The Application Timing of a Cytokinin B-Mo-based Product Affects the Characteristics of Rooted Cuttings and Nonstructural Carbohydrates of Coleus (Plectranthus scutellarioides cv. Wild Lime) during Adventitious Root Development

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Mayra A. Toro-Herrera Department of Plant Science and Landscape Architecture, University of Connecticut, 1376 Storrs Road, Storrs, CT 06269, USA

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Rosa E. Raudales Department of Plant Science and Landscape Architecture, University of Connecticut, 1376 Storrs Road, Storrs, CT 06269, USA

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Abstract

A large group of horticulture species are propagated vegetatively through shoot-tip cuttings harvested from stock plants and planted to form adventitious roots. Adventitious rooting leads to establishing a carbohydrate sink in the region of root regeneration that is highly dependent on energy and carbon skeletons. We hypothesized that the timing of exogenous applications of cytokinin (CK) and boron (B)–molybdenum (Mo)-based products during adventitious root development can affect the flow of sugars from leaves to sinks, carbon allocation to the adventitious roots, and the quality of rooted cuttings. During this project, we aimed to determine if the application time of a CK/B-Mo-based product during the adventitious root development of unrooted cuttings would impact the source-to-sink relationship and, hence, affect plug growth and quality. A sink-strengthening commercial product based on cytokinin, B, and Mo was applied at four plug development stages plus a negative control as follows: T1, plants without product (control); T2, sticking stage (starting 24 hours after the sticking); T3, callus formation stage; T4, root development stage; and T5, toning stage. The root and shoot lengths and dry matter, number of leaves, leaf chlorophyll content, root-to-shoot ratio (based on dry matter), and nonstructural carbohydrate contents were measured. The timing of the application of the product impacted the root development, quality of the cuttings, and nonstructural carbohydrate content. Product application during the adventitious root dedifferentiation and induction phases (T2) resulted in the shortest root and shoot lengths, lowest dry matter accumulation, lowest nonstructural carbohydrate contents, and some phytotoxicity. Application during the initiation phase (T3) resulted in greater root length, total dry matter, and total soluble sugar contents compared with the control. Application during the expression phase (T4) resulted in the largest root length and mass and the highest sucrose contents. Applying the product when the roots had grown and reached all the edges of the growing media (T5) did not have any benefits compared with the control. This study provides new insights into the application timing of exogenous CKs, B, and Mo to generate a well-toned rooted coleus cutting and potential explanations in relation to nonstructural carbohydrate metabolism.

Propagative floriculture materials represent 21% of the US commercial floriculture wholesale industry (US Department of Agriculture 2023). Annual bedding and garden plants, potted herbaceous perennials, potted flowering plants, foliage plants, cut flowers, and cut cultivated greens are, to a large extent, propagated vegetatively, usually from shoot-tip cuttings harvested from stock plants, and planted to form adventitious roots (Druege 2020).

Because a well-rooted cutting is essential for optimal growth, development, and high-quality plants, the formation of adventitious roots plays a central role in the propagation stage. Adventitious rooting involves a complex interaction of physiological processes and successive developmental phases that require different hormonal signals and other internal and external factors. Those processes lead to roots formed de novo from differentiated cells (e.g., of stem or leafy tissues) with the establishment of a complete and autonomous plant (Atkinson et al. 2014; da Costa et al. 2013; De Klerk et al. 1999; Druege 2020; Li et al. 2009). The current knowledge of these phases and internal and external factors has been possible because of significant progress in understanding plant regeneration over the past century. This has been made possible through extensive research of and experimentation involving various subjects, including the induction of cell division and “self-renewal” activity resulting from tissue nonhormone-mediated and hormone-mediated wound responses, callus-like tissue development, the ability of cells to differentiate into specialized cell types, the formation of promeristems and primordia, and the outgrowth of newly formed roots or shoots (Kutschera and Ray 2022).

Adventitious rooting is an energy-intensive process that demands carbon skeletons for root growth and development (da Costa et al. 2013; Klopotek et al. 2010). The carbon budget of the cutting changes over the rooting process. In general, the separation of a cutting from the stock plant disturbs the influx of nutrients, inducing a key metabolic event that determines the establishment of a carbohydrate sink in the region of root regeneration (Ahkami et al. 2009; Klopotek et al. 2010). Until the cutting’s nutrient uptake capacity is restored, the nutritional status is determined by the initial endogenous pool of nutrients that were provided by the stock plant (Druege et al. 2004, 2019). Later, current photosynthesis plays an important role in the actual carbon budget, influencing the carbohydrate allocation and distribution within the cutting (Rapaka et al. 2005). Therefore, the capacity of the source to meet the sink demand and the sink strength with efficient utilization and partitioning of carbohydrates are determinants in the adventitious root formation (da Costa et al. 2013).

Some factors have been widely studied to improve the understanding of the carbon source and sink relationships during adventitious rooting, especially from the perspective of the leaf carbohydrate source capacity. For example, nitrogen levels (Druege et al. 2000; Zerche and Druege 2009), the interplay between initial carbohydrate reserves and current photosynthesis (Druege 2020; Rapaka et al. 2005), and light intensity and quality (Christiaens et al. 2015; Currey and Lopez 2015; Klopotek et al. 2010; Lopez and Runkle 2008; Tombesi et al. 2015) have been assessed to determine their influence on adventitious rooting. However, the translocation and sink perspectives have been comparatively less explored. Our research explored this side of the carbohydrate network by using a cytokinin (CK), boron (B)–molybdenum-based product that aims to strengthen the plant’s source-to-sink relationship and assess whether shifting the movement of sugars toward the sink organs can favor the adventitious root development process. Therefore, the focus was on the efficiency of the translocation of sugars and how they are allocated and used by sinks rather than on enhancing the carbohydrate source capacity.

The interaction between plant growth regulators and mineral nutrition is one factor that controls adventitious root development and plant regeneration (Brondani et al. 2012; Kutschera and Ray 2022). Therefore, we implemented the use of a product with CK, B, and Mo that influences the sugar translocation process in plants and has shown a fundamental role during adventitious root development. The CKs comprise a group of adenine derivatives biosynthesized in roots and transported via the xylem to shoots and leaves, where they promote organogenesis and mainly regulate cell division and differentiation (Arya et al. 2022; Oshchepkov et al. 2020; Wu et al. 2021). Additionally, CKs have mixed results when used in adventitious root development. The antagonistic effect between CK and auxins results in the inhibition of adventitious root formation. The expression of cytokinin-related genes is downregulated during adventitious rooting, and its exogenous application inhibits root elongation because of a decrease in the number of dividing cells and the size of the root meristem (Mao et al. 2019; Oshchepkov et al. 2020). During vegetative propagation protocols, positive regulation has been widely observed to promote organogenesis in different horticultural species such as Coleus (Krishna et al. 2010; Reddy et al. 2001; Sivakumar et al. 2021), eucalyptus (Negishi et al. 2014), olive (Fazeli-Nasab et al. 2021), and thymus (El Ansari et al. 2019).

One essential micronutrient for plants is B (Warington 1923), which is necessary for the growth of meristematic tissues, cell division, membrane function, and assimilate partitioning (Pereira et al. 2021; Rehman et al. 2018; Shireen et al. 2018). In the roots, B is concentrated in the root apex, thus influencing the growth of newly formed tissues (Poza-Viejo et al. 2018; Shimotohno et al. 2015). During adventitious root development, B translocates from the shoot apex of the cutting to the roots and other sink areas (Josten and Kutschera 1999; Svenson and Davies 1995). Under B-deficient conditions, the plant phloem transport is affected, thus reducing the sink capacity, which alters assimilate translocation and distribution to growing regions (Dugger 1983; Marschner 1995; Rehman et al. 2018; Wimmer and Eichert 2013).

Additionally, Mo is a trace element required for plant growth that is predominantly found as an integral part of an organic pterin complex called the Mo cofactor (Moco). Furthermore, Moco binds to Mo-requiring enzymes, such as nitrogen assimilation and nitrogen-fixing enzymes (Kaiser et al. 2005; Rana et al. 2020). The concentration of Mo in the basal portions of unrooted stem cuttings increases during the root initiation stage, suggesting a critical role for early root primordia formation or other concurrent growth processes (Svenson and Davies 1995).

We hypothesized that the timing of exogenous applications of CK and B–Mo-based products during adventitious root development can affect the flow of sugars from leaves to sinks, carbon allocation to the adventitious roots in formation, and the quality of rooted cuttings. During this project, we tracked the growth, dry matter accumulation, and partitioning of nonstructural carbohydrates (NSCs) when Sugar Mover® Premier (Stoller USA, Houston, TX, USA), a product containing CK, B, and Mo, was applied at different stages of the adventitious root development in unrooted cuttings of Coleus (Plectranthus scutellarioides cv. Wild Lime) to determine if the application time influenced adventitious rooting.

Materials and Methods

Plant growth conditions.

Excised herbaceous stem-tip unrooted cuttings of P. scutellarioides cv. Wild Lime (Plant Source International, Rogers, MN, USA) with four to five leaves of similar size were placed in 51-plug plastic trays with a 3 × 17 configuration and a depth of 1.62 inches. The trays were filled with soilless substrate with 80% to 90% sphagnum peatmoss, perlite, and calcitic/dolomitic limestone (PROMIX FPX B; Premier Tech Horticulture, Quebec, Canada). Trays containing cuttings were placed in a polycarbonate greenhouse in the Floriculture Greenhouse building at the University of Connecticut, Storrs, CT, USA. The greenhouse included a heating setpoint of 20 °C and a ventilation setpoint of 25 °C under a natural photoperiod from 9 Mar to 6 Apr 2023, for experimental run one, and from 27 Apr to 25 May 2023, for experimental run two. Each experimental run lasted 4 weeks.

The trays were distributed on five benches inside the greenhouse with an overhead mist system. For each bench, the irrigation consisted of three microemitters (Vibronet; Netafim Irrigation Inc., Fresno, CA, USA) with a flow rate of 40 L⋅h−1 controlled by an environmental computer (WS2 Argus Titan Weather Station; Argus Controls Systems Ltd., Surrey, British Columbia, Canada) as a function of time and solar radiation. Twelve seconds of misting were provided every time the pyranometer reached a value of 150 W⋅m−2, and the accumulation threshold was increased to 300 W⋅m−2 as the plants rooted out. At night, an irrigation period started every hour to keep the substrate moist. The uniformity between the five irrigation lines was evaluated through ¾-inch stainless steel pulse output water meters (SPWM-075-CF; EKM Metering Inc., Santa Cruz, CA, USA) linked with data loggers (HOBO, Bourne, MA, USA). Plants were irrigated with a nutrient solution containing (mg⋅L−1): 100 nitrogen, 6.7 phosphorus, 83 potassium, 46.2 calcium, 23.1 magnesium, 0.1 B, 0.05 copper, 0.5 iron, 0.25 manganese, 0.05 Mo, and 0.25 zinc (20–10–20; JR Peters Inc., Allentown, PA, USA). The pH of the substrate was between 5.8 and 6.2, and the electrical conductivity (EC) was approximately 1.2 mS/cm.

Commercial product.

The Sugar Mover® Premier (Stoller USA, Houston, TX, USA) is an Environmental Protection Agency-registered product containing CK (0.003% as kinetin), B (8%), and Mo (0.004%). The product was prepared following the recommended dose on the label for watering plants when transplanting; 15 mL of the product was diluted in 3.8 L of water, resulting in 0.001 mg⋅L−1 CK, 3.2 mg⋅L−1 Bo, and 0.002 mg⋅L−1 Mo. The solution was applied as a foliar spray at a rate of 25 mL on a 5 ½-inch × 21 ¼-inch tray using a hand pump sprayer (420–2L, Solo®; Newport News, VA, USA).

Treatments and experimental design.

The treatments consisted of the application of the product at the same concentration at five different developmental stages of the plug development: T1, plants without product (control); T2, sticking stage (24 h after the sticking); T3, callus formation stage; T4, root development stage; and T5, toning stage. The application time was determined during the experimental period by using additional plants under the same growing conditions for destructive sampling and verification of the developmental stage. Five cuttings were randomly selected daily to verify the rooting status and determine when a new treatment should start. A photographic record of the process was maintained throughout the experimental period (Fig. 1 and Supplementary Fig. 1). The sticking stage was defined as 24 h after sticking the unrooted cutting into the growing media. The callus formation stage encompassed the time when the callus was visible before any visible root emergence. Callusing is the first step in protecting the wound after a cutting is harvested and is primarily composed of undifferentiated cells or a cluster of irregular proliferation at the wound site (Kennedy 2008; Owen and Lopez 2017; Vallejo et al. 2018). Our visual indicator of callus formation was the tissue at the base of the wound, which tended to swell and whiten. Root development was considered the stage when the first roots emerged until the roots reached the edge of the tray. The toning stage was defined as the time when the plug could be pulled without any loose roots.

Fig. 1.
Fig. 1.

Visual of Plectranthus scutellarioides cv. Wild Lime from the sticking stage at 0 days after sticking (DAS) until the roots reached the edge of the tray at 22 DAS.

Citation: HortScience 59, 6; 10.21273/HORTSCI17756-24

The experiment had a complete randomized design with five treatments (four application times plus a negative control). The experimental unit consisted of a plug tray with 51 plants, with one experimental unit per repetition and four repetitions per treatment. From each experimental unit, 15 plants from the middle and center of the tray were selected to measure growth and dry matter. A total of 60 plants per treatment were measured (n = 60) during each experimental run for the growth and dry matter parameters, and the experiment was run twice (n = 120). After separately weighing the shoot and root dry matter of the 60 plants per treatment, the samples were homogenized, ground, and sieved to determine the partitioning of nonstructural carbohydrates. Five technical replicates of 0.2 g were taken from the homogenized material for each organ (shoot/root) from 10 samples per treatment and 50 samples per experimental run.

Plant measurements.

All plants were harvested after 29 d. The roots were carefully washed with pure water, and the root length and root dry matter were measured. The shoot length, number of leaves, leaf chlorophyll content, and dry matter were determined from the shoots. The leaf chlorophyll content was indirectly estimated by a handheld chlorophyll meter (model SPAD 502 Plus chlorophyll meter; Spectrum Technologies, Inc., Aurora, IL, USA) in three fully expanded leaves from each cutting. The dry matter was obtained by putting the shoot and root in paper bags inside an oven at 72 °C for 24 h and determining the dry weights on a calibrated digital analytical balance. The root-to-shoot ratio was determined by considering the root dry matter divided by the shoot dry matter (root dry matter/shoot dry matter). Macromolecules were quantified in both types of organs (leaves and roots) separately.

Quantification of macromolecules.

The proteins, amino acids, reducing sugars, total soluble sugars, starch, and sucrose levels were quantified to determine the proportion of the total biomass stored in nonstructural carbon. Carbohydrates were extracted from the samples (leaves and roots) by homogenizing 0.2 g of dry, ground, and sieved material in 10 mL of 0.1 M potassium phosphate buffer (pH = 7); then, they were subjected to a water bath at 40 °C for 30 min. The homogenate was centrifuged at 5000 gn for 10 min. The supernatant was collected to determine the levels of proteins, amino acids, reducing sugars, and total soluble sugars (Zanandrea et al. 2009). For starch extraction, the pellet was stored for resuspension with 200 mm potassium acetate buffer (pH = 4.8) (Zanandrea et al. 2009). The methods used for quantification were the anthrone reagent method (Dische 1962) for total soluble sugars, starch, and sucrose, the dinitrosalicylic acid method (Miller 1959) for reducing sugars, the ninhydrin method (Yemm et al. 1955) for amino acids, and the Bradford protein assay method (Bradford 1976) for proteins.

Statistical analysis.

Statistical analyses were conducted using RStudio statistical software version 2023.06.0 (Posit, Boston, MA, USA). Normality was evaluated using the Shapiro-Wilk test. Because some of the variables were not normally distributed, the homogeneity of variances between runs was determined using Bartlett’s and Fligner-Killeen’s tests for variables that were normally and non-normally distributed, respectively. To establish the significance of the effects of all factors (α = 0.05), data were analyzed by performing an analysis of variance (ANOVA). The Kruskal-Wallis one-way ANOVA was performed for growth and dry matter data, and a two-way ANOVA was performed for the nonstructural carbohydrates content. In the two-way ANOVA, the sources of variation and their levels were the treatments (T1–T5) and organs (leaves and roots). Dunn’s multiple comparison post hoc test with the Bonferroni correction was performed following a significant test. Additionally, a Pearson statistical correlation analysis (P < 0.05) was performed to analyze the correlation between the variables. Because there was homogeneity between runs for most measurements (P < 0.05), both experimental runs were analyzed together.

Results

Rooting process.

According to the results of daily monitoring performed to evaluate the rooting stage for both runs, callus formation occurred 4 to 5 days after sticking (DAS), root initiation occurred 8 DAS, and the roots reached all edges of the rooting media in the tray at 22 DAS. Plants were harvested and destructively sampled at 29 DAS. Figure 2 displays the visual appearance of the cuttings during harvest time at 29 DAS.

Fig. 2.
Fig. 2.

Visual representation of shoot growth and roots of Plectranthus scutellarioides cv. Wild Lime at 29 days after sticking (DAS) in experimental run two. From left to right: T1, plants without product (control); T2, application of the product in the sticking stage; T3, application of the product in the callus formation stage; T4, application of the product in the root development stage; and T5, application of the product in the toning stage.

Citation: HortScience 59, 6; 10.21273/HORTSCI17756-24

Plant measurements.

The application of Sugar Mover® Premier at different stages had a statistically significant effect (P < 0.05) on all the growth variables evaluated. The root length was shortest when the product was applied during sticking and longest when applied during root development, with differences of −3.4 cm and +1 cm compared with the negative control, respectively (Fig. 3). In turn, the shoot length was, on average, 1.22 cm smaller for T2 and T5 compared with the control group, whereas T3 (+1 cm) and T4 (+0.63 cm) had larger shoots (Fig. 3C). For T3, although the shoot length represents a magnitude greater length, it may be visually unfavorable because the plants looked like elongated cuttings with few leaves (Fig. 2).

Fig. 3.
Fig. 3.

(A) SPAD index, (B) root length (RL) (cm), (C) shoot length (SL) (cm), (D) the number of leaves (NL), (E) shoot dry matter (SDM) (g), (F) root dry matter (RDM) (g), (G) total dry matter (TDM) (cm), and (H) root-to-shoot ratio (RSR) (g) of Plectranthus scutellarioides cv. Wild Lime cuttings applied with Sugar Mover® Premier in four rooting stages. Boxplots (medians) with the same letters are not significantly different according to the Kruskal-Wallis rank-sum test and Dunn’s post hoc analysis at P < 0.05 (n = 60). T1 = control; T2 = sticking stage; T3 = callus formation stage; T4 = root development stage; and T5 = toning stage.

Citation: HortScience 59, 6; 10.21273/HORTSCI17756-24

The cuttings had, on average, four leaves when the experiment started. Both root (r = 0.4; P < 0.001) and shoot length (r = 0.3; P = 0.0015) were positively correlated with the number of leaves by the end of the experiment at 29 DAS (Fig. 3). T4, with the highest shoot length, had the highest number of leaves with greater dispersion and cuttings with up to 13 leaves. T5, which had the lowest shoot length, had the lowest number of leaves (Fig. 3D). The relative greenness or SPAD index indicating foliar chlorosis or greenness had no significant correlation with the growth or dry matter accumulation variables. However, T3, which had the highest shoot length, had the highest SPAD compared with the control group, whereas T2, which had the lowest root and shoot lengths, also had the lowest SPAD values (Fig. 3A).

The root-to-shoot ratio was higher for T3, T4, and T5 compared with the control treatment, showing an root-to-shoot ratio median value of approximately 1.6 (Fig. 3H), indicating a root with almost twice the biomass of the shoot, suggesting higher carbon investment in root growth and development. This result concurs with the negative correlation observed between the root-to-shoot ratio and number of leaves (r = −0.2; P = 0.0198) and the positive correlation between the root-to-shoot ratio and root length (r = 0.2; P = 0.0328) and root dry matter (r = 0.4; P < 0.001) (Fig. 4). Following this dynamic, the root dry matter of cuttings was higher than the control by 87%, 111%, and 46% for T3, T4, and T5, respectively (Fig. 3F). Likewise, the shoot dry matter of cuttings was lower than the control by 14%, 12%, and 23% for T3, T4, and T5, respectively (Fig. 3E). T2 showed values equal to or lower than that of the control treatment for all dry matter variables. The biomass allocated to the roots was lower by 29%, and the shoot dry matter was equivalent; hence, the root-to-shoot ratio indicates more biomass allocation to shoot growth, with a median value of approximately 0.4.

Fig. 4.
Fig. 4.

Pearson’s correlation analysis matrix between the variables analyzed. NL = number of leaves; RDM = root dry matter; RL = root length; RSR = root-to-shoot ratio; SDM = shoot dry matter; SL = shoot length; SPAD = SPAD index; TDM = total dry matter; AA = amino acids content; RS = reducing sugars content; TSS = total soluble sugars content. Circles represent positive (blue circles) or negative (red circles) statistically significant correlations (P ≤ 0.05). White boxes represent statistically nonsignificant interactions (P > 0.05).

Citation: HortScience 59, 6; 10.21273/HORTSCI17756-24

Quantification of macromolecules.

The application of Sugar Mover® Premier in different rooting stages had a statistically significant effect on the factor treatments (P < 0.05) for all biochemical variables except for protein (P = 0.5478), and on the factor organ (P < 0.05) for all the biochemical variables except for sucrose (P = 0.4798) (Fig. 5). There was no statistically significant interaction between the two factors for any variables.

Fig. 5.
Fig. 5.

(A) Protein [μg⋅g−1 dry matter (DM)], (B) amino acids (μmol⋅g−1 DM), (C) reducing sugars (RS) (μmol⋅g−1 DM), (D) total soluble sugars (TSS) (μmol⋅g−1 DM), (E) sucrose (μmol⋅g−1 DM), and (F) starch (μmol⋅g−1 DM) contents of leaves and roots of Plectranthus scutellarioides cv. Wild Lime cuttings with Sugar Mover® Premier applied in four rooting stages. Boxplots (medians) with the same letters are not significantly different according to the Kruskal-Wallis rank-sum test and Dunn’s post hoc analysis at P ≤ 0.05 (n = 10). T1 = control; T2 = sticking stage; T3 = callus formation stage; T4 = root development stage; and T5 = toning stage.

Citation: HortScience 59, 6; 10.21273/HORTSCI17756-24

Reducing sugars (Fig. 5C) and total soluble sugars (Fig. 5D) were higher in the roots than in the leaves. These two variables were positively correlated with each other (r = 0.7; P < 0.001) and with most of the root growth variables (Fig. 4). Both reducing sugars (r = −0.2; P = 0.0158) and total soluble sugars (r = −0.4; P < 0.001) correlated negatively with the shoot dry matter; additionally, total soluble sugars had a significant positive correlation with the root length (r = 0.2; P = 0.0345), root dry matter (r = 0.2; P = 0.0328), and root-to-shoot ratio (r = 0.6; P = 0.0036). Protein (Fig. 5A), amino acid (Fig. 5B), and starch (Fig. 5F) contents were higher in the leaves than in the roots. However, they were positively correlated with most of the root growth variables. Maintaining the same trend as reducing sugars and total soluble sugars, both amino acids (r = −0.2; P = 0.0479) and protein (r = −0.3; P = 0.0040) correlated negatively with the shoot dry matter. Additionally, there is a significant positive correlation between protein and root-to-shoot ratio (r = 0.3; P = 0.0088), and starch is significantly positively correlated with the root dry matter (r = 0.2; P = 0.0143), root length (r = 0.3; P = 0.0033), and root-to-shoot ratio (r = 0.3; P = 0.0036) (Fig. 4).

Regarding the treatments, T2 showed the lowest values for biochemical variables compared with the control and the other treatments (Fig. 5). This trend was also observed for some growth variables of T2, which had the lowest values among all the treatments (Fig. 3). In contrast, for all variables, T3, T4, and T5 had values equal to or greater than those of the control. T5 had the highest amino acids and reducing sugars values, whereas T4 had the highest sucrose values. Regarding total soluble sugars, T3, T4, and T5 had higher values than the control group (Fig. 5). Total soluble sugars was the variable that had the most significant correlations with the other variables, and it was positively correlated with all the biochemical variables, thus highlighting the high correlation with reducing sugars (r = 0.7; P < 0.001). Considering that total soluble sugars includes reducing sugars plus sucrose, this high correlation and the quantified values could indicate that most total soluble sugars present in the samples could be reducing sugars. Additionally, a high correlation with the root-to-shoot ratio (r = 0.6; P < 0.001) and a negative correlation with the shoot dry matter (r = −0.4; P < 0.001) were evident, suggesting a fundamental role of total soluble sugars in root growth (Fig. 4).

Sucrose and starch positively correlated with most root growth variables, root dry matter, root length, and root-to-shoot ratio (Fig. 4). This suggests the fundamental role of these compounds in adventitious root formation. Sucrose had the highest values for T3 and T4, which also had the highest values of dry matter accumulation in the roots and the plant.

Discussion

During this study, we assessed whether Sugar Mover® Premier, a commercial sink-strengthening product, altered the source-to-sink relationship during the adventitious root development process in unrooted cuttings, which is a high carbon requirement process. Adventitious rooting demands an adequate supply of carbohydrates for energy sources, structural and storage compounds, osmolytes, and signal molecules that regulate the allocation and fluxes of sugars (da Costa et al. 2013; Klopotek et al. 2010; Rapaka et al. 2005). The dynamics of sugars over time are measured by quantifying the concentration of nonstructural carbohydrates in plant tissues and organs (Hartmann et al. 2020; Hartmann and Trumbore 2016). Our results provide insights into how the cuttings manage nonstructural carbohydrates resources through the adventitious rooting and are discussed based on the four main phases that have been identified in the adventitious root development of stem cuttings (Fig. 6): dedifferentiation, induction, initiation, and expression (Agulló-Antón et al. 2014; da Costa et al. 2013; De Klerk et al. 1999; Druege et al. 2019).

Fig. 6.
Fig. 6.

General scheme of the results of growth and metabolism of nonstructural carbohydrates (NSCs) according to the stage of development and rooting of Plectranthus scutellarioides cv. Wild Lime cuttings with Sugar Mover® Premier applied in four rooting stages. DAS = days after sticking; NL = number of leaves; RDM = root dry matter; RL = root length; RSR = root-to-shoot ratio; SDM = shoot dry matter; SL = shoot length; SPAD = SPAD index; TDM = total dry matter; AA = amino acids content; RS = reducing sugars content; TSS = total soluble sugars content; Suc = sucrose. The scheme was adapted to the adventitious root development phases proposed by da Costa et al. (2013).

Citation: HortScience 59, 6; 10.21273/HORTSCI17756-24

Growth and nonstructural carbohydrate content in adventitious root formation.

During the first hours after excision, in the dedifferentiation and induction phases of adventitious root development (Fig. 6), there is physical isolation of resources and signals from the stock plant, thus generating an accumulation above the cut site of some substances generally transported downward, such as auxins, some phenolic compounds, and some mobile nutrients (Druege et al. 2019; Hilo et al. 2017; Svenson and Davies 1995). This marks the beginning of a new developmental program in the cells at the base of the stem, which is associated with cellular division, the reprogramming of target cells, and the establishment of a carbohydrate sink characterized by apoplastic unloading of sucrose (De Klerk et al. 1999). When Sugar Mover® Premier was applied in the sticking stage (T2), the cuttings were in the induction phase, some had symptoms of phytotoxicity, and they had the shortest root and shoot lengths (Fig. 1), along with the lowest accumulation of root dry matter (Fig. 3). During the induction phase, cuttings of various species have shown a negative correlation between the increase of CKs or the CK:auxin ratio at the base of the stem and the adventitious root formation (Agulló-Antón et al. 2014; De Klerk et al. 2001; Druege 2020; Villacorta-Martín et al. 2015). Therefore, the exogenous application of CK during this phase may have caused a decrease in adventitious root formation. However, it is not possible to determine with certainty whether the values are attributable to the inhibitory effect of CKs on adventitious root development, a secondary effect derived from phytotoxicity, or both. Additionally, the lowest values of sugars, starch, and amino acids were also presented, which may be related to the decrease in the photosynthetic area in the regions of the leaves affected by toxicity, which could cause a decrease in the production of sugars by photosynthesis during that and later stages of adventitious root development in which the most significant accumulation of sugars occurs (Ahkami et al. 2009).

In the next phase, the initiation phase, subsequent cell divisions and differentiation lead to the formation of small clusters of cells into dome-shaped root primordia (Fig. 6). Cell division and differentiation during organogenesis and adventitious root development have been reported to be B-dependent in Arabidopsis thaliana (Poza-Viejo et al. 2018; Reguera et al. 2019), Phaseolus aureus (Jarvis et al. 1984; Middleton et al. 1978), and Helianthus annuus (Josten and Kutschera 1999; Kutschera and Niklas 2017). In those species, B depletion affected cell division and altered the root system architecture, revealing disorganization of the root apical meristem. Additionally, root initiation and elongation are also influenced by Mo through their role in nitrate reductase regulation, nitrogen assimilation, and nitric oxide biosynthesis (Imran et al. 2019, 2021; Kovács et al. 2015). Nitrate reductase plays a key role in the nitrogen acquisition and assimilation through the regulation of the expression of nitrate transporters (Manoli et al. 2014; Sun et al. 2015) and the production of nitric oxide (Chamizo-Ampudia et al. 2017; Sun et al. 2014). The inability to synthesize Moco can significantly reduce the activity of these critical nitrogen-reducing and assimilatory enzymes (Kaiser et al. 2005). Previous studies have suggested that all Mo enzymes in higher plants are involved in nitric oxide production (Chamizo-Ampudia et al. 2017). Nitric oxide plays a vital role in regulating various aspects of root development, such as root initiation (Correa-Aragunde et al. 2006; Sun et al. 2015), root meristem growth (Yuan and Huang 2016), and root function under abiotic stress conditions (Sun et al. 2014).

The maintenance of a structurally intact root apical meristem depends on the balance between cell division and differentiation rates and is regulated by hormone signaling and transcriptional regulation (Poza-Viejo et al. 2018). Among the hormonal networks, CKs are key hormones that regulate the establishment and maintenance of meristem size during postembryonic development. With the early stimulation of the cell cycle in the transition zone through endocycling events, CKs regulate the transition from proliferation to the differentiation stage, which is essential to promote cell differentiation in the root meristem (Dello Ioio et al. 2007; Takatsuka and Umeda 2014). Additionally, in B-deficient conditions, the addition of CKs can lead to an increase in root meristem size (Poza-Viejo et al. 2018). The regulation of the balance between cell division and differentiation (mitosis and endoreduplication) may indicate a cytokinin response that antagonizes the auxin response wherein cell differentiation is promoted at the expense of cell division (Dello Ioio et al. 2008; Schaller et al. 2014). When we applied Sugar Mover® Premier in the callus stage (T3), cuttings showed greater root dry mass, total dry mass, and root-to-shoot ratio compared with those of the untreated control treatment. Therefore, the interplay between B, Mo, and CKs in this phase seems to improve the root primordia formation with the concomitant effect of a greater biomass accumulation in the roots.

The last phase, the expression phase, corresponds to the establishment of a mature root primordium with vascular connectivity to the stem, which grows and emerges from the stem as a functional and differentiated root (Agulló-Antón et al. 2014; da Costa et al. 2013; De Klerk et al. 1999) (Fig. 6). The re-establishment of cell connection between the newly formed root and the original stem cutting allows the symplastic transport of sugars from source leaves, which, until then, had been performed in an apoplastic way with the joint action of cell wall invertases and apoplastic sugar transporters (Ahkami et al. 2009, 2013). According to our results, applying Sugar Mover® Premier from the callus to the toning stage (T3–T5) showed higher total soluble sugars contents compared with that of the control group, especially in the roots. Additionally, at the root emergence stage (T4), which is part of the expression phase, cuttings showed greater root length, number of leaves, root dry matter, total dry matter, root-to-shoot ratio (Fig. 3), and sucrose content compared with the control and other treatments (Fig. 5). Sucrose, which is the primary sugar transported from the source leaves, can be used immediately in catabolic processes or transiently stored as starch as an intermediate carbohydrate depot close to the adventitious root-forming cells or to support developmental functions (Druege et al. 2019).

Root primordia elongation has been associated with an increased concentration of B (Svenson and Davies 1995) and high levels of soluble sugars and starch (Ahkami et al. 2009). Additionally, B has been associated with sugar translocation through the formation of soluble B complexes with polyols (sorbitol and mannitol) in the phloem of polyol-translocating plant species (Hu et al. 1997) and sucrose (Stangoulis et al. 2010). These complexes facilitate the “shuttle” of compounds across the plasma membrane, resulting in the efflux of sugars in the cytoplasm (Dannel et al. 2002; Perica et al. 2001). Additionally, several studies have found reduced assimilate translocation to growing regions on B deficiency (Rehman et al. 2018). Furthermore, as root tissues form, endogenous CK production increases to prevent uncontrolled cell division and promote the emergence and elongation of adventitious roots (Steffens and Rasmussen 2016). The Mo supply might initiate various nitric oxide-dependent physiological processes associated with root elongation. In addition to the root initiation and meristem size, nitric oxide has been associated with enhanced adventitious root elongation (Correa-Aragunde et al. 2004; Manoli et al. 2014) by increasing the activity of some enzymes involved in adventitious root development, such as polyphenol oxidase and indoleacetic acid oxidase, and the content of water-soluble carbohydrates and nitrogen (Liao et al. 2010). Therefore, it is possible that during this phase, the interplay between B, Mo, and CKs enhances the translocation of assimilates from the source to the sinks, thus improving root elongation and dry matter accumulation because of a high translocation rate and discharge of sucrose and its subsequent cleavage into hexoses for immediate use as a source of energy instead of for use for further root initiation.

When we applied the product in the toning stage (T5), when the roots had grown and reached all the edges of the growing media, cuttings showed smaller shoots and a smaller number of leaves compared with those of the control treatment, with high levels of total soluble sugars, mainly in the reducing sugars form, and high levels of amino acids. By this stage, all the photosynthetic and absorption processes of the new plant have been wholly re-established. The high content of total soluble sugars and reducing sugars may be related to the flow and cleavage of sucrose that was translocated from the source leaves into hexoses that are metabolized in the sinks or growing roots. The catabolism of the sugars produced yields ATP as a source of energy and amino acids for synthesizing proteins that participate in fundamental processes such as nitrogen assimilation.

Conclusion

Our study of the use of a commercial product to strengthen the source-to-sink relationship during adventitious root development in coleus stem cuttings provides new insights into the nonstructural carbohydrate’s metabolism and the role of CKs and B in generating a well-toned rooted cutting. During the adventitious root development induction phase (T2), it was unclear whether the decline in growth and dry matter accumulation was caused by the antagonistic function of CKs during the determination of root founder cells or caused by an effect of the phytotoxicity. During the initiation phase (T3), the interplay between B and CKs seems to maintain an appropriate balance between cell division and differentiation, thus maintaining the organization of the meristem while promoting the translocation of assimilates for the formation of the root primordia. During the extension phase (T4), the best growth and dry matter accumulation results were achieved. Additionally, B and CKs possibly facilitated a high translocation rate, leading to assimilates being directed toward root elongation. Finally, applying the product in T5 when the roots had grown and reached all the edges of the growing media did not have any benefit compared with the control. In practice, the application timing of commercial products impacts the outcome of the quality of the rooted cutting. In the future, underexplored factors such as anatomical changes, enzymatic activities, and genetic expression should be explored to improve the physiological understanding of the application of Sugar Mover® Premier or similar products/compounds in the rooting process of species of horticultural interest.

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  • Fig. 1.

    Visual of Plectranthus scutellarioides cv. Wild Lime from the sticking stage at 0 days after sticking (DAS) until the roots reached the edge of the tray at 22 DAS.

  • Fig. 2.

    Visual representation of shoot growth and roots of Plectranthus scutellarioides cv. Wild Lime at 29 days after sticking (DAS) in experimental run two. From left to right: T1, plants without product (control); T2, application of the product in the sticking stage; T3, application of the product in the callus formation stage; T4, application of the product in the root development stage; and T5, application of the product in the toning stage.

  • Fig. 3.

    (A) SPAD index, (B) root length (RL) (cm), (C) shoot length (SL) (cm), (D) the number of leaves (NL), (E) shoot dry matter (SDM) (g), (F) root dry matter (RDM) (g), (G) total dry matter (TDM) (cm), and (H) root-to-shoot ratio (RSR) (g) of Plectranthus scutellarioides cv. Wild Lime cuttings applied with Sugar Mover® Premier in four rooting stages. Boxplots (medians) with the same letters are not significantly different according to the Kruskal-Wallis rank-sum test and Dunn’s post hoc analysis at P < 0.05 (n = 60). T1 = control; T2 = sticking stage; T3 = callus formation stage; T4 = root development stage; and T5 = toning stage.

  • Fig. 4.

    Pearson’s correlation analysis matrix between the variables analyzed. NL = number of leaves; RDM = root dry matter; RL = root length; RSR = root-to-shoot ratio; SDM = shoot dry matter; SL = shoot length; SPAD = SPAD index; TDM = total dry matter; AA = amino acids content; RS = reducing sugars content; TSS = total soluble sugars content. Circles represent positive (blue circles) or negative (red circles) statistically significant correlations (P ≤ 0.05). White boxes represent statistically nonsignificant interactions (P > 0.05).

  • Fig. 5.

    (A) Protein [μg⋅g−1 dry matter (DM)], (B) amino acids (μmol⋅g−1 DM), (C) reducing sugars (RS) (μmol⋅g−1 DM), (D) total soluble sugars (TSS) (μmol⋅g−1 DM), (E) sucrose (μmol⋅g−1 DM), and (F) starch (μmol⋅g−1 DM) contents of leaves and roots of Plectranthus scutellarioides cv. Wild Lime cuttings with Sugar Mover® Premier applied in four rooting stages. Boxplots (medians) with the same letters are not significantly different according to the Kruskal-Wallis rank-sum test and Dunn’s post hoc analysis at P ≤ 0.05 (n = 10). T1 = control; T2 = sticking stage; T3 = callus formation stage; T4 = root development stage; and T5 = toning stage.

  • Fig. 6.

    General scheme of the results of growth and metabolism of nonstructural carbohydrates (NSCs) according to the stage of development and rooting of Plectranthus scutellarioides cv. Wild Lime cuttings with Sugar Mover® Premier applied in four rooting stages. DAS = days after sticking; NL = number of leaves; RDM = root dry matter; RL = root length; RSR = root-to-shoot ratio; SDM = shoot dry matter; SL = shoot length; SPAD = SPAD index; TDM = total dry matter; AA = amino acids content; RS = reducing sugars content; TSS = total soluble sugars content; Suc = sucrose. The scheme was adapted to the adventitious root development phases proposed by da Costa et al. (2013).

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Supplementary Materials

Mayra A. Toro-Herrera Department of Plant Science and Landscape Architecture, University of Connecticut, 1376 Storrs Road, Storrs, CT 06269, USA

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Rosa E. Raudales Department of Plant Science and Landscape Architecture, University of Connecticut, 1376 Storrs Road, Storrs, CT 06269, USA

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Contributor Notes

We thank the US Department of Agriculture-Agricultural Research Service (USDA-ARS) Floriculture and Nursery Research Initiative for partially supporting this research.

R.E.R. is the corresponding author. E-mail: rosa.raudales@uconn.edu.

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  • Fig. 1.

    Visual of Plectranthus scutellarioides cv. Wild Lime from the sticking stage at 0 days after sticking (DAS) until the roots reached the edge of the tray at 22 DAS.

  • Fig. 2.

    Visual representation of shoot growth and roots of Plectranthus scutellarioides cv. Wild Lime at 29 days after sticking (DAS) in experimental run two. From left to right: T1, plants without product (control); T2, application of the product in the sticking stage; T3, application of the product in the callus formation stage; T4, application of the product in the root development stage; and T5, application of the product in the toning stage.

  • Fig. 3.

    (A) SPAD index, (B) root length (RL) (cm), (C) shoot length (SL) (cm), (D) the number of leaves (NL), (E) shoot dry matter (SDM) (g), (F) root dry matter (RDM) (g), (G) total dry matter (TDM) (cm), and (H) root-to-shoot ratio (RSR) (g) of Plectranthus scutellarioides cv. Wild Lime cuttings applied with Sugar Mover® Premier in four rooting stages. Boxplots (medians) with the same letters are not significantly different according to the Kruskal-Wallis rank-sum test and Dunn’s post hoc analysis at P < 0.05 (n = 60). T1 = control; T2 = sticking stage; T3 = callus formation stage; T4 = root development stage; and T5 = toning stage.