*Article* **Effect of Foliar Supplied PGRs on Flower Growth and Antioxidant Activity of African Marigold (***Tagetes erecta L.***)**

**Sadia Sadique 1,†, Muhammad Moaaz Ali 2,3,†, Muhammad Usman 4, Mahmood Ul Hasan 3, Ahmed F. Yousef 2,5, Muhammad Adnan 6, Shaista Gull 3,7 and Silvana Nicola 8,\***


**Abstract:** Marigold is one of the commercially exploited flowering crops that belongs to the family Asteraceae. The production of economical yield and better quality of marigold flowers requires proper crop management techniques. Crop regulation is an important technique to make the marigold production profitable. This can be done by adopting application of plant growth regulators (PGRs). The present study was designed to investigate the effect of PGRs on flowering and antioxidant activity of two cultivars of African marigold (*Tagetes erecta L.*) viz. "Pusa Narangi Gainda" (hereinafter referred to as Narangi) and "Pusa Basanthi Gainda" (hereafter referred to as Basanthi). Plants were sprayed with abscisic acid (ABA), N-acetyl thiazolidine (NAD), gibberellic acid (GA3), salicylic acid (SA), indole-3-butyric acid (IBA) and oxalic acid (OA) at the concentrations of 100, 150, 250, 300 and 800 mg·L<sup>−</sup>1, each. Results revealed that the plants treated with 500–600 mg·L−<sup>1</sup> IBA exhibited maximum increase in floral diameter (34–51%). The use of 500–550 mg·L−<sup>1</sup> IBA exhibited maximal enhancement in flower fresh weight (21–92%). The exogenously applied OA significantly (*p* ≤ 0.05) improved flower dry weight, total phenolic contents, total flavonoid contents and reducing power ability of marigold plants. Overall, "Narangi" performed better than "Basanthi", in terms of flowering and antioxidant activity. Conclusively, the results suggest that foliar application of PGRs favors flowering and antioxidant activity of African marigold.

**Keywords:** plant growth regulators; salicylic acid; oxalic acid; DPPH; antioxidant activity; reducing power ability

#### **1. Introduction**

African marigold (*Tagetes erecta L.*) belongs to family Asteraceae and is one of the major and important commercial flower crops and widely grown for loose flower production [1]. It is an ornamental plant species with known medicinal use due to the high content of carotenoids and phenolics in flower petals [2]. It is popular throughout the world because of its wide spectrum of attractive colours, shape and good keeping quality. Marigold has gained popularity on account of its easy cultivation, wide adaptability and production throughout the year [3]. Apart from beautification, its flower petals are also being used

**Citation:** Sadique, S.; Ali, M.M.; Usman, M.; Hasan, M.U.; Yousef, A.F.; Adnan, M.; Gull, S.; Nicola, S. Effect of Foliar Supplied PGRs on Flower Growth and Antioxidant Activity of African Marigold (*Tagetes erecta L.)*. *Horticulturae* **2021**, *7*, 378. https:// doi.org/10.3390/horticulturae7100378

Academic Editor: Piotr Salachna

Received: 16 September 2021 Accepted: 6 October 2021 Published: 8 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for xanthophyll production which is a major carotenoid fraction and accounts for 80–90% of lutein [4,5]. The flowers are also used for religious rituals and social functions because of their wide adoptability to varying soil and climatic conditions and long duration of flowering [6].

In many countries, research was conducted to improve flowering growth of ornamental plants by treating them with environment friendly substances, e.g., gibberellic acid, oxalic acid and salicylic acid, and success was achieved to a certain level [7]. Plant growth regulators (PGRs) play an important role in flower production, which in small amounts promotes or inhibits or quantitatively modifies growth and development. Gibberellic acid has proved to be very effective in manipulating growth and flowering in chrysanthemum (*Chrysanthemum morifolium*) [8] and petunia (*Petunia hybrida*) [9]. Gibberellic acid and NAA enhance the elongation and cell division by promoting the DNA synthesis in the cell. They reduced the juvenile phase due to increase in photosynthesis and respiration with enhanced CO2 fixation in the plant [10]. The flowering growth of marigold was reported as increased under the influence of abscisic acid [6]. Indole-3-butyric acid played a vital role in increasing floral diameter of rose species (*Rosa* spp.) by increasing cell division [11]. However, limited research was conducted in Pakistan using these PGRs to enhance growth and development of flowering plants [12].

Commercially, plant growth regulators are used for suppressing apical dominance retarding vegetative growth, lateral buds induction and the production of a large number of flowers in various crops resulting in a higher flower yield and easy cultivation [13–15]. There are many examples of utilization of plant growth hormones to regulate the flowering in aromatic plants [16]. This research is of great interest and importance for flower merchants, growers, and scientists in Pakistan. The traditionally used chemicals have negative impacts on the environment due to their nonbiodegradable characteristics [17]. Considering that there are not extensive studies about the effects of PGRs on flowering and antioxidant activity of African marigold, a pot experiment was designed to evaluate the floral growth and antioxidant response of two cultivars ("Pusa Narangi Gainda", (hereafter referred to as Narangi, and "Pusa Basanthi Gainda", hereafter referred to as Basanthi) of African marigold to exogenously applied different doses (100, 150, 250, 300 and 800 mg·L−1) of PGRs, including abscisic acid (ABA), N-acetyl thiazolidine (NAD), gibberellic acid (GA3), salicylic acid (SA), indole-3-butyric acid (IBA) and oxalic acid (OA).

#### **2. Materials and Methods**

#### *2.1. Plant Material and Experimental Site*

The experiment was conducted at the research farm of the Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan (31◦26 03.3 N 73◦04 28.1 E) from 16 July 2020 to 15 November 2020. One-month old seedlings of marigold cultivars "Pusa Narangi Gainda" and "Pusa Basanthi Gainda" were purchased from Qadir Bakhsh Nursery (Pvt. Ltd.), Faisalabad-38000, Pakistan, and transplanted to plastic pots (30 × 33 cm), one seedling per pot. Before transplanting, the pots were filled with coconut coir, sand and well-pulverized soil collected from the field of the research farm with the ratio of 1:3:3, respectively. After filling growing media into pots, the moisture was applied up to field capacity. The pots media had pH, EC, available phosphorus and potassium of 6.3, 0.424 dS m<sup>−</sup>1, 14.92 mg·L−<sup>1</sup> and 347.57 mg·L−1, respectively. After transplanting, pots were placed in a greenhouse. The greenhouse climate data during the complete execution of the experiment is given in Figure 1.

**Figure 1.** Microclimate conditions inside the greenhouse during the experiment (16 July to 15 November 2020) at research station of University of Agriculture, Faisalabad, Punjab, Pakistan.

#### *2.2. PGRs Treatments*

Marigold plants of both cultivars were sprayed with six different plant growth regulators (Merck KGaA, Darmstadt, Germany), namely abscisic acid (ABA), N-acetylthiazolidine (NAD), gibberellic acid (GA3), salicylic acid (SA), indole-3-butyric acid (IBA) and oxalic acid (OA) at five different doses (i.e., 100, 150, 250, 300 and 800 mg·L−1, each), twice a week from blooming, when each plant had ≥3 flowers. Blooming initiated in both cultivars at same time (<2 days difference). Each cultivar was sprayed with PGRs at the same time. The plants were sprayed one week after first bloom. Marigold plants were foliar sprayed with PGRs early in the morning using 1 L electronic sprayer (T Tovia, Ningbo, China) operated at a constant speed. Each treatment received 1 L of PGRs solution per spray. Control plants were treated with distilled water and maintained for comparison (0 mg·L−1). Each treatment was replicated thrice, and each replication contained 10 pots, thus 10 plants.

#### *2.3. Flowering Attributes*

Flowering attributes, i.e., floral diameter, flower fresh weight and flower dry weight, were measured at full flower physiological maturity—determined by visual observation— 123 days after transplanting, after 4 weeks from the first spray, that is after 8 sprays. These parameters were calculated by randomly picking 10 physiologically mature flowers from 10 plants per replicate and per treatment. Floral diameter was measured with digital Vernier callipers (DR-MV0100NG, Ningbo Dongrun Imp. & Exp. Co., Ltd., Ningbo, China), whereas flower weight was measured with digital weighing balance (MJ-W176P, Panasonic, Osaka, Japan). Fresh flowers were dried in hot air dehydrator (Ultimate 4000, Fowlers Vacola Australia Pty Ltd., Melbourne, Australia) at 65 ◦C for 72 h.

#### *2.4. Antioxidant Attributes*

#### 2.4.1. Sample Preparation

The oven dried flowers were grinded and mixed in methanol to prepare sample solution (1:15 *w*/*v*). The mixture was stirred for 2 h and kept at room temperature for 24 h. Then, it was filtered and kept in sealed bottles in the dark [18].

#### 2.4.2. Total Phenolic Contents

The 1.0 mL of each sample solution and gallic acid standard solution (20, 40, 60, 80 and 100 mg·L−1), 5 mL of Folin–Ciocalteu reagent and 4 mL sodium carbonate (7% *w*/*v*) were added in a flask and shaken to mix the components completely. After keeping

all the samples in the dark for 30 min, absorbance was measured at 765 nm using a spectrophotometer (T60 U Spectrophotometer, PG Instruments Ltd., Leicestershire, UK). Reagent solution was used as a blank. The amount of total phenolics was expressed as gallic acid equivalent in milligram per gram plant dry weight [19].

#### 2.4.3. Total Flavonoid Contents

The 1.0 ml of sample or catechin standard solution (20, 40, 60, 80 and 100 mg·L<sup>−</sup>1) was mixed with 4.0 mL of water in 10 mL volumetric flask followed by addition of 0.3 mL of 5% NaNO2. After 5 min, 0.3 mL of 10% AlCl3 was added and after waiting for one more min, 2 mL of 1 M NaOH were added, and total volume was made up to 10 mL using deionized distilled water (DDW). After mixing the solution properly, the absorbance reading was measured at 510 nm using reagent as blank. The amount of total flavonoids was expressed as catechin equivalent in milligram per gram plant dry weight [20].

#### 2.4.4. DPPH Free Radical Scavenging Activity

The 1,1-diphenyl-2-picrylhydrazine (CAS No. 1707-75-1, ≥95% purity, Sigma-Aldrich, Milwaukee, WI, USA) scavenging activity was carried out by adding DPPH solution (1.0 mL, 0.3 M) to 2.5 mL solution of plant extract or gallic acid standard. Then samples and standards were incubated at room temperature in the dark for 20 min. Finally, absorbance was recorded at 518 nm. The control solution was prepared by adding 1.0 mL of methanol to 2.5 mL of extract solution without DPPH, while the positive control was prepared by adding 1.0 mL of DPPH solutions to 2.5 mL of gallic acid. The DPPH scavenging activity was calculated using the following expression [21].

$$\text{DPPH saving acting activity } (\%) = 100 - \left[ \frac{\text{Absorbance of sample}}{\text{Absorbance of control}} \times 100 \right]$$

#### 2.4.5. Plant Reducing Power Ability

The plant extract (1.0 mL) or gallic acid standard solution (20, 40, 60, 80 and 100 mg·L<sup>−</sup>1) was mixed with 2.3 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide (K3[Fe(CN)6]). The mixture was incubated at 37 ◦C for 20 min. Then, 10% trichloroacetic acid (2.5 mL) was added to the mixture and centrifuged for 10 min at 1000 rpm, the supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl3. After keeping the solution for 10 min, the absorbance was measured at 700 nm [22].

#### *2.5. Experimental Design and Statistical Data Analysis*

The experiment was designed under bi-factorial completely randomized design (Cultivar × PGRs doses) with three replicates. Collected data were submitted to analysis of variance (ANOVA) and found that cultivars and PGR doses had significant (*p* ≤ 0.05) interaction, except in the case of flower diameter when treated with ABA and SA. Fisher's LSD (interaction) technique was used to compare treatment means (*p* ≤ 0.05) using the analytical software package "Statistix 8.1". Principle component analysis and correlation coefficient values were determined through Pearson (*n*) technique using "XLSTAT ver. 2019".

#### **3. Results**

#### *3.1. Flowering Attributes*

Marigold plants of "Basanthi" exhibited more floral diameter as compared to "Narangi". In case of ABA application, marigold plants of "Basanthi" had a bigger floral diameter than "Narangi" (*p* ≤ 0.05). The maximum floral diameter (5.24 cm) was observed in plants of "Basanthi" treated with 250 mg·L−<sup>1</sup> ABA which was 1.34-fold greater than those of untreated plants (Figure 2A). Similarly, marigold plants of "basanthi" receiving foliar application of 100, 300 and 800 mg·L−<sup>1</sup> NAD exhibited a significant (*<sup>p</sup>* ≤ 0.05) increase in the floral diameter. However, for NAD application above than 250 mg·L−1, a sharp

decrease in the average was obtained for floral diameter, reaching 2.5 cm for the maximum dose applied (800 mg·L−1) (Figure 2B). Regardless of the concentration, plants of "Basanthi" showed enhanced floral diameter under the influence of GA3, while the plants of "Narangi" showed 28% increase in the floral diameter when treated with 800 mg·L−<sup>1</sup> GA3, as compared with control (Figure 2C). Floral diameter of both cultivars under the influence of foliar application of SA was non-significantly improved (Figure 2D). Conversely, exogenously applied IBA significantly enhanced the floral diameter of "Basanthi". Maximum floral diameter (6.25 cm) was recorded in the plants of the same cultivar receiving foliar application of 150 mg·L−<sup>1</sup> IBA, followed by 800 mg·L−<sup>1</sup> IBA which was 1.61 and 1.52-fold greater than those of untreated plants, respectively. The plants of "Narangi" showed non-significant but comparable results to control, except when treated with 300 and 800 mg·L−<sup>1</sup> IBA (Figure 2E). In the case of OA treatment, the plants treated with 300 mg·L−<sup>1</sup> OA showed maximum floral diameter (5.59 cm "Narangi", 4.95 cm "Basanthi") in both cultivars as compared to control (Figure 2F).

**Figure 2.** Floral diameter (cm) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses (mg·L−1) of ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

In case of flower fresh weight, "Narangi" showed significant response to ABA application. Plants of "Narangi" treated with 300 mg·L−<sup>1</sup> ABA exhibited 62.53% increase in flower fresh weight as compared to control (Figure 3A). Under the influence of NAD, the plants of "Basanthi" receiving foliar application of 300 and 800 mg·L−<sup>1</sup> NAD exhib-

ited 85.74 and 43.88% increase in flower fresh weight, respectively. "Narangi" exhibited 93.55% increase in flower fresh weight when received 100–250 mg·L−<sup>1</sup> NAD as compared to control (Figure 3B). In case of GA3 application, "Narangi" showed maximum flower fresh weight in plants treated with 800 mg·L−<sup>1</sup> (9.93 g) which was 145.26% greater than untreated plants. Meanwhile, the flower fresh weight of "Basanthi" increased up to 15% when treated with GA3 except when treated with 300 mg·L−<sup>1</sup> GA3 (Figure 3C).

**Figure 3.** Flower fresh weight (g) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses (mg·L−1) of ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

The foliar application of 250 mg·L−<sup>1</sup> SA showed maximum flower fresh weight (9.29 g "Narangi", 5.59 g "Basanthi") in both cultivars as compared to control (Figure 3D). Similarly, the plants of "Narangi" and "Basanthi" showed 110.23 and 92.80% increase in flower fresh weight when treated with 300 and 150 mg·L−<sup>1</sup> IBA, respectively (Figure 3E). The plants treated with foliar application of 300 mg·L−<sup>1</sup> OA showed maximum flower fresh weight (8.25 g "Narangi", 6.24 g "Basanthi") followed by 100 mg·L−<sup>1</sup> (7.83 g "Narangi", 5.19 g "Basanthi") in both cultivars (Figure 3F).

"Basanthi" exhibited a 6.16% increase in flower dry weight when treated with 250 mg·L−<sup>1</sup> ABA, while "Narangi" showed a 1.45-fold better response under the influence of 800 mg·L−<sup>1</sup> ABA as compared to untreated plants (Figure 4A). The plants of "Basanthi" receiving foliar application of 100, 300 and 800 mg·L−<sup>1</sup> NAD exhibited 3.90, 9.43 and 7.62% increase in flower dry weight, while the plants treated with 150 and 250 mg·L−<sup>1</sup> NAD showed a

1.47 and 0.845 decrease in flower dry weight as compared to control. Flower dry weight of "Narangi" increased up to 10% with 100–300 mg·L−<sup>1</sup> NAD as compared to control (Figure 4B). In case of GA3 application, "Narangi" and "Basanthi" showed maximum flower dry weight when treated with 800 and 100 mg·L<sup>−</sup>1, which were 8.15 and 3.91% more than those of untreated plants (Figure 4C). The foliar application of 150 and 250 mg·L−<sup>1</sup> SA showed 12.84 and 6.61% increment in flower dry weight of "Narangi" and "Basanthi", respectively (Figure 4D). Similarly, 800 and 300 mg·L−<sup>1</sup> IBA induced 6.09 and 6.47% increase in flower dry weight of the plants of "Narangi" and "Basanthi", respectively (Figure 4E). The plants treated with 300 mg·L−<sup>1</sup> OA exhibited maximum flower dry weight (1.41 g "Narangi", 0.84 g "Basanthi") (Figure 5F).

**Figure 4.** Flower dry weight (g) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses (mg·L<sup>−</sup>1) of ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

**Figure 5.** Total phenolic contents (mg·g−1) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses (mg·L<sup>−</sup>1) of ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

#### *3.2. Antioxidant Attributes*

Marigold plants of "Basanthi" treated with 150 mg·L−<sup>1</sup> ABA showed enhanced phenolic contents by 2.02-fold comparing with untreated plants (Figure 5A). Similarly, plants receiving foliar application of 150–800 mg·L−<sup>1</sup> NAD exhibited up to a 121.87% increase in total phenolics. Conversely, the plants treated with 100 NAD showed decreased phenolic contents by 8.11% as compared to control (Figure 5B). "Basanthi" and "Narangi" showed an 116.36 and 368.82% increase in total phenolics when treated with 150 and 100 mg·L−<sup>1</sup> GA3, respectively (Figure 5C). Plants of "Basanthi" receiving a foliar application of 250 mg·L−<sup>1</sup> SA exhibited maximum phenolic contents (229.63 mg·g<sup>−</sup>1) which were 103.53% more than those of untreated plants (Figure 5D). IBA enhanced the phenolics of both cultivars in a concentration dependent manner. Maximum phenolics (237.47 mg·g−1) were recorded in the plants of "Basanthi" receiving foliar application of 800 mg·L−<sup>1</sup> IBA followed by the plants of same cultivar treated with 300 mg·L−<sup>1</sup> IBA (Figure 5E). "Basanthi" and "Narangi" showed 397.63 and 70.46% increase in phenolics when treated with 150 and 300 mg·L−<sup>1</sup> SA, respectively (Figure 5F).

The flavonoid contents of "Narangi" were recorded 69.56% more as compared to control when treated with 250 mg·L−<sup>1</sup> ABA (Figure 6A). The plants of both cultivars receiving foliar application of 100, 150, 800 mg·L−<sup>1</sup> NAD exhibited a significant increase in total flavonoids, while the plants of "Narangi" treated with 250 and 300 NAD showed decreased flavonoid contents as compared to the control (Figure 6B). The plants of "Basanthi" and "Narangi" treated with 300 and 100 mg·L−<sup>1</sup> GA3 showed a 2.85- and 2.04-fold increase in flavonoid contents as compared to untreated plants (Figure 6C). In case of SA application, total flavonoids of "Basanthi" significantly increased with the application of 250 and 300 mg·L−<sup>1</sup> SA, while "Narangi" exhibited enhanced flavonoids as the result of 100 and 800 mg·L−<sup>1</sup> SA (Figure 6D). IBA at 250 and 800 mg·L−<sup>1</sup> enhanced the flavonoid contents of both cultivars by 1.89 and 2.88-fold, respectively. Maximum flavonoids (98.34 mg·g<sup>−</sup>1) were recorded in the plants of "Basanthi" receiving foliar application of 800 mg·L−<sup>1</sup> IBA (Figure 6E). The plants of "Basanthi" treated with 250 mg·L−<sup>1</sup> OA and "Narangi" treated with 150 mg·L−<sup>1</sup> OA showed maximum level of flavonoid contents (75.32 and 100.51 mg·g<sup>−</sup>1, respectively) (Figure 6F).

**Figure 6.** Total flavonoid contents (mg·g−1) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses (mg·L<sup>−</sup>1) of ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

The maximum floral DPPH free radical scavenging activity (29.32%) was observed in plants of "Basanthi" treated with 800 mg·L−<sup>1</sup> ABA which was 24.65% more than those of untreated plants (Figure 7A). The plants of "Narangi" treated with 100, 150 and 800 NAD, and "Basanthi" treated with 100 and 250–800 showed increase in DPPH activity up to 55 and 88%, respectively, as compared to control (Figure 7B). In case of GA3, plants of "Narangi" showed reduction in DPPH activity, while the plants of "Basanthi" showed increased DPPH activity in a dose dependent manner (Figure 7C). The graph of SA (Figure 7D) showed opposite trend among both cultivars. The plants of "Basanthi" receiving foliar application of 800 mg·L−<sup>1</sup> IBA exhibited maximum DPPH scavenging activity (35.73%) followed by the plants of "Narangi" receiving 150 mg·L−<sup>1</sup> IBA (30.77%), which were 169.25 and 28.20% more than those of untreated plants of respective cultivars (Figure 7E). The exogenously applied OA also enhanced the DPPH of both cultivars except when applied at the concentration of 800 and 150 mg·L−<sup>1</sup> in "Basanthi" and "Narangi", respectively (Figure 7F).

**Figure 7.** The DPPH free radical scavenging activity (%) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses of (mg·L−1) ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

ABA enhanced the reducing power ability (RPA) of the plants of "Basanthi" by 1.67-fold when treated with 100 mg·L−<sup>1</sup> (Figure 8A). Similarly, plants receiving foliar application of NAD exhibited 1.6-times more RPA as compared to control (Figure 8B). In the case of GA3 treatment, plants of "Basanthi" treated with 300 mg·L−<sup>1</sup> and plants "Narangi" treated with 100 mg·L−<sup>1</sup> showed maximum RPA (84.72 and 84.45 mg·g−1, respectively), which were 59.03 and 44.48% more than those of untreated plants (Figure 8C). The RPA of

both cultivars under the influence of foliar application of SA was significantly improved. Plants of "Basanthi" receiving foliar application of 250 mg·L−<sup>1</sup> SA exhibited a 59.21% increase in RPA as compared to the control, which was maximum among all other dose of SA (Figure 8D). The exogenously applied IBA enhanced the RPA of both cultivars except when treated at the concentration of 800 mg·L<sup>−</sup>1. Maximum RPA (84.09 mg·g−<sup>1</sup> "Narangi", 78.83 mg·g−<sup>1</sup> "Basanthi") was recorded in the plants receiving a foliar application of 150 mg·L−<sup>1</sup> IBA (Figure 8E). In the case of OA treatment, "Basanthi" and "Narangi" were treated with 300 and 150 mg·L−1, respectively, showed maximum RPA (77.23 and 80.36 mg·g<sup>−</sup>1, respectively) (Figure 8F).

**Figure 8.** The reducing power ability (mg·g<sup>−</sup>1) of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda") as affected by different doses of (mg·L<sup>−</sup>1) ABA (**A**); NAD (**B**); GA3 (**C**); SA (**D**); IBA (**E**) and OA (**F**). Same letters indicate non-significant difference among treatments under Fisher's least significant difference test (*p* ≤ 0.05). Vertical bars indicate average ± standard error (three replications, under bi-factorial CRD).

#### *3.3. Principle Component Analysis*

The efficacy of PGRs to modulate plant physiology depends on its concentration, application method and plant genetics [15]. Results from this study also showed that the response of marigold flower growth and antioxidant capacity to PGRs application changed with a change in the concentration and type of hormone. So, principal component analysis was conducted to delineate the concentration and PGR-dependent effects (Figure 9). Based on the highest squared cosine value corresponding to factors F1, F2 or F3, flower growth and quality attributes were clustered around PGR treatments. Factor F1, covering

20.23% variability in data (eigenvalue 2.833), showed clustering of flower diameter, flower fresh weight, flower dry weight and DPPH activity of "Basanthi" with 150–300 mg·L−<sup>1</sup> NAD, 100 mg·L−<sup>1</sup> SA, 150 mg·L−<sup>1</sup> IBA, 300 mg·L−<sup>1</sup> IBA and 150 mg·L−<sup>1</sup> OA suggesting a positive influence of these treatments on these parameters. The second factor, covering 18.81% variability in data (eigen value 2.634), showed clustering of flower diameter, flower fresh weight and flower dry weight of "Narangi" with 150 mg·L−<sup>1</sup> ABA, 800 mg·L−<sup>1</sup> NAD, 150 mg·L−<sup>1</sup> GA3, 800 mg·L−<sup>1</sup> GA3 and 800 mg·L−<sup>1</sup> OA. However, the distribution of clusters in two distinct groups on opposite sides of F1 axis indicated that application of the aforementioned PGRs had strong negative correlations with flower diameter, flower fresh weight and flower dry weight of "Narangi". The third factor of principal component analysis, covering 13.41% variability in data (eigenvalue 1.877; not shown), showed clustering of total phenolics and reducing power ability of "Narangi" with foliar application of 300 mg·L−<sup>1</sup> ABA, 100 mg·L−<sup>1</sup> NAD, 100 mg·L−<sup>1</sup> GA3 and 250–300 mg·L−<sup>1</sup> OA. Thus, principal component analysis helped to delineate individual roles of PGRs with respect to their concentrations in regulating various aspects of flower growth and antioxidant attributes of African marigold.

**Figure 9.** Principal component analysis among PGRs treatments and various flower growth and antioxidant attributes of two cultivars of marigold ("Pusa Narangi Gainda" and "Pusa Basanthi Gainda"). The treatments are shown in green colour, while variables of "Narangi" and "Basanthi" are indicated by red and blue coloured labels, respectively. Abbreviations: FD—flower diameter; FFW—flower fresh weight; FDW—flower dry weight; TP—total phenolics; TF—total flavonoids; DPPH—DPPH free radical scavenging activity; RPA—reducing power ability.

#### **4. Discussion**

#### *4.1. Flowering Attributes*

Plant growth regulators play an important role in flower production, which in small amounts promotes or inhibits or quantitatively modifies growth and development [23]. In the current study, the impact of different PGRs on floral size, weight, and some antioxidant attributes of two cultivars of African marigold, i.e., "Basanthi" and "Narangi" was evaluated. In terms of floral diameter, both cultivars differentially responded to different PGRs. "Basanthi" exhibited a 60% increase in floral diameter under the influence of 150 mg·L−<sup>1</sup> IBA, while "Narangi" showed its maximum response (48% enhancement) under 300 mg·L−<sup>1</sup> OA. ABA, NAD, GA3, and SA also increased the floral diameter of marigold up to 35, 48, 34 and 34%, respectively (Figure 2). Riaz et al. [6], Mitchell and Stewart [24], and Dhuma et al. [25] reported similar results in marigold and tuberose under the influence of ABA and NAD. The GA3-induced increment in floral diameter might be due to more synthate translocation to only a fewer sink. A similar effect of GA3 was reported earlier in marigold and chrysanthemum [26–30]. The increase in floral diameter under the influence of SA was stated as the result of increased CO2 assimilation, photosynthetic rate and mineral uptake as supported by previous studies [31]. Some researchers reported similar findings in calendula and marigold [32,33]. IBA plays a vital role in increasing cell division [11], and hence found a promising way to improve floral diameter of marigold. Our results about the influence of IBA and OA on floral diameter of marigold are supported by previous findings in marigold, red firespike (*Odontonema strictum*), and henna (*Lawsonia Inermis*) [34–37].

It was reported earlier that ABA is consistently effective at reducing water loss and increasing flower fresh weight of bedding plants [38]. NAD represents one of the cornerstones of cellular oxidation and is essential for plant growth and development [39]. In our findings, "Narangi" exhibited 145% more fresh weight while receiving 800 mg·L−<sup>1</sup> GA3, as compared to untreated plants, 57% more than the maximum observed in the plants of "Basanthi". The plants of both cultivars treated with PGRs exhibited more flower fresh weight as compared to untreated plants (Figure 3). The increase in fresh weight of marigold flowers with GA3 might be due to the production of bigger sized flowers. This might be attributed to rapid synthesis in the cell, increase in cell size, cell elongation and rapid translocation of assimilates to sink under the influence of phytohormones [40]. SA is an emerging plant growth regulator that acts as signaling molecule in plants under biotic and abiotic stresses in marigold. SA also exerts a stimulatory effect on different physiological processes of plant growth [41]. In case of IBA treatment, the plants of both cultivars treated with 150 mg·L−<sup>1</sup> IBA exhibited maximum flowers fresh weight. The reason behind the increase in flowers' fresh weight might be the enhancement of photosynthesis and maximum accumulation of photosynthates due to IBA application [42]. Similar results were also reported in marigold [35,43,44].

In terms of flower dry weight, "Narangi" responded better to PGRs as compared to "Basanthi". The maximum flower dry weight under the influence of PGRs was observed in the plants of "Narangi" when treated with 300 mg·L−<sup>1</sup> OA. The plants of "Basanthi" receiving similar dose of NAD exhibited 69% more flower dry weight as compared to those of untreated plants. Other applied PGRs i.e., ABA, GA3, SA and IBA induced a significant increase in flower dry weight of marigold up to 45, 53, 84 and 47%, respectively (Figure 4). The results about ABA and GA3-induced floral dry weight are supported by previous findings, stating the role of PGRs in reducing water loss in the plants [28,45–49]. The results about IBA-induced flower dry weight was supported by Pacheco et al. [50] and Choudhary et al. [51] in marigold, and Pal [52] in calendula. Moreover, it was also reported that dry weight is well known to exhibit a high positive correlation with fresh weight of marigold flowers [53,54].

#### *4.2. Antioxidant Attributes*

Phenolic compounds are the secondary metabolites acting as antioxidants due to their hydroxyl group [55]. PGRs play an important role in improving antioxidant capacity of the plants. ABA was reported as having a key role in the enhancement of antioxidant capacity, anthocyanins and phenolic content of bedding plants (i.e., *Impatiens walleriana*, *Pelargonium hortorum*, *Petunia hybrida*, *Tagetes patula*, *Salvia splendens*, and *Viola wittrockiana*) [38]. In the current study, the total phenolics (floral extract) of both cultivars of *Tagetes erecta* were significantly influenced by different PGRs. Moreover, "Narangi" responded better to PGRs as compared to "Basanthi". The maximum phenolics were observed in the plants of "Narangi" and "Basanthi" under the influence of 300 mg·L−<sup>1</sup> OA and 800 mg·L−<sup>1</sup> NAD (Figure 5).

The effect of different PGRs on total flavonoid contents of floral extract of *Tagetes erecta* was found significant. ABA, NAD, GA3, SA, IBA and OA increased floral flavonoids of marigold up to 70, 114, 185, 114, 188 and 120%, respectively. Marigold plants treated with maximum dose (800 mg·L−1) of PGRs found having maximum flavonoids among other PGR doses (Figure 6). The increase in flavonoid contents was earlier reported in *Taraxacum officinale* [56] and *Zinnia elegans* [57] under the influence of GA3 and SA application, respectively. IBA stimulated the production of total flavonoids in *Thymus vulgaris* and *Origanum vulgare*, but decreased it in *Ocimum basilicum* [58,59]. Likewise, OA-induced flavonoid contents were reported in *Bellis perennis* [60].

The DPPH free radical scavenging activity is considered as an acceptable mechanism to evaluate the antioxidant activity of plants [61]. The plants of "Basanthi" showed a variation of DPPH activity from the lowest value (7%) to the highest value (35.8%). The plants treated with 100 and 800 mg·L−<sup>1</sup> IBA showed the highest value of free radical scavenging activity. Similarly, the plants of "Narangi" also showed varied DPPH activity from the lowest value (10%) to the highest value (35.8%). The plants of "Narangi" treated with 300 mg·L−<sup>1</sup> OA exhibited maximum DPPH activity (Figure 7). Previously, it was reported that methanolic extract of *Tagetes erecta* exhibited 15.63 to 95.34% DPPH scavenging activity [62]. The antioxidant activity of plant extracts was found strongly associated with the reducing power of bioactive compounds [63]. Thus, the reducing power ability of marigold plants were determined. Our results suggest that the exogenously supplied PGRs significantly improved the reducing power ability of both cultivars (Figure 8). Similar findings in marigold were reported by some researchers [62,64].

#### **5. Conclusions**

PGRs play an important role in flower production, which in small amount promotes or inhibits or quantitatively modifies growth and development. In the present study, foliar application with different concentrations (0, 100, 150, 250, 300 and 800 mg·L−1) of ABA, NAD, GA3, SA, IBA and OA proved to be successful for enhancing flowering and antioxidant activity of two cultivars of African marigold. This was evidenced by the improved diameter, fresh weight, dry weight, phenolics, flavonoids, DPPH free radical scavenging activity and reducing power ability of flowers, in which the doses were increased to a certain extent, after then a detrimental effect, although not lethal, was registered. Since foliar applications of PGRs differentially regulate distinct aspects of flowers growth, specific concentrations of PGRs may help to achieve some specific quality objectives of the flowers, commercially valuable. Among the foliar applied PGRs, 150 mg·L−<sup>1</sup> IBA proved to be superior in terms of maximum floral diameter of "Basanthi", whereas the maximal flower fresh weight was recorded in the plants of "Narangi" receiving foliar application of 800 mg·L−<sup>1</sup> GA3. Regardless of the concentration, OA significantly improved flower dry weight, total phenolic contents, total flavonoid contents and reducing power ability of "Narangi". The plants of "Basanthi" treated with 100 mg·L−<sup>1</sup> IBA exhibited maximum DPPH free radical scavenging activity. The highest dose of the PGRs viz. 800 mg·L−<sup>1</sup> was applied to evaluate its lethal effects on plant health. The results proved that this dose was not harmful for plants, indicating the African marigold as a hardy plant. Overall, PGRs

at specific concentrations may be used as an effective exogenous application strategy to improve the flowering and antioxidant capacity of marigold flowers.

**Author Contributions:** Conceptualization, S.S., M.M.A. and S.N.; methodology, S.S. and M.U.; statistical analysis, M.M.A. and A.F.Y.; data curation, M.M.A.; writing—original draft preparation, S.S. and M.M.A.; writing—review and editing, M.U.H., M.A., S.G. and S.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Authors would like to thank all field technicians at Institute of Horticultural Sciences, University of Agriculture, Faisalabad.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Studies of Vegetative Growth, Inflorescence Development and Eco-Dormancy Formation of Abscission Layers in** *Streptocarpus formosus* **(Gesneriaceae)**

**Cherise Christina Viljoen, Muhali Olaide Jimoh and Charles Petrus Laubscher \***

Department of Horticultural Sciences, Faculty of Applied Sciences, Cape Peninsula University of Technology, P.O. Box 1906, Bellville, Symphony Way, Cape Town 7535, South Africa; C.Viljoen@sanbi.org.za (C.C.V.); Jimohm@cput.ac.za (M.O.J.)

**\*** Correspondence: LaubscherC@cput.ac.za

**Abstract:** *Streptocarpus formosus* (Hilliard & B.L. Burtt) T.J. Edwards is a flowering herbaceous perennial indigenous to South Africa and is part of the rosulate group of herbaceous acaulescent plants within the Gesneriaceae family. According to the National Assessment database for the Red List of South African Plants version 2020.1., the plant is listed as rare. The ornamental use of *S. formosus* has untapped commercial potential as a flowering indoor pot plant, an outdoor bedding plant for shade and as a cut flower for the vase, all of which are limited by a five-month eco-dormancy period during the late autumn and all through the cold season in the short-day winter months. Viable commercial production will require cultivation techniques that produce flowering plants all year round. This study investigated the effectiveness of applying root zone heating to *S. formosus* plants grown in deep water culture hydroponics during the eco-dormancy period in preventing abscission layer formation and in encouraging flowering and assessed the growth activity response of the plants. The experiment was conducted over eight weeks during the winter season in the greenhouse at Kirstenbosch Botanical garden in water reservoirs, each maintained at five different experimental temperature treatments (18, 22, 26—control, 30 and 34 ◦C) applied to 10 sample replicates. The results showed that the lowest hydroponic root zone temperature of 18 ◦C had the greatest effect on the vegetative growth of *S. formosus*, with the highest average increases in fresh weight (1078 g), root length (211 cm), overall leaf length (362 cm) and the number of newly leaves formed (177 = n), all noted as statistically significant when compared with the other water temperature treatments, which yielded negative results from reduced vegetative growth. Findings from the study also revealed that while all heated solutions significantly prevented the formation of abscission layers of *S. formosus*, they had a less significant effect on inflorescence formation, with only 18 ◦C having the greatest positive effect on flower development.

**Keywords:** abscission; cape primrose; eco-dormancy; flowering pot plant; hydroponics; Gesneriaceae; root zone heating; phyllomorphy; *Streptocarpus formosus*

#### **1. Introduction**

Within the Gesneriaceae, Streptocarpus form part of an economically important ornamental plant group with other significant members such as *Saintpaulia* spp. (African Violets), *Gloxinia* spp. and *Sinningia* spp. [1], all of which are herbaceous perennials known for the beauty of their flowers [2,3]. In its wild habitat in the Eastern Cape province of South Africa, *Streptocarpus formosus* flowers only in long-day, warm, summer months of the year [1,4]. *S. formosus* grows naturally in a summer rainfall locality with very little irrigation through precipitation during the cold season [4]. This abiotic combination of reduced water and low temperatures triggers a survival tactic where the nutrients and carbohydrate reserves in the leaves are transported and remobilized to actively growing

**Citation:** Viljoen, C.C.; Jimoh, M.O.; Laubscher, C.P. Studies of Vegetative Growth, Inflorescence Development and Eco-Dormancy Formation of Abscission Layers in *Streptocarpus formosus* (Gesneriaceae). *Horticulturae* **2021**, *7*, 120. https://doi.org/ 10.3390/horticulturae7060120

Academic Editor: Piotr Salachna

Received: 25 March 2021 Accepted: 28 April 2021 Published: 21 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

parts of the plant causing yellowing of the part, or all, of the leaves [5,6] before the plants enter a survival state of eco-dormancy [7].

This annual process in *Streptocarpus* with flowering occurring mostly under 15 h long days as compared to 8 h short days [8] and combined with the slowed short-day growth processes of eco-dormancy and the shedding of leaf mass through unsightly abscission layers severely limits the ornamental commercial use of *Streptocarpus formosus.* Therefore, cultivation methods to keep plants looking attractive in active growth and to extend the flowering season are required [8,9]. The manipulation of flowering is an important aspect of the cultivation of many horticultural crops [10]. There is a high demand for flowering pot plants during all seasons, even in winter when temperatures are below optimum for flower production [11] and annual plant senescence due to low temperature causes yield reduction that results in significant economic losses to growers [12–14]. Root temperature is one of the key environmental factors that control plant growth and physiological activities in cold seasons [15,16]. Manipulating root zone temperature to keep plant crops and ornamentals actively growing for commercial out-of-season production has been comprehensively researched purposely to meet market demands [17–20].

Growth cessation, abscission formation and dormancy development, all of which are exhibited by *S. formosus*, are considerably affected by temperature [21]. Leaf loss is a physiological strategy for the avoidance of water stress in plant species adapted to drought, reducing the transpiring surface of the foliage and thereby lessening water demand [21,22]. However, leaf senescence is mainly caused by cold and less commonly by high temperature [5,23]. In *Streptocarpus formosus* with no predetermined abscission zone, leaves are either shed entirely or a 2–3 mm wide demarcation line [24] forms on the leaves and a visible difference between the basal and distal sections of the lamina is distinguishable with a dark green base and a bright yellow upper section [25]. This partial senescence in *Streptocarpus* is a perennation mechanism that ensures the protection of the basal meristem [24,26]. When the distal leaf section is completely brown and dry, this part breaks away cleanly along the abscission layer [27,28] and the leaf can continue to lengthen with new growth from the base [3,27].

The chlorophyll content is an important criterion when evaluating the ornamental value of a pot plant [29]. The degradation of chlorophyll is the cause of leaf yellowing during senescence [28,30,31]. Various studies have shown the positive effect of heated water on the retention of chlorophyll and the increased amount present within leaves [11,16,32]. The optimum temperature of the growth medium can contribute beneficially to plant physiological processes such as chlorophyll pigment formation, the accumulation of phenolic compounds and an increase in photosynthetic capacity [11].

Heat is required to increase growth to expand plant production during the cold season and can be provided with the use of greenhouses [30], and it can be combined with hydroponic growing which has become common practice to improve winter yields and to obtain optimum production under periods of suboptimal climatic conditions [11,31]. Additional benefits of heating the nutrient solution are the provision of the energy requirements for plant development, activating metabolism [32] and a reduction in pathogenic activity [33]. The application of root zone heating in a closed hydroponic system enables the volume of water to buffer temperature and contributes to energy savings compared with the expense of heating entire greenhouse structures [11,33]. With a notable global increase in the scarcity of resources and climate change, hydroponics offers workable solutions by achieving optimal growth yield and good quality crops due to the precise control of nutrition and growing conditions [17,34,35]. Yields in hydroponics average at 20–25% higher than in conventional soil cultivation and have demonstrated significantly more growth and development in root systems, which also improves the nutrient uptake ability of the plants which, in turn, leads to better shoot and leaf growth [34].

This study was designed to investigate the possibility of achieving optimum growth during the winter season by determining how the application of root zone heat could viably facilitate the ornamental production of *S. formosus*, and to evaluate the effects of different regimes of root zone temperature on abscission layers activated by eco-dormancy and the earlier formation of *S. formosus* flowers. It was also envisaged that this study would assist in determining an optimal temperature for the active growth and inflorescence formation of *S. formosus* to produce consistently high vegetative growth and flowers for cultivating superior quality pot plants in hydroponics to benefit the ornamental and floriculture industries.

#### **2. Materials and Methods**

#### *2.1. Greenhouse Experiment*

The experiment was conducted over 8 weeks during winter in the greenhouse facility at the Kirstenbosch National Botanical Garden (KNBG), Cape Town, South Africa (33◦98 56.12 S, 18◦43 60.25 E) from mid-June 2019 to mid-August 2019. Plants were grown under natural daylight conditions which provided the short-day photoperiod, 9:59:26 hr day length (15th June) to 10:54:49 hr day length (15th August), required for the experiment as *S. formosus* is then in the eco-dormancy period of its annual vegetative growth [1]. An overhead Aluminet shade net screen provided 40% shading and minimized temperature fluctuations. Maximum day temperatures ranged between 13 ◦C and 18 ◦C and night temperatures between 3 ◦C and 7.8 ◦C, with an average relative humidity between 77 and 81%.

#### *2.2. Plant Preparation*

Fifty genetically identical *S. formosus* plantlets were propagated vegetatively (Figure 1a) from one *S. formosus* mother plant. After the rooting period of four months (Figure 1b), the plantlets were thoroughly rinsed to remove the rooting media and all foreign matter from their leaves and roots. They were then potted into lattice-net plastic pots filled with 4–10 mm lightweight expanded clay aggregate (LECA) and placed in the hydroponic system with only their roots submerged in water. LECA was the preferred soilless growth medium for this study because its lightweight properties, with added porosity, would not degrade in the water while its pH remained neutral with the additional advantage of protecting the roots with its thermal insulation properties [36].

(**a**) Leaf cuttings freshly done (**b**) Leaf cuttings matured with plantlets

**Figure 1.** Leaf cuttings of *S. formosus* provided n = 50 plants cultivated from one initial mother plant obtained from Kirstenbosch National Botanical Garden, Cape Town (Photos: C. Viljoen).

#### *2.3. Hydroponic Cultivation*

A closed deep water hydroponic system with an air stone and a circulating pump was used based on the recommendations, discussions and methodologies of [11,37,38]. Deepwater hydroponics allows for methods of heating the nutrient solution to the required temperature and maintains a consistent nutrient supply and temperature over the entire root surface area of the replicates. Closed hydroponics systems allow for the reuse of nutrient solution, reducing the negative environmental impacts such as leaching of fertilizers, soil and groundwater pollution, and water wastage while saving on labor. LECA provided support for the plants needing to be suspended in the nutrient solution while providing excellent aeration qualities [11,36].

Five identical deep water hydroponic systems were constructed and placed onto wire mesh tables. Each system consisted of one 70 L capacity low-density polyethylene (LDPE) reservoir filled with 60 L of aqueous nutrient solution. Each reservoir was covered with an LDPE sheet into which holes were cut to hold the 10 lattice-net (7.5 cm) plastic pots suspended (Figure 2). The pot size and depth ensured that the root zones of the plants were submerged in the nutrient solution without wetting the plant's leaf crowns, avoiding possible crown rot. To prevent oxygen deficiency and the limitations this would place on the plant growth, root aeration is essential in a hydroponic system, especially in deep water culture where there is limited air-water exchange capacity and particularly when heating the solution as there is a direct correlation between the temperature of water and the amount of oxygen it contains [35,39]. As water temperature increases, less oxygen becomes available to the roots [40], so to increase aeration all the solutions were aerated using one electromagnetic air compressor (BOYU ACQ-003) linked to each system's single air stone (50 mm), which bubbled the air up through the nutrient solution at a rate of 50 L per minute, supplying oxygen to the roots of the plants. To assist with the even distribution of both the additional air (O2) and the heated water [37], each system's solutions were circulated using an 800 L/h hour HT submersible pump (HJ-941).

**Figure 2.** The closed hydroponic deep water culture system used for this study with air stone and circulating pump, and plants in lattice-net pots filled with LECA aggregate held suspended in nutrient solution (Diagram by J.D. Viljoen).

The solution comprised of ozone-treated borehole water containing Nutrifeed at a dilution rate of 1:500 (120 g in 60 L), as specified by the manufacturer Starke Ayres Pty. Ltd. Hartebeefontein Farm, Bredell Rd, Kaalfontein, Kempton Park, Gauteng, 1619, South Africa. This nutrient product supplied all the essential macro and micronutrients (6.5% Nitrogen, (N), 2.7% Phosphorous (P), 13.0% Potassium (K), 7.0% Calcium (Ca), 2.2% Magnesium (Mg), 7.5% Sulphur (S), plus Iron, Manganese, Boron, Zinc, Copper and Molybdenum) required for healthy plant growth as hydro-soluble fertilizer salts [38]. As the experiment would fall within a two-month growth period, it was decided that replacing the nutrient solution to overcome the build-up of phototoxic substances in the nutrient solution would not be required, to prevent potential disturbance damage to the roots [41,42].

The pH levels of all the nutrient solutions were monitored biweekly using a calibrated hand-held digital pH meter (HM Digital PS PH-200) and kept within a range of 6.4–7.0, a slightly acidic level recommended by [43]. The pH was adjusted accordingly using either sodium hydroxide (NaOH) to raise the pH, or hydrochloric acid (HCL) to lower

the pH [42]. The various temperatures of the five test solutions were also measured for monitoring consistency. The electrical conductivity (EC) level of each system was kept within a 0.9–1.1 dSm-1 range as suggested for *Streptocarpus* by [44] and was used as a measure of the nutrient concentration of the solution. The EC levels and temperatures of all the nutrient solutions were monitored biweekly using a calibrated handheld (PS COM-100) EC and temperature meter produced by HM Digital Inc., Culver City, CA, USA 90230. For decreasing the EC of aqueous nutrient solutions, ozone-treated borehole water was added into reservoirs, while adding 1:500 diluted Nutrifeed™ solution increased EC levels.

#### *2.4. Water Temperature Treatments and Experimental Design*

The experiment consisted of five different hydroponic solution temperatures which were applied to 50 plants of *S. formosus* using a completely randomized block design (Figure 3; Table 1). Each temperature treatment consisted of 5 treatments with 10 replicates (n = 10), one per pot suspended in a closed deep water culture system. Pots were individually numbered and arranged randomly. The five test solutions were heated using submersible EHEIM (Plochinger Str. 54 73779 Deizisau, Germany) thermo control manually adjustable heaters as standard aquarium equipment.

**Figure 3.** A completely randomized experimental block design used for the investigation (Diagram: J.D. Viljoen).



The water temperature range applied was based on the ideal temperature recommendations for growing *Streptocarpus* [45], Gesneriad *Sinningia cardinalis*, [46] and other perennials [11,16], which were proven beneficial to both vegetative and inflorescence development in each case. The mean annual temperature at Port St Johns, which is *S. formosus*' natural habitat, is 19.9 ◦C as recorded between 1961 and 1990, with a mean summer minmax of 17.1 ◦C–27.6 ◦C when the plants are in full growth and flowering, as opposed to the mean winter min-max of 7.4 ◦C–20.5 ◦C when the plants are eco-dormant [47]. This experiment focused on applying a similar summer temperature range to the root zone to test whether the plants could thus be stimulated into active growth and flowering during the colder winter months, and WT3 at 26 ◦C was selected for the control as the literature reviewed indicated this to be both the ideal ambient air temperature for *Streptocarpus* under non-experimental circumstances, and a common root zone median temperature for root to shoot ratios under experimental conditions for a selection of perennial crops [11,16,32,48,49].

#### *2.5. Vegetative Growth and Data Collection*

Various measurements were taken to determine plant growth response to different nutrient solution temperatures on leaf quantity, leaf lengths, root lengths and fresh weights. Data capturing took place pre-planting and at the time of planting the plants into the quantitative research experiment system, and again post-harvest after a two-month growth period.

Before planting, each entire plantlet (roots and shoots together) was weighed for a fresh wet measurement, using an electronic laboratory scale (Sartorius Analytical Balance Scale Model type 1518) with 0.001 g readability. Additionally, at this time the lengths of both the shortest and longest leaves, as well as root length for each plantlet, were measured using a ruler [50] and recorded. The measurement of leaves was taken from the growth media level to the apex point of each leaf. All present and emerging leaves were measured, but not if less than 2 mm in length. The root length of each plant was measured by the points at which roots emerged from the stem to the tip of the root mass. Immediately after being transplanted into the LECA filled pots and placed in the deep water culture solutions, the total number of present and emerging leaves on each plantlet was then counted, but not if less than 2 mm in length, and recorded. At post-harvest, these same measurements were repeated, and the data recorded.

#### *2.6. Inflorescence Data Collection*

Immediately after being transplanted into the LECA filled pots and placed in the hydroponic test solutions, measurement of various floral parts was performed. The numbers of inflorescence stalks per plant were counted and recorded, including all present and emerging pedicels, but not if less than 2 mm in length, and the numbers of flower buds and flowers per plant were counted and recorded. After a two-month growth period, at postharvest, these same counts were repeated and the data recorded and analyzed to determine inflorescence development in response to different nutrient solution temperatures.

#### *2.7. Eco-Dormancy Data Collection*

Immediately after being transplanted into the LECA filled pots and placed in the hydroponic test solutions, the number of abscission layers present in the leaves per plant was counted and recorded. At post-harvest, the presence or absence of abscission layers was recounted and the data recorded.

#### *2.8. Statistical Analysis*

All data collected were statistically analyzed using one-way analysis of variance (ANOVA) and computed by the software program TIBC STATISTICA Version 13.6.0. The ANOVA test was used to determine if there was a statistically significant difference between each group of water temperature's mean value. A within-between ANOVA mixed model was applied to examine for potential differences in a continuous level variable between the treatment and the control group, and over time with pre and post-tests. The occurrence of statistical difference was determined by using the Fisher Protected Least Significance Difference (L.S.D.), a pair-wise comparison technique for the comparison of two means, at values of *p* < 0.05; *p* < 0.01 and *p* < 0.001 levels of significance [51].

#### **3. Results**

#### *3.1. Total Leaf Number*

There was an interaction between hydroponic root zone temperature and the final numbers of leaves produced by the plants. The increase in the number of leaves was highly significant (*F*1,4 = 34.27, *p* ≤ 0.0001), and the WT1 (18 ◦C) treatment showed a greater increase in leaves compared to the control WT3 (26 ◦C) treatment by week 8 of

the experiment (Table 2). The greatest increase in leaf numbers occurred at the lowest temperature treatment 18 ◦C, with a mean of 17.7 when compared to the 26 ◦C control with a mean of 6.7. Leaf numbers also displayed notably poorer results in WT2 (22 ◦C), mean 13.3, and WT4 (30 ◦C), mean 3.3. WT5 (34 ◦C) resulted in almost complete leaf fatality.

**Table 2.** The interaction of various root zone water temperatures on the overall vegetative growth of *S. formosus.*


Note: Values presented are means ± SE. The mean values followed by different letters are significantly different at *p* ≤ 0.001 (\*\*\*) as calculated by Fisher's least significant difference and those followed by the same letter are not significantly different.

#### *3.2. Total Leaf Length*

There was an interaction between root zone water temperature and final leaf length produced by the plants with a highly significant *F*-statistic (*F*1,4 = 49.02, *p* ≤ 0.0001). The 18 ◦C (WT1) treatment with a mean of 36.22 cm had the highest reading compared to the control 26 ◦C (WT3) treatment, mean 6.68 cm, or any of the other treatments by the final 8th week of the experiment (Table 2). There was thus a significant reduction in the rate of leaf length development at both WT2 22 ◦C (15.85 cm) and WT3 (6.68 cm), with a further reductive loss in leaf length in WT4 30 ◦C (−2.18 cm). This sharp decline was visually observed in the leaf health, quality and length, as temperature increases to 34 ◦C (WT5) compared to the control WT1 of 18 ◦C (Figure 4) yielded the largest increase in leaf length.

**Figure 4.** The visible effect of the escalating hydroponic root zone temperatures on the vegetative aerial parts of *S. formosus* evident through simple observation over the experimental period with a directly proportional reduction in leaf numbers and lengths at increasingly higher temperatures (Photos: C. Viljoen).

#### *3.3. Total Root Growth*

The statistical analysis in Table 2 indicates the greater significant values (*F*1,4 = 69.63, *p* ≤ 0.0001) with root growth than with the aerial parts of the plant, and indicates that WT1 (18 ◦C) demonstrated a notable 210.5 cm overall increase in root length, compared with only 4.5 cm at WT2 (22 ◦C), versus the overall negative growths of −5.4 cm for the control WT3 (26 ◦C) and −23.3 cm at WT4 (30 ◦C) treatment, with the complete death of the roots at WT5 (34 ◦C) treatment. Figure 5 presents the treatment interaction effect on the total root growth of the *S. formosus* plants and it indicates that heat in the root zone is a severely limiting factor when heating above a critical temperature range. Root zone temperatures at 22 ◦C resulted in poor root development and all the temperatures above resulted in no development and sharply declining growth or death of the *S. formosus* plant's root system.

**Figure 5.** The relationship between root zone water temperature treatments and the relative rates of increase and decrease in root length.

#### *3.4. Total Fresh Weight*

Combined root and leaf fresh weights, as shown statistically in Table 2, were significantly affected by the root zone temperature (*F*1,4 = 23.75, *p* ≤ 0.0001), and the results show that incremental increases in water temperature treatments from 18 ◦C to 34 ◦C decreased fresh weight, to the point of notable fatality at the highest temperatures of 30 ◦C and 34 ◦C, clearly visible in Figure 4. The WT1 (18 ◦C) treatment offered the highest significant increase in overall fresh weight and vegetative growth when compared to the control WT3 (26 ◦C) and all the other treatments: WT2 (22 ◦C), WT4 (30 ◦C) and WT5 (34 ◦C). Findings from this study established that increasing hydroponic root zone solution temperature beyond the 18 ◦C–20 ◦C range did not promote the overall growth and development of *S. formosus* when compared with the significant increase in biomass growth in WT1, 18 ◦C, yielding a total of 107.90 g, which is equivalent to a 400% increase over 8 weeks.

#### *3.5. Flowering in Response to Five Different Temperature Regimes in Hydroponics*

The interaction between root zone heating and the inflorescence development of *S*. *formosus* was found to be statistically significant (Table 3), in the flower and bud formation (*F*1,4 = 4.72, *p* ≤ 0.01) as well as the pedicel development (*F*1,4 = 4.72, *p* ≤ 0.001). The highest individual mean value was evident in treatment WT1 18 ◦C (Figure 6); both for numbers of flowers and buds (mean 2.5) and the number of pedicels (mean 5), indicating

that higher root zone temperatures WT2 (22 ◦C), WT3 (26 ◦C), WT4 (30 ◦C) and WT5 (34 ◦C) incrementally decreased inflorescence formation.

**Table 3.** The effect of various root zone water temperatures on the total flower development of *S. formosus.*


Values presented are means ±SE. The mean values followed by different letters are significantly different at *p* ≤ 0.01 (\*\*) and at *p* ≤ 0.001 (\*\*\*) as calculated by Fisher's least significant difference and those followed by the same letter are not significantly different.

**Figure 6.** The relationship between root zone water temperatures and inflorescence formation. Note: The bars presented here are means ± SE. Bars with different letters are significantly different at *p* ≤ 0.01 (\*\*) (total number of flower and buds; overall inflorescence formation) and *p* ≤ 0.001 (\*\*\*) for the total number of pedicels as calculated by Fisher's least significant difference.

Conversely, the lowest temperature of 18 ◦C (WT1) significantly increased the inflorescence formation of *S. formosus.* Flowers were evident at lower root zone temperatures of 18 ◦C and 22 ◦C compared to the control treatment at 26 ◦C (WT3) or the higher temperatures. Increasing water temperature in the range from 26 ◦C to 34 ◦C not only decreased inflorescence formation but led to total fatality of the plants at the highest temperature of 34 ◦C (WT5), as seen in Figure 6. A positive finding is that at 18 ◦C (WT1) flowers did develop during colder short-day periods, which indicates a strong possibility that manipulating the growing temperatures could induce *S. formosus* to flower earlier in the season, thereby extending the flowering period for an all-round year commercial marketing period.

#### *3.6. Reduction in Abscission Layers in Response to Five Different Temperature Regimes in Hydroponics*

As shown in Figure 7, the effects of root zone water temperature on the reduction in abscission layers already present on the *S. formosus* replicates' leaves were statistically significant at a value (*F*1,4 = 19.85, *p* < 0.0005). The few abscission layers that were present at the time of the experiment's inception all disappeared (Figure 8); however, more significantly, no abscission layers formed on any plants in the heated treatments during the winter period as would usually naturally occur (Figure 9a,b). Treatments applied in this study indicate that root zone heating is a viable method for overcoming and preventing the formation of abscission layers.

**Figure 7.** Root zone heating has the effect of minimizing abscission layers on *S. formosus.* Note: The line graph presented here depicts means of reduction in abscission layers ± SE. The mean values followed by the same letters are not significantly different (ns) at *p* ≤ 0.05 as calculated by Fisher's least significant difference.

**Figure 8.** The correlation between all root zone water temperature treatments and the decrease in abscission layers that were present at the start, as compared to the complete absence of abscission layers at the end of the study. Note: Bar graphs presented here are means of the number of abscission layers ±SE. The mean values followed by the same letters are not significantly different (ns) at *p* ≤ 0.05 as calculated by Fisher's least significant difference.

(**b**) Applied to (n) = G.

**Figure 9.** Postharvest photos display the effect of increasing root zone temperature across the temperature range 18 ◦C to 34 ◦C on flower and abscission layer formation on both n = A and n = G (Photos: C. Viljoen).

#### **4. Discussion**

High vegetative, flower and fruit yields in quality greenhouse crops are possible with hydroponics due to the precise control of growing conditions and required nutrients [42,52]. Nutrient solution temperature is easily controllable in hydroponics and may be manipulated to control plant growth and maximize the production of plants and flowering during winter periods [11]. Two cultivars of *Saintpaulia* (Gesneriaceae) subjected to a root zone

heating range of 17 ◦C–25 ◦C exhibited a 10–15% reduced cultivation time and a significant increase in the rate of flower formation [53]. Root zone heating has shown significant results in herbaceous leafy crops, increasing flower numbers by increasing nutrient uptake [20,48]. *Chrysanthemum* responded positively when grown in a soilless culture system with a heated solution and produced flowers earlier with optimum results at 24 ◦C [49]. In woodier crops, such as apple, a root zone temperature of 15 ◦C proved to be optimal for flowering with a distinct reduction at 30 ◦C [54], and roses grown in a heated soilless culture system showed an increase in the number of blooms produced over the production season [55].

*Streptocarpus formosus* responded in various ways to different temperature regimes with a clear trend that resulted in the death of the leaves and roots at higher temperatures (26 ◦C to 34 ◦C), with the most optimal growth at the lower temperature of 18 ◦C. It is also clear that the roots were more sensitive than the shoots. Treatments applied in this investigation had a significant effect on the vegetative root and leaf growth as well as the overall fresh weight of *S. formosus.* The results obtained from this research disagree with various previous studies which yielded positive results in other leafy perennials and crops at higher temperature ranges such as 24 ◦C–28 ◦C for spinach [56]; 25 ◦C–30 ◦C for tomatoes and lettuce [20]; 25 ◦C–45 ◦C for muskmelon plants [57]; and 15 ◦C–30 ◦C, for *Chrysanthemum* with an optimum temperature of 24 ◦C [49].

Several other studies performed on soft shrubs, such as roses, indicated that shoot growth was reduced at root temperatures lower than 18 ◦C [55] and, at this specific temperature or above, heat in the root zone was beneficial [58]. For *Euphorbia pulcherrima* cuttings, the optimum temperature range for rooting was 25 ◦C–28 ◦C [59]. Results for conifer seedlings, such as pine (temperature range 8 ◦C–20 ◦C), had significantly new root growth at 20 ◦C [60]. In [19], the authors showed that lowering the temperature from 21.4 ◦C to 16.8 ◦C for *Disa* spp. had a negative effect on root growth and fresh weight, which agrees with this study where optimum vegetative growth was recorded at root temperatures lower than 18 ◦C.

The vegetative growth responses of *S. formosus* in this study contradict the results of research performed on cooler root zone temperature ranges, indicating that lower temperature ranges can restrict photosynthetic, respiration, metabolic and osmotic activities [20,61,62]. However, findings from this study concur with research performed on *Streptocarpus* hybrid leaf cuttings in the laboratory, which produced the most roots and buds at 12 ◦C and 18 ◦C [63], as well as with research performed on cucumbers, where the lowest temperature within the range 22 ◦C–33 ◦C yielded the best results [16].

*Streptocarpus* species only naturally produce flowers during the long-day summer months [3,25]. This study, however, showed that *S. formosus* was able to produce flower buds during the winter short-day period at lower temperatures. The importance of increasing flowers and regulating the timing of flowering in pot plant production can support the production of the species [61]. As confirmed in Table 3 earlier, the effect of various root zone temperatures on the total flower development of *S. formosus* was statistically significant at *p* ≤ 0.05.

In *S*. *formosus*, the tips of the leaves often slowly die back to an abscission layer when stressed by drought, low temperature or when overwintered [3]. Growth cessation, abscission formation and dormancy development are considerably affected by temperature [21]. Leaf loss is a strategy for the avoidance of water stress in plant species adapted to drought because it reduces the transpiring surface of the foliage and therefore lessens the water demand [21,22]. Leaf senescence is mainly caused by cold and less commonly by high temperature [5,23]. In winter deciduous species, leaf senescence is an indication of the change from an active to a dormant growth stage [21,64]. When climatic conditions become unfavorable and the plants experience a state of stress, phytohormones react and leaf abscission that can lead to complete senescence is often the result [21]. Some significant abiotic factors affecting leaf abscission are nutrient availability, temperature and water supply [5,21,23], all of which can be managed within hydroponic cultivation systems [17,38]. Ref. [44] recommends that during colder months sub-irrigation should be used with minimal overhead

irrigation as water that is considerably colder than the average leaf temperature causes unsightly leaf damage with yellow spots or blotches on *Streptocarpus* leaves.

In [65], the authors reported that abscission occurred due to short photoperiods in some *Streptocarpus* spp. In [66], the authors also stated that photoperiod and temperature are the main cues controlling leaf senescence in winter deciduous species, with water stress imposing an additional influence [21]. Although manipulating light and photoperiod could prevent eco-dormancy in *S. formosus* in the cold season short days, this study, however, proved that manipulating root zone temperature significantly affected the vegetative growth and flower development. A lack of water or nutrients results in leaf yellowing in many plant species, which can be reversed in some species upon removing the stress [6]. In *Streptocarpus*, it is still possible for the reversal of the formation of the abscission layer and the senescence processes even if the leaves are displaying a distal depletion of chloroplasts [65] if the plants are maintained under conditions of high temperature, nutrient levels and humidity [24]. Leaf senescence can be delayed by warming as photoperiodic triggers and growth proficiency could increase because of a slower speed or prevention of leaf senescence [21,62]. Plant growth can be controlled by the direct correlation between nutrient solution temperatures around the root zone and the uptake of nutrients, with increased plant growth at elevated root temperature correlated with higher nutrient absorption [15,67].

In this study, *S. formosus* responded in various ways to different root zone temperatures but showed the most significant vegetative mass increases in both shoots and roots at the lower temperature of 18 ◦C indicating that a low-temperature heating range can be used to keep *S. formosus* in active growth during the cold season. Treatments applied in this investigation had a significant effect on the flowering formation of *S. formosus.* Plants responded better to the lower root zone temperatures of 18 ◦C and 22 ◦C compared to the higher temperature intervals, and flowers were formed during the colder short-day periods, which indicates a strong possibility that manipulating the growing temperatures could induce *S. formosus* to flower earlier in the season, thereby increasing its annual commercial marketing period. These findings agree with [49] and [53], in which root zone heating increased blooms and extended the flowering period into the cold season months or encouraged earlier flowering. Moreover, treatments applied in this research also had a significant effect on the abscission layer formation of *S. formosus*. This indicates that root zone heating is a viable method for preventing the formation of abscission layers.

#### **5. Conclusions**

It is concluded that high root-zone temperatures decreased the vegetative growth of *S. formosus.* The results showed that the cooler root-zone temperature of 18 ◦C improved growth (leaf number, leaf and root lengths and fresh weight). This study further established that increasing root-zone temperatures did not promote the flowering of *S. formosus*; however, plants responded positively to flowering at decreased temperatures from 22 ◦C–18 ◦C. *S. formosus* has commercial potential as an indoor flowering pot plant and a flowering landscape perennial, and shows potential within the cut-flower trade. Therefore, these results will contribute to developing optimal cultivation protocols for cultivating *Streptocarpus* spp. and its hybrids and guide commercial growers in the cultivation of *S. formosus* in particular.

**Author Contributions:** Conceptualization, C.C.V. and C.P.L.; Data curation, C.C.V. and M.O.J.; Formal analysis, C.C.V. and M.O.J.; Funding acquisition, C.C.V. and C.P.L.; Investigation, C.C.V.; Methodology, C.C.V. and C.P.L.; Project administration, C.P.L.; Resources, C.C.V. and C.P.L.; Supervision, C.P.L.; Validation, M.O.J. and C.P.L.; Writing—original draft, C.C.V.; Writing—review & editing, C.C.V., M.O.J. and C.P.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was funded by the South African National Biodiversity Institute.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Communication* **Evaluation of Carrageenan, Xanthan Gum and Depolymerized Chitosan Based Coatings for Pineapple Lily Plant Production**

**Piotr Salachna \* and Anna Pietrak**

Department of Horticulture, West Pomeranian University of Technology, 3 Papieza Pawła VI Str., ˙ 71-459 Szczecin, Poland; pa37778@zut.edu.pl

**\*** Correspondence: piotr.salachna@zut.edu.pl; Tel.: +48-91-449-6359

**Abstract:** Some natural polysaccharides and their derivatives are used in horticulture to stimulate plant growth. This study investigated the effects of coating bulbs with carrageenan-depolymerized chitosan (C-DCh) or xanthan-depolymerized chitosan (X-DCh) on growth, flowering, and bulb yield as well as physiological and biochemical attributes of pineapple lily (*Eucomis autumnalis*). The results showed that treatment with C-DCh or X-DCh significantly increased all growth parameters, bulb yield, greenness index, stomatal conductance, total N, total K, and total sugar content of bulbs and accelerated anthesis as compared with untreated bulbs. The positive impact of coatings on plant growth and physiological attributes depended on the type of biopolymer complexes. The X-DCh treatment exhibited the greatest plant height, fresh weight, daughter bulb number, greenness index, stomatal conductance, total N, K, and sugar content. However, this treatment induced a significant decrease in L-ascorbic acid, total polyphenol content and antioxidant activity. Overall, the results of this study indicated high suitability of C-DCh and X-DCh as bulb coatings for pineapple lily plant production.

**Keywords:** biostimulants; polysaccharides; bulb coating; plant enhancement; metabolites

#### **1. Introduction**

Currently, biostimulants are used to improve growth and development of horticultural plants [1,2]. Biostimulants are a broad group of substances and microorganisms with high biological activity [3–5]. Of particular interest are biodegradable polysaccharides and their depolymerized derivatives exhibiting multi-directional actions in plants [6–10]. Chitosan is one of the best known natural polysaccharides with biostimulatory properties obtained in the process of chitin de-N-acetylation [11,12]. Chitosan and its oligomeric forms have stimulated plant growth and flowering, increased photosynthesis and nutrient uptake, and protected plants against stress [13–16]. Many studies have reported the benefits provided by chitosan on various ornamental plants, such as *Begonia* × *hiemalis* Fotsch [17], *Chrysanthemum morifolium* Ramat [18], *Eucomis bicolor* Baker [19], *Freesia* × *hybrida* [20], and *Petunia* × *atkinsiana* D. Don [21]. In practice, chitosan solution is applied as a spray or drench [22–24] as well as hydrogels for coating seeds, but hydrogels from "pure" chitosan have low stability and durability [25]. Moreover, the wider use of chitosan is limited due to its poor water solubility [26]. The application of depolymerized chitosan with low molecular weight and ionic biopolymers in the form of hydrogel coating formed on the surface of plant organs based on polyelectrolyte complexes may be the solution to the problem [27]. This type of coating formed by chitosan and ionic polymers can positively affect plant growth and flowering [28]; however, it is reasonable to conduct broader research, including evaluation of the effectiveness of various biopolymers as coating components [29]. Carrageenans are a family of anionic polymers extracted from red algae used as plant biostimulants [10,30,31]. Carrageenans and their breakdown products can stimulate plant productivity and root system development, and enhance net photosynthesis, basal, and secondary metabolisms [32–36]. Among natural biopolymers, xanthan gum, an

**Citation:** Salachna, P.; Pietrak, A. Evaluation of Carrageenan, Xanthan Gum and Depolymerized Chitosan Based Coatings for Pineapple Lily Plant Production. *Horticulturae* **2021**, *7*, 19. https://doi.org/10.3390/ horticulturae7020019

Academic Editor: Douglas D. Archbold Received: 31 December 2020 Accepted: 27 January 2021 Published: 29 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

anionic, high-molecular-weight exo-polysaccharide secreted by the bacterium *Xanthomonas campestris* is also known [37]. Application of xanthan gum can influence plant growth and physiology, content of phenolic compounds, and antioxidant activity [38–40]. Xanthan gum used in micropropagation as an alternative to agar has a positive effect on the regenerative potential of some plants [41], which may indicate its biostimulative action. However, no information is available regarding the effect of xanthan gum as a biostimulant on plant growth and flowering.

Pineapple lily (*Eucomis autumnalis* (Mill.) Chitt. Asparagaceae) is a prospective bulbous ornamental plant grown in gardens, for cut flowers, and as a potted plant for indoor display [42–44]. The bulbs produce a rosette of smooth leaves and original decorative raceme-type inflorescences with a tuft of leaf-like bracts on top, composed of star-shaped white flowers with a pleasant scent. After flowering, the plants set decorative and durable green capsules. Besides its ornamental use, pineapple lily is one of the most popular plant species in traditional medicine in southern Africa [45]. The extracts of pineapple lily exert multidirectional effects, including antioxidant, anti-inflammatory, bactericidal, fungicidal and cytostatic effects [45,46]. The species is threatened with extinction in its natural habitat due to the excessive collection of bulbs for medicinal purposes as well as low vegetative propagation rate [45]. Thus, proper production methods of pineapple lily using various plant biostimulants is needed [47,48].

Previous work [49] reported that oligochitosan and sodium alginate can be successfully used for the preparation of hydrogel coatings for the bulbs of pineapple lily. However, systematic study of the effects of other biopolymers on growth, plant physiological status, and biochemical parameters of pineapple lily remains to be investigated. The current study was aimed to compare the effects of coatings containing hydrogels based on carrageenan or xanthan gum with depolymerized chitosan on the growth characteristics, flowering, bulb yield, physiological parameters, nutrients, L-ascorbic acid, and total polyphenol content, as well as antioxidant activity of pineapple lily. It was hypothesized that a coating treatment with polysaccharides would enhance the growth and bulb production of pineapple lily.

#### **2. Materials and Methods**

#### *2.1. Plant Culture and Treatment*

Bulbs of pineapple lily (*E. autumnalis*) with 12–14 cm circumference were imported from The Netherlands by Ogrodnictwo Wi´sniewski Jacek Junior (Góraszka, Poland) and treated for 30 min in a suspension of 0.7% Topsin M 500 SC and 1% Captan 50 WP fungicides. Before planting, the uniform bulbs were coated according to the technology described by Startek et al. [29] in hydrogels based on 1% (*w/v*) carrageenan or 1% (*w/v*) xanthan gum in which bulbs were dipped for 30 s, and 0.2% (*w/v*) depolymerized chitosan in which the bulbs were soaked for 10 min. Control bulbs were soaked in distilled water. Depolymerized chitosan obtained by controlled free radical degradation [28] had a molecular weight of 154, 500 g mol−1, the number-average molecular weight of 22,800 g mol−1, and deacetylation degree of 85%. Carrageenan-depolymerized chitosan (C-DCh) and xanthan-depolymerized chitosan (X-DCh) were produced. Iota-carrageenan and xanthan gum were purchased from Sigma-Aldrich. Polysaccharides were prepared by solubilization using a magnetic stirrer. Each treatment was replicated four times and each replicate had 10 bulbs.

Coated bulbs were planted in a randomized block design on 15 April 2016 and 13 April 2017 into polyethylene boxes (60 × 40 × 19 cm) filled with peat substrates (pH 6.3) supplemented with a fertilizer Hydrocomplex (12% N, 4.5% P, and 15% K plus micronutrients; Yara International ASA, Oslo, Norway) at a dose of 3 g L−1. Each box contained 10 bulbs. The boxes were transferred to a non-heated tunnel covered with a double layer of plastic located in the area of West Pomeranian University of Technology in Szczecin (53◦25 N, 14◦32 E; 25 m a.s.l.). Air temperature inside the tunnel was controlled with vents that were opened when the temperature exceeded 20 ◦C.

#### *2.2. Measurement of Growth Parameters*

The number of days to anthesis was recorded. When the first flowers opened in the raceme, plant height, diameter of the plant, and inflorescence length were recorded. At the end of the flowering period, the number of florets in the inflorescence were counted and fresh weight of the excised aboveground part was measured. On 3 October 2016 and 6 October 2017, the plants were removed from boxes, and fresh weight of bulbs per plant and the number of daughter bulbs were determined.

#### *2.3. Measurement of Physiological Parameters*

At the flowering stage, relative leaf chlorophyll content measurements were performed using a SPAD-502 Chlorophyll Meter (Minolta, Osaka, Japan) and stomatal conductance was assessed with a SC-1 Leaf Porometer (Dekagon Device, Pullwan, WA, USA). SPAD and stomatal conductance measurements were calculated based on four readings of four uniform leaves selected from five plants of each treatment.

#### *2.4. Total N, P, K, and Total Sugar Content in Bulb Determination*

At the end of the growing season, the bulb samples were collected, dried at 65 ◦C for 72 h and ground. Powdered samples (2.0 g) were digested in 17 mL concentrated 96–97% H2SO4. The total forms of N, P, and K were determined as outlined by Ostrowska [50]. Total N was determined according to the Kjeldahl method, P with colorimetric method according to Barton, and K by flame photometry [50]. The content of total sugar in samples of fresh bulbs was determined following the Luff-Schoorl method [51]. Nutrients and total sugar content were determined using three replicates per treatment.

#### *2.5. L-Ascorbic Acid, Total Polyphenol Content, and Antioxidant Activity Determination*

At the flowering stage, fully developed leaves were taken for biochemical analyses. Before homogenization, leaves were washed with water to remove soil, cut into slices, and dried in a circulating-air oven (35 ◦C ± 2 ◦C). Vitamin C was determined as L-ascorbic acid by the Tillman's titration method of the reduction of 2.6-dichlorophenolindophenol [52]. The preparation of plant extracts for the determination of the total polyphenol content and antioxidant activities was performed using the method of Wojdyło et al. [53] with some modifications. The sample of leaves was treated with 70% aqueous methanol (MeOH). Total polyphenol content was analyzed spectrophotometrically using the Folin–Ciocalteu colorimetric method as described by Wojdyło et al. [53]. The absorbance was measured at 760 nm. Antioxidant activity of leaves on DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was determined according to the procedure of Yen and Chen [54], and DPPH inhibition percentage was calculated according to the formula provided by Rossi et al. [55]. All determinations were carried out in three replicates.

#### *2.6. Statistical Analysis*

Data were normally distributed and passed Levene's test (*α* ≤ 0.05) for homogeneity of variance. Data were statistically analyzed by one-way ANOVA using Statistica™ Professional 13.3.0 software (TIBCO Statistica, Palo Alto, CA, USA). After checking the goodness of fit of the model, post hoc comparisons were done using the Duncan's Multiple Range Test (DMRT) at *α* ≤ 0.05. The results are presented as a mean from two years of the study.

#### **3. Results**

The effect of coating with carrageenan-depolymerized chitosan (C-DCh) or xanthandepolymerized chitosan (X-DCh) on growth and flowering of pineapple lily is shown in Table 1. The C-DCh and X-DCh applications significantly increased plant height by 8% and 16%, respectively, plant width by 39% and 40%, respectively, fresh weight of the aboveground part by 71% and 95%, respectively, inflorescence length by 32% and 25%, respectively, and the number of florets by 8% and 9%, respectively, as well as accelerated flowering by 17 and 13 days, respectively. Statistically significant differences were observed

between X-DCh and C-DCh treatments. Bulbs coated in X-DCh were taller by 7% and had a greater fresh weight of the aboveground part by 16%, compared with bulbs coated in C-DCh.

**Table 1.** Growth and flowering parameters of pineapple lily treated with carrageenan-depolymerized chitosan (C-DCh) or xanthan-depolymerized chitosan (X-DCh).


<sup>z</sup> Means (±SD) followed by the same small letter in the same row did not differ by Duncan's Multiple Range Test at *α* ≤ 0.05.

The fresh weight of bulbs, number of daughter bulbs, total N, K, and total sugar content in the pineapple lily bulbs were significantly affected by C-DCh or X-DCh complexes (Table 2). The coating of bulbs with C-DCh and X-DCh enhanced fresh weight of bulbs by 39% and 61%, respectively, and number of daughter bulbs by 24% and 48%, respectively. Moreover, the application of C-DCh and X-DCh increased levels of N by 49% and 54%, respectively, K by 46% and 57%, respectively, and total sugar content by 12% and 17%, respectively, in comparison with the control. The treatment with X-DCh resulted in the greatest fresh weight of bulbs, number of daughter bulbs, and total N, K, and sugar content. Bulb treatment with C-DCh and X-DCh did not affect total P content.

**Table 2.** Fresh weight of bulbs, number of daughter bulbs, total N, P, K, and total sugar content in bulb of pineapple lily treated with carrageenan-depolymerized chitosan (C-DCh) or xanthandepolymerized chitosan (X-DCh).


<sup>z</sup> Means (±SD) followed by the same small letter in the same row did not differ by Duncan's Multiple Range Test (DMRT) at *α* ≤ 0.05.

As shown in Figure 1, SPAD chlorophyll meter measurements and stomatal conductance were significantly increased due to C-DCh and X-DCh treatment in comparison to control. The C-DCh increased SPAD and stomatal conductance by 11% and 55%, and X-DCh by 7% and 31%, respectively. The SPAD and stomatal conductance of plants treated with X-DCh were 3% and 19%, respectively, greater than that of C-DCh treatment.

Figure 2 shows the effects of bulb coatings on the content of L-ascorbic acid and total polyphenols and the antioxidant activity. In comparison with the control the application of C-DCh or X-DCh significantly decreased total polyphenol content by 13% and 17%, respectively. Furthermore, the application of X-DCh significantly decreased L-ascorbic acid content by 33% and free DPPH radicals by 56%. The plant L-ascorbic acid content and antioxidant activity showed no statistically significant differences between control and C-DCh treatment.

**Figure 1.** Relative leaf chlorophyll content (SPAD) (**a**) and stomatal conductance (**b**) of pineapple lily treated with carrageenan-depolymerized chitosan (C-DCh) and xanthan-depolymerized chitosan (X-DCh). Data are presented as means (±SD) and bars with different letters in each graph are significantly different by Duncan's Multiple Range Test (DMRT) at *α* ≤ 0.05.

**Figure 2.** L-ascorbic acid (**a**) and total polyphenol content (**b**) and antioxidant activity (DPPH) (**c**) of pineapple lily treated with carrageenan-depolymerized chitosan (C-DCh) and xanthan-depolymerized chitosan (X-DCh). Data are presented as means (±SD) and bars with different letters in each graph are significantly different by Duncan's Multiple Range Test (DMRT) at *α* ≤ 0.05.

#### **4. Discussion**

Most studies on the application of biostimulant coatings focus on seeds, while data on coating other plant organs such as bulbs, tubers or rhizomes are far less available [56]. Our study is the first to use two biostimulant complexes, containing depolymerized chitosan and carrageenan (C-DCh) or xanthan (X-DCh), for coating pineapple lily bulbs. We found a stimulatory effect of both types of coatings on plant growth and development manifested in accelerated flowering and clearly higher yield of flowers and bulbs. In addition, plants grown out of biostimulant-coated bulbs featured more efficient gas exchange and better nutrient content. Some results of the current study are in agreement with our earlier work [29]. We reported a considerable improvement in plant growth, as assessed by morphological and physiological parameters and nutrient content in *E. autumnalis* when the bulbs were coated with oligochitosan and sodium alginate, and in *Ornithogalum saundersiae* Baker when the bulbs were coated with chitooligosaccharide and sodium alginate, carrageenan, gellan gum, or xanthan gum [28,49]. Stimulating effects of the coatings were probably due to the fact that their components enhanced plant tolerance to stresses from the beginning of their development, similar to that noted with seed coating [56]. Chitosan can improve growth and structure of a plant root system by limiting the presence of soil pathogens [11,12]. As a source of carbon for microorganisms, it also indirectly boosts soil microbial activity and thus improves absorption of minerals and water by plant roots [15]. In consequence, plants grow faster and stronger and produce better yield. Carrageenan may act as an elicitor that induces plant defense response against viroids, viruses, bacteria, or fungi, and it also may improve plant growth by controlling numerous metabolic processes including photosynthesis and assimilation of nitrogen and sulfur [10,30,35]. Xanthan gum is also capable of inducing local and systemic resistance against diseases and shows the same efficiency in plant protection against some phytopathogens as fungicides [38,57]. Soil application of xanthan gum may increase root biomass production and plant tolerance to drought and other environmental stresses [40].

The results presented in this paper demonstrated that the positive effects of coating pineapple lily bulbs on plant growth depended on the type of biopolymer complexes. The strongest plant growth stimulation was observed in plants obtained from bulbs treated with X-DCh complex. We assume that joint application of the biostimulants in X-DCh coatings may induce a stronger synergistic effect than C-DCh coating alone. It is commonly known that many biologically active substances change their properties when interacting with other substances [58,59]. Interestingly, the stimulating effect of X-DCh on the growth of the aboveground plant tissues and bulb biomass and the content of N, K, and total sugars in pineapple lily was accompanied by a clear drop in the levels of L-ascorbic acid and total polyphenols and by reduced antioxidant activity, responses not recorded in plants treated with C-DCh. The inhibitory effect of X-DCh treatment on secondary metabolite production may be a result of a trade-off between the production of plant biomass and secondary metabolism [60]. It is well known that defense and plant growth cannot usually be successfully executed at the same time [61,62]. Another possible interpretation, in line with previously cited research, is that xanthan gum induced a reduction in polyphenol content due to activation of some cellular biochemical mechanisms involved in plant resistance [57]. Still, further studies are necessary to validate either of these hypotheses.

#### **5. Conclusions**

The biostimulant complexes carrageenan-depolymerized chitosan (C-DCh) and xanthandepolymerized chitosan (X-DCh) used for bulb coating improved plant productivity, allowing growers to speed up the production cycle in protected culture and to obtain higher quality flowers and bulbs of pineapple lily. Particularly strong biostimulant activity was shown for coatings containing derivatives of X-DCh. Bulb coatings in biostimulants seems a prospective, efficient, and environmentally friendly method of improving plant growth that can be recommended for sustainable production of ornamental plants.

**Author Contributions:** Conceptualization, methodology, formal analysis and investigation, P.S.; writing, P.S., A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Environmental Analysis of Sustainable Production Practices Applied to Cyclamen and Zonal Geranium**

**Jaco Emanuele Bonaguro, Lucia Coletto, Paolo Sambo, Carlo Nicoletto and Giampaolo Zanin \***

Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padova, Viale dell'Università, 16, 35020 Legnaro, Italy; jaco.bonaguro@gmail.com (J.E.B.); colettolucia@gmail.com (L.C.); paolo.sambo@unipd.it (P.S.); carlo.nicoletto@unipd.it (C.N.) **\*** Correspondence: paolo.zanin@unipd.it; Tel.: +39-049-827-2902

**Abstract:** Italian floriculture is facing structural changes. Possible options to maintain competitiveness of the involved companies include promotion of added values, from local production to environmental sustainability. To quantify value and benefits of cleaner production processes and choices, a holistic view is necessary and could be provided by life cycle assessment (LCA) methodology. Previous studies on ornamental products generally focused on data from one company or a small sample. The aim of this study was a gate-to-gate life cycle assessment of two ornamental species, cyclamen (*Cyclamen persicum* Mill.) and zonal geranium (*Pelargonium* × *hortorum* Bailey), using data from a sample of 20 companies belonging to a floriculture district in the Treviso, Veneto region. We also assessed the potential benefits of the environmental impact of alternative management choices regarding plant protection and reuse of composted waste biomass. Life cycle impact assessment showed higher impact scores for the zonal geranium, mainly as a consequence of greenhouse heating with fossil fuels. This factor, along with higher uniformity of production practices and technological levels of equipment, translated to a lower variability in comparison with cyclamen production, which showed a wider results range, in particular for eutrophication, acidification and human toxicity potential. The application of integrated pest management with cyclamen had significant benefits by reducing acidification and human toxicity, while reducing use of mineral nutrients through amending growing media with compost resulted in a reduction in eutrophication potential. Similar achievable benefits for zonal geranium were not observed because of the dominant contribution of energy inputs.

**Keywords:** life cycle impact assessment (LCIA); plant protection; compost; sustainable greenhouse production

#### **1. Introduction**

Ornamental plant production is a specialized and intensive agricultural sector that includes a wide range of outputs, such as cut flowers, nursery stock, potted flowering or leafy plants, bulbs and tubers. Europe is the largest consumer market, with Germany, the United Kingdom, France and Italy as leading consumers. Italy is also an important producer, having over 14,000 companies with a GSP of over 1125 million € [1]. This sector has a complex structure, with a few regions having districts specialized in some sections of the production chain.

The Veneto region of Italy is home to some important districts, located in Padova, Treviso, Vicenza and Rovigo provinces. Data from 2016 [2] showed a total of 1490 companies, with a total area of 2730 ha, and a GSP of 206 million €. The overall trend compared to the previous five years highlights how the sector is facing structural changes to cope with the ongoing stagnation in domestic demand as a consequence of the current economic crisis; the number of companies is steadily decreasing, averaging −2% per year, with 4–5% peak losses in some districts (Rovigo, Vicenza). The GSP value of marketable pot plants decreased slightly (−0.5%) until 2016, then the trend started to increase. The

**Citation:** Bonaguro, J.E.; Coletto, L.; Sambo, P.; Nicoletto, C.; Zanin, G. Environmental Analysis of Sustainable Production Practices Applied to Cyclamen and Zonal Geranium. *Horticulturae* **2021**, *7*, 8. https://doi.org/10.3390/ horticulturae7010008

Received: 20 December 2020 Accepted: 14 January 2021 Published: 15 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

nursery production of ornamental, vegetable and orchard plants is stable. Regarding marketing areas, local and regional sales fell (from 34.3% to 29.1% and from 22.6% to 20.2% over five years, respectively), but a slight increasing share of sales to other Italian regions (+3.9% in five years) or EU countries (+4.7% in five years). Indeed, an increased number of companies have obtained the Certificate of Conformity required for sales in EU member countries, to 264 (+18%) in 2018. The changes highlighted by these data are partly related to increased competitiveness from emerging countries [3], and partly to the shift in consumer preferences. Italian companies operating in the northern, high-cost regions are generally small and family-run, and cannot tackle sudden changes in international markets with cost reductions and technological improvements only. Possible options for maintaining competitiveness could focus on the promotion of added value, such as typical/local productions, range and variety of choice, seasonal products and "eco-friendly" choices in production systems. For countries within the EU, sustainable or cleaner production are becoming a requirement rather than an encouraged practice, even the agricultural sector which is often regarded as a polluting activity [4].

Cleaner production is defined by the United Nations Environmental Program [5] as the continuous application of an integrated preventive environmental strategy to processes, products and services, to increase overall efficiency and reduce risks to humans and the environment. Five main components of cleaner production are related to conservation of raw materials, water and energy, eliminating toxic and dangerous emissions and reducing waste. Plastic waste, fertilizer use, peat-based growing media and heating requirements are usually perceived as major contributors to protected crop impacts on environment. Some of the above-mentioned issues have been addressed by researchers, such as integrated or biological crop protection [6–9], use of slow-release fertilizers [10–13], irrigation plans based on crop needs [14], and cultivation of native low energy demanding species [15]. Use of alternative containers such as biodegradable pots for the cultivation of ornamental pot plants were evaluated in various studies [16,17]; also peat substitution with composted materials, or other agro-industrial by-products rich in nutrients, has been widely evaluated in several trials with container grown plants, such as shrubs [18], poinsettia [17], geranium [19–21] and other bedding plants [22–24]. Efficient use of energy in greenhouses has received great focus [25–27]. Many trials aimed at reducing the energy consumption of greenhouses have focused on ventilation processes and the effects of thermal energy and mass transfer [28–31].

To quantify the potential impacts and assess the efficiency of reduction measures on specific crops and production systems, a life cycle methodology should be used. Life cycle assessment (LCA) is a material and energy balance applied to the production of goods or services (ISO 14040 [32]). This methodology has been applied to some ornamental commodities and production systems [33–36]. Previous studies on potted plants under protected cultivation highlighted some of the processes and materials involved in the production of certain emissions, such as energy for heating and artificial lighting, greenhouse frames and covers, plastic containers and peat [35,37–40]. Most assessments, except for a study on nursery production conducted by Lazzerini et al. [39], analyzed data sourced from one representative company and from specific literature or databases.

#### *Objective of the Study*

The aim of this study was to assess the environmental aspects of the cultivation of two ornamental species, using data from a sample of nurseries in the Treviso production district. While trying to define average impact results for the most important categories, we analyzed how different management choices and production practices affect final results. In the following sections we present the functional units, data collection processes and the alternative scenarios we chose to assess.

#### **2. Materials and Methods**

#### *2.1. Goal and Scope*

The goal of this research was to characterize the final cultivation phase of two potted flowering plant species, cyclamen and zonal geranium, from an environmental point of view, defining a range of results representative of the most common practices in the investigated floriculture district. We also assessed the potential environmental benefits achievable with specific practices or management choices that have been adopted by individual growers independently.

Other practices that apply to all the investigated companies, such as collection and recycling of plastic materials, have been implemented in the system models. The scenarios that we investigated concern typical environmental bottlenecks of protected cultivation, such as fertilizer use, plant protection and waste management (biomass).

The scope of our study included production, installation, use and disposal of capital goods (greenhouse frame and cover, as well as heating systems and auxiliary equipment for fertigation) and production, transport, use and disposal of crop inputs. The model system we describe was based on the production practices of a sample of nurseries sited in Treviso province. Since our goal was to describe and assess common practices and average structure and technology, comparison of different company sizes or sale types were outside the scope of this study.

We used open LCA software version 1.5.0 and EcoInvent database version 3.3 to input and model the Life Cycle Inventory data. Life cycle impact assessment (LCIA) was performed with CML-2015 method (CML-Centrum Voor Milieukunde Der Rijksuniversiteit Leiden), first created by the University of Leiden in the Netherlands in 2001. It has been published in a handbook with several authors [41]. The impacts were described using the baseline method impact categories. Acidification potential (AP) measures the increase of the acidity in water and soil systems due to the acidifying effects of anthropogenic emissions nitrogen oxides (NOx) and sulfur oxides (SOx). Acidification potential is expressed using the reference unit, kg SO2 equivalents. Global warming potential (GWP) measures the alteration of global temperature caused by greenhouse gases released by human activities; characterization of the model was based on factors developed by the UN's Intergovernmental Panel on Climate Change (IPCC). Factors are expressed as global warming potential over the time horizon of 100 years (GWP100), measured in the reference unit kg CO2 equivalents. Eutrophication is the build-up of a concentration of chemical nutrients in an ecosystem which leads to abnormal productivity. Emissions of ammonia, nitrates, nitrogen oxides and phosphorous to air or water all have an impact on eutrophication. This category is expressed using the reference unit, kg PO4 <sup>3</sup><sup>−</sup> equivalents. Direct and indirect impacts of fertilizers are included in the method. The direct impacts are from production of the fertilizers and the indirect ones are calculated using the IPCC method to estimate emissions to water causing eutrophication. Environmental toxicity is measured as two separate impact categories which examine freshwater and terrestrial emission of some substances, such as heavy metals, which can have impacts on the ecosystem. Assessment of toxicity is based on maximum tolerable concentrations in water for ecosystems. The calculation method provides a description of fate, exposure and the effects of toxic substances on the environment. Characterization factors are expressed using the reference unit, kg 1,4-dichlorobenzene equivalents (1,4-DB). The human toxicity potential is a calculated index that reflects the potential harm of a unit of chemical released into the environment, and it is based on both the inherent toxicity of a compound and its potential dose. This impact category is measured in 1,4-DB equivalents. The normalization step is necessary to analyze and describe the relevance of single contributions to an impact category, and to calculate the order of magnitude of the category indicator results relative to a reference information (i.e., total impacts for the selected category in a specific area). LCIA results were normalized with factors for the EU 25 area.

#### *2.2. Data Collection*

Data were collected through a survey conducted through questionnaires and interviews with 20 floriculture companies belonging to the Florveneto association, representing ornamental plant growers in Treviso province. The questionnaires were administered in person to the owners, at the company, so that the data collected could be, at least in part, verified. A questionnaire, which had previously been submitted to and validated by two pilot companies, was used to collect information on general production practices, greenhouse structures and equipment. The questionnaire with examples of compilation is available as Supplementary Material questionnaire.

#### Functional Units and System Boundaries

The functional unit was a single marketable plant in a 14-cm pot. The investigated species, zonal geranium and cyclamen, were chosen for several reasons: First, their economic relevance (they comprise 20% and 22% of the Italian flower market, respectively); second, they represent part of an ideal crop sequence for the average nursery. Lastly, given the seasonality of their production cycles, they are crops with different climate control needs and energy demands. System boundaries include all operations and inputs from transplant to market-ready flowering plants. The plug production phase was also included, even if specific information on seedling or cutting production for the considered species were not collected. This is also motivated by considerable differences concerning the choices of variety and young plant producers found among the surveyed companies.

#### *2.3. System Description: Cyclamen*

Cyclamen (*Cyclamen persicum* Mill.) plants are usually grown in structures with a plastic cover (single layer) over a galvanized steel frame. Average plastic cover replacement rate is 6 years, while supporting structure lifetime is 30 years. Potting of young plants occurs from May to mid-July. With an average growing period of 14–16 weeks, early potted plants bloom in September. Optimal temperature in the first period of growth is around 18–20 ◦C. During flower development normal temperatures should be between 15 and 20 ◦C. To promote cooler temperatures, shading from 30% to 50% is applied in summer months, together with lateral and roof ventilation. Active cooling systems, like fogging or fan-and-pad are installed and operating in only three nurseries. Cyclamen seedlings are transplanted into 14-cm pots, filled with a substrate composed of white peat with a coarse, porous texture (40% *v*/*v*), black peat (45–50% *v*/*v*) and expanded perlite (10–15% *v*/*v*). Plants are irrigated using overhead spray irrigation (no added fertilizer) for 1–2 weeks, then a fertilizer solution (N:P:K at 1:0.4:1.2) is applied. In some cases, overhead spray irrigation is still preferred at this stage, while most growers (14 out of 20) start fertigation with a spaghetti tube system. Fertilizer solutions applied during the growing period have increasing ratios of potassium to phosphorus to promote flowering and plant resistance to both disease and environmental stress (typical formulations: 17N-3.05P-14.2K; 20N-8.29P–23.3K). Plants are spaced after one month to allow air circulation and canopy growth. Fungal diseases include *Botrytis* and *Fusarium*, anthracnose and powdery mildew. Most are limited by prevention practices and improved breeding, yet between one and three fungicide treatments (classes: Carbamate, thiadiazole, amide, aromatic organic compounds) are reported by most growers. Common cyclamen pests are thrips (*Frankliniella occidentalis*; *Echinotrips americanus*), aphids (*Aphys gossypii*, *Aulacortum circumflexum*), vine weevil (*Otiorhynchus sulcatus*) and mites (*Steneotarsonemus pallidus*, *Tetranichus urticae*). Insecticides (active ingredient classes: Neonicotinoids, organophosphate, pyrethroids or avermectine) are applied from 2 to 5 times during the growing cycle. Growth regulators (chlormequat or daminozide) are applied once or twice to inhibit petiole elongation by 14 growers. See Supplementary Material questionnaire for the complete list of the inputs that were considered.

#### *2.4. System Description: Zonal Geranium*

Zonal geranium (*Pelargonium* × *hortorum* Bailey) plants are usually grown in structures with a plastic cover (double layer, air inflated) and galvanized steel frame, or in glasshouses with a steel frame. Average replacement rate of a plastic cover is 6 years while glass and supporting structures lifetime often exceeds 30 years which was the value assumed for calculations. The most widely used heating system consists of diesel-powered fanburners generating hot air while only two companies use gas boilers and a network of polypropylene pipes to deliver hot water under cultivation benches. In the first 10–15 days after seedling transplant, optimal temperature is around 18 ◦C in the daytime and 16 ◦C at night. After this phase, diurnal temperatures are kept around 16 ◦C and night temperatures around 14 ◦C. No artificial lighting is applied during this growth phase. Growing media are usually comprised of peat moss (80–85% *v*/*v*) blended with porous materials such as perlite or expanded clay (10–15% *v*/*v*). Plants are fertigated using overhead spray irrigation for a period ranging from 6 days to 3 weeks, depending on the individual choices made by growers. After this period, until marketable size is attained, plants are placed on benches and fertigated with ebb-and-flow or with spaghetti-tubing irrigation systems. Fertilizer solutions applied during the first period have a N:P:K ratio of 1:0.5:1. To promote flower quality, potassium concentration is increased during the final growth phase (N:P:K at 0.8:0.3:1.2). Common diseases are *Xanthomonas campestris* pv. *pelargonii* (wilt and spots), *Ralstonia* (wilt), *Pythium*, and *Botrytis*. Bacterial diseases are best fought with prevention practices and early detection, and soil-borne fungal diseases can be prevented by avoiding excessive air and substrate humidity, facilitating canopy air movement and raising night temperatures. Besides prevention practices, plants are usually treated one to three times with fungicides (active ingredient classes: Dichlorophenyl dicarboximide, aromatic organic compounds, amide). As a typical spring crop, zonal geranium is very sensitive to thrips; aphids (*Acyrthosiphon malvae*) can also be a problem and cause small, distorted leaves and black sooty mold. Insecticides are applied preventively in 40% of cases; most common active ingredients belong to the carbamate, organochlorine and pyrethroid classes. Along with other ornamentals such as petunias (*Petunia* spp.) and calibrachoas (*Calibrachoa* spp.), pelargoniums can be affected by budworms (*Geraniums bronze*, *Cacyreus marshalli*) during the last growth stages. These worms can devastate geraniums by tunneling into young buds and destroying the flower. Neonicotenoid or pyrethroid insecticides are applied to control this pest. See Supplementary Material questionnaire for the complete list of the inputs that were considered.

#### *2.5. Assumptions*

Data for background processes such as material manufacturing and disposal activities were sourced from the Ecoinvent 3.3 database, and modeled with OpenLCA ver. 1.5.0. Direct emissions were calculated by using estimation models, which are flexible and allow for an estimation of mitigating options. For fertilizer use, we estimated nitrate (NO3 −) emissions with the Swiss agricultural life cycle assessment (SALCA) method, assuming a draining fraction of 25% for open-loop systems, which is a common leaching value applied to prevent root zone salinization. Phosphate (PO4 <sup>3</sup>−) emissions were calculated according to SALCA-P emission model [42]. Plant protection products applied were modeled as emissions to agricultural soil.

#### *2.6. Description of Alternative Practices*

As mentioned earlier, during the data collection it was noticed that, even if close similarities were recorded in most of the interviewed nurseries regarding structure types, technological level of growing equipment, management decisions and cultivation inputs for the studied crops, the choices made by some growers led to significant differences in the reported input levels. Management decisions could in turn lead to different emission patterns and levels. These practices mainly included plant protection practices, fertigation management and recycling of waste biomass.

#### 2.6.1. Integrated Pest Management and Biological Plant Protection

Monitoring of insect presence (with chromotropic traps or visual inspection) is a known, yet not very widespread practice. Objective assessment of infestation and potential damage is also very difficult for crops with aesthetic value as their main feature. Despite this, the application of integrated pest management (IPM) and biological control agents is receiving growing attention, also because many active ingredients registered for use on ornamental species have recently been revoked or are no more available [43].

Due to the greater effort required, and uncertainties linked to these practices, most growers are delaying their application and still rely heavily on chemical control.

Based on information from four growers using IPM strategies, we assessed the potential impact of less chemical input and use (manufacturing of raw materials and soil emissions) as compared to an average production scenario. For cyclamen production, we considered that prevention practices at the transplant phase, with inoculation of a biological antagonist to *F. oxysporum* in the growth medium, can the reduce need for fungicide treatment to 1 per crop cycle, while improved insect scouting and monitoring reduces insecticide sprays to 2 per crop cycle. Zonal geranium benefits from biological prevention and control both at the transplant phase and in the first stages with *T. harzianum* and *B. subtilis* strains, while preventive insecticide spraying is integrated with antifeedant treatments (Azadirachtin); for this scenario we considered no fungicide treatment and a reduction of 40% in insecticide use (active substance).

#### 2.6.2. Management and Reuse of Waste Biomass

Protected soilless crops generate a significant amount of waste, due to material requirements for growing media, containers, benches, irrigation pipes and plug trays. These materials need to be disposed of at their end-of-life and several options are available from incineration to landfilling, or composting, depending on material segregation practices, regulations and grower's choices. Recycling of plastic material is a common and well-established practice among the interviewed growers, thanks to good awareness and coordinated efforts by the Florveneto association. Management of biowaste differs between growers. The amount of non-yield biomass in ornamental containerized crops is lower than in other protected crops, yet a certain amount of unsold or discarded plants are produced and must be disposed of confined windrow composting and reuse in situ could be an option, and one grower reported to have adopted this practice. However, in this case chemical and physical properties as well as direct emissions are probably highly variable and difficult to measure, and we therefore chose to model an alternative option, where compost is produced from miscellaneous green waste in a composting facility and used in growth media preparation as a substitute for peat. The considered rate of compost addition to the growth medium is 20% (*v*/*v*); this was chosen in accordance with growth trials of containerized plants on compost amended substrates reported in several studies [19,44,45]. For different plant species, supporting effects on growth with compost rates up to 20% were reported in these studies, but different effects were found for higher substitution rates. An analysis from a local composting plant shows the chemical composition and nutrient content of composted garden waste (Table 1). Two options are considered for the offset of mineral fertilizers: NPK content of compost does not replace fertilizers (option 1); NPK content replaces part of the mineral fertilizers applied through fertigation (option 2). The following rates of nutrient content available for the crop were considered: 20% for N, 50% for P and 50% for K. These values were taken from Boldrin et al. [46], and were reduced to account for the limited length of the growing period for the considered species.


**Table 1.** Chemical and physical properties of the garden waste compost considered for the evaluation of the impacts.

#### **3. Results and Discussion**

The considered inputs (Supplementary Material questionnaire) were grouped into six main categories, which include production, use and end-of-life phases: Greenhouse structures and covering materials, fertilizers, plant protection products, pots (including only the containers used in the cultivation phase), growing media and heating. Looking at absolute values (Table 2) for the assessed impact categories, we can note how the heated crop (zonal geranium) scored higher results for all indicators, even by several orders of magnitude for AP and GWP categories. As highlighted in the analysis of relative contributions (Figures 1 and 2), heating with fossil fuels contributed the greatest to the inputs of production. This factor, together with the greater uniformity found for some management choices in zonal geranium, also influenced the variability, which showed minor fluctuations around average values compared to cyclamen. For geranium, to better highlight the contribution of the main groups of input to the different impact categories we excluded the most impacting one (heat) even if this is beyond both the scope of the work and the meaning of the LCA analysis. With the exclusion of heating, the categories with the greatest weight were the greenhouse structure and coverage that contributed over 60% in the fresh water aquatic ecotoxicity (FWAE), human toxicity (HT) and terrestrial ecotoxicity (TE) categories, the pot that contributed over 51% in the acidification potential (AP) and 64% in the GWP categories, the substrate accounted for about 20% in three categories (AP, GWP and HT), and the fertilizers which contributed 75% in the Eutrophication potential (EP) category.


**Table 2.** Absolute values and standard deviation (in percentage) for the assessed impact categories for flowering potted plants of cyclamen (*Cyclamen persicum* Mill.) and zonal geranium (*Pelargonium* × *hortorum* Bailey).

<sup>z</sup> 1,4-dichlorobenzene (DB).

**Figure 1.** Relative contribution of different inputs for cyclamen potted plant production. The impact categories assessed are: Acidification potential (AP), global warming potential (time horizon of 100 years) (GWP), eutrophication potential (EP), fresh water aquatic ecotoxicity (FWAE), human toxicity (HT) and terrestrial ecotoxicity (TE).

**Figure 2.** Relative contribution of different inputs for zonal geranium potted plant production. The impact categories assessed are: AP, GWP, EP, FWAE, HT and TE.

Relative contributions in the impact categories are depicted graphically in Figures 1 and 2. The reported percentages refer to average sample values. The contribution of some materials or structures showed little variation, given the relative uniformity of supply chain and input choices among the growers. Other inputs with less standardization showed significant differences in their contribution to impact categories, which will be discussed in the following paragraphs.

#### *3.1. Cyclamen*

Plastic containers were the major contributor (60.5%) for the GWP category, but also accounted for a significant share of impacts in AP (35.2%) and FWAE (17.6%) (Figure 1). All burdens were associated with material production, since no emissions were considered for use and end-of-life phases. Growing media components had an important share of impacts in the AP (21.3%), GWP (21.6%), FWAE (18%), HT (16.5%) and TE (17%) categories. Expanded perlite production and disposal was an important source of emissions for HT, TE and FWAE; emissions related to peat roadway transport from Baltic countries contributed mainly to GWP and AP categories (Figure 1). Greenhouse structure shared major burdens in FWAE (53.5%), HT (37.6%) and TE (70.4%) categories, mostly linked to production and disposal of steel frame and electricity consumption. Emissions related to production and use of plant protection products mainly influenced HT (39.4%) and AP (28.6%) categories; depending on chemical products type and frequency of treatments their contribution varied between 35.6% and 22.7% for AP, and between 43.8% and 32.5% for HT (Figure 1). Emissions related to fertilizer and water use contributed mainly (68.5%) to EP category results. The release of nitrate and phosphate in ground and surface water was directly linked to fertigation method and discharge mode and rate of nutrient solutions; the overall contribution of this phase varied between 44.6% for closed systems with no overhead application to 72% for open systems with frequent overhead applications (Figure 1). Fertigation management of cyclamen plants with the latter method was prevalent among the interviewed growers. Most studies on the environmental impact of potted plants have focused mainly on climate change (GWP) [38,39], while few studies conducted complete LCIA including other impact categories [34,37]. In accordance with our results, when referring to unheated crops with no artificial lighting, factors influencing GWP are mainly linked to manufacturing of plastic materials (containers and greenhouse cover) and growing media components (peat and expanded perlite). Fertilizer contribution to the EP category on the overall production process of cyclamen potted plants was also highlighted by Russo and De Lucia Zeller [37]. Their finding is in line with our results, suggesting that management practices aimed at reducing fertilizer use and leaching have the best chances for impact reduction in this category. The significant contribution of greenhouse structures to TE and FWAE categories is in line with similar studies on ornamental productions [34].

#### *3.2. Zonal Geranium*

Emissions deriving from production and use of diesel fuel burned to heat the greenhouse contributed a major share of impacts in all considered categories, accounting for over 91.3% of overall emissions in GWP and 84.7% in AP (Figure 2). Production and disposal of greenhouse frames contributed significantly to FWAE (28.7%), HT (17.9%) and TE (32.1%) categories (Figure 2). Fertilizer and water use contributed 40% of the impacts in the EP category. Since zonal geranium is often fertigated with ebb and flow systems, which allow for a reduction of direct emissions of both water and fertilizers, the contribution of this step was less variable than in cyclamen and ranged from 36.4% to 43.9% (Figure 2). Plastic pot contribution averaged 9.7% for FWAE, 7.7% for AP and 5.65% for GWP categories. The share of environmental burden from application of plant protection products and fertigation was not relevant for the selected impact categories, except for HT (4.8%) (Figure 2). These results are in line with other studies on protected crops that require energy inputs to actively control the greenhouse environment (light, temperature) or for preservation purposes [40]; the overall impact dramatically increases [34] and is almost entirely attributable to energy demand, as in the case of zonal geranium.

#### *3.3. Effect of Alternative Practices on Cyclamen and Zonal Geranium Impact Assessment Results*

Sensitivity analysis is a tool for studying the variability of LCIA results to input parameters and data. In the following sections we use it to assess the effects of different scenarios (management choices) on the environmental profile of our functional units. Since the chosen unit was a single potted plant, absolute impact values and variations observed in the analysis were extremely small. For this reason, the relevant differences are expressed in percentage on the impact potential.

Table 3 shows the results for the chosen categories of average production practices and for the alternative scenarios for cyclamen plants, highlighting the achievable impact. The reduction in chemical inputs attained through the application of integrated pest and pathogen management programs for cyclamen plants resulted in an overall reduction of potential impacts, which is relevant in particular for HT (−25%) and AP (−16.3%) categories (IPM in Table 3 vs. actual scenario in Table 2). For HT, this result was due primarily to reduction of soil emissions and manufacturing of active ingredients with fungicide activity, achieved through application of biological control agents and careful fertigation management.

**Table 3.** Sensitivity analysis for one cyclamen plant subjected to alternative practices. In relation to garden waste compost addition to the growing medium in option 1, NPK content of compost does not replace fertilizers and in option 2 NPK content replaces part of the mineral fertilizers applied through fertigation. IPM = integrated pest management.


<sup>z</sup> 1,4-dichlorobenzene (DB).

Use of compost as growing media component without changes in fertilizer application rate (option 1) showed relatively small further reduction potential compared to the option in which fertilization was also considered (option 2), linked mostly to reduced peat extraction and transport. Another study in which the environmental aspects of compost substitution was assessed [46] reported lower impact values for different categories, including climate change (another expression used for GWP), acidification potential, eutrophication potential and photochemical ozone formation. In this study, leaching tests for soil application suggested a potential higher impact of composts when considering potential impacts on human toxicity via water and soil, because of high release rates of heavy metals. These considerations partly support our results, since application of compost, that substitutes a 20% volume of peat in the growth medium, results in a slight reduction of several indicators, including GWP, that was reduced by only 7.4% and 6.4% in options 2 and 1, respectively. However, the reduction achieved by this practice had a limited relevance on the overall impact of the functional units. This can be explained by the small amount of peat replaced, the relative importance of growing media components in the assessed categories, and finally because of the impacts related to the compost production process. When considering also nutrient release from the compost amendment and subsequent reduction of fertigation needs, a significant reduction for the EP category (−19.6%) was observed, which can be explained both by reduction of fertilizer production and decreased leaching. We highlight that the minimum value of EP observed for cyclamen was very similar to that obtained for this scenario. This result is justified by data on cultivation with closed-loop fertigation systems with nutrient solution recirculation. To maximize impact reduction from nutrient production and leaching to surface and groundwater, a combination of fertigation management and use of nutrient-rich amendments in the growth medium could be a useful indication for best management practices.

Table 4 shows the impact of average production practices and the alternative scenarios for zonal geranium plants. We highlight how the potential for impact reduction was strongly limited by the major burdens linked to heating in all impact categories. Application of IPM programs achieved a moderate reduction of results for HT (−2.4%) category. Use of compost, not considering nutrient supply, achieved a reduction exceeding 1% of impact results for only FWAE (1.08%), TE (1.11%) and HT (1.63%) categories.

**Table 4.** Sensitivity analysis for one zonal geranium plant subjected to alternative practices. In relation to garden waste compost addition to the growing medium in option 1, NPK content of compost does not replace fertilizers and in option 2 NPK content replaces part of the mineral fertilizers applied through fertigation.


<sup>z</sup> 1,4-dichlorobenzene (DB).

When considering mineral fertilizing offsets, the differences increased, in particular for EP that shows a 14% reduction in the final result. This value was lower than the observed minimum, highlighting the higher uniformity and technological level adopted for zonal geranium fertigation. In a trial on geranium bedding plants [20], compost from selected materials had a supporting effect on growth of geranium plants, providing an increased nutrient budget in the growing media and an increased uptake and nutrient content in plant tissues. The use of peat-free substrate increases production risk and requires expertise, and often alternative substrates cannot be adopted [36]; however, the addition of compost to growing media for geranium growth may be increased to 40%, providing a large part of its nutrient requirements, as evidenced by Perner et al. [44] in growth trials conducted with potted geranium. The adoption of this practice therefore shows a potential for impact reduction in the EP category, if mineral fertilizer inputs are accordingly reduced. The alternative practice we investigated falls among the priorities in pollution prevention listed as Best Agricultural Practices for protected crops in Mediterranean Climates [47], yet their improvement potential differs greatly depending on the set of impact categories and technological level, material and energy requirements of the investigated production system. For low energy-input crops such as cyclamen, the decrease in fertilizer and pesticide use can result in a significant impact reduction for most of the selected categories. The potential benefit resulting from combined application was 32% for HT, 20% for AP and EP, 12.5% for FWAE and 10% for GWP.

For zonal geranium, we highlight how reduction of energy input is the first priority for soilless heated crops, since best practices for other highly impacting materials (plastic containers and cover) have already been adopted. The reduced amount of fertilizer and plant protection product translates to a relatively irrelevant contribution, except for the EP category.

#### **4. Conclusions**

In this study we investigated the environmental impact aspects of the cultivation of cyclamen and zonal geranium starting from data coming from different greenhouse farms located in the Treviso province of Italy. Given the fragmented structure of the production chain for floriculture products in this region, the definition of common practices and their characterization should be linked to a variability measure in order to include the complexity and plurality of structures and management choices in the final results. In the case of cyclamen production, technological level and management choices can greatly affect the values obtained for different environmental indicators, in particular with regard to fertigation management and use of plant protection products. The results of the analysis also highlighted how the efficiency of reduction measures should always be checked with a life cycle study on the production or process to address (e.g., potted ornamental plants). While "sustainable" choices such as composting and reuse of waste biomass and reduction of chemical treatments have a significant benefit when applied to crops grown in a passive greenhouse, energy saving and changes in fuel type should be the main concern when aiming to reduce the impacts for crops requiring active control of the growth environment, as in the case of zonal geranium.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2311-752 4/7/1/8/s1, questionnaire.

**Author Contributions:** Conceptualization, J.E.B., P.S. and G.Z.; methodology, J.E.B. and L.C.; formal analysis, J.E.B., L.C. and G.Z.; investigation, J.E.B., C.N. and L.C.; resources, C.N. and G.Z.; data curation, J.E.B., C.N. and G.Z.; writing—original draft, J.E.B.; writing—review and editing, P.S., C.N. and G.Z.; visualization, J.E.B., L.C. and P.S.; supervision, G.Z.; funding acquisition, P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Measure 124 of the Rural Development Program 2007–2013 of the Veneto Region (Italy), Project "REFF".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available on request due to privacy restrictions.

**Acknowledgments:** The authors are grateful to the Florveneto association and to the 20 farmers who participated in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Overview of the Dynamic Role of Specialty Cut Flowers in the International Cut Flower Market**

**Anastasios Darras**

Laboratory of Floriculture and Landscape Architecture, Department of Agriculture, University of Peloponnese, 24100 Kalamata, Greece; a.darras@uop.gr; Tel.: +30-27210-45199

**Abstract:** The global cut flower industry has faced serious challenges over the years, but still remains an important sector of agriculture. Floriculture businesses seek new, innovative trends and niches to help increase product sales. Specialty cut flower (SCF) production has increased in the past 20 years in the US, Australia, Africa, and Europe. SCF production and sales could increase further if these new products were supported by dynamic marketing campaigns that focus on their strengths compared to the traditional cut flowers (TCF) such as roses, carnations, gerberas, and chrysanthemums. The major strength of SCF is the eco-friendly profile, which is associated to low CO2 footprints and environmental outputs. This contrasts TCF cultivation, which is associated to high energy inputs, especially at the traditional production centres (e.g., The Netherlands). It is suggested that environmental legislations, production costs, and customer demand for eco-friendly products will positively affect future SCF cultivation and sale.

**Keywords:** roses; gerberas; chrysanthemums; sustainability; floriculture; environmental impact; CO2 footprint

#### **1. Introduction**

Global cut flower production and consumption has overcome serious challenges in the past 20 years, especially those related to global economic recessions. The EU holds the first place in cut flower and ornamental potted plants sales with 31.0% of the global value, with China and the USA in second and third place, holding 18.6% and 12.5%, respectively [1]. Within the EU, in 2016, the Netherlands had the most sales of cut flowers and ornamental plants, with France and Italy in second and third places, respectively [1]. Cut flower and ornamental plant sales in the EU increased by 7% (approx. 1.4 billion euro) from 2006 to 2016, indicating a slow, but steady increase, despite the elaborate global economic status [1]. On the contrary, the number of cut flower producers in the USA declined significantly from 2007 to 2015 [2], with many of them forced out from the floriculture industry during the 2008–2009 recessionary shakeout period [3]. Cut flower production in the USA showed a modest increase from 2015 to 2018 [4,5]. The reductions recorded by the USDA between 2007 and 2015 reached 30%, indicating that cut flower production has shifted to new worldwide players such as China, Colombia, and Ecuador [6]. In 2017, China came first in sales of cut flowers and first in cut flower exports to the EU.

The aim of this review was to analyse the dynamic role of specialty cut flowers (SCF) in a market overwhelmed by traditional cut flowers (TCF) holding the majority of sales. A critical analysis provides evidence that SCFs might serve as the environmentally friendly alternatives to TCFs and claim future higher production volumes and market shares globally.

#### **2. Exploitation of the Endemic Flora by the Floriculture Sector**

New specialty ornamental crops and cut flowers are often introduced from the endemic flora. Native species leaving their natural environment may become global market trends. More than 6000 species native to Asia, Europe, North America, South America,

**Citation:** Darras, A. Overview of the Dynamic Role of Specialty Cut Flowers in the International Cut Flower Market. *Horticulturae* **2021**, *7*, 51. https://doi.org/10.3390/ horticulturae7030051

Academic Editor: Piotr Salachna

Received: 22 February 2021 Accepted: 12 March 2021 Published: 14 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Australia, Africa, and New Zealand were found in gardens in the USA [7]. The migration of ornamental plants has a deep historic background (18th century) and is still active to the present day.

Many South African native plants such as agapanthus (*Agapanthus africanus*), gerbera (*Gerbera jamesonii*), gladiolus (*Gladiolus* hybrids), gloriosa (*Gloriosa rothschildiana*), freesia (*Freesia hybrida*), leucadendron (*Leucadendron* sp.), leucospermum (*Leucospermum* sp.), ornithogalum (*Ornithogalum arabicum*), and protea (*Protea* sp.), were among the best-selling cut flower species during the previous decades [8]. Most of them are now considered as TCFs of great commercial success and are cultivated globally all-year round. They have been crossed or hybridised to produce numerous cultivars which gained commercial success [8]. The modern-day interest for South African native species lies on the breeding programs and/or on adaptations to various growing conditions. South Africa is regarded as a "hotspot" of diversity and an important source of new ornamental plants with the potential for commercial exploitation [8].

Cunningham et al. [9] presented many vital steps as part of a strategy to address and overcome serious challenges in the production of endemic cut flowers in Australia. Issues addressed were: (a) The implementation of existing national and international protocols for genetic resources protection, (b) the development of cultural branding and certification as marketing tools, (c) building business expertise and producer associations, and (d) increasing the reliability of supply chain of species such as *Geraldton waxflower*, *Anigozanthos* sp. *Boronia heterophylla, Leptospermum* sp., and *Grevillea* sp. [9]. In order to increase the economic and social impact of the native Australian SCF production and sale, local growers must improve their business and marketing skills, induce collaboration, exploit communication technologies, and increase product reliability and branding, while ensuring governmental support on protection of the initial genetic material. Similar challenges should be addressed by the South African floriculture markets [10]. The political isolation, the lack of complete distribution channels, the reduced quality compared to high grade European products, and the lack of export orientation were considered as the main barriers to valorisation of the South African native SCF. Although there is a dynamic increase in SCF production in the African continent, the labour organisations need to set labour standards for workers in production facilities [11].

#### **3. Cultivation of TCF and SCF in a Globalised Market**

The definition of SCF cannot be clear and precise, although scientists have tried to introduce a terminology that includes the following:


All-year round, intensive greenhouse cultivation of cut flowering stems may separate SCF from TCF. Species such as roses, gerberas, carnations, chrysanthemums, freesias, gypsophylla, eustoma, alstroemeria, phalaenopsis, anthurium etc. all fall into the TCF group (Table 1). The main differences between TCF vs. SCF could be related to:


**Table 1.** List of selected annual, perennial, and bulb specialty cut flowers (SCF) grown mainly outdoors at certain times of the year and traditional cut flowers (TCF) cultivated all-year round mainly inside greenhouses at different parts of the world [8,12–14].


The above mentioned lists of SCF and TCF are dynamic and change over the years as a result of changing worldwide production and marketing strategies by companies in the floriculture sector. In this view, SCF cultivated all-year round due to their increased market demands may be considered as TCF. In a globalised market increased popularity may define the crop as SCF or TCF. Growers may cultivate SCF and TCF simultaneously to satisfy local and/or international market demands.

Cultivation of SCF may increase local growers' income [14–18]. Growers located in North America found great potential in growing "alternative forest crops" such as salix species (i.e., *Salix matsudana*, *S. caprea*, *S. purpurea*), cornus (i.e., *Cornus sericea*), forsythias (*Forsythia* sp.), and various other flowering bunches [17]. In the North America wholesale markets, more than 1 million bunches of curly and pussy willow were sold in 2001. An additional 152,000 forsythia stems and more than 140,000 *Cornus* sp. bunches were also sold [17]. Willow cut stem growers in North America had great experience on cultivation, but they were not aware or informed about fertilization, pest management, and postharvest handling [19]. More research is required on SCF production and postharvest handling to provide solutions to growers and sellers [4,19].

In Australia, SCF production involved a wide range of native species dominated by the waxflower (*Chamelaucium uncinatum*), the kangaroo paw (*Anigozanthos* spp.), and the thryptomene (*Thryptomene* spp.) [9]. At least 64 other countries produce endemic Australian cut flowers with Israel, USA (California), South Africa, Ecuador, and Colombia being among them. *C. uncinatum* is a fast growing, evergreen shrub cultivated outdoors that produces flowering stems during winter after going through the short-day autumn period [20]. Australian native acacia species such as *A. dealbata*, *A. retinodes*, and *A. baileyana* are grown commercially in Australia and in the Mediterranean (i.e., Italy, Israel) for their impressive inflorescences [9,21]. Many SCF such as *Antirrhinum majus*, *Echinacea purpurea, Helianthus annuus, Limonium sinuatum, Matthiola incana*, *Scabiosa atropurpurea*, *Zinnia elegans*, and many more have increased their share in the US market [14,15]. According to the study presented by Starman et al. [15], 16 out of the 19 field grown SCF crops were profitable for commercial cultivation. Grower's income from *Achillea filipedulina*, *Liatris spicata*, *Veronica spicata*, and *Centranthus ruber* production increased linearly with increasing price/bunch sales. Stem prices varied during the year, and generally peaked around major holidays [15]. Among those species, *Cosmos bippinatus* cut flowering stems gave the highest income to growers (e.g., \$10.63–\$13.62). *Zinnia elegans*, *Scabiosa atropurpurea*, and *Antirrhinum majus* were also profitable for growers with incomes ranging from \$3.36 to \$4.51 [15]. Byczynski [22] stated that SCF growers could profit \$25,000 to \$35,000 per year, per ha of cultivation. Locally cultivated *Helianthus annuus* "Firecracker" plants would potentially be sold to wholesalers, retailers, and consumers in prices similar to those of the imported *H. annuus* flowering stems [18].

Although there is great potential in SCF cultivation, TCF hold the major part of sales in the local and international markets. In FloraHolland, the largest market of floricultural products worldwide, roses and chrysanthemums are the first and second best-selling cut flowers, respectively. The tulips, the gerberas, and the liliums came 3rd, 4th, and 5th in the top-5 list of cut flowers sold in 2019 [23]. Tulips may be considered as the best-selling SCF in the world, produced mainly in the Netherlands and presented as their national species.

The SWOT analysis shown in Table 2 provides useful comparisons between SCF and TCF. Although, growers' and sellers' decisions on cultivation and trade are complex and are often related to several factors and idiosyncrasies such as social and environmental legislations, infrastructures, environmental conditions, labour, and transportation costs. In a constantly changing global market, the TCF cultivation is the assured solution for growers and sellers, which, however, shows weaknesses associated with environmental legislations, CO2 footprints, pesticide residues, and increased energy demands (Table 2). On the contrary, production of SCF could serve as the new alternative choices for retailers and florists who always seek niche markets to sell their products [16]. This was the case for Oklahoma (US) ornamental-horticulture and cut flower retailers that indicated the positive outcome in retail by using a greater variety of species [16]. Local production for domestic markets could be another strength of SCF (Table 2). In the US, production of local SCF increased the past 20 years and challenged imports of TCF grown in South America or

other locaters [4,24]. Consumers who bought locally grown products had the perception of benefiting the local economy [25].


#### **4. Sustainable Production of SCF vs. TCF**

As the green industry continues to mature, differentiation is an increasingly important business strategy [26]. One way to accomplish this is by adopting environmentally friendly behaviours that will attract consumers with environmental awareness [27–29]. These potential consumers are more likely to purchase environmental friendly products with reduced CO2 footprints [3,26]. There is a small, but considerable, percentage of people who were willing to pay more money for agricultural products associated with sustainable, eco-friendly cultivation procedures [25,30]. Mainstream consumers were willing to buy eco-friendly products, but only at a modest price difference. Special attention should be given to consumer education and other promotion-related programs based on partnership between universities and private bodies (i.e., Texas Superstar®) to increase sales of new cut ornamental products [31]. Growers were also willing to adopt eco-friendly practices in cultivation, although they were sceptical on the implementation within their current cultivation system [32]. Back in 2010, Dennis et al. [32] reported that none of the grower respondents in their survey were certified as sustainable.

The recognition of floricultural products as "sustainable" is complex and demanding. Sustainable production is achieved via the implementation of strict environmental and social protocols as defined by the national and international directives [11,33]. Restrictions on CO2 footprints and global warming potential (GWP) may affect production of cut flowers in the future [33,34]. Over a public demand for cleaner agricultural products, the sustainable SCF cultivation may serve as the environmental friendly alternative option. This can be a major strength of SCF compared to TCF (Table 2). Wandl and Haberl [35]

showed that summer and spring SCF grown outdoors had <0.1 kg CO2 eq, while rose cultivation produced up to 13-fold more kg CO2. The main differences in CO2 production between the SCF and TCF were associated with excessive heating and electricity use "offseason" (i.e., the cold days of the year). Life cycle assessments (LCA) showed that increased CO2 outputs for the production of roses, chrysanthemums, and gerberas were profound in Central and Northern European countries such as the Netherlands, Germany, and Austria [35–38]. Significant differences in CO2 footprints, acidification, global warming, human toxicity, marine ecotoxicity, terrestrial ecotoxicity, and phytochemical oxidation were reported for roses produced in Dutch greenhouses compared to those produced in Ecuadorian facilities [33,38]. While CO2 for roses produced in Kenya and Ethiopia ranged between 0.4 and 3.7 kg CO2 eq, the Dutch roses released 16–29 kg CO2 eq [37]. In Greece, the cultivation of carnations in non-heated greenhouses produced only 0.316 kg CO2 eq [39], indicating that heating during winter is the single most important factor contributing to greenhouse gas (GHG) emissions. As a result, future environmental legislations will apply limitations to TCF cultivation at traditional production centres, or may help in shifting production of TCF to countries in the African continent or at South America [33,34,40–42].

Fertilization and agrochemical use both contribute to the environmental outputs during cultivation. Growers of TCF support the integrated nutrition management (INM) and integrated pest and disease management (IPDM) programs to reduce their environmental footprints [33]. However, pesticide residue levels in roses, gerberas, and chrysanthemums highly concerned authorities and consumers in the EU in the past decade. In a study conducted in Belgium, 107 active ingredients were detected in harvested rose, gerbera, and chrysanthemum bunches [43]. Among them, roses were the most contaminated flowers with 14 distinct substances detected per sample and a total concentration of 26 mg kg−<sup>1</sup> for a single rose sample. Substances such as acephate, methiocarb, monocrotophos, methomyl, deltamethrin, etc., could generate direct toxic effects to the nervous system of florists and consumers [43]. No research was found on pesticide residues detected on SCF. Generally, SCF suffer fewer fungal and pest contaminations during production, and therefore require minimal amounts of phytochemicals. SCF crop rotation and seasonal production might be the key to less infections and herbivore attacks. In every case, the implementation of IPDM to ornamental crop production may eventually reduce pesticide residues and improve the profile of the floricultural products.

#### **5. Postharvest Performance and Quality**

Vase life (VL) of cut flowers is considered as the single most important factor affecting consumers' buying choices. The shorter the VL of flowers purchased, the lower the possibility for a repeated buying [44,45]. While TCF hybrids show exceptional VL, the SCF show shorter longevities [14]. Environment conditions and genotype were the most important preharvest factors that contributed to inflorescence VL [46]. For example, increased RH levels inside the greenhouse decreased stomatal conductance of rose plants during the day. It was shown that stomatal responsiveness could be improved by adjusting the humidity levels either during the day or at night. Mortensen and Gislerød [47] showed that severe drought stress during growth of roses at high RH increased their VL. Longevity of vars. "Akito" and "First Red" roses decreased in winter as a result of higher humidity levels inside the greenhouses [48].

Harvest time and stage of development significantly affected VL of TCF and SCF [49]. Harvest procedures vary among species. For example, species of the Asteraceae family (i.e., chrysanthemums, gerberas, zinnias, etc.) should be in full maturity, whereas inflorescences with multiple buds are harvested at 25–50% open flowers [12,14]. Harvesting the SCF inflorescences *Eremurus* "Line Dance" and "Tap Dance" at the stage of 0-florets open resulted in significantly longer VL compared to inflorescences harvested at 1–2 and at 3–5 floret rows open [50]. VL of cut *Capsicum* "Rio Light Orange" stems was affected by harvest stage with "partly mature fruit stage" being the best for harvest [51]. Both *Celosia argentea* and *Antirrhinum majus* inflorescences had maximum VL at early harvest stage of short

head diameter and 0-florets open, respectively [52]. On the contrary, VL of *Leucocoryne coquimbensis* [53], *Viburnum tinus* [54], and *Spartium junceum* [55] inflorescences were not affected by the harvest stage, indicating that different responses are recorded among different genotypes. Stem length, leaf number, and harvest time significantly affected VL of various cut flower species [46]. Leaf number and stem length affected transpiration, water balance of cells, and water transport in xylem vessels, respectively. These were identified as the most crucial factors of flower wilting. Day-light and temperature during cultivation may also affect VL in response to stem hydraulic conductivity and carbohydrate storage [56,57].

Short-period storage and handling significantly affect VL of TCF and SCF [49]. Many SCF may respond well to low temperature storage [14]. SCF responded differently to postharvest handling (i.e., storage time and temperature, wet or dry storage). For example, wet or dry storage of *Eremurus* [50], *Matthiola incana* [58], *Capsicum* ornamental peppers [51], *Celosia argentea*, and *Antirrhinum majus* [52] was effective for up to 2 weeks. *Achillea filipendulina*, *Buddleia davidii*, *Cercis canadensis*, *Cosmos bipinnatus*, *Echinacea purpurea*, *Helianthus maximilianii*, *Penstemon digitalis*, and *Weigela* sp. were all positively affected by 1–2 week storage at temperatures ranging from 2.0 to 7.0 ◦C [59]. *S. junceum* inflorescences were successfully stored for 30 d at 3 ◦C, without any loss in VL [55]. Likewise, *Curcuma alismatifolia* was stored for 6 d at 7 ◦C without any loss in VL [60]. *Anigozanthos* spp. could be stored for up to 2 weeks at 4 ◦C without showing symptoms of wilt or petal discolouration [61].

The presence of ethylene inside storage facilities during handling or transport may severely reduce VL and quality of climacteric TCF and SCF. The SCF *Achillea filipendulina*, *Celosia argentea, Helianthus maximilianii*, *Penstemon digitalis*, and *Weigela* sp. were negatively affected by the presence of <1 μL L−<sup>1</sup> ethylene inside the storage rooms [59]. Exposure of *Boronia heterophylla* flowers to 10 μL ethylene L−<sup>1</sup> significantly reduced VL by 2.6 d compared to the un-exposed flowers [62]. *Curcuma alismatifolia* showed significant reductions in VL and bud opening after exposure to 0.5 - 2.0 μL L−<sup>1</sup> ethylene [60]. *Spartium junceum* inflorescences suffered a detrimental flower and organ fall when exposed to 5 or 10 μL L−<sup>1</sup> ethylene [55]. Treatments with 1-MCP, STS, Ag+, and α-aminoisobutyric acid alleviated ethylene suffering and reduced aging symptoms. 1-MCP was found to be effective in various SCF including *Boronia heterophylla* [62], *Curcuma alismatifolia* [60], *Viburnum tinus* [54], *Celosia argentea, Antirrhinum majus* [52,63], *Eremurus* [50], and *Spartium junceum* [55]. 1-MCP increased the VL in ethylene-present or ethylene-free environments and delayed the aging process. Likewise, STS or Ag+ blocked ethylene-induced flower abscission and wilt and facilitated VL increases in most of the ethylene-sensitive SCF (i.e., *V. tinus, A. majus, B. heterophylla*).

#### **6. Conclusions**

Analysing the strengths and weaknesses of SCF and TCF, there are arguments over sustainability, marketing, and promotion of those cut flower groups. TCF are highly recognizable floricultural products, and biotechnology centres back-up their development with new varieties. On the contrary, SCF may be the eco-friendly alternative species to provide sustainable solutions to growers and consumers. SCF cultivation and sales have gained volumes over the last 15 years. SCF production can be sustainable with minimum energy and agrochemical inputs, although more research is required on VL extension and postharvest quality care.

**Funding:** This research received no external funding

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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