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2023 Volume 3
Article Contents
Abstract
Introduction
Methods
Bioinformatics analysis of the ARF18 gene
Plant materials and cultivation methods
Plasmid construction and plant transformation
Extracting plant genomic DNA and RNA
Real-Time quantitative Polymerase Chain Reaction (qPCR)
GUS staining
Determination of nitrate content, nitrate reductase activity
Yeast one-hybrid (Y1H) assay
Dual luciferase assays
Protein degradation assays in vitro
Data analysis
Results
Auxin regulates nitrogen uptake and utilization in apple
Transcript levels of MdARFs are induced by nitrate
Phylogenetic relationships and multiple sequence alignment of MdARF18
Overexpression of MdARF18 affects root development
Overexpression of MdARF18 inhibits nitrate uptake in plants
MdARF18 targets genes related to nitrogen uptake and utilization
MdARF18 binds to the promoter of MdNRT1.1 to restrain its expression
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Polyploidy induction of raspberry species (Rubus) using colchicine and oryzalin

  • Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA

More Information
  • Raspberry (Rubus spp.) is a fruit crop used in many products across the United States. Commercial production of raspberry in North Dakota is challenging due to lack of cultivars with winter hardiness. In this study, two diploid red raspberry cultivars 'Prelude' and 'Polana' that are reported hardy in USDA hardiness zone 5 were used to induce polyploid raspberries that may have benefits of greater growth vigor, enhanced stress tolerance, larger fruit size, and higher yield. The effects of cultivar, anti-mitotic agent and its concentration, and the treatment duration on in vitro and ex vitro polyploidy induction were determined. 'Prelude' succeeded in polyploidy induction through the colchicine treatment of in vitro explants (apical and nodal buds) and ex vitro root dipping. There were three verified tetraploid 'Prelude' plants. One tetraploid plant of 'Prelude' was developed when in vitro explants were treated with 160 mM colchicine for 96 h and the other two were developed when the root system was dipped in the 10 mM colchicine for 8 h. No polyploid plants of 'Polana' were developed. Oryzalin, another anti-mitotic agent, did not succeed in polyploidy induction in this study. This study provides useful information on polyploidy induction in raspberry species, which could benefit future germplasm enhancement of raspberry and other Rubus species.

  • Introduction

    Crops require a variety of nutrients for growth and nitrogen is particularly important. Nitrogen is the primary factor limiting plant growth and yield formation, and it also plays a significant role in improving product quality[1−4]. Nitrogen accounts for 1%−3% of the dry weight of plants and is a component of many compounds. For example, it is an important part of proteins, a component of nucleic acids, the skeleton of cell membranes, and a constituent of chlorophyll[5,6]. When the plant is deficient in nitrogen, the synthesis process of nitrogen-containing substances such as proteins decrease significantly, cell division and elongation are restricted, and chlorophyll content decreases, and this leads to short and thin plants, small leaves, and pale color[2,7,8]. If nitrogen in the plant is in excess, a large number of carbohydrates will be used for the synthesis of proteins, chlorophyll, and other substances, so that cells are large and thin-walled, and easy to be attacked by pests and diseases. At the same time, the mechanical tissues in the stem are not well developed and are prone to collapse[3,8,9]. Therefore, the development of new crop varieties with both high yields and improved nitrogen use efficiency (NUE) is an urgently needed goal for more sustainable agriculture with minimal nitrogen demand.

    Plants obtain inorganic nitrogen from the soil, mainly in the form of NH4+ and nitrate (NO3−)[10−13]. Nitrate uptake by plants occurs primarily in aerobic environments[3]. Transmembrane proteins are required for nitrate uptake from the external environment as well as for transport and translocation between cells, tissues, and organs. NITRATE TRANSPORTER PROTEIN 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family (NPF), NRT2, CHLORIDE CHANNEL (CLC) family, and SLOW ACTIVATING ANION CHANNEL are four protein families involved in nitrate transport[14]. One of the most studied of these is NRT1.1, which has multiple functions[14]. NRT1.1 is a major nitrate sensor, regulating many aspects of nitrate physiology and developmental responses, including regulating the expression levels of nitrate-related genes, modulating root architecture, and alleviating seed dormancy[15−18].

    There is mounting evidence that plant growth and development are influenced by interactions across numerous phytohormone signaling pathways, including abscisic acid, gibberellins, growth hormones, and cytokinins[3,19,20]. To increase the effectiveness of plant nitrogen fertilizer application, it may be possible to tweak the signaling mediators or vary the content of certain phytohormones. Since the 1930s, research on the interplay between growth factors and N metabolism has also been conducted[3]. The Indole acetic acid (IAA) level of plant shoots is shown to decrease in early studies due to N shortage, although roots exhibit the reverse tendency[3,21]. In particular, low NO3− levels caused IAA buildup in the roots of Arabidopsis, Glycine max, Triticum aestivum, and Zea mays, indicating that IAA is crucial for conveying the effectiveness of exogenous nitrogen to the root growth response[20,22,23].

    Studies have shown that two families are required to control the expression of auxin-responsive genes: one is the Auxin Response Factor (ARF) and the other is the Aux/IAA repressor family[24−26]. As the transcription factor, the ARF protein regulates the expression of auxin response genes by specifically binding to the TGTCNN auxin response element (AuxRE) in promoters of primary or early auxin response genes[27]. Among them, rice OsARF18, as a class of transcriptional repressor, has been involved in the field of nitrogen utilization and yield[23,28]. In rice (Oryza sativa), mutations in rice salt tolerant 1 (rst1), encoding the OsARF18 gene, lead to the loss of its transcriptional repressor activity and up-regulation of OsAS1 expression, which accelerates the assimilation of NH4+ to Asn and thus increases N utilization[28]. In addition, dao mutant plants deterred the conversion of IAA to OxIAA, thus high levels of IAA strongly activates OsARF18, which subsequently represses the expression of OsARF2 and OsSUT1 by directly binding to the AuxRE and SuRE promoter motifs, resulting in the inhibition of carbohydrate partitioning[23]. As a result, rice carrying the dao has low yields.

    Apples (Malus domestica) are used as a commercially important crop because of their high ecological adaptability, high nutritional value, and annual availability of fruit[29]. To ensure high apple yields, growers promote rapid early fruit yield growth by applying nitrogen. However, the over-application of nitrogen fertilizer to apples during cultivation also produces common diseases and the over-application of nitrogen fertilizer is not only a waste of resources but also harmful to the environment[29]. Therefore, it is of great significance to explore efficient nitrogen-regulated genes to understand the uptake and regulation of nitrogen fertilizer in apples, and to provide reasonable guidance for nitrogen application during apple production[30]. In this study, MdARF18 is identified which is a key transcription factor involved in nitrate uptake and transport in apples and MdARF18 reduces NO3− uptake and assimilation. Further analysis suggests that MdRF18 may inhibit the transcriptional level of MdNRT1.1 promoter by directly binding to its TGTCTT target, thus affecting normal plant growth.

    Methods

    Bioinformatics analysis of the ARF18 gene

    The protein sequence of apple MdARF18 (MD07G1152100) was obtained from The Apple Genome (https://iris.angers.inra.fr/gddh13/). Mutant of arf18 (GABI_699B09) sequence numbers were obtained from the official TAIR website (www.arabidopsis.org). The protein sequences of ARF18 from different species were obtained from the protein sequence of apple MdARF18 on the NCBI website. Using these data, a phylogenetic tree with reasonably close associations was constructed[31].

    Protein structural domain prediction of ARF18 was performed on the SMART website (https://smart.embl.de/). Motif analysis of ARF18 was performed by MEME (https://meme-suite.org/meme/tools/meme). Clustal was used to do multiple sequence comparisons. The first step was accessing the EBI web server through the Clustal Omega channel. The visualization of the results was altered using Jalview, which may be downloaded from www.jalview.org/download.[32]

    Plant materials and cultivation methods

    The apple 'Orin' callus was transplanted on MS medium containing 1.5 mg·L−1 6-benzylaminopurine (6-BA) and 0.5 mg·L−1 2,4 dichlorophenoxyacetic acid (2,4-D) at 25 °C, in the dark, at 21-d intervals. 'Royal Gala' apple cultivars were cultured in vermiculite and transplanted at 25 °C every 30 d. The Arabidopsis plants used were of the Columbia (Col-0) wild-type variety. Sowing and germinating Arabidopsis seeds on MS nutrient medium, and Arabidopsis seeds were incubated and grown at 25 °C (light/dark cycle of 16 h/8 h)[33].

    The nutrient solution in the base contained 1.0 mM CaCl2, 1.0 mM KH2PO4, 1.0 mM MgSO4, 0.1 mM FeSO4·7H2O 0.1 mM Na2EDTA·2H2O, 50 μM MnSO4·H2O, 50 μM H3BO3, 0.05 μM CuSO4·5H2O, 0.5 μM Na2MoO4·2H2O, 15 μM ZnSO4·7H2O, 2.5 μM KI, and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl· 6H2O. 2H2O, 15 μM ZnSO4·7H2O, 2.5 μM KI and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl·6H2O, supplemented with 0.5 mM, 2 mM, and 10 mM KNO3 as the sole nitrogen source, and added with the relevant concentrations of KCl to maintain the same K concentration[33,34].

    For auxin treatment, 12 uniformly growing apple tissue-cultured seedlings (Malus domestica 'Royal Gala') were selected from each of the control and treatment groups, apple seedlings were incubated in a nutrient solution containing 1.5 mg·L−1 6-BA, 0.2 mg·L−1 naphthalene acetic acid, and IAA (10 μM) for 50 d, and then the physiological data were determined. Apple seedlings were incubated and grown at 25 °C (light/dark cycle of 16 h/8 h).

    For nitrate treatment, Arabidopsis seedlings were transferred into an MS medium (containing different concentrations of KNO3) as soon as they germinated to test root development. Seven-day-old Arabidopsis were transplanted into vermiculite and then treated with a nutrient solution containing different concentrations of KNO3 (0.5, 2, 10 mM) and watered at 10-d intervals. Apple calli were treated with medium containing 1.5 mg·L−1 6-BA, 0.5 mg·L−1 2,4-D, and varying doses of KNO3 (0.5, 2, and 10 mM) for 25 d, and samples were examined for relevant physiological data. Apple calli were subjected to the same treatment for 1 d for GUS staining[35].

    Plasmid construction and plant transformation

    To obtain MdARF18 overexpression materials, the open reading frame (ORF) of MdARF18 was introduced into the pRI-101 vector. To obtain pMdNRT1.1 material, the 2 kb segment located before the transcription start site of MdNRT1.1 was inserted into the pCAMBIA1300 vector. The Agrobacterium tumefaciens LBA4404 strain was cultivated in lysozyme broth (LB) medium supplemented with 50 mg·L−1 kanamycin and 50 mg·L−1 rifampicin. The MdARF18 overexpression vector and the ProMdNRT1.1::GUS vector were introduced into Arabidopsis and apple callus using the flower dip transformation procedure. The third-generation homozygous transgenic Arabidopsis (T3) and transgenic calli were obtained[36]. Information on the relevant primers designed is shown in Supplemental Table S1.

    Extracting plant genomic DNA and RNA

    Plant DNA and RNA were obtained using the Genomic DNA Kit and the Omni Plant RNA Kit (tDNase I) (Tiangen, Beijing, China)[37].

    Real-Time quantitative Polymerase Chain Reaction (qPCR)

    cDNA was synthesized for qPCR by using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). The cDNA for qPCR was synthesized by using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). Quantitative real-time fluorescence analysis was performed by using the UltraSYBR Mixture (Low Rox) kit (ComWin Biotech Co. Ltd., Beijing, China). qRT-PCR experiments were performed using the 2−ΔΔCᴛ method for data analysis. The data were analyzed by the 2−ΔΔCᴛ method[31].

    GUS staining

    GUS staining buffer contained 1 mM 5-bromo-4-chloro-3-indolyl-β-glutamic acid, 0.01 mM EDTA, 0.5 mM hydrogen ferrocyanide, 100 mM sodium phosphate (pH 7.0), and 0.1% (v/v) Triton X-100 was maintained at 37 °C in the dark. The pMdNRT1.1::GUS construct was transiently introduced into apple calli. To confirm whether MdNRT1.1 is activated or inhibited by MdARF18, we co-transformed 35S::MdARF18 into pMdNRT1.1::GUS is calling. The activity of transgenic calli was assessed using GUS labeling and activity assays[33,38].

    Determination of nitrate content, nitrate reductase activity

    The specimens were crushed into fine particles, combined with 1 mL of ddH2O, and thereafter subjected to a temperature of 100 °C for 30 min. The supernatant was collected in a flow cell after centrifugation at 12,000 revolutions per minute for 10 min. The AutoAnalyzer 3 continuous flow analyzer was utilized to measure nitrate concentrations. (SEAL analytical, Mequon, WI, USA). Nitrate reductase (NR) activity was characterized by the corresponding kits (Solarbio Life Science, Beijing, China) using a spectrophotometric method[31].

    Yeast one-hybrid (Y1H) assay

    Y1H assays were performed as previously described by Liu et al.[39]. The coding sequence of MdARF18 was integrated into the pGADT7 expression vector, whereas the promoter region of MdNRT1.1 was included in the pHIS2 reporter vector. Subsequently, the constitutive vectors were co-transformed into the yeast monohybrid strain Y187. The individual transformants were assessed on a medium lacking tryptophan, leucine, and histidine (SDT/-L/-H). Subsequently, the positive yeast cells were identified using polymerase chain reaction (PCR). The yeast strain cells were diluted at dilution factors of 10, 100, 1,000, and 10,000. Ten μL of various doses were added to selective medium (SD-T/-L/-H) containing 120 mM 3-aminotriazole (3-AT) and incubated at 28 °C for 2−3 d[37].

    Dual luciferase assays

    Dual-luciferase assays were performed as described previously[40]. Full-length MdARF18 was cloned into pGreenII 62-SK to produce MdARF18-62-SK. The promoter fragment of MdNRT1.1 was cloned into pGreenII 0800-LUC to produce pMdNRT1.1-LUC. Different combinations were transformed into Agrobacterium tumefaciens LBA4404 and the Agrobacterium solution was injected onto the underside of the leaves of tobacco (Nicotiana benthamiana) leaves abaxially. The Dual Luciferase Reporter Kit (Promega, www.promega.com) was used to detect fluorescence activity.

    Protein degradation assays in vitro

    Total protein was extracted from wild-type and transgenic apple calli with or without 100 μM MG132 treatment. The purified MdARF18-HIS fusion protein was incubated with total protein[41]. Samples were collected at the indicated times (0, 1, 3, 5, and 7 h).

    Protein gel blots were analyzed using GST antibody. ACTIN antibody was used as an internal reference. All antibodies used in this study were provided by Abmart (www.ab-mart.com).

    Data analysis

    Unless otherwise noted, every experiment was carried out independently in triplicate. A one-way analysis of variance (ANOVA) was used to establish the statistical significance of all data, and Duncan's test was used to compare results at the p < 0.05 level[31].

    Results

    Auxin regulates nitrogen uptake and utilization in apple

    To investigate whether auxin affects the effective uptake of nitrate in apple, we first externally applied IAA under normal N (5 mM NO3−) environment, and this result showed that the growth of Gala apple seedlings in the IAA-treated group were better than the control, and their fresh weights were heavier than the control group (Fig. 1a, d). The N-related physiological indexes of apple seedlings also showed that the nitrate content and NR activity of the root part of the IAA-treated group were significantly higher than the control group, while the nitrate content and NR activity of the shoot part were lower than the control group (Fig. 1b, c). These results demonstrate that auxin could promote the uptake of nitrate and thus promotes growth of plants.

    Figure 1.  Auxin enhances nitrate uptake of Gala seedlings. (a) Phenotypes of apple (Malus domestica 'Royal Gala') seedlings grown nutritionally for 50 d under IAA (10 μM) treatment. (b) Nitrate content of shoot and root apple (Malus domestica 'Royal Gala') seedlings treated with IAA. (c) NR activity in shoot and root of IAA treatment apple (Malus domestica 'Royal Gala') seedlings. (d) Seedling fresh weight under IAA treatment. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    To test whether auxin affects the expression of genes related to nitrogen uptake and metabolism. For the root, the expression levels of MdNRT1.1, MdNRT2.1, MdNIA1, MdNIA2, and MdNIR were higher than control group (Supplemental Fig. S1a, f, h−j), while the expression levels of MdNRT1.2, MdNRT1.6 and MdNRT2.5 were lower than control group significantly (Supplemental Fig. S1b, d, g). For the shoot, the expression of MdNRT1.1, MdNRT1.5, MdNRT1.6, MdNRT1.7, MdNRT2.1, MdNRT2.5, MdNIA1, MdNIA2, and MdNIR genes were significantly down-regulated (Supplemental Fig. S1a, c−j). This result infers that the application of auxin could mediate nitrate uptake in plants by affecting the expression levels of relevant nitrate uptake and assimilation genes.

    Transcript levels of MdARFs are induced by nitrate

    Since the auxin signaling pathway requires the regulation of the auxin response factors (ARFs)[25,27], it was investigated whether members of ARF genes were nitrate responsive. Firstly, qPCR quantitative analysis showed that the five subfamily genes of MdARFs (MdARF9, MdARF2, MdARF12, MdARF3, and MdARF18) were expressed at different levels in various organs of the plant (Supplemental Fig. S2). Afterward, the expression levels of five ARF genes were analyzed under different concentrations of nitrate treatment (Fig. 2), and it was concluded that these genes represented by each subfamily responded in different degrees, but the expression level of MdARF18 was up-regulated regardless of low or high nitrogen (Fig. 2i, j), and the expression level of MdARF18 showed a trend of stable up-regulation under IAA treatment (Supplemental Fig. S3). The result demonstrates that MdARFs could affect the uptake of external nitrate by plants and MdARF18 may play an important role in the regulation of nitrate uptake.

    Figure 2.  Relative expression analysis of MdARFs subfamilies in response to different concentrations of nitrate. Expression analysis of representative genes from five subfamilies of MdARF transcription factors. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
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    Phylogenetic relationships and multiple sequence alignment of MdARF18

    MdARF18 (MD07G1152100) was predicted through The Apple Genome website (https://iris.angers.inra.fr/gddh13/) and it had high fitness with AtARF18 (AT3G61830). The homologs of ARF18 from 15 species were then identified in NCBI (www.ncbi.nlm.nih.gov) and then constructed an evolutionary tree (Supplemental Fig. S4). The data indicates that MdARF18 was most closely genetically related to MbARF18 (Malus baccata), indicating that they diverged recently in evolution (Supplemental Fig. S4). Conserved structural domain analyses indicated that all 15 ARF18 proteins had highly similar conserved structural domains (Supplemental Fig. S5). In addition, multiple sequence alignment analysis showed that all 15 ARF18 genes have B3-type DNA-binding domains (Supplemental Fig. S6), which is in accordance with the previous reports on ARF18 protein structure[26].

    Overexpression of MdARF18 affects root development

    To explore whether MdARF18 could affect the development of the plant's root system. Firstly, MdARF18 was heterologously expressed into Arabidopsis, and an arf18 mutant (GABI_699B09) Arabidopsis was also obtained (Supplemental Fig. S7). Seven-day-old MdARF18 transgenic Arabidopsis and arf18 mutants were treated in a medium with different nitrate concentrations for 10 d (Fig. 3a, b). After observing results, it was found that under the environment of high nitrate concentration, the primary root of MdARF18 was shorter than arf18 and wild type (Fig. 3c), and the primary root length of arf18 is the longest (Fig. 3c), while there was no significant difference in the lateral root (Fig. 3d). For low nitrate concentration, there was no significant difference in the length of the primary root, and the number of lateral roots of MdARF18 was slightly more than wild type and arf18 mutant. These results suggest that MdARF18 affects root development in plants. However, in general, low nitrate concentrations could promote the transport of IAA by NRT1.1 and thus inhibit lateral root production[3], so it might be hypothesized that MdARF18 would have some effect on MdNRT1.1 thus leading to the disruption of lateral root development.

    Figure 3.  MdARF18 inhibits root development. (a) MdARF18 inhibits root length at 10 mM nitrate concentration. (b) MdARF18 promotes lateral root growth at 0.5 mM nitrate concentration. (c) Primary root length statistics. (d) Lateral root number statistics. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    Overexpression of MdARF18 inhibits nitrate uptake in plants

    To investigate whether MdARF18 affects the growth of individual plants under different concentrations of nitrate, 7-day-old overexpression MdARF18, and arf18 mutants were planted in the soil and incubated for 20 d. It was found that arf18 had the best growth of shoot, while MdARF18 had the weakest shoot growth at any nitrate concentration (Fig. 4a). MdARF18 had the lightest fresh weight and the arf18 mutant had the heaviest fresh weight (Fig. 4b). N-related physiological indexes revealed that the nitrate content and NR activity of arf18 were significantly higher than wild type, whereas MdARF18 materials were lower than wild type (Fig. 4c, d). More detail, MdARF18 had the lightest fresh weight under low and normal nitrate, while the arf18 mutant had the heaviest fresh weight, and the fresh weight of arf18 under high nitrate concentration did not differ much from the wild type (Fig. 4b). Nitrogen-related physiological indexes showed that the nitrate content of arf18 was significantly higher than wild type, while MdARF18 was lower than wild type. The NR activity of arf18 under high nitrate did not differ much from the wild type, but the NR activity of MdARF18 was the lowest in any treatment (Fig. 4c, d). These results indicate that MdARF18 significantly inhibits plant growth by inhibiting plants to absorb nitrate, and is particularly pronounced at high nitrate concentrations.

    Figure 4.  Ectopic expression of MdARF18 inhibits Arabidopsis growth. (a) Status of Arabidopsis growth after one month of incubation at different nitrate concentrations. (b) Fresh weight of Arabidopsis. (c) Nitrate content of Arabidopsis. (d) NR activity in Arabidopsis. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    In addition, to further validate this conclusion, MdARF18 overexpression calli were obtained and treated with different concentrations of nitrate (Supplemental Fig. S8). The results show that the growth of overexpressed MdARF18 was weaker than wild type in both treatments (Supplemental Fig. S9a). The fresh weight of MdARF18 was significantly lighter than wild type (Supplemental Fig. S9b), and its nitrate and NR activity were lower than wild type (Supplemental Fig. S9c, d), which was consistent with the above results (Fig. 4). This result further confirms that MdARF18 could inhibit the development of individual plants by inhibiting the uptake of nitrate.

    MdARF18 targets genes related to nitrogen uptake and utilization

    Nitrate acts as a signaling molecule that takes up nitrate by activating the NRT family as well as NIAs and NIR[3,34]. To further investigate the pathway by which MdARF18 inhibits plant growth and reduces nitrate content, qRT-PCR was performed on the above plant materials treated with different concentrations of nitrate (Fig. 5). The result shows that the expression levels of AtNRT1.1, AtNIA1, AtNIA2, and AtNIR were all down-regulated in overexpression of MdARF18, and up-regulated in the arf18 mutant (Fig. 5a, h−j). There was no significant change in AtNRT1.2 at normal nitrate levels, but AtNRT1.2 expression levels were down-regulated in MdARF18 and up-regulated in arf18 at both high and low nitrate levels (Fig. 5b). This trend in the expression levels of these genes might be consistent with the fact that MdARF18 inhibits the expression of nitrogen-related genes and restricts plant growth. The trend in the expression levels of these genes is consistent with MdARF18 restricting plant growth by inhibiting the expression of nitrogen-related genes. However, AtNRT1.5, AtNRT1.6, AtNRT1.7, AtNRT2.1, and AtNRT2.5 did not show suppressed expression levels in MdARF18 (Fig. 5c−g). These results suggest that MdARF18 inhibits nitrate uptake and plant growth by repressing some of the genes for nitrate uptake or assimilation.

    Figure 5.  qPCR-RT analysis of N-related genes. Expression analysis of N-related genes in MdARF18 transgenic Arabidopsis at different nitrate concentrations. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    In addition, to test whether different concentrations of nitrate affect the protein stability of MdARF18. However, it was found that there was no significant difference in the protein stability of MdARF18 at different concentrations of nitrate (Supplemental Fig. S10). This result suggests that nitrate does not affect the degradation of MdARF18 protein.

    MdARF18 binds to the promoter of MdNRT1.1 to restrain its expression

    To further verify whether MdARF18 can directly bind N-related genes, firstly we found that the MdNRT1.1 promoter contains binding sites to ARF factors (Fig. 6a). The yeast one-hybrid research demonstrated an interaction between MdARF18 and the MdNRT1.1 promoter, as shown in Fig. 6b. Yeast cells that were simultaneously transformed with MdNRT1.1-P-pHIS and pGADT7 were unable to grow in selected SD medium. However, cells that were transformed with MdNRT1.1-P-pHIS and MdARF18-pGADT7 grew successfully in the selective medium. The result therefore hypothesizes that MdARF18 could bind specifically to MdNRT1.1 promoter to regulate nitrate uptake in plants.

    Figure 6.  MdARF18 binds directly to the promoter of MdNRT1.1. (a) Schematic representation of MdNRT1.1 promoter. (b) Y1H assay of MdARF18 bound to the MdNRT1.1 promoter in vitro. 10−1, 10−2, 10−3, and 10−4 indicate that the yeast concentration was diluted 10, 100, 1,000, and 10,000 times, respectively. 3-AT stands for 3-Amino-1,2,4-triazole. (c) Dual luciferase assays demonstrate the binding of MdARF18 with MdNRT1.1 promoter. The horizontal bar on the left side of the right indicates the captured signal intensity. Empty LUC and 35S vectors were used as controls. Representative images of three independent experiments are shown here.
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    To identify the inhibition or activation of MdNRT1.1 by MdARF18, we analyzed their connections by Dual luciferase assays (Fig. 6c), and also analyzed the fluorescence intensity (Supplemental Fig. S11). It was concluded that the fluorescence signals of cells carrying 35Spro and MdNRT1.1pro::LUC were stronger, but the mixture of 35Spro::MdARF18 and MdNRT1.1pro::LUC injected with fluorescence signal intensity was significantly weakened. Next, we transiently transformed the 35S::MdARF18 into pMdNRT1.1::GUS transgenic calli (Fig. 7). GUS results first showed that the color depth of pMdNRT1.1::GUS and 35S::MdARF18 were significantly lighter than pMdnNRT1.1::GUS alone (Fig. 7a). GUS enzyme activity, as well as GUS expression, also indicated that the calli containing pMdnNRT1.1::GUS alone had a stronger GUS activity (Fig. 7b, c). In addition, the GUS activity of calli containing both pMdNRT1.1:GUS and 35S::MdARF18 were further attenuated under both high and low nitrate concentrations (Fig. 7a). These results suggest that MdARF18 represses MdNRT1.1 expression by directly binding to the MdNRT1.1 promoter region.

    Figure 7.  MdARF18 inhibits the expression of MdNRT1.1. (a) GUS staining experiment of pMdNRT1.1::GUS transgenic calli and transgenic calli containing both pMdNRT1.1::GUS and 35S::MdARF18 with different nitrate treatments. (b) GUS activity assays in MdARF18 overexpressing calli with different nitrate treatments. (c) GUS expression level in MdARF18 overexpressing calli with different nitrate treatments. Bars represent the mean ± SD (n = 3). Different numbers of asterisk above the bars indicate significant differences using the LSD test (*p < 0.05 and **p< 0.01).
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    Discussion

    Plants replenish their nutrients by absorbing nitrates from the soil[42,43]. Previous studies have shown that some of the plant hormones such as IAA, GA, and ABA interact with nitrate[25,44−45]. The effect of nitrate on the content and transport of IAA has been reported in previous studies, e.g., nitrate supply reduced IAA content in Arabidopsis, wheat, and maize roots and inhibited the transport of IAA from shoot to root[20,21]. In this study, it was found that auxin treatment promoted individual fresh weight gain and growth (Fig. 1a, b). Nitrate content and NR activity were also significantly higher in their root parts (Fig. 1c, d) and also affected the transcript expression levels of related nitrate uptake and assimilation genes (Supplemental Fig. S1). Possibly because IAA can affect plant growth by influencing the uptake of external nitrates by the plant.

    ARFs are key transcription factors to regulate auxin signaling[46−49]. We identified five representative genes of the apple MdARFs subfamily and they all had different expression patterns (Supplemental Fig. S2). The transcript levels of each gene were found to be affected to different degrees under different concentrations of nitrate, but the expression level of MdARF18 was up-regulated under both low and high nitrate conditions (Fig. 2). The transcript level of MdARF18 was also activated under IAA treatment (Supplemental Fig. S3), so MdARF18 began to be used in the study of the mechanism of nitrate uptake in plants. In this study, an Arabidopsis AtARF18 homolog was successfully cloned and named MdARF18 (Supplemental Figs S4, S5). It contains a B3-type DNA-binding structural domain consistent with previous studies of ARFs (Supplemental Fig. S6), and arf18 mutants were also obtained and their transcript levels were examined (Supplemental Fig. S7).

    Plants rely on rapid modification of the root system to efficiently access effective nitrogen resources in the soil for growth and survival. The plasticity of root development is an effective strategy for accessing nitrate, and appropriate concentrations of IAA can promote the development of lateral roots[7,44]. The present study found that the length of the primary root was shortened and the number of lateral roots did increase in IAA-treated Gl3 apple seedlings (Supplemental Fig. S12). Generally, an environment with low concentrations of nitrate promotes the transport of IAA by AtNRT1.1, which inhibits the growth of lateral roots[14]. However, in the research of MdARF18 transgenic Arabidopsis, it was found that the lateral roots of MdARF18-OX increased under low concentrations of nitrate, but there was no significant change in the mutant arf18 (Fig. 3). Therefore, it was hypothesized that MdARF18 might repress the expression of the MdNRT1.1 gene or other related genes that can regulate root plasticity, thereby affecting nitrate uptake in plants.

    In rice, several researchers have demonstrated that OsARF18 significantly regulates nitrogen utilization. Loss of function of the Rice Salt Tolerant 1 (RST1) gene (encoding OsARF18) removes its ability to transcriptionally repress OsAS1, accelerating the assimilation of NH4+ to Asn and thereby increasing nitrogen utilization[28]. During soil incubation of MdARF18-OX Arabidopsis, it was found that leaving aside the effect of differences in nitrate concentration, the arf18 mutant grew significantly better than MdARF18-OX and had higher levels of nitrate and NR activity in arf18 than in MdARF18-OX. This demonstrates that MdARF18 may act as a repressor of nitrate uptake and assimilation, thereby inhibiting normal plant development (Fig. 4). Interestingly, an adequate nitrogen environment promotes plant growth, but MdARF18-OX Arabidopsis growth and all physiological indexes were poorer under high nitrate concentration than MdARF18-OX at other concentrations. We hypothesize that MdARF18 may be activated more intensively at high nitrate concentrations, or that MdARF18 suppresses the expression levels of genes for nitrate uptake or assimilation (genes that may play a stronger role at high nitrate concentrations), thereby inhibiting plant growth. In addition, we obtained MdARF18 transgenic calli (Supplemental Fig. S8) and subjected them to high and low concentrations of nitrate, and also found that MdARF18 inhibited the growth of individuals at both concentrations (Supplemental Fig. S9). This further confirms that MdARF18 inhibits nitrate uptake in individuals.

    ARF family transcription factors play a key role in transmitting auxin signals to alter plant growth and development, e.g. osarf1 and osarf24 mutants have reduced levels of OsNRT1.1B, OsNRT2.3a and OsNIA2 transcripts[22]. Therefore, further studies are needed to determine whether MdARF18 activates nitrate uptake through different molecular mechanisms. The result revealed that the transcript levels of AtNRT1.1, AtNIA1, AtNIA2, and AtNIR in MdARF18-OX were consistent with the developmental pattern of impaired plant growth (Fig. 5). Unfortunately, we attempted to explore whether variability in nitrate concentration affects MdARF18 to differ at the protein level, but the two did not appear to differ significantly (Supplemental Fig. S10).

    ARF transcription factors act as trans-activators/repressors of N metabolism-related genes by directly binding to TGTCNN/NNGACA-containing fragments in the promoter regions of downstream target genes[27,50]. The NRT family plays important roles in nitrate uptake, transport, and storage, and NRT1.1 is an important dual-affinity nitrate transporter protein[7,50−52], and nitrogen utilization is very important for apple growth[53,54]. We identified binding sites in the promoters of these N-related genes that are compatible with ARF factors, and MdARF18 was found to bind to MdNRT1.1 promoter by yeast one-hybrid technique (Fig. 6a, b). It was also verified by Dual luciferase assays that MdARF18 could act as a transcriptional repressor that inhibited the expression of the downstream gene MdNRT1.1 (Fig. 6c), which inhibited the uptake of nitrate in plants. In addition, the GUS assay was synchronized to verify that transiently expressed pMdNRT1.1::GUS calli with 35S::MdARF18 showed a lighter staining depth and a significant decrease in GUS transcript level and enzyme activity (Fig. 7). This phenomenon was particularly pronounced at high concentrations of nitrate. These results suggest that MdARF18 may directly bind to the MdNRT1.1 promoter and inhibit its expression, thereby suppressing NO3− metabolism and decreasing the efficiency of nitrate uptake more significantly under high nitrate concentrations.

    Conclusions

    In conclusion, in this study, we found that MdARF18 responds to nitrate and could directly bind to the TGTCTT site of the MdNRT1.1 promoter to repress its expression. Our findings provide new insights into the molecular mechanisms by which MdARF18 regulates nitrate transport in apple.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Liu GD; data collection: Liu GD, Rui L, Liu RX; analysis and interpretation of results: Liu GD, Li HL, An XH; draft manuscript preparation: Liu GD; supervision: Zhang S, Zhang ZL; funding acquisition: You CX, Wang XF; All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32272683), the Shandong Province Key R&D Program of China (2022TZXD008-02), the China Agriculture Research System of MOF and MARA (CARS-27), the National Key Research and Development Program of China (2022YFD1201700), and the National Natural Science Foundation of China (NSFC) (32172538).

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

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    Hermanson AL, Dai W. 2023. Polyploidy induction of raspberry species (Rubus) using colchicine and oryzalin. Technology in Horticulture 3:22 doi: 10.48130/TIH-2023-0022
    Hermanson AL, Dai W. 2023. Polyploidy induction of raspberry species (Rubus) using colchicine and oryzalin. Technology in Horticulture 3:22 doi: 10.48130/TIH-2023-0022
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Polyploidy induction of raspberry species (Rubus) using colchicine and oryzalin

  • Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA

  • Received: 11 June 2023
  • Accepted: 04 September 2023
  • Published online: 02 November 2023
Technology in Horticulture  3,  Article number: 22  (2023)  |  Cite this article

Abstract: Raspberry (Rubus spp.) is a fruit crop used in many products across the United States. Commercial production of raspberry in North Dakota is challenging due to lack of cultivars with winter hardiness. In this study, two diploid red raspberry cultivars 'Prelude' and 'Polana' that are reported hardy in USDA hardiness zone 5 were used to induce polyploid raspberries that may have benefits of greater growth vigor, enhanced stress tolerance, larger fruit size, and higher yield. The effects of cultivar, anti-mitotic agent and its concentration, and the treatment duration on in vitro and ex vitro polyploidy induction were determined. 'Prelude' succeeded in polyploidy induction through the colchicine treatment of in vitro explants (apical and nodal buds) and ex vitro root dipping. There were three verified tetraploid 'Prelude' plants. One tetraploid plant of 'Prelude' was developed when in vitro explants were treated with 160 mM colchicine for 96 h and the other two were developed when the root system was dipped in the 10 mM colchicine for 8 h. No polyploid plants of 'Polana' were developed. Oryzalin, another anti-mitotic agent, did not succeed in polyploidy induction in this study. This study provides useful information on polyploidy induction in raspberry species, which could benefit future germplasm enhancement of raspberry and other Rubus species.

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    Introduction

    • Raspberry, belonging to the genus Rubus (known as brambles) in the Rosaceae family, is a fruit species producing small fruits that are usually marketed towards fresh produce, juice, jelly, jams, wine, desserts, tea, and processed or frozen products[1,2]. Raspberry is a woody perennial shrub with biennial canes. The current year cane is called primocane that becomes floricane in the next year. The floricane dies after it produces fruit. Based on the fruiting habit, raspberries can be classified into three types: floricanes that produce the fruit in summer, primocanes that produce the fruit in the fall, and double-cropping ones produce the fruit on both floricanes and primocanes. The raspberry fruit is an aggregate that can be red, black, purple, or yellow/golden with a large variety in fruit size[3]. A USDA (United States Department of Agriculture) survey showed that 6,758 hectares in the United States were dedicated to raspberry production and a total of 81,147 megagrams (89,450 tons) raspberry were produced in 2022 with a market value of 531 million US dollars[4].

      Commercial raspberry production in the US is mainly in the Pacific Northwest region including northern California, Washington, and Oregon[5]; however, the demand of locally grown produce/products that are sold at farmer's markets and direct markets has been increasing due to concerns over food quality, nutrition, and food safety[6]. Raspberry is one of the most popular fruit crops for local markets nationwide including North Dakota and other Upper Midwest area. Due to the harsh winters and short growing season in North Dakota, limited raspberry cultivars that are winter hardy with good quality and high yield fruits are available to local growers. The cold climate restricts raspberry production and loss of yield due to cold injury is often observed in the Midwest and Northeast part of the United States[7]. The short growing season in North Dakota also discourages growing primocane raspberry because a large number of berries cannot be harvested before the first fall frost that often occurs in early October (The authors' personal observation). Therefore, the development of cold hardy floricane raspberry or early ripening primocane raspberry with large and flavorful fruit may help increase local raspberry production in North Dakota and other cold regions. Polyploidy plants are those that carry more than two sets of chromosomes that are heritable[8]. Polyploids have been used for plant breeding since 1930s due to the breeding implications and benefits that the polyploidy may have, such as tolerance to pests and diseases, thicker/darker leaves, larger flowers, longer-lasting flowers[8], increased frost tolerance[9], drought tolerance[10], greater chlorophyll content, greater phenolic compound content, greater carotenoid content, increased photosynthesis rate[11], larger fruit size, and different fruit shape[12]. In fruit crop breeding, increasing fruit size and yield as well as improving resistance to biotic and abiotic stresses are often pursued. Thus, the objective of this study was to induce polyploidy of red raspberry cultivars 'Prelude' and 'Polana' using colchicine and oryzalin, two most commonly used anti-mitotic agents for chromosome doubling in plants. 'Prelude' was bred at Cornell University, New York, USA and patented in 2001 mainly for fresh market production[13]. 'Prelude' is one of the earliest-ripening red raspberry cultivars. The fruit on floricanes can be harvested as early as late June in North Dakota. 'Prelude' is a double-cropping cultivar that can also produce fruit on current year growth (primocanes). The fruit on primocanes can be harvested from early September to early November in North Dakota. Canes of 'Prelude' are winter hardy and no severe winter damages were observed in recent ten years in Fargo, North Dakota (USDA zone 4a, −30o F) (unpublished information). 'Polana' is a red raspberry cultivar bred in Poland at the Research Institute of Pomology and Floriculture[14]. 'Polana' is one of the earliest-ripening primocane raspberry cultivars. In North Dakota, the 'Polana' fruit are harvested from late August to late October, making it a good primocane raspberry in the cold region. The ploidy of 'Prelude' and 'Polana' was determined to be diploid (2n = 2x = 14) (unpublished information) using the chromosome counting method[15]. We expect that increase ploidy level of raspberry species may result in improvement of winter hardiness, fruit quality, and yield.

    Materials and methods

      Plant material

    • Two red raspberry (Rubus idaeus L.) cultivars, 'Prelude' and 'Polana', were used in this research. Tissue cultures of the raspberries were developed in Dr. Dai's lab at North Dakota State University (USA)[16]. Tissue cultures of the two cultivars were maintained by sub-culturing every four weeks in Murashige and Skoog medium (MS)[17] supplemented with 30 g·l−1 sucrose, 5.5 g·l−1 agar (Caisson Labs Inc., Smithfield, UT, 2009), and 3 g·l−1 activated charcoal (Merck KGaA, Darmstadt, Germany). Medium pH was adjusted to 5.7−5.8 before autoclaving at 121 °C and 15 psi for 15 min. All raspberry tissue cultures were maintained in the culture room with a photoperiod of 16/8 h of day/night length, a light intensity of 55 µmol·m−2·s−1 PPF (photosynthetic photon flux), and a day/night temperature of 24/18 °C. Tissue culture conditions in this study were identical unless otherwise noted.

      In vitro bud treatment with colchicine and oryzalin for chromosome doubling

    • A total of ten concentrations of colchicine (0, 20, 40, 80, and 160 mM) and oryzalin (0, 80, 160, 320, and 640 µM) solutions were made before the treatment. In brief, 100 ml of liquid MS medium supplemented with 30 g·l−1 sucrose and 1 ml·l−1 PPMTM (Plant Preservative Mixture) (Plant Cell Technology, Inc., Washington, DC, USA) was added to a 300 ml Erlenmeyer flask and autoclaved at 121 °C and 15 psi for 15 min. After cooling, appropriate amounts of 100 mM colchicine (in DMSO) (Sigma-Aldrich, St. Louis, MO, USA) or 100 mM oryzalin came from premixed with a surfactant at 40.4% (Monterey Lawn and Garden, Fresno, CA, USA) were added to the flask to make the final concentration of each solution. Six apical buds (~1 cm) and six nodal buds (~1 cm) were collected from in vitro plants of 'Prelude' or 'Polana' and placed in each of ten flasks. The flasks were covered with aluminum foil, labeled, and placed on an SK-300 Lab Companion shaker (Jeio Tech Inc., Billerica, MA, USA) for a constantly shaking at 110 rpm. After each treatment duration ended (24, 48, and 96 h), two apical buds and two nodal buds were taken from each flask, triple rinsed in autoclaved water and patted dry. Treated buds were then transferred to the 100 ml baby food jar (Sigma-Aldrich, St. Louis, MO, USA) containing 25 ml of MS medium supplemented with 1 ml·l−1 of PPM, 30 g·l−1 sucrose, 5.5 g·l−1 agar, and 10 µM BA for shoot development in the tissue culture room.

      In vitro shoots developed from the bud were subjected to ex vitro rooting. Shoots were dipped in a commercial rooting powder Take Root® (United Industries Corporation, St. Louis, MO, USA) for 5 s and inserted into ProMix BX+ (Premier Horticulture Inc., Quakertown, PA, USA) in a 48-hole plug tray covered with a plastic lid. After 4 weeks, plants were being acclimatized by replacing the initial lid (hole-less) with a series of lids with an increasing number of holes on it during a period of four weeks. The rooted and acclimatized plants were subjected to determination of chromosome number.

      The experiment of in vitro bud treatment with colchicine and oryzalin was conducted in a randomized complete block design (RCBD) with four replicates in a 2 × 5 × 3 factorial arrangement of two cultivars (with nested tissue apical buds and nodal segments), five concentrations of each agent, and three treatment durations. There were 30 treatments and each treatment had four buds and was repeated four times; therefore, a total of 480 buds were used in this experiment.

      Ex vitro root-dipping in colchicine for chromosome doubling

    • Colchicine solutions for root dipping were prepared by adding 100 ml of distilled water into (300 ml) Erlenmeyer flask, then adding appropriate amount of the colchicine stock solution. Plants with roots of 'Prelude' and 'Polana' were pulled out from the ex vitro rooting medium. The lower leaves of the plant were removed and kept only the top 2−3 leaves. The plant roots were trimmed to 5 cm long, gently rinsed with running water for 1 min, and blotted dry. Ten ml of each of four colchicine solutions (0, 0.1, 1.0, and 10.0 mM) was added to a 50 ml disposable plastic tube (Corning Incorporated, Corning, NY, USA). Two plants were inserted into a tube placed in a test tube rack with the root submerged in colchicine solution for a 4 or 8 h colchicine treatment. The tube rack was covered with aluminum foil. There were 16 treatments (2 cultivars × 4 concentrations × 2 durations) and three tubes per treatment (treated as replication). After treatment, plant roots were rinsed three times with distilled water in three 300 ml magenta boxes with 10-min intervals. Rinsed plants were then planted in a seedling tray filled with ProMix BX+ mycorrhizae (Premier Horticulture Inc., Quakertown, PA, USA). The trays were placed in the tissue culture room for plant re-growth.

      The ex vitro root dipping experiment was conducted as a RCBD with three replications in a 2 × 4 × 2 factorial arrangement with two cultivars ('Polana' or 'Prelude'), four colchicine concentrations (0, 0.1, 1.0, or 10.0 mM), and two durations (4 and 8 h). There were 16 treatments and each treatment had one plant with three replications; therefore, a total of 48 plants were used in this experiment.

      Determination of chromosome number

    • New emerging shoot tips from the plants recovered from the chemical treatment of either in vitro buds or ex vitro root dipping were collected. The meristem of the shoot tip was squashed on a microscope viewing plate covered with a slide (VWR international, Randnor, PA, USA). Acetocarmine (Sigma-Aldrich, St. Louis, MO, USA) solution was used to stain the chromosomes. Chromosome detection was carried out on OLYMPUS CX41 microscope (Leeds Precision Instruments Inc., Minneapolis, MN, USA) at 40× view. After, plants confirmed to be polyploidy were prepared for DAPI (Vectashield®, Burlingame, CA, USA) image capture following the seven-step process. Step 1, slides with a good image of chromosome profile were dipped in liquid nitrogen for 10 s till the nitrogen stopped boiling around the slide. Step 2, put the slide down on a flat surface and pop off the cover slide using a blade on the corner. Step 3, put the frozen slide into 70% ethanol first for 5 min and air dry using clean blotting paper (Fisher Scientific, Pittsburgh, PA, USA) and repeated the previous step with 95% and 100% ethanol solutions (Decon Labratories Inc., King of Prussia, PA, USA). Step 4, after slide dried, load 13 µl of the DAPI antifade mounting solution to where the original center of the slide was, placed fresh cover slide flat on the DAPI droplet, and covered with a blotting paper. Step 5, one min after the DAPI antifade mounting solution spread, pressed gently on the corner of the cover slide to remove excess solution. Step 6, placed the finished slide into a shadow box to protect the DAPI dye from light, and then stored in the fridge until the image capture, and Step 7, took the shadow box with prepared slides into a dark room and captured the image with OLYMPUS BX51 (Olympus America Inc., Center Valley, PA, USA).

      Data collection and analysis

    • Data were collected after new bud growth was detected. The percent of initial buds developing new shoots were recorded as the survival rate. The number of plants with double the chromosome number of all survived plants was recorded as the polyploidy induction rate. Data in the in vitro bud treatment were tested for normality assumptions using PROC UNIVARIATE and homogeneity of variances among main factors (cultivar, chemicals, concentration, tissue type) were tested using PROC GLM for Hovtest (i.e. Levene). All data were analyzed by PROC GLIMMIX (SAS version 9.0401M6, SAS Institute, Cary NC, USA) for survival and polyploidy rates that were separated by the LSMEANS for factors and their interactions with Options lines (α = 0.05) for simple effects differences. Since PROC GLIMMIX was used to specify for specific distribution for correct analysis, only Type III tests of fixed effects for each response variables of ANOVA were calculated rather than the sum of squares and their means square.

      Results

      Effects of cultivar, concentration, and treatment duration on the survival and polyploidy induction of in vitro buds

    • Results of the in vitro bud treatment showed that the effect of cultivar on the survival rate was significant, whereas no significance was found in either concentration or treatment duration of both colchicine and oryzalin. There were no significant interactions between the cultivar and the concentration and treatment duration of colchicine or oryzalin on explant survival rate (Table 1) and polyploidy induction (Table 2).

      Table 1.  Type III tests of fixed effects of cultivar, concentration, and duration on the explant survival of red raspberry cultivars.

      Source of variationNumerator
      degrees of
      freedom      		Denominator
      degrees
      of freedom      		F ValueP > F
      Colchicine
      Cultivar12014.150.0430*
      Concentration42010.770.5443
      Duration260.710.4700
      Cultivar × Concentration42010.470.5867
      Cultivar × Duration22010.280.2800
      Concentration × Duration82010.680.6800
      Cultivar × Concentration × Duration82010.800.8000
      Oryzalin
      Cultivar12027.580.0064*
      Concentration42020.710.5860
      Duration261.280.2807
      Cultivar × Concentration42021.410.3146
      Cultivar × Duration22020.460.6292
      Concentration × Duration82020.390.9267
      Cultivar × Concentration × Duration82020.490.8631
      * Significant at α < 0.05.

      Table 2.  Type III tests of fixed effects of cultivar, concentration and duration on polyploidy induction of red raspberry cultivars.

      Source of variationNumerator
      degrees
      of freedom      		Denominator
      degrees
      of freedom      		F ValueP > F
      Colchicine
      Cultivar1670.320.3209
      Concentration4670.410.4139
      Duration2NANANA
      Cultivar × Concentration4670.410.4139
      Cultivar × Duration2NANANA
      Concentration × Duration8NANANA
      Cultivar × Concentration × Duration8NANANA
      Oryzalin
      Cultivar1NANANA
      Concentration4NANANA
      Duration2NANANA
      Cultivar × Concentration4NANANA
      Cultivar × Duration2NANANA
      Concentration × Duration8NANANA
      Cultivar × Concentration × Duration8NANANA
      NA: SAS could not compute data analysis due to no polyploidy values being present at different treatment levels.

      The effect of cultivar on the survival rate in colchicine treatments was significantly different (Table 1). 'Prelude' had a better survival rate (12.95%) than 'Polana' (7.08%) across all colchicine treatments (Table 3). In the oryzalin treatment, the effect of cultivar on the explant survival rate was also significant (Table 1). Unlike the cultivar response to colchicine, 'Polana' (14.17%) has a higher survival rate than 'Prelude' (6.15%) across all oryzalin treatments (Table 3). There were no significant interactions among oryzalin concentration, cultivar, and treatment duration on explant survival rate (Table 1). Due to no polyploidy plants being produced with oryzalin, no probability rate was calculated in SAS ANOVA (Table 2).

      Table 3.  Effect of cultivar on the explant survival rate in the colchicine and oryzalin treatment.

      CultivarSurvival rate (%)
      Colchicine treatmentOryzalin treatment
      'Prelude'12.95a*6.15b
      'Polana'7.08b14.17a
      * The same letter followed behind indicates no significance at α < 0.05.

      A tetraploid plant of 'Prelude' was identified from the treatment of 160 mM colchicine for 96 h with a sample size of 16 plants. Chromosome counting indicated that the plant contains 28 chromosomes in the meristem cell (2n = 4x = 28) (Fig. 1).

      Figure 1. 

      DAPI comparison of (a) diploid 'Prelude' (2n = 2x =14) and (b) tetraploid 'Prelude' (2n = 4x = 28). Bar = 10 µm.

      Effects of cultivar, colchicine concentration, and treatment duration on plant survival and polyploidy induction in root dipping treatment

    • Results showed that there were no significant differences in plant survival rate among cultivar, colchicine concentration, and treatment duration. No interactions between these factors were detected (Table 4). There was a trend of decreasing survival rate as an increase of colchicine concentration (Table 5). However, significant effects of cultivar (α = 0.1) and colchicine concentration (α = 0.5) on polyploidy induction were detected and an interaction between cultivar and concentration was also significant at α = 0.05 (Table 6).

      Table 4.  Type III tests of fixed effects of cultivar, concentration, and treatment duration on the plant survival in the root dipping treatment of raspberry cultivars.

      Source of variationNumerator
      degrees of
      freedom      		Denominator
      degrees of
      freedom      		F ValueP > F
      Cultivar13201.0000
      Concentration3321.890.1513
      Duration1320.330.5677
      Cultivar × Concentration3320.220.8802
      Cultivar × Duration1321.330.2568
      Concentration × Duration3321.000.4055
      Cultivar × Concentration × Duration3320.670.5787

      Table 5.  The effect of colchicine concentration on plant survival rate in root dipping treatment of raspberry.

      Colchicine (mM)Plant survival rate (%)
      0100a*
      0.1100a
      1.097.92a
      1089.58a
      * The same letter followed behind indicates no significance at α < 0.05.

      Table 6.  Type III tests of fixed effects of cultivar, concentration, and treatment duration on the polyploidy induction in the root dipping treatment of raspberry cultivars.

      Source of variationNumerator
      degrees of
      freedom      		Denominator
      degrees of
      freedom      		F valueP > F
      Cultivar1324.000.0540**
      Concentration3324.000.0159*
      Duration13201.0000
      Cultivar × Concentration3324.000.0159*
      Cultivar × Duration13201.0000
      Concentration × Duration33201.0000
      Cultivar × Concentration × Duration33201.0000
      * Significant at α < 0.05. ** Significant at α < 0.1.

      In the root dipping study, a polyploidy induction rate of 16.67% was achieved in the treatment of 10 mM colchicine for 'Prelude' (Table 7), indicating the different response of cultivars to colchicine. The result also indicates that an increase of colchicine concentration is needed for future polyploidy induction in red raspberry cultivars when ex vitro root dipping method is used.

      Table 7.  The effect of cultivar × colchicine concentration on the polyploidy rate in root dipping treatment of raspberry cultivars.

      Colchicine (mM)Polyploidy induction rate (%)
      Cultivar ‘Prelude’Cultivar ‘Polana’
      00b*0b
      0.10b0b
      1.00b0b
      1016.67a0b
      * The same lowercase letter indicates no significance at α < 0.05.
    Discussion

    • A polyploidy plant is a plant that carries more than double sets of chromosomes that are heritable[8]. Polyploidy plants possess certain traits, such as tolerance to pests and diseases[8], increased frost tolerance[9], drought tolerance[10], increased photosynthesis rate[11], larger fruit size, and different fruit shape[12], that benefit raspberry breeding and germplasm enhancement. A survey on the field-grown tetraploid red raspberry seedlings showed variations in flower size and fertility, fruit size, seed size, and leaf morphology between diploid and tetraploid raspberries[18]. Such polyploidy benefits could be utilized to potentially increase the plant yield quickly compared to standard breeding procedures[8]. The Rubus species are cytologically complex and their ploidy levels range from 2x to 14x with the basic chromosome number being seven[19−21]. However, in the subgenus Idaeobatus (raspberries), polyploidy is not common and most species are diploid[21] as well as in raspberry cultivars[22]. In this study, a polyploidy induction system was developed through testing two anti-mitotic agents, colchicine and oryzalin and two red raspberry cultivars 'Prelude' and 'Polana'. The ultimate goal of this research was to develop polyploid raspberries with enhanced winter hardiness, good yield, and fruit quality that can be used as new cultivars or unique germplasm for breeding new raspberries for the Upper Midwest and other cold regions of the United States.

      In this study, two different ways were adopted for polyploidy induction: in vitro and ex vitro. In the in vitro way, both apical and lateral (nodal) buds were collected from in vitro plants and treated with colchicine or oryzalin under in vitro conditions. Treated buds were then placed back to tissue culture medium for recovery. New shoots grew from the bud were ex vitro rooted and subject to chromosome counting. In the ex vitro way, in vitro shoots were ex vitro rooted and acclimated. Polyploidy induction was conducted by dipping the root system in a colchicine solution under ex vitro conditions. Treated plants were recovered ex vitro and subject to chromosome counting. This study indicates that polyploidy induction in raspberry species can be achieved from both in vitro and ex vitro methods. However, differences in cultivars responding to the anti-mitotic agents and their concentrations were found. One tetraploid plant of 'Prelude' was produced in the in vitro treatment of 160 mM colchicine at 96 h duration that was a combination of the highest concentration and longest treatment time. No polyploid plants were developed from other colchicine treatments and from all treatments of colchicine and oryzalin for 'Polana', indicating the differential responses of raspberry genotypes to anti-mitotic agents and the concentration and treatment time, which agrees with findings in other research that polyploidy induction can be influenced by genotype[23]. Touchell et al.[8] reported that higher concentrations of colchicine and oryzalin lead to a greater polyploidy rate, which was confirmed in this research with colchicine. An increase of the concentration and/or the treatment time may result in a high polyploid induction rate in raspberry species, which should be considered in future research. The result from the root dipping treatment showed a higher polyploidy induction rate (16.67%) than in vitro bud treatment. Two tetraploid plants of 'Prelude' were developed from the treatment of 10 mM colchicine that was the highest colchicine concentration in the ex vitro root dipping treatment, indicating that increasing colchicine concentration is necessary for polyploidy induction using root dipping method. Although there were no significant factors affecting survival rate that might be caused by small sample number and less replicates, a trend where survival rate decreased with an increase of colchicine concentrations in root dipping was observed; therefore, increasing the sample and replicate numbers, optimizing concentration and treatment duration of colchicine (or oryzalin) is also necessary in future endeavor of polyploidy induction of raspberry species.

      In this study, all three tetraploid plants of 'Prelude' were induced by colchicine. While colchicine is the most used anti-mitotic agent for ploidy manipulation in plants, oryzalin, an herbicide, is also one of the anti-mitotic agents used for chromosome doubling, particularly in woody or semi-woody plants, including several species in the Rosacea family[8,24] although oryzalin had no success creating polyploidy of 'Polana' and 'Prelude' raspberry in this study. Thus, the applicability of oryzalin to ploidy manipulation of raspberry species needs to be further investigated because oryzalin is more cost efficient and environment friendly than colchicine.

      In conclusion, the effects of anti-mitotic agent (colchicine and oryzalin), treatment method, and treatment duration on polyploidy induction of two red raspberry cultivars were determined. A total of three tetraploid plants of 'Prelude' were induced using colchicine and their ploidy was verified using chromosome counting method. These tetraploid plants can potentially be used as new cultivars or breeding materials; therefore, this study can benefit germplasm enhancement as well as genetic understanding of raspberry and other Rubus species.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Dai W, Hermanson A; data collection: Hermanson A; analysis and interpretation of results: Hermanson A, Dai W; draft manuscript preparation: Hermanson A, Dai W. Both 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.

    Acknowledgments

    • The authors would like to thank Dr. Jawahar Jyoti for his help with statistical analyses.

      Conflict of interest

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

      Rights and permissions
      • Copyright: © 2023 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/.
    Figure (1)  Table (7) References (24)
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    Cite this article
    Hermanson AL, Dai W. 2023. Polyploidy induction of raspberry species (Rubus) using colchicine and oryzalin. Technology in Horticulture 3:22 doi: 10.48130/TIH-2023-0022
    Hermanson AL, Dai W. 2023. Polyploidy induction of raspberry species (Rubus) using colchicine and oryzalin. Technology in Horticulture 3:22 doi: 10.48130/TIH-2023-0022
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  • Introduction
  • Materials and methods

  • Plant material

  • In vitro bud treatment with colchicine and oryzalin for chromosome doubling

  • Ex vitro root-dipping in colchicine for chromosome doubling

  • Determination of chromosome number

  • Data collection and analysis

  • Results

  • Effects of cultivar, concentration, and treatment duration on the survival and polyploidy induction of in vitro buds

  • Effects of cultivar, colchicine concentration, and treatment duration on plant survival and polyploidy induction in root dipping treatment

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