fescue seed yield attributable to TE were the result of increased seed number m−2[17]. Previous studies on the application of PGRs to maize (Zea mays)[28], wheat (Triticum aestivum)[29], and papaya (Chaenomeles sinensis)[30] reported that PGRs not only affected the seed yield but also affected the content of endogenous hormones in seeds, such as IAA, ABA, Z + ZR, GA, etc., and the activity of amylase. Although the content of relevant endogenous hormones and enzymes were not determined in this experiment, but the use of TE and CCC might have certain effects on the endogenous hormones and amylase of seeds, according to the results of previous studies.

      Under different plant growth regulators, the relationship between the morphological and reproductive traits and the yield of orchardgrass was different. In the control treatment, plant height and yield were negatively correlated. In comparison, plant height and yield were positively correlated when treated with plant growth regulators. In alfalfa, spraying of CCC promoted reproductive development and increased seed yield by reducing plant height[31]. Besides that, the relationship between plant height and seed yield was different under different treatments. It might be based on the premise that plant growth regulators significantly reduced the plant height of orchardgrass. Reduced plant height might lead to high seed yield. Under this circumstance, lodging probably was no longer the main factor affecting seed yield. Chynoweth et al. found that reduced stem length was associated with increased perennial ryegrass seed yield[32]. In this experiment, there was a significant positive correlation between internode length and yield of orchardgrass under the treatment of CCC and TE, but there was no significant correlation between internode number and internode length and seed yield under the treatment of the mixture of CCC and TE. These results showed that internode number and internode length might not be the main factors affecting seed yield under the mixture of CCC and TE. Flag leaf and top second leaf are the important functional leaves of plants for seed production yield[33]. It has been reported that flag leaf size had a significant positive effect on the seed yield of Festuca pratensis[34]. In this experiment, under different plant growth regulators, the correlation between the width and length of the flag leaf and the top second leaf and actual seed yield were different. For instance, under the treatment of CCC and TE, the length of the top second leaf had a significant positive correlation with the actual seed yield, while under the mixed treatment of CCC + TE, had a negative correlation with the seed yield. This might be caused by the simultaneous use of CCC + TE which had some antagonistic effects on the traits of orchardgrass[35].

      The decrease in seed yield was correlated with a decrease in reproductive tiller number, fewer spikelets per reproductive tiller, and fewer seeds per spikelet[36]. In a specific concentration range, different plant growth regulator treatments significantly increased the number of fertile tillers·m−2, spikes per fertile tillers, and seed setting rate. However, when the concentration exceeded a certain range, it would reduce the seed yield of orchardgrass, such as the concentration of CCC at 1,250 g·hm−2 and the concentration of TE at 400 g·hm−2, there was a decrease in seed yield. In this experiment, under different plant growth regulators, the number of fertile tillers·m2 was significantly positively correlated with the yield, and under the treatment of TE and the mixture of CCC + TE, the number of spikelets was also significantly positively correlated with the yield of orchardgrass. These results showed that different plant growth regulators might mainly increase the seed yield of orchardgrass by increasing the number of fertile tillers·m−2 and spikelets. The study by Tao et al. reported a similar results[37].

      Zhang et al. showed that the seed yield of alfalfa could be increased by spraying CCC[31]. In an experiment conducted in Oregon (USA), the two-year experimental data showed that the yield of Trifolium pratense increased by 18.7% after spraying TE[38]. In addition, Rolston et al. proved that the application of TE could increase seed yield of perennial ryegrass seed crops[39]. TE is beneficial to increase the tiller number of perennial ryegrass[40]. In a certain concentration range, different plant growth regulator treatments significantly increased the number of fertile tillers·m−2, spikes per fertile tillers, and seed setting rate, but when the concentration exceeded a certain range, it would reduce the seed yield of orchardgrass, such as the seed yield decreased when the concentration of CCC was 1,250 g·hm−2 and the concentration of TE was 400 g·hm−2. PGRs effectually control lodging during pollination and set filling, resulting in better light interception. However, a high application rate combined with drought might leave seed crops standing upright and exposed to wind[9]. In the 2-year experiment, under high concentrations of CCC, the seed yield of orchardgrass decreased. In 2017, high concentrations of TE also reduced the seed yield. In western Oregon (USA), the application of paclobutrazol at low rates can improve seed yields in Festuca rubra and Festuca arundinacea, but not in orchardgrass[41]. However, in this experiment, the results showed that low doses of plant growth regulators increased the seed yield of orchardgrass. The conflicting results might be due to the difference in plant growth regulators used for both experiments. In the experiment by Young et al., although paclobutrazol did not affect fertile tiller numbers in other years, it increased the number of seeds per unit area in 1985[41]. During the production of gramineous plant seeds, lodging could reduce the seed yield during the growth stages of plants. However, the good effects of the sclerenchymal tissue thickness and the number of vascular bundles on plant lodging resistance according to a lot of studies[42,43], is positively correlated with plant lodging resistance[44]. In this study, the number of vascular bundles and sclerenchyma tissue thickness of orchardgrass increased after treatment with CCC, TE, and CCC + TE. Therefore, the application of PGRs might be indirectly beneficial to the lodging resistance in the seed production of orchardgrass. In addition, it might be due to the improvement of the vascular bundle cell populations, which in turn promotes the transportation of assimilating substances to the grain storage, to directly or indirectly improve the yield of orchardgrass seeds[45,46].

      In terms of seed yield in two years, the yield-increasing effect of the mixture of CCC and TE was bigger than that of the single plant growth regulator. During the actual seed production in 2017−2018, when the mixture concentration of CCC and TE was 500 + 150 g·hm−2, the seed production reached its maximum, with an increase of 47.9% compared with the control. This is consistent with the results from previous studies which showed that the effect of mixed application of the same type of growth regulators on seed yield was bigger than that of single application of a regulator[47]. The comparison experiment of single and mixed application of chlormequat chloride and choline chloride was also carried out on winter wheat. The experiment proved that the mixed application further increased the winter wheat yield by about 8%[48]. In the mixed application of CCC + TE, the increase in seed yield of orchardgrass was mainly achieved by increasing the number of fertile tillers·m−2, florets per spikelet, and seed setting rate. Different plant growth regulators have significantly improved the theoretical seed yield and actual seed yield, but they are very different. The spring defoliation management of white clover seed crops had been shown to have a significant influence on the seed yield potential[49]. Procedures such as harvesting, handling, picking, and threshing during forage seed harvesting or even diseases will cause seed loss, thereby reducing actual seed yield. Therefore, spraying of the plant growth regulator in combination with good management practices can maximize the actual seed yield.

    Conclusions

    • Plant growth regulators play an important role in forage seed production. 'Dianbei' Dactylis as a high-quality forage species, significantly increased its actual seed yield after spraying with certain concentrations of PGRs. Through the effects of different PGRs and different concentrations on the characteristics and seed yield of orchardgrass, it was found that the plant height of orchardgrass was significantly reduced by spraying two PGRs alone, and the stem diameter of Dactylis glomerata increased by spraying a mixture of CCC + TE. Different PGRs had different effects on the functional leaves (flag leaf and the top second leaf) of orchardgrass under different concentrations. Spraying two types of PGRs together in a certain range of concentrations could significantly improve the seed yield, and the microstructure of the stem, but when the concentration was too high, the seed yield was reduced. Among them, the mixture of CCC + TE at a concentration of 500 + 150 g·hm−2, resulted in the largest seed yield. Therefore, this PGR mixture is recommended to promote seed yield in orchardgrass production.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Zhang R, Zhang X; data collection: Zhang R, Zhang X, Yang Z; analysis and interpretation of results: Zhang R, Rolston P, Yang Z, Feng G; draft manuscript preparation: Zhang R; supervision, resources, funding acquisition: Huang L, Zhang XQ, Nie G. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article and its supplementary information files, and are available from the corresponding author upon reasonable request.

      Acknowledgments

      • This research work was supported by the earmarked fund for CARS (CARS-34), and the Sichuan Province Breeding Research grant (Grant No. 2021YFYZ0013).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Supplementary information
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      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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    Zhang R, Zhang X, Rolston P, Yang Z, Feng G, et al. 2024. Effects of different plant growth regulators on phenotypic variation and seed yield of Dactylis glomerata. Grass Research 4: e022 doi: 10.48130/grares-0024-0021
    Zhang R, Zhang X, Rolston P, Yang Z, Feng G, et al. 2024. Effects of different plant growth regulators on phenotypic variation and seed yield of Dactylis glomerata. Grass Research 4: e022 doi: 10.48130/grares-0024-0021
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  • Introduction
  • Materials and methods

  • Plant material and experimental design

  • Treatments

  • Plant measurements

  • Statistical analysis

  • Results

  • Effect of plant growth regulators on phenotypic variation of Dactylis glomerata

  • Effects of plant growth regulators on seed yield and yield component of Dactylis glomerata

  • Correlation between the morphological and reproductive traits of Dactylis glomerata

  • Principal component analysis of the traits

  • Effects of PGRs at different concentrations on actual seed yield of Dactylis glomerata

  • Effects of PGRs at different concentrations on the microstructure of vascular bundle cells of Dactylis glomerata

  • Discussion
  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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