Transcriptome analysis reveals the effects of root development across different ginger generations on plant morphology and yield

Transcriptome analysis reveals the effects of root development across different ginger generations on plant morphology and yield | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 1 April 2025 V1 Latest version Share on Transcriptome analysis reveals the effects of root development across different ginger generations on plant morphology and yield Authors : Xiaoqin Zhao , Jinlian Yuan , Xiaodong Cai , Zhen Zeng , Lijuan Wei 0009-0000-4473-6237 [email protected] , and Yiqing Liu Authors Info & Affiliations https://doi.org/10.22541/au.174348751.14411766/v1 386 views 169 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Ginger ( Zingiber officinale Rosc.) holds significant culinary and medicinal value. Tissue-cultured ginger can markedly enhance rhizome yield after two years of growth. This study investigates the field performance variations and reveals the influence mechanism of root on plant morphology and yield among different ginger generations. The results indicate that the T1, T2 and T3 gingers exhibit strong genetic stability. Compared to T1 and T2 gingers, T3 ginger show superior performance in both aboveground and belowground biomass, photosynthetic capacity, yield and quality. However, anatomical analysis reveals that T3 ginger has a smaller diameter for fibrous roots and a lower cortex-to-stele thickness ratio in the fleshy roots compared to T1 and T2 gingers. Additionally, transcriptomic analysis elucidates the relationship between root development and metabolic regulation across different ginger generations. Compared to T2 roots, T3 roots exhibit significantly higher expression levels of DEGs associated with starch and sucrose metabolism, along with increased activity of starch-degrading enzymes (BAM), which ultimately influence the accumulation and conversion of starch and sucrose. Meanwhile, key enzymes activity and DEGs expression in flavonoid biosynthesis were downregulated, resulting in a significant reduction of flavonoid content in T3 roots. Furthermore, levels of ABA, TZR and SA were markedly lower in T3 roots, whereas the concentrations of IAA, JA and GA 3 were significantly higher. In conclusion, this study reveals notable differences in morphology, root development and rhizome yield across various generations of ginger. Specifically, T3 ginger exhibit superior yield and quality with weaker root development, which may be related to the dynamic regulations of starch and sucrose metabolism, flavonoid biosynthesis and phytohormone levels. These findings provide valuable theoretical insights and practical recommendations for optimizing the field cultivation of tissue-cultured ginger. Transcriptome analysis reveals the effects of root development across different ginger generations on plant morphology and yield Xiaoqin Zhao 1,2# , Jinlian Yuan 1# , Xiaodong Cai 1 , Zhen Zeng 1 , Lijuan Wei 1 , Yiqing Liu 1,2 1 Hubei Key Laboratory of Spices and Horticultural Plant Germplasm Innovation and Utilization, College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, China 2 College of Smart Agriculture, Chongqing University of Arts and Sciences, Yongchuan Chongqing 402160, China * Correspondence: Lijuan Wei ( [email protected] ) and Yiqing Liu ( [email protected] ) # Both authors contribute equally to this work. Abstract: Ginger ( Zingiber officinale Rosc.) holds significant culinary and medicinal value. Tissue-cultured ginger can markedly enhance rhizome yield after two years of growth. This study investigates the field performance variations and reveals the influence mechanism of root on plant morphology and yield among different ginger generations. The results indicate that the T1, T2 and T3 gingers exhibit strong genetic stability. Compared to T1 and T2 gingers, T3 ginger show superior performance in both aboveground and belowground biomass, photosynthetic capacity, yield and quality. However, anatomical analysis reveals that T3 ginger has a smaller diameter for fibrous roots and a lower cortex-to-stele thickness ratio in the fleshy roots compared to T1 and T2 gingers. Additionally, transcriptomic analysis elucidates the relationship between root development and metabolic regulation across different ginger generations. Compared to T2 roots, T3 roots exhibit significantly higher expression levels of DEGs associated with starch and sucrose metabolism, along with increased activity of starch-degrading enzymes (BAM), which ultimately influence the accumulation and conversion of starch and sucrose. Meanwhile, key enzymes activity and DEGs expression in flavonoid biosynthesis were downregulated, resulting in a significant reduction of flavonoid content in T3 roots. Furthermore, levels of ABA, TZR and SA were markedly lower in T3 roots, whereas the concentrations of IAA, JA and GA 3 were significantly higher. In conclusion, this study reveals notable differences in morphology, root development and rhizome yield across various generations of ginger. Specifically, T3 ginger exhibit superior yield and quality with weaker root development, which may be related to the dynamic regulations of starch and sucrose metabolism, flavonoid biosynthesis and phytohormone levels. These findings provide valuable theoretical insights and practical recommendations for optimizing the field cultivation of tissue-cultured ginger. Keywords: Ginger, Anatomical features, Starch and sucrose, Flavonoids, Hormones, Root characteristics Introduction Ginger ( Zingiber officinale Rosc.) is an important agricultural crop with significant economic value, as well as high edible and medicinal properties (Zhang et al., 2021; Shahrajabian et al., 2019). In cultivation, ginger primarily relies on mature rhizomes for asexual reproduction (Jiang et al., 2017), whereas this method results in a low propagation rate. And long-term monoculture has exacerbated soil-borne diseases, such as ginger bacterial wilt, which leads to reduced vigor, seed degeneration and declines in both yield and quality (Zhao et al., 2022; Yu et al., 2024; Dohroo et al., 2016). Plant propagation methods are typically categorized into sexual reproduction (via seeds) and asexual reproduction (e.g., cuttings, grafting and air-layering) (Awotedu et al., 2021; Megersa, 2017). In recent years, culture techniques in vitro have emerged as a highly effective alternative for large-scale production of high-quality plants, with widespread application across various crops (Taji et al., 2024; Hussain et al., 2012). Studies have demonstrated that, compared to traditional propagation methods, in vitro cultured plants offer several advantages in the field, including enhanced growth vigor (Wójcik et al., 2021), superior product quality (Irshad et al., 2023), increased metabolite production (Chatterjee et al., 2022; Cheng et al., 2024), improved disease and stress resistance (Hussain et al., 2012; Brown and Thorpe, 1995) and higher crop yields (Wójcik et al., 2021). These benefits have contributed to the gradual replacement of conventional propagation techniques in vitro culture, thereby enhancing overall agricultural productivity. For instance, Wójcik et al. (2021) found that micropropagated gooseberry ( Ribes grossularia L.) exhibited significantly better growth vigor and yield compared to those propagated conventionally. However, research by Ren et al. (2020) and Tetteh (2024) indicated that while culture in vitro shows significant potential for ginger production, it generally requires approximately two years of growth to reach the same economic productivity as conventionally propagated plants. Consequently, further research is needed to examine the field performance of cultured plants in vitro across multiple generations and to identify the underlying factors contributing to these differences. Root development is essential for plant growth and is closely linked to various physiological factors, including plant hormones, flavonoids, sugars and starch (Ullah et al., 2024; Gao et al., 2022). Jeong et al. (2010) demonstrated that the overexpression of OsNAC10 specifically in the roots of rice ( Oryza sativa L.) not only expanded the root system, but also significantly enhanced drought resistance and increased yield. In a similar vein, Davies et al. (2005) showed that flavonoid (formononetin) treatment in Peruvian potatoes ( Solanum tuberosum L.) improved the root-to-shoot ratio and reduced the leaf-to-tuber ratio, thus promoting root development and boosting yield. Furthermore, Xin et al. (2021) found that nitrogen-efficient rice varieties ( Oryza sativa L.) stimulated root development by regulating plant hormone levels, resulting in increased total root length, enhanced root oxidative activity and a larger active absorption of surface area, while reducing root diameter. This ultimately led to improved nitrogen accumulation and higher yield. Thus, based on these findings, the present study examines the differences in field performance across generations of ginger using morphological, genetic stability and anatomical approaches. Besides, the study investigates the impact of root development on plant morphology and yield. Through transcriptomic analysis, we explore the key roles of plant hormones, flavonoids, sugars and starch in regulating root morphology and physiological function in different ginger generation. These results offer new theoretical insights and practical guidance for the field application of ginger tissue culture. Materials and methods 2.1 Plant materials All materials used in this study were different generations of disease-free “Fengtou” ginger developed by the Spice Crops Research Institute of Yangtze University. Specifically, the materials included: breeder seed (T1), consisting of tissue-cultured plants obtained by shoot tip culture; foundation seed (T2), which was ginger harvested from the T1 ginger; and cultivar seed (T3), which was ginger harvested from T2 ginger. The plants were planted in the experimental field of Yangtze University, Hubei Province, China (Latitude: 30°21’N, Longitude: 112°09’E, Elevation: 28 m). The growing season lasted from early-May to early-December in 2023 and 2024. 2.2 Morphological measurements and root architecture characteristics After 120 days of grown, morphological parameters including main stem height, plant height, main tiller diameter, number of tillers per plant, number of main tiller leaves, leaf length and leaf width were recorded. Additionally, the aboveground biomass, rhizome biomass and root biomass were quantified. The average root diameter, root surface area, root volume and number of root tips for T1, T2 and T3 gingers were determined using a WinRhizo root scanner (Regent Instruments Inc., Quebec, Canada). Five plants were randomly selected from each group and all measurements were repeated three times. 2.3 Photosynthetic characteristics of leaves The third fully expanded leaves from the T1, T2 and T3 gingers were sampled, and chlorophyll and carotenoid contents were extracted using the acetone extraction method described by Arnon et al. (1949). Between 9:00 and 11:00 AM on clear days, net photosynthetic rate ( P n ), stomatal conductance ( G s ), intercellular CO 2 concentration ( C i ) and transpiration rate ( T r ) were measured with a portable photosynthesis and transpiration system (Yaxin-1102). Chlorophyll fluorescence parameters, including maximal photochemical efficiency of PSII ( F v / F m ), non-photochemical quenching ( NPQ ), photochemical quenching ( q P ) and actual optical quantum efficiency ( Φ PSII ), were determined using the FlourCam multispectral fluorescence imaging platform (PSI, Czech Republic). All treatments were replicated five times. 2.4 Rhizome biomass and quality The main yield traits including rhizome diameter, rhizome length, number of rhizomes and fresh rhizome weight per plant were measured. The 6-gingerol content in the rhizomes was determined using a Shimadzu LC-20A HPLC system (Kyoto, Japan) with a Waters Sep-pak C18 column (Milford, Massachusetts, USA). Soluble protein content in the rhizomes was quantified using the Coomassie Brilliant Blue G-250 method. 2.5 Paraffin sections and anatomical analysis of roots and leaves According to Li et al. (2017), the third fully expanded leaves and fleshy and fibrous roots were fixed in FAA solution (absolute ethanol: acetic acid: formaldehyde = 90:5:5) for at least 24 hours. The samples were then dehydrated through a graded ethanol series, embedded in paraffin and sectioned transversely into 8 μm thick slices. These sections were stained with 0.1% (w/v) Fast Green FCF and 0.1% (w/v) Safranin O (Solarbio, Beijing, China). After staining, the sections were mounted with neutral resin and examined using a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan). Quantitative anatomical characteristics of the leaves and roots were measured using NIS-Elements Documentation 4.50 software (Nikon, Tokyo, Japan). For leaf sections, six anatomical parameters were analyzed: leaf thickness, midrib thickness, thickness of the upper and lower epidermis and thickness of the palisade and spongy mesophyll. For fibrous and fleshy roots, four parameters were measured: root diameter, cortex thickness, stele diameter and average xylem vessel area, along with vascular bundle count. Five randomly selected sections each sample were analyzed, with at least six fields of view per section. 2.6 Determination of starch, sucrose, flavonoids and endogenous hormones contents After freeze-drying with an Eyela FDU-2110 freeze dryer (EYELA, Tokyo, Japan), starch and sucrose contents in the rhizomes of T1, T2 and T3 gingers were determined using a Bio Tek EON multifunctional microplate reader (Bio Tek Instruments, Winooski, VT, USA) following the plant starch and sucrose assay kit (Tongwei Bio, Shanghai, China). As described by Zhang et al. (2024), 2 g of freeze-dried rhizomes were sonicated in 10 mL of 70% ethanol for 1 hour. After centrifugation at 10,000 × g for 20 minutes, the supernatant was collected for flavonoid content analysis. Plant hormone levels, including Indole-3-acetic acid (IAA), Abscisic acid (ABA), trans-Zeatin riboside (TZR), Salicylic acid (SA), Jasmonic acid (JA) and Gibberellin A3 (GA 3 ), were quantified in ginger rhizomes by enzyme-linked immunosorbent assay (ELISA) as outlined by Ren et al. (2023). 2.7 Determination of enzyme activities The activities of SPS (sucrose phosphate synthase), SUS (sucrose synthase) and BAM (beta-amylase) enzymes were measured according to the method described by Huang et al. (2024). Briefly, enzyme extracts were prepared from the freeze-dried ginger rhizomes, and enzyme assays were conducted under optimized conditions. SPS activity was determined by measuring the production of UDP-glucose, SUS activity was quantified based on the conversion of sucrose to UDP-glucose and fructose and BAM activity was assessed by monitoring the release of maltose from starch hydrolysis. CHI (Chalcone isomerase), CHS (Chalcone Synthase) and DFR (Dihydroflavonol 4-Reductase) activities were determined using Lai Er Bio’s (Hefei, China) enzyme assay kits. All assays were performed in triplicate and results were analyzed statistically. 2.8 Flow cytometry analysis and chromosome counting As described by Zhao et al. (2022), the relative nuclear DNA content of T1, T2 and T3 gingers was measured using a Beckman CytoFLEX flow cytometer (Suzhou, China), with three replicates per sample and at least 3000 particles recorded per run. Root apical meristem tissue was prepared according to Zhao et al. (2022) and stained with carbol-fuchsin solution (Solarbio, Beijing, China). The slides were then observed and photographed using a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan). 2.9 Transcriptome sequencing, identification and functional annotations of DEGs RNA was isolated from the T2 and T3 ginger roots using the RNA Extraction Kit (Invitrogen, Nottingham, UK). The RNA purity was assessed using a NanoPhotometer spectrophotometer and RNA integrity was precisely utilized with the Agilent 2100 bioanalyzer. The construction and quality inspection of the cDNA library were conducted following the methods described by Liu et al. (2022). After passing the quality c inspection criteria, the libraries were pooled based on their effective concentrations and target offline data volume requirements. Subsequently, the paired-end RNA-seq sequencing was conducted using the Illumina HiSeq2500 platform at Shanghai Origingene Bio-pharm Technology Co., Ltd. The clear reads were mapped to the Zingiber officinale (Li et al., 2021) reference genome using HISAT2 software. To gain insights into the functional annotation of the DEGs, we conducted Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. 2.10 Quantitative real-time PCR analysis The RNA samples of T2 and T3 ginger roots were confirmed by qRT-PCR using CFX 96 Real-Time PCR system (Bio-Rad). The primer sequences were designed using Primer Premier 5.0. The relative expression levels of candidate genes were calculated by the 2−∆∆Ct method using RBP as internal standards (Li et al., 2022a). 2.11 Statistical ‘analysis Statistical differences between T1, T2 and T3 gingers were compared using a two-tailed Student’s t-test at significance levels of 0.05, 0.01 and 0.001. Pearson’s correlation analysis was performed to assess the relationship between growth parameters, rhizome biomass and root characteristics. Graphs were generated using Origin 2022 (Origin Lab Inc., Northampton, MA, USA). 3. Results 3.1 Growth parameters and root architecture characteristics across different ginger generations After a 120-day cultivation period, the different generations of ‘Fengtou’ ginger displayed significant variations in growth parameters and root architectural characteristics (Table 1; Fig. 1). The plant height, main tiller diameter and leaf biomass of the T1, T2 and T3 gingers showed a significant increase ( P < 0.05) with successive planting years. However, the number of main tiller leaves exhibited a significant decrease ( P < 0.05). Furthermore, the main stem height, leaf length, leaf width and above-ground stem biomass in T2 and T3 gingers were significantly higher compared to T1 ginger. The proportion of different plant components also varied significantly (Fig. 1B). The above-ground biomass and root biomass ratio in T3 ginger was significantly lower than that in T1 and T2 gingers, whereas the rhizome biomass ratio of T3 ginger was markedly higher compared to T1 and T2 gingers . Additionally, the root biomass ratio in T2 ginger was significantly higher than that in both T3 and T1 gingers (Fig. 1A, B). Root architectural characteristics, including the total number of root tips, root surface area, root projection area and total root length, exhibited notable declines across the ginger generations (T1, T2 and T3 gingers) (Fig. 1D-G). Specifically, the total number of root tips and total root length in T3 ginger were 953.50 and 1052.76 cm, respectively, which were lower than the T2 ginger (Fig. 1D and G). Overall, compared to T1 and T2 gingers, the T3 ginger exhibited the highest aboveground biomass and rhizome biomass, but the poorest root development. 3.2 Rhizome biomass and quality across different ginger generations The results presented in Table 2 indicate that the number of rhizome knobs per plant in T1 ginger was significantly higher than T2 and T3 gingers, with T3 ginger producing only 15 rhizome knobs per plant. Conversely, the rhizome length and height of the T1 ginger were the lowest, significantly lower than T2 and T3 gingers. Notably, the average rhizome biomass in T3 ginger was 677.47 grams, which was significantly higher than T1 and T2 gingers. Furthermore, th e contents of 6-gingerol and soluble protein in T3 ginger were significantly higher than T1 and T2 gingers. Overall, the T3 ginger exhibited the highest yield and quality compared to the T1 and T2 3.3 Anatomical characteristics of fibrous and fleshy roots across different ginger generations The transverse sections of middle region of fibrous roots in different ginger generations revealed a similar anatomical structure, comprising the stele, cortex and epidermis arranged from the interior to the exterior (Fig. S1). The epidermis and endodermis are composed of uniseriate cells, while the cortex consists of irregularly shaped, thin-walled cells. The stele includes the pericycle, vascular column, xylem, phloem and pith (Fig. S1). As shown in Table 3, the T1 ginger exhibited an average cortex thickness of 527.13 μm, which was significantly higher than that observed in T2 and T3 gingers ( p < 0.05). Additionally, the cortex/stele ratio in T2 and T3 gingers , measured at 1.62 and 1.63 respectively, was significantly lower than that of T1 ginger , with statistical significance ( p < 0.05). Other anatomical parameters, including root diameter, stele diameter, average xylem vessel area and the number of vascular bundles, did not display significant variations among the different generations. As depicted in Fig. 2, in comparison to fibrous roots, the fleshy roots of T1, T2 and T3 gingers showed a pronounced accumulation of starch granules within the cortical regions and central areas (Fig. 2B, D and F). Furthermore, as indicated in Table 4, the root diameter of T2 and T3 gingers was significantly higher than that of T1 ginger ( P < 0.05). 3. 4 Photosynthetic and leaf anatomical characteristics across different ginger generations As shown in Table 5, the SPAD value, along with the contents of chlorophyll a, chlorophyll b, total chlorophyll (a + b) and carotenoids significantly increased in T2 and T3 gingers compared to T1 ginger ( P < 0.05) . The net photosynthetic rate and stomatal conductance in T1 and T2 gingers were lower compared to T3 ginger. In contrast, the transpiration rate and intercellular CO 2 concentration in T1 and T2 gingers were higher than T3 ginger ( P < 0.05) . For chlorophyll fluorescence characteristics, T3 ginger demonstrated significantly higher maximum photochemical efficiency and actual quantum efficiency than T1 and T2 gingers. Conversely, the photochemical quenching coefficient and non-photochemical quenching coefficient of T3 ginger were significantly lower compared to T1 and T2 gingers ( P < 0.05) . As illustrated in Fig. S2, the leaf anatomical structure of T1, T2 and T3 gingers demonstrates notable similarities, comprising epidermal cells, mesophyll cells and vascular bundles. As presented in Table 6, the anatomical characteristics of leaf structure reveal that the midrib thickness, adaxial epidermis thickness, palisade tissue thickness and spongy tissue thickness of T3 ginger were significantly higher than T1 and T2 gingers ( p < 0.05). Moreover, the ratio of palisade tissue to spongy tissue showed significant differences between T1 and T2 gingers. In summary, compared to T1 and T2 gingers, the T3 ginger demonstrated the highest photosynthetic capacity. 3.5 Correlation analysis of root characteristics, plant growth parameters and rhizome biomass across different ginger generations The correlation analysis of growth parameters and root architecture characteristics revealed highly significant negative correlations between plant height (PH), main tiller diameter (MTD), leaf length (LL) and leaf width (LW) with total root length (TRL), root projection area (RPA), root surface area (RSA) and number of root tips (NRT). All these correlation coefficients (r) less than -0.85. In contrast, the number of tillers per plant (NT) and the number of leaves on the main tiller (NLT) exhibited significant positive correlations with the aforementioned root architecture traits. The correlation coefficients (r) were greater than 0.81 (Fi g. 3A ). Additionally, the correlation analysis between root architecture characteristics and rhizome biomass revealed a statistically significant negative correlation between total root length (TRL), root projection area (RPA), root surface area (RSA) and number of root tips (NRT) with the number of ginger balls (NGB), rhizome length (RL), rhizome height (RH) and rhizome biomass (RhB), where the r were all less than -0.76 (Fig . 3B ). 3.6 Ploidy analysis across different ginger generations The analysis of T1, T2 and T3 gingers using flow cytometry and chromosome counting is presented in Fig. 4. In histograms A, C and E, a single, distinct peak of fluorescence intensity was observed, precisely located around 1.9 × 10 5 . Complementary cytogenetic analysis further showed that T1, T2 and T3 gingers exhibit identical chromosome numbers, which are clearly denoted as 2n = 2x = 22 (Fig . 4B, 4D and 4F ). 3.7 Transcriptomic profiling analysis of T2 and T3 roots To investigate the molecular network underlying root development, we performed RNA-seq analysis on the roots of T2 and T3 gingers. The results showed that a total of 1598 differentially expressed genes (DEGs) were identified between the T2 and T3 roots. Among them, 787 DEGs were upregulated and 811 DEGs were downregulated (Fig . 5A ). GO annotation analysis revealed that the 1598 DEGs were associated with 2715 GO terms. The most prominent terms for biological processes (such as biological regulation, cellular processes and metabolic processes), cellular components (including cells, cell parts and organelles) and molecular functions (such as binding and catalytic activities) are illustrated in Fig . 5B. Through comparison with the KEGG database, a total of 1598 DEGs were mapped to 110 KEGG pathways ( p < 0.05 ). The top 20 KEGG pathways corresponding to the most abundant DEGs are presented in Fig . 5C . The results indicate that the majority of DEGs were significantly enriched in these pathways, such as ” starch and sucrose metabolism”, ”plant hormone signal transduction”, ”flavonoid biosynthesis” and ”biosynthesis of secondary metabolites” pathways. These findings suggest that the root development of ginger may be closely linked to these metabolic pathways. We randomly selected nine DEGs to validate the reliability of the RNA-seq data using quantitative real-time PCR (qRT-PCR) (Table. S1) . As shown in Fig . 5D , compared to T2 roots, the relative expression levels of SPP (maker00023454), glgC (maker00001019), AUX (maker00028125), GH3 (maker00020403), ETR (maker00069328) and CHS (maker0007506) were significantly increased in T3 roots, which is consistent with the RNA-seq data. This consistency supports the reliability of the RNA-seq sequencing data. 3.8 Metabolic analysis of the starch and sucrose in gingers A total of 23 DEGs were significantly enriched in the starch and sucrose metabolism pathway. These DEGs mainly involves the starch biosynthesis and degradation, sucrose metabolism and the interconversion between sugar and starch (Fig. 6A ). SPS and sucrose phosphate phosphatase (SPP) are pivotal enzymes in sucrose biosynthesis and transport. In this study, we observed that the expression of SPP was significantly elevated in T3 roots compared to T2 roots, while the expression of SPS was markedly reduced in T3 roots relative to T2 roots. Furthermore, SPS activity in T3 roots was also significantly lower than T2 roots ( P < 0.01). Sucrose metabolism and transport are regulated by SUS, beta-fructofuranosidase (INV), hexokinase (HK), endo-β-glucanase (EG) and beta-glucosidase (BGL). As depicted in Fig. 6A and 6C , the INV , HK , BGL and EG exhibited significantly reduced expression levels in T3 roots compared to T2 roots, but SUS showed significantly higher expression in T3 roots. Additionally, we found that the sucrose content and SUS activity were substantially higher in T3 roots than T2 roots (Fig . 6B , P < 0.001). Starch synthase (SS) plays a crucial role in starch biosynthesis in plants. Our analysis revealed that the expression of SS was significantly lower in T3 roots compared to T2 roots. Starch could be degraded either directly by amylase or starch phosphorylase. In this study, we identified genes encoding BAM, Alpha-amylase (AMY) and trehalose 6-phosphate phosphatase (otsB). The data presented in Fig. 6A and 6C indicate that the expression levels of these three DEGs were significantly upregulated in T3 roots relative to T2 roots, accompanied by an increase in BAM activity, which contributed to a significant reduction in sucrose content in T3 roots compared to T2 roots ( P < 0.01) (Fig. 6B and Table. S2 ). In summary, the differential expression of DEGs related to starch and sucrose metabolism in T3 ginger leads to significantly higher sucrose content compared to T2 ginger, along with increased starch degradation enzyme activity, thereby modulating the accumulation and conversion of starch and sucrose. 3.9 Metabolic analysis of flavonoid biosynthesis in gingers A total of 19 DEGs were significantly enriched in the flavonoid biosynthesis pathway. These DEGs include key enzymes that regulate flavonoid biosynthesis, such as CHI, CHS and DFR. As shown in Fig. 7A and 7B, T3 roots exhibited significantly reduced enzyme activities of CHI, CHS and DFR ( P < 0.01), with CHI activity showing a highly significant decrease ( P < 0.001) compared to T2 roots. Furthermore, Fig. 7A and 7C demonstrated that the expressions of DEGs related to CHI (1 DEG), CHS (5 DEGs) and DFR (3 DEGs) were also markedly reduced in T3 roots (Table. S3) . Lastly, our results revealed that T2 roots contained significantly higher levels of flavonoids than T3 roots. Thus, the downregulation of key enzymes and DEGs associated with flavonoid biosynthesis in T3 roots results in a marked reduction in flavonoid levels compared to T2 roots. 3.10 Metabolic analysis of plant hormone signal transduction in gingers As depicted in Fig. 8A, 35 DEGs in plant hormone signaling transduction pathways are enriched, which involved in the auxin, cytokinin (CK), ABA, gibberellin (GA), ethylene (ET), JA, brassinosteroid (BR) and SA. Specifically, in the auxin signaling pathway, three upregulated DEGs ( maker00028125 , maker00008618 , maker00020403 ) and one downregulated DEG ( maker00045379 ) were identified. In the ABA pathway, one DEG was upregulated ( maker00033289 ) and two DEGs were downregulated ( maker00000587 , maker00020644 ). In the GA pathway, two DEGs were upregulated ( maker00001365 , maker00022853 ), while one DEG was downregulated ( maker00068764 ). Fewer DEGs were detected in the CK, ET, JA, BR and SA signaling pathways. In these pathways, all DEGs in the GA ( maker00022853 , maker00068764 ), ET ( maker00069328 ) and JA ( maker00034679 ) pathways were upregulated, whereas all DEGs in the SA pathway ( maker00015142 ) were downregulated ( Fig. 8C and Table. S4) . As shown in Fig. 8B, compared to T2 roots, T3 roots exhibited significantly lower levels of IAA, ABA, TZR and SA, with the reductions in TZR and SA reaching highly significant levels ( P < 0.001). Conversely, the contents of JA and GA 3 in T3 roots were significantly higher ( P < 0.01). 4. Discussion Ginger is o ne of the most widely recognized spice and medicinal vegetables worldwide (Pázmándi et al., 2024). Its mature rhizomes have extensive applications in biopharmaceuticals, food preservation and health food development (Edo et al., 2024; Mao et al., 2019). The primary bioactive compounds in ginger, such as gingerol and soluble proteins, play a significant role in its nutritional value and flavor (Chen et al., 2024; Sanwal et al., 2010; Pázmándi et al., 2024). Studies have demonstrated that plant tissue culture offers an efficient and viable method for the large-scale production of disease-free ginger (Zhao et al., 2022; Bhattacharya et al., 2006; Zahid et al., 2021). After field planting, the rhizome yield in tissue-cultured ginger is positively correlated with aboveground morphological traits, while negatively correlated with root characteristics in field cultivation (Lincy et al., 2008; Zhao et al., 2022; Yu et al., 2024). In the present study, T3 ginger exhibited greater aboveground biomass compared to T1 and T2 gingers, along with the highest rhizome yield, gingerol content and soluble protein levels (Fig. 1 and Tables 1 and 2). The root architecture of T3 ginger, including average root diameter, root surface area, root volume and root tip number, shows a significant reduction compared to T1 and T2 gingers (Fig. 1). These findings align with prior research, suggesting that ginger yield and quality is closely linked to aboveground morphological traits. These results are consistent with the research results of Zhang et al. (2021) and Wójcik et al. (2021), who found that the key aboveground factors such as increased plant height, stem diameter and reduced tiller number were essential for improving ginger yield and quality. Biomass allocation across plant organs is influenced by a variety of factors, including plant species, age, size, water availability and light conditions (Poorter, 2012). Moreover, plant growth is closely linked to the distribution of biomass among the roots, stems and leaves. For instance, under drought stress, plants tend to allocate more biomass to the root system to improve water uptake. In the development of the T1 and T2 gingers, the limited nutrient supply from the underground parts leads plants to prioritize the growth of aboveground tissues and roots, resulting in a reduced storage capacity of the rhizomes (Fig. 1). Tilman et al.’s (1985) resource ratio hypothesis provides a critical theoretical framework for understanding the interplay between aboveground and belowground processes and their contribution to plant productivity. To gain deeper insight into this phenomenon, it is essential to explore the relationships and regulatory mechanisms between aboveground characteristics, root systems and rhizomes. Specifically, it is crucial to investigate the genetic and physiological pathways that regulate this balance, such as hormone signaling (e.g., auxins and cytokinins), nutrient allocation (e.g., nitrogen and phosphorus transport) and environmental factors (e.g., water availability) (Wardle, 2004). Leaves are critical organs for respiration, photosynthesis and transpiration in plants (Adams et al., 2018; Lopez et al., 2024). Alterations in leaf anatomical structure can significantly influence photosynthetic efficiency, thereby affecting plant growth, development and crop yield (Martins et al., 2017; Xiao et al., 2024). In this study, T3 generation of ginger exhibited significantly larger leaf length, width, main vein thickness, adaxial epidermal thickness, palisade parenchyma thickness and spongy parenchyma thickness compared to T1 and T2 gingers (Fig. S2, Tables 5 and 6). Previous studies by Martins et al. (2017) and Xiao et al. (2024) have shown that thicker palisade and spongy mesophyll layers enhanced light penetration and facilitated carbon dioxide diffusion to chloroplasts, thereby increasing the net photosynthetic rate. In line with these findings, this study observed that T3 ginger exhibited significantly higher chlorophyll content, net photosynthetic rate and maximum photochemical efficiency compared to T1 and T2 gingers (Table 5). These results confirm that changes in leaf anatomical structure may improve gas exchange efficiency, thereby enhancing photosynthetic capacity and ultimately supporting greater plant growth and development (Leverett, 2024). The root structure and its spatial distribution play a crucial role in the absorption of water and nutrients by plants (Ullah et al., 2024). Key root parameters, including root length, root diameter, root surface area and the number of root tips, are influenced by external environmental factors (Ullah et al., 2024), genotype (Li et al., 2023) and propagation methods (Rahman et al., 2003). Research by Guo et al. (2022) demonstrated that larger root systems and increased root surface area enhanced a plant’s ability to acquire soil resources. Studies by Albrecht et al. (2020) and Li et al. (2022b) indicated significant differences in the root characteristics of tissue-cultured plants, leading to substantial changes in morphology and biomass production. In this study, compared to the T1 and T2 gingers, T3 ginger exhibited a marked decline in root characteristics, including reduced root biomass, average root diameter, root surface area, root volume and number of root tips (Fig. 1). Zhao et al. (2022) and Yu et al. (2024) further found that tissue-cultured ginger grown in the field had stronger roots and superior root characteristics, whereas produced lower underground rhizome yields. These findings suggest a potential interrelationship between root development and underground stem yield in ginger. The ability of roots to absorb water and nutrients is closely linked not only to their morphological characteristics but also to their anatomical structure (Sidhu et al., 2023; Ullah et al., 2024). Sidhu et al. (2023) and Li et al. (2024) have shown that root anatomical features directly reflected the developmental state of the root system and have a significant impact on plant growth and crop yield. In this study, compared to the T2 and T3 gingers, T1 ginger exhibited a marked thickening of fibrous root cortex and a significant increase in the diameter of the fleshy roots (Figs. 2 and S1, Tables 3 and 4). Zhang et al. (2023) noted that the root cortex is primarily responsible for nutrient and water absorption. Pan et al. (2023) and Li et al. (2024) suggested that a reduction in cortex thickness or an increase in the diameter of the stele can improve the efficiency of nutrient and water transport. Moreover, Li et al. (2024) indicated that higher root activity and optimized anatomical structures (such as a thicker cortex) could significantly enhance grain yield. These findings highlight the critical role of root anatomical features in nutrient absorption and yield enhancement. Zhao et al. (2022) and Yu et al. (2024) found a significant negative correlation between rhizome biomass and root biomass in tissue-cultured plants. Our study also revealed a significant negative correlation between rhizome biomass and root biomass parameters, such as TRL, RPA, RSA and NRT with NGB, RL and RH (Fig. 3). This result aligns with the findings of Wei et al. (2024), indicating that plants with more developed root systems do not necessarily exhibit higher yields. This may be due to the fact that an increase in root biomass could reduce the efficiency of nutrient utilization in the aboveground parts, consequently affecting rhizome yield (Siddique et al., 1990). Therefore, despite the larger root biomass in the T1 ginger, the increased assimilation efficiency in the aboveground parts led to a reduction in rhizome yield (Fig. 3B). The ploidy level of plants plays a crucial role in determining their morphology, cellular characteristics and physiological processes (Li et al., 2020; Wang et al., 2014; Cao et al., 2018). Zhao et al. (2022) reported that tissue-cultured ginger exhibited somatic chromosomal ploidy variations, which resulted in changes in root quantity and morphology, ultimately affecting rhizome yield. A similar phenomenon was observed in the microrhizome-propagated plants (Yu et al., 2024). In the present study, flow cytometry and chromosome counting were employed to verify the ploidy of the T1, T2 and T3 gingers, demonstrating high stability (Fig. 4). However, genetic variation in plants is not limited to changes in number and structure of chromosome but also involves nucleotide sequence variations, gene copy number alterations, transposon activation and alterations in DNA methylation patterns (Bairu et al., 2011; Krishna et al., 2016). Therefore, future research should further investigate whether other patterns of genetic variation influence the traits and growth development between T1, T2 and T3 gingers. Carbohydrates, particularly starch and sucrose, serving as the primary energy sources directly affect root growth and differentiation (Gaddam et al., 2021; Esparza-Reynoso et al., 2021). Research has shown that the conversion of starch into available sugars promotes root growth and development by regulating the proliferation and differentiation of root cells (Mishra et al., 2007; Kicher et al., 2012). Mitsui et al. (2015) analyzed the radish genome and identified several genes associated with sucrose and starch metabolism that are involved in root development. Ren et al. (2023) demonstrated that differential expression of genes encoding enzymes involved in starch and sucrose metabolism resulted in significant accumulation of starch and soluble proteins in the roots, while soluble sugar content decreases. In the present study, we observed that the expressions of several sucrose metabolism-related genes ( SPP , SUS , BGL , bglB , glgC , otsB , etc.) were significantly upregulated in the T3 ginger, promoting the efficient conversion of sucrose. This process not only facilitated starch accumulation but may also enhanced energy supply to roots, thereby supporting higher rhizome yield (Fig. 6). Flavonoids as key secondary metabolites in plants, play a crucial role in root development (Wang et al., 2016; Wan et al., 2018). Research has demonstrated that flavonoids are not only transported within plant cells and intercellular spaces, but also specifically released into the rhizosphere via roots, contributing to plant interactions and allelopathic effects (Weston et al., 2013; Hassan et al., 2012). In this study, the expressions of flavonoid biosynthesis-related genes ( CYP75A , CHS , CYP75B1 , DFR , F3H and ANR ) in T3 ginger were significantly lower than in the T2 ginger. Moreover, the activities of enzymes such as DFR, CHI and CHS were notably reduced, leading to a substantial decrease in flavonoid content in the T3 roots (Fig. 7) . Previous studies have shown that flavonoids could influence root development by regulating the balance of endogenous hormones (Maloney et al., 2014; Zeng et al., 2010; Xu et al., 2019). For instance, Xu et al. (2019) demonstrated that quercetin treatment of rice primary roots influenced cell wall remodeling, redox homeostasis and auxin level by regulating the transcription of genes related to phenylpropanoid biosynthesis, glutathione metabolism and plant hormone signal transduction. These transcriptional changes lead to a reduction in cell division in the root meristematic zone, promoting cell elongation in the elongation zone. Burbulis et al. (1996) reported that a mutation in the CHS gene ( tt4 , transparent testa 4 ) severely impaired IAA transport. Subsequent studies by Maloney et al. (2014) and Zeng et al. (2010) suggested that flavanol accumulation could regulate auxin transport in tomato root tips, thereby promoting lateral root formation. Therefore, the reduced flavonoid content in T3 ginger may impair the transport of endogenous auxins, leading to significantly lower root development-related parameters compared to the T2 ginger (Figs. 1 and 7). In conclusion, flavonoids likely play an essential role in ginger root development by regulating the transport and signaling pathways of endogenous hormones. Endogenous hormones play a critical role in root growth and development (Gao et al., 2022; Bagautdinova et al., 2022; Vanstraelen, 2012). Various hormones regulate root development through distinct mechanisms and interact synergistically at different stages of root growth. Specifically, IAA promotes lateral root formation and regulates cell elongation and division (Aloni et al., 2006; Gao et al., 2022). GA 3 is essential for cell elongation and root development (Aloni et al., 2006; Wang et al., 2023), while cytokinins inhibit primary root elongation and regulate lateral root development (Nishimura et al., 2004; Lohar et al., 2004). Additionally, ABA modulates root development by inhibiting the activity of lateral root meristem cells (Zhang and Forde, 2000; Razem et al., 2006). JA suppresses root growth and reduces the activity of root meristem cells through modulation of specific genes, such as JAZ and PLT2 (Ghorbel et al., 2021; Chen et al., 2011). In contrast, SA is associated with the promotion of root growth and enhancement of root density (Bagautdinova et al., 2022). Singh et al. (2016) compared the root transcriptomes of two indica rice varieties, IR-64 (OsAS83) and IET-16348 (OsAS84) and found that differences in root structure were primarily due to that DEGs involved in abiotic stress response, hormone signaling and nutrient acquisition or transport. In the present study, we observed a significant increase in the levels of JA and GA 3 in the T3 ginger, while IAA, ABA, TZR and SA levels were significantly reduced. We hypothesize that these hormonal changes may regulate root growth and development by modulating the differential expression of genes involved in hormone signaling pathways, such as AUX1 , AUX/IAA , PYP/PYL , ETR , JAZ , GH3 , PP2C , NPR1 , B-BRR , DELLA and TF (Fig. 8). These findings are consistent with those of Ren et al. (2023), who highlighted the importance of hormone signaling pathways and those associated with starch and sucrose metabolism in regulating root development in Chinese cabbage. Similarly, Song et al. (2016) identified differential regulation of genes involved in auxin/ethylene signaling, carbohydrate metabolism and cell wall biosynthesis (e.g., XTH, lipids, flavonoids and lignin) in soybean roots. In our transcriptome analysis of the T2 and T3 roots, we found that DEGs were highly enriched in starch and sucrose metabolism, flavonoid biosynthesis and plant hormone signaling pathways. This suggests that, despite the differences in the regulatory pathways of root development across plant species, certain regulatory mechanisms are conserved, indicating a potential common regulatory network between hormone signaling, metabolic pathways and gene expression in root development across different plants. 5. Conclusions Overall, the study demonstrates that the T3 ginger exhibits superior yield and quality. However, its impaired root development may be closely associated with the regulation of starch, sucrose, flavonoid metabolism and hormone levels (Fig. 9). Specifically, the T1, T2 and T3 gingers display strong genetic stability (Fig. 4), with T3 ginger showing significantly greater aboveground and underground biomass, photosynthetic efficiency, yield and quality compared to the T1 and T2 gingers (Fig. 1, Tables 1 and 2). However, root development in T3 ginger is relatively weaker (Fig. 1). Transcriptome analysis reveals a significant upregulation in the expression of DEGs related to starch and sucrose metabolism, as well as an increase of key enzyme activities (Fig. 6), whereas DEGs related to flavonoid biosynthesis and the enzyme activities are significantly downregulated (Fig. 7). Root development in T3 ginger is notably influenced by hormonal levels (Fig. 8). Therefore, future studies should investigate the molecular mechanisms underlying the regulation of root development by starch, sucrose, flavonoids and plant hormones, which could provide valuable insights for enhancing ginger rhizome yield. Acknowledgments This work was supported by the Natural Science Foundation of Hubei Province (2023AFB1001), Hubei International Science and Technology Cooperation Project (2024EHA011), Yongchuan District of Chongqing Natural Science Foundation Project (2024yc-cxfz30091). Conflicts of interest The authors declare there is no conflict of interest. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Adams III, W. W., & Terashima, I. (Eds.). (2018). The leaf: a platform for performing photosynthesis (Vol. 44). Cham: Springer International Publishing. Albrecht, U., Bodaghi, S., Meyering, B., & Bowman, K. D. (2020). Influence of rootstock propagation method on traits of grafted sweet orange trees. HortScience , 55(5), 729-737. Aloni, R., Aloni, E., Langhans, M., & Ullrich, C. I. (2006). 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Field performance of disease-free plants of ginger produced by tissue culture and agronomic, cytological, and molecular characterization of the morphological variants. Agronomy , 13(1), 74. Fig. 1 Growth performance and root architecture of ‘Fengtou’ ginger across different generations after 120 days of growth. T1: breeder seed; T2: foundation seed; T3: cultivar seed. Fig. 2 Anatomical comparisons (transverse section) of the middle region of the fleshy roots across different ginger generations. A and B: Anatomic structures of fleshy roots (A) and stele region (B) of T1 ginger; C and D: Anatomic structures of fleshy roots (C) and stele region (D) of T2 ginger; E and F: Anatomic structures of fleshy roots (E) and stele region (F) of T3 ginger. Co: cortex; En: endodermis; Ep: epidermis; Pe: pericycle; Ph: phloem; Pi: pith; Sg: starch granules; St: stele; Xy=xylem. Fig. 3 Correlation analysis between root characteristics and plant growth parameters (A) and root characteristics and rhizome biomass across different ginger generations. MSH: main stem height; PH: plant height; MTD: main tiller diameter; NT: No. of tillers per plant; NLT: No. of leaves of the main tiller; LL: leaf length; LW: leaf width; TRL: total root length; RPA: root projection area; RSA: root surface area; NRT: number of root tips; NGB: number of ginger balls; RL: rhizome length; RH: rhizome height; RB: root biomass; RhB: rhizome biomass. *, **, and *** indicate significant correlation at 5%, 1% and 0.1% level, respectively. Same as below. Fig. 4 Flow cytometry analysis and chromosome counting of the TI (A and B), T2(C and D) and T3(E and F) gingers. FI: Fluorescence intensity. Bars = 20 μm. Fig. 5 Tran scriptome analysis of T2 roots and T3 roots. A: Total number of DEGs in T2 and T3 roots; The red and blue dots represent upregulated and downregulated genes, respectively. B: GO enrichment analysis of common DEGs. Distinct regions represent different functional categories based on GO terms. C: KEGG pathway enrichment analysis of common DEGs. The x-axis indicates the proportion of DEGs in each KEGG pathway relative to the total number of DEGs. Pathways are listed on the y-axis. Dot size corresponds to the number of DEGs within each pathway, and dot color represents the adjusted p-value. D: qRT-PCR validation of DEGs expression levels observed in the transcriptomic analysis. Fig. 6 Starch and sucrose concentrations, biosynthetic key enzyme activities and differential gene expression in starch and sucrose metabolism pathway. A: Starch and sucrose metabolism pathway; B: Starch and sucrose concentrations and biosynthetic key enzyme activities; C: Expression levels of genes involved in starch and sucrose metabolism. Fig. 7 Total flavonoid content, biosynthetic key enzymatic activities and differential gene expression in flavonoid biosynthesis pathway. A: Flavonoid biosynthesis pathway; B: Total flavonoid content and biosynthetic key enzymatic activities; C: Expression levels of genes involved in flavonoid biosynthesis pathways. Fig. 8 Endogenous hormone concentrations and differential gene expression in plant hormone signal transduction pathway. A: Plant hormone signal transduction pathway; B: Endogenous hormone content; C: Expression levels of genes involved in plant hormone signal transduction pathways. Fig. 9 Mechanisms of growth and development across different ginger generations. Parameters T1 T2 T3 Main stem height (cm) 60.35±0.83 b 80.70±3.65 a 83.38±3.64 a Plant height (cm) 81.83±1.57 c 94.33±3.40 b 100.25±4.19 a Main tiller diameter (mm) 7.22±0.54 c 8.83±0.41 b 9.65±0.49 a No. of tillers per plant 23.33±1.37 a 13.75±0.50 b 12.50±1.29 b No. of leaves of the main tiller 27.00±2.19 a 23.25±2.06 b 19.25±0.96 c Leaf length (cm) 20.07±0.72 b 23.70±1.09 a 24.68±0.85 a Leaf width (cm) 2.35±0.12 b 2.68±0.17 a 2.85±0.06 a Above-ground stems biomass (g) 103.06±6.08 b 248.90±7.15 a 247.23±5.59 a Leaf biomass (g) 48.72±3.61 c 96.93±11.25 b 121.94±11.95 a Values (mean ± standard deviation) within columns followed by different superscript letters are significantly different according to Duncan’s multiple range tests at 5% level. Same as below. Parameters T1 T2 T3 No. of rhizome knobs per plant 27.00±3.16 a 17.00±1.00 b 13.83±1.17 c Rhizome length (cm) 14.68±0.82 c 17.40±1.20 b 25.50±1.00 a Rhizome height (cm) 3.28±0.25 c 9.45±0.71 b 15.43±1.21 a Rhizome fresh weight per plant (g) 52.21±7.96 c 241.45±5.09 b 677.47±22.22 a Soluble protein (mg g -1 FW) 5.64±0.16 b 5.85±0.56 b 8.36±0.07 a 6-gingerol 0.31±0.01 b 23.31±0.02 b 0.95±0.01 a Anatomical characteristics T1 T2 T3 Root diameter (μm) 1561.97±14.07 a 1578.18±27.05 a 1575.55±36.10 a Cortex thickness (μm) 527.13±15.87 a 479.83±18.78 b 485.29±17.49 b Stele diameter (μm) 591.54±13.65 a 595.53±6.64 a 599.11±7.79 a Cortex/stele ratio 1.78±0.06 a 1.61±0.07 b 1.62±0.04 b Average xylem vessel area (μm 2 ) 4671.23±148.58 a 4693.72±57.89 a 4706.81±213.04 a No. of vascular bundles 22.83±1.94 a 22.00±1.79 a 22.00±1.55 a Anatomical characteristics T1 T2 T3 Root diameter (μm) 5045.27±71.52 a 5136.77±17.42 b 5152.79±26.95 b Cortex thickness (μm) 2084.77±27.73 a 2087.19±23.75 a 2091.27±46.79 a Stele diameter (μm) 896.14±7.99 a 902.46±5.82 a 896.22±22.33 a Cortex/stele ratio 4.65±0.08 a 4.63±0.08 a 4.67±0.19 a Average xylem vessel area (μm 2 ) 2760.66±129.81 a 2775.80±142.18 a 2794.64±194.48 a No. of vascular bundles 18.33±0.82 a 18.00±0.89 a 17.83±0.75 a Parameters T1 T2 T3 Leaf SPAD value 26.35±2.41 c 30.73±1.24 b 35.08±0.44 a Chlorophyll a [mg g -1 (FW)] 3.72±0.12 c 5.81±0.07 b 7.88±0.05 a Chlorophyll b [mg g -1 (FW)] 1.74±0.12 c 2.48±0.02 b 5.10±0.15 a Total chlorophyll [mg g -1 (FW)] 5.46±0.07 c 8.28±0.08 b 12.98±0.19 a Chlorophyll a/b 2.15±0.21 a 2.35±0.02 a 1.55±0.04 b Carotenoid [mg g -1 (FW)] 1.41±0.05 c 2.06±0.01 b 2.74±0.01 a Net photosynthetic rate (μmol m -2 s -1 ) 7.60±0.37 c 10.58±0.43 b 13.48±0.16 a Stomatal conductance (mol m -2 s -1 ) 91.04±0.72 c 120.89±1.80 b 142.31±2.13 a Transpiration rate (mmol m -2 s -1 ) 4.29±0.02 a 3.46±0.04 b 2.70±0.06 c Intercellular CO 2 concentration (μmol m -1 ) 342.55±2.49 a 338.84±1.83 b 331.51±2.20 c Max photochemical efficiency 0.71±0.02 c 0.79±0.02 b 0.93±0.02 a Photochemical quenching coefficient 0.63±0.01 a 0.55±0.02 b 0.50±0.01 c Actual optical quantum efficiency 0.32±0.02 b 0.34±0.01 b 0.42±0.02 a Non-photochemical quenching coefficient 0.71±0.01 a 0.59±0.02 b 0.47±0.03 c Anatomical characteristics T1 T2 T3 Leaf thickness (μm) 332.15±4.25 a 341.48±13.52 a 346.17±16.90 a Midrib thickness (μm) 804.88±18.44 c 875.64±17.13 b 901.85±7.52 a Abaxial epidermis thickness (μm) 46.98±4.46 a 47.31±2.52 a 47.18±3.63 a Adaxial epidermis thickness (μm) 93.63±3.43 c 109.25±5.50 b 115.99±2.61 a Palisade parenchyma thickness (μm) 45.84±4.59 c 49.96±1.68 b 56.22±1.99 a Spongy parenchyma thickness (μm) 36.00±0.83 c 43.56±1.15 b 46.68±3.24 a Palisade/spongy ratio 1.27±0.13 a 1.15±0.05 b 1.21±0.08 ab Supplementary Material File (1 figures.docx) Download 3.73 MB Information & Authors Information Version history V1 Version 1 01 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords flavonoids growth hormones root characteristics starch and sucrose transcriptome Authors Affiliations Xiaoqin Zhao Yangtze University View all articles by this author Jinlian Yuan Yangtze University View all articles by this author Xiaodong Cai Yangtze University View all articles by this author Zhen Zeng Yangtze University View all articles by this author Lijuan Wei 0009-0000-4473-6237 [email protected] Yangtze University View all articles by this author Yiqing Liu Yangtze University View all articles by this author Metrics & Citations Metrics Article Usage 386 views 169 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Xiaoqin Zhao, Jinlian Yuan, Xiaodong Cai, et al. Transcriptome analysis reveals the effects of root development across different ginger generations on plant morphology and yield. Authorea . 01 April 2025. 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