# Cutting propagation of Bornmuellera tymphaea, a nickel hyper accumulator for use in agromining: Effects of rooting media and auxins on stem cuttings

Dimitrios Kyrkas, Nikolaos Mantzos, George Patakioutas, Eleni Lampraki, Evangelos Filis, Panayiotis G Dimitrakopoulos, Guillaume Echevarria, Maria Konstantinou

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Dimitrios Kyrkas, Nikolaos Mantzos, George Patakioutas, Eleni Lampraki, Evangelos Filis, et al. Cutting propagation of Bornmuellera tymphaea, a nickel hyperaccumulator for use in agromining: Effects of rooting media and auxins on stem cuttings. Ecological Research, 2024, 39 (4), pp.471-478. 10.1111/1440-1703.12459 . hal-04666256

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SPECIAL FEATURE

Ultramafic Ecology: Proceedings of the 10th International Conference on Serpentine Ecology

ECOLOGICAL
RESEARCH

WILEY

# Cutting propagation of *Bornmuellera tymphaea*, a nickel hyperaccumulator for use in agromining: Effects of rooting media and auxins on stem cuttings

Dimitrios Kyrkas<fnref n="1" />,<fnref n="2" /> ID

Nikolaos Mantzos<fnref n="1" />

George Patakioutas<fnref n="1" />

Eleni Lampraki<fnref n="1" />

Evangelos Filis<fnref n="3" /> ID

Panayiotis G. Dimitrakopoulos<fnref n="2" /> ID

Guillaume Echevarria<fnref n="4" />,<fnref n="5" /> ID

Maria Konstantinou<fnref n="3" /> ID

**Correspondence**

Dimitrios Kyrkas, Department of Agriculture, University of Ioannina, Arta, Greece.

Email: dkyrkas@uoi.gr

**Abstract**

The nickel hyperaccumulator *Bornmuellera tymphaea* has been evaluated for use in agromining in Greece with very promising results. This study examines propagation of this species based on cuttings, which provides rapid multiplication and clonal propagation of genotypes with useful genetic traits and offers obvious advantages over propagation by seed. Apical cuttings, 10 cm long, obtained from plants in the field, were propagated on mist benches under greenhouse conditions. Four treatments based on the rooting medium were evaluated: (a) perlite, (b) perlite + peat 1:1 (v/v), (c) ultramafic soil, and (d) ultramafic soil + perlite 1:1 (v/v). Completely randomized blocks were used in a 4 × 2 factorial design, using 4 different propagation rooting media and 2 levels of auxins (with or without the use of indole-3-butyric acid [IBA]), with 3 replicates of 28 cuttings. The response to perlite medium was best in terms of rooting: 75% without IBA and 71.33% with IBA, and the dry weight of roots was 0.18 g and of shoots 0.34 g. Perlite + peat 1:1 (v/v) medium also resulted in rooting in more than 55% of cuttings whereas cuttings grown in ultramafic soil showed very low rooting (<27%). Based on the findings, *B. tymphaea* has an aptitude for vegetative propagation by cuttings with survival values and rooting greater than 70%. Perlite combined with the application of auxin (IBA) is recommended.

**KEYWORDS**

Brassicaceae, indole-3-butyric acid (IBA), peat, perlite, ultramafic soil

# 1 | INTRODUCTION

The ability to hyperaccumulate 1 or more elements has been detected in 721 taxa, with the largest number of

them being members of the family Brassicaceae (Jakovljević et al., 2022; Reeves et al., 2018). Nickel is the element most frequently hyperaccumulated by plants growing in ultramafic soils at concentrations above the

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

© 2024 The Author(s). *Ecological Research* published by John Wiley & Sons Australia, Ltd on behalf of The Ecological Society of Japan.

<footnote n="1">¹Department of Agriculture, University of Ioannina, Arta, Greece</footnote>

<footnote n="2">²Department of Environment, University of the Aegean, Mytilene, Greece</footnote>

<footnote n="3">³Department of Agriculture, International Hellenic University, Sindos (Thessaloniki), Greece</footnote>

<footnote n="4">⁴Laboratoire Sols et Environnement, Université de Lorraine, INRAE, Nancy, France</footnote>

<footnote n="5">⁵ECONICK, Lunéville, France</footnote>
nominal threshold (>1000 µg g⁻¹; van der Ent et al., 2013). Hyperaccumulation of nickel has been detected in 532 taxa that occur on ultramafic soils (Reeves et al., 2018). These soils are typically characterized by elevated concentrations of nickel, chromium, cobalt, iron, and in some cases manganese. Additionally, they often exhibit a low Ca/Mg ratio, and a deficiency in essential macronutrients such as nitrogen, potassium, or phosphorus (Echevarria, 2021; Kazakou et al., 2008; Proctor & Woodell, 1975). Agricultural land situated on ultramafic soils is often unproductive and unsuitable for conventional farming practices due to these geochemical characteristics. A pioneering agricultural method, known as nickel agromining, has emerged as an innovative approach to transform low-productivity agricultural land derived from ultramafic bedrock into a source of income. This involves cultivating appropriate nickel hyperaccumulator species to harness the economic potential of these challenging soil conditions (Chaney et al., 2021; Nascimento et al., 2022; Nkrumah et al., 2016; van der Ent et al., 2015).

Research results up to date indicate the existence of two basic groups of Ni hyperaccumulators based on their distribution: one main group with tropical and subtropical distribution (predominantly New Caledonia, Cuba, and Indonesia) and the other one with distribution in the Mediterranean and Eurasian regions (extending from Portugal eastward across the Apennines and the Balkan Peninsula to Turkey) (Jakovljević et al., 2022). Within Europe, the Balkans possess extensive coverage of ultramafic rocks, more than in any other part of the continent. Furthermore, nickel hyperaccumulators native to Europe are concentrated in the Balkan peninsula, totaling 27 taxa (Jakovljević et al., 2022; Stevanović et al., 2003; van der Ent et al., 2013). Most of them, comprising 24 taxa (77.4%), belong to the Brassicaceae family (Bergmeier et al., 2009; Jakovljević et al., 2022; Kidd et al., 2018; Stevanović et al., 2003). This concentration of nickel hyperaccumulators in the Balkan Peninsula establishes it as a potential hotspot for nickel agromining activities (Bani et al., 2021; Bani & Echevarria, 2019; Bani, Echevarria, Sulçe, & Morel, 2015; Bani, Echevarria, Zhang, et al., 2015; Brooks, 1987; van der Ent et al., 2015).

*Bornmuellera tymphaea* (Hausskn.) Hausskn. is a Greek endemic with high biomass, perennial nickel hyperaccumulator of the Brassicaceae family, an obligate serpentinophyte, and closely related to ultramafic substrates of the Pindus mountain range (Dimopoulos et al., 2023; Jakovljević et al., 2022; Stevanović et al., 2003). The highest foliar Ni concentration measured up to date in *B. tymphaea* when growing in its natural environment is 31.2 g kg⁻¹ DW (Jakovljević et al., 2022; Reeves et al., 1980, 1983). Recently, *B. tymphaea* has been studied for its potential in Ni

agromining in the Mediterranean area (Chardot et al., 2005; Hipfinger et al., 2022; Kidd et al., 2018; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos, et al., 2019; Pardo et al., 2018). The highest nickel yield recorded for *B. tymphaea* is 149 kg ha⁻¹, which was achieved from field experiments in its natural habitat in Greece (Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos & Konstantinou, 2019; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Tognacchini, et al., 2019). The agromonic practices followed to date in Greek field trials showed that *B. tymphaea* is a promising candidate for use in Ni agromining (Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Dimitrakopoulos & Konstantinou, 2019; Kyrkas, Echevarria, Benizri, Mantzos, Patakioutas, Kidd, Morel, Simonnot, Tognacchini, et al., 2019).

One of the most important stages in crop development is the preparation of the propagative material. Propagation through cuttings is a method of producing a large amount of planting material in a short period, maintaining the genetic purity of the plants (Siqueira et al., 2020). It is often followed in the reproduction of horticultural, ornamental, and protected or endangered plant species (Baul et al., 2011; Jiang et al., 2017; Siqueira et al., 2020). Although *B. tymphaea* can reproduce via seeds (Kyrkas et al., 2023) since it is an endemic species, the collection of seeds from the wild could affect the size of local populations. Additionally, the exploitation of native plant species should only be carried out with the certainty of their conservation. Therefore, it is necessary to investigate the asexual propagation methods for the sustainable exploitation of *B. tymphaea*. In this context, creating cuttings is one of the most suitable techniques for large-scale asexual propagation (Siqueira et al., 2020).

Synthetic auxins such as indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) are commonly used to promote root development in vegetative propagation. Auxins promote starch hydrolysis as well as the mobilization of sugars and nutrients at the base of the cuttings during the regeneration of adventitious roots (Nanda et al., 1974). Although auxin is produced naturally in the shoots and young leaves of plants, the use of synthetic auxin is recommended to ensure rooting of cuttings and prevent them from dying (Daskalakis et al., 2018; Galavi et al., 2013).

The rooting media are another important factor in the propagation system as directly influence the rooting percentage and quality of the roots produced. A rooting medium must provide nutrients and water, allow gas exchange to and from the roots and must also provide support to the plants (Kumar et al., 2019, 2021; Riaz et al., 2008). The selection and preparation of the
medium are particularly important for the rooting response and plant growth. Mixed rooting media are also widely used, in order not to shock the plants after rooting, because the nutrients are already available in the media (Chaudhari et al., 2018).

This study aims to verify the possibility of propagating *B. tymphaea* through cuttings, testing different rooting media and using auxins as this species is a significant candidate for large-scale cultivation in agricultural land derived from ultramafic bedrocks.

# 2 | MATERIALS AND METHODS

## 2.1 | Experimental design

The experiment was carried out from October to December 2019 in the glass greenhouse of the Department of Agriculture of the University of Ioannina, at the campus of Arta. (39°07′15.7″ N, 20°56′44.7″ E). Apical softwood cuttings obtained from plants in the field were propagated in benches under misting and natural photoperiod. The temperature was set at 21/18.3°C day/night. The irrigation system was intermittent misting, and the relative air humidity was kept above 90%.

The experiment was laid out in randomized complete block design with three replications of 28 cuttings, in four different rooting media which were used pure or in mixture, with or without the use of IBA rooting hormone. Therefore, a total of 672 apical softwood cuttings were used. The rooting media applied were: (a) perlite, (b) perlite + peat, (c) ultramafic soil, and (d) ultramafic soil + perlite. In all rooting media mixtures used the ratio was 1:1 (v/v). The ultramafic soil (Ni concentration 2368 mg kg⁻¹ dry soil) was sandy clay and was collected from the field at 20 cm depth. The peat used in the experiment had a pH of 5.5–6.5 and an electrical conductivity of 0.1–0.3 mS cm⁻¹ (100–300 µS cm⁻¹). The perlite used was for hydroponic cultivations with grain size between 0.63 and 4 mm (with only 4% below 0.63 mm).

## 2.2 | Setting of the cuttings

Branches of *B. tymphaea* were collected in situ, early in the morning, from a population from Smixi, Municipality of Grevena, Greece (40°03′40.8″ N, 21°06′54.3″ E, alt. 1932 m a.s.l.), in an area with ultramafic soil dominated by grasses prone to erosive processes. Immediately after the collection, the branches were put in a portable cooler and transferred to the laboratory. In the afternoon of the same day, apical cuttings, of approximately 10 cm in length, with 10–15 leaves each, were treated with auxin

rooting hormone (IBA, 0,066% w/w, in powder form, trade name Radicin, Phytorgan, Greece) and planted into four different rooting media.

## 2.3 | Measurements

The evaluation of cuttings was performed 72 days after the experiment was set up. Six plants from each treatment were used to determine the following data: rooting percentage, fresh and dry weight of the roots and shoots of rooted plants. For the measurement of root and shoot dry weight, the difference of mass (in %) before and after drying the material in an oven at 85°C for 3 days was calculated. During the experiment, the percentage of survival of the cuttings (percentage of survived cuttings regardless of whether they had rooted) in relation to the time was also calculated.

## 2.4 | Statistical analysis

The data were analyzed with analysis of variance (ANOVA) using IBM SPSS software (Version 28). The percentage of survival of the cuttings of *B. tymphaea* was the response variable tested for significant differences between treatments. The design included the following factors: time, rooting medium, auxin use, and the two- or three-way interactions between them. When fresh and dry weight of shoots and roots used as dependent variable, rooting medium, auxin use, and individual plant served as factors. To test for pairwise differences, Bonferroni multiple comparisons were used at a significance level of 0.05.

# 3 | RESULTS AND DISCUSSION

Both rooting medium and hormone treatments influence the survival and rooting percentage of the cuttings as well as the fresh and dry weight of the roots and shoots of rooted plants. The percentage of survival of the cuttings was reduced during the experiment ($F_{9,160} = 56.868, p < 0.001$ for the main effect of time; Table 1 and Figure 1). A significant "rooting medium × auxin" interaction ($F_{3,160} = 43.398; p < 0.001$; Table 1) emerged, indicating that the effect of rooting medium on cuttings' survival varied with the use of auxin (Figure 1). Cuttings grown on ultramafic soil showed the lowest survival rates regardless of the addition of auxin (Figure 1), but differed significantly only from those on the peat + perlite rooting medium ($p = 0.007$).

Only the rooting medium has a significant effect on fresh ($F_{3,91} = 3.441, p = 0.020$; Figure 2) and dry weight
<table><thead><tr><th>Source of variation</th><th>SS</th><th>df</th><th>MS</th><th>F</th><th>p-value</th></tr></thead><tbody><tr><td>Time</td><td>96,267.287</td><td>9</td><td>10,696.365</td><td>56.868</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Rooting medium</td><td>2247.013</td><td>3</td><td>749.004</td><td>3.982</td><td><strong>0.009</strong></td></tr><tr><td>Auxin</td><td>862.604</td><td>1</td><td>862.604</td><td>4.586</td><td><strong>0.034</strong></td></tr><tr><td>Rooting medium × auxin</td><td>24,488.613</td><td>3</td><td>8162.871</td><td>43.398</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Time × rooting medium × auxin</td><td>14,055.813</td><td>63</td><td>223.108</td><td>1.186</td><td>0.198</td></tr><tr><td>Error</td><td>30,094.667</td><td>160</td><td>188.092</td><td></td><td></td></tr><tr><td>Total</td><td>168,015.996</td><td>239</td><td></td><td></td><td></td></tr></tbody></table>

Note: Sum of squares (SS), degrees of freedom (df), mean squares (MS), F-ratio, and *p*-values for each variable are shown. Significant values (*p* < 0.05) are in bold.

<caption>TABLE 1 Summary of analysis of variance (ANOVA) results for differences in the percentage of survival of *Bornmuellera tymphaea* cuttings as a function of different rooting media, auxin use, and time after planting.</caption>

<figure><img src="image_2.png" /><figcaption>FIGURE 1 Percentage of survival of the cuttings of *Bornmuellera tymphaea* (initial size: 28 cuttings) across the experiment (72 days) in relation to rooting medium and auxin use. Values are mean values of three replicates per treatment and auxin level. IBA, indole-3-butyric acid.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>FIGURE 2 Fresh weight (g) of *Bornmuellera tymphaea* shoots (*n* = 18, 6 plants × 3 replicates) measured at the end of the experiment (Day 72) in relation to rooting medium and auxin use. Different lowercase letters indicate significant differences (at *p* < 0.05) among rooting media using the Bonferroni multiple comparison test.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>FIGURE 3 Dry weight (g) of *Bornmuellera tymphaea* shoots (*n* = 18, 6 plants × 3 replicates) measured at the end of the experiment (Day 72) in relation to rooting medium and auxin use. Different lowercase letters indicate significant differences (at *p* < 0.05) among rooting media using the Bonferroni multiple comparison test.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

FIGURE 4 Fresh weight (g) of *Bornmuellera tymphaea* roots ($n = 18$, 6 plants × 3 replicates) measured at the end of the experiment (Day 72) in relation to rooting medium and auxin use. Different lowercase letters indicate significant differences (at $p < 0.05$) among rooting media using the Bonferroni multiple comparison test.

<figure><img src="image_6.png" /></figure>

FIGURE 5 Dry weight (g) of *Bornmuellera tymphaea* roots ($n = 18$, 6 plants × 3 replicates) measured at the end of the experiment (Day 72) in relation to rooting medium and auxin use. Different lowercase letters indicate significant differences (at $p < 0.05$) among rooting media using the Bonferroni multiple comparison test.

($F_{3,91} = 10.316$, $p < 0.001$; Figure 3) of the shoots of rooted plants. A significant "rooting medium × auxin" interaction arose for both fresh ($F_{3,91} = 3.786$; $p = 0.013$; Figure 4) and dry weight ($F_{3,91} = 3.392$; $p = 0.021$; Figure 5) of roots of the species. This indicates that the effect of rooting medium on fresh or dry root weight varied with auxin use. For example, the effect of auxin on fresh and dry root weight was more pronounced when cuttings were grown on perlite (Figures 4–6).

These findings substantiate those of Sogoni et al. (2022) on the vegetative propagation by nodal stem cuttings of *Tetragonia decumbens*, a wild vegetable, who studied four different rooting media and IBA. The results indicated significant growth differences in various rooting media, whereas the IBA treatment and its interaction with rooting media were only significant in rooting

<figure><img src="image_7.png" /></figure>

FIGURE 6 Rooted stem cuttings of *Bornmuellera tymphaea* after 72 days in the rooting phase, in four different growing media: (a) perlite; (b) perlite + peat; (c) ultramafic soil; (d) ultramafic soil + perlite.

percentage and height of the stem cuttings. Zheng et al. (2020) investigated the effects of rooting substrate and auxin concentration on the adventitious rooting of the stem cuttings of *Dianthus caryophyllus* (carnation), an ornamental plant. They concluded that the appropriate rooting substrate combined with the application of the certain auxin's concentrations promoted the rooting percentage of the cuttings. According to the results of Jiang et al. (2017), who studied the asexual cutting propagation of two ornamental species *Silene chalcedonica* and *Silene coronaria*, using two different propagation substrates and three different root-promoting hormone products, the rooting success of stem cuttings was highly species related. *Silene coronaria* cuttings rooted readily in vermiculite without an auxin treatment whereas the rooting percentage of *S. chalcedonica* cuttings was improved by using an auxin and grown in vermiculite. The application of external hormone was essential for achieving high rooting capacity of summer softwood leafy cuttings of the *Cornus sanguinea* (Karapatzak et al., 2023).

The rooting medium is one of the most significant factors affecting root initiation and ensures a good balance between the water-holding capacity and air/water permeability. The different rooting media tested in the present study showed significant differences in root formation. Perlite and peat rooting media stood out over rooting media containing ultramafic soil, the latter being observed as the least efficient for the induction of roots from plant cuttings. When the amount of ultramafic soil
in the rooting media was increased, a decrease in root formation and greater mortality of cuttings was observed (Figure 6). This is likely due to the sandy clay texture of the ultramafic soil, leading to poor aeration. Poor aeration is a fundamental problem in waterlogged situations, leading to the decay of cuttings before root initiation (Schmitz et al., 2013). In the present study, perlite combined with the application of auxin (IBA) gave the best results for the propagation of *B. tymphaea* by cuttings.

## 4 | CONCLUSIONS

Based on the findings, *B. tymphaea* has an aptitude for vegetative propagation by cuttings with survival values and rooting greater than 70%. Regarding the use of auxin, a beneficial effect was observed on the fresh and dry weight of roots of the species only in the treatment with perlite, while in the above-ground part, respectively, no significant effect was observed. Therefore, the use of perlite as a rooting medium in combination with the use of auxin is recommended for the vegetative propagation through cuttings of the nickel hyperaccumulator *B. tymphaea*. Further research is recommended to investigate other auxins such as NAA and other rooting media, type of cuttings, and genotype to achieve good quality plants with a high nickel yield that can be used in agromining.

### CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

### ORCID

Dimitrios Kyrkas https://orcid.org/0000-0003-0267-3121

Evangelos Filis https://orcid.org/0000-0001-8726-9077

Panayiotis G. Dimitrakopoulos https://orcid.org/0000-0002-8374-4392

Guillaume Echevarria https://orcid.org/0000-0003-2124-1447

Maria Konstantinou https://orcid.org/0000-0002-4163-1736

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**How to cite this article:** Kyrkas, D., Mantzos, N.,

Patakioutas, G., Lampraki, E., Filis, E., Dimitrakopoulos, P. G., Echevarria, G., & Konstantinou, M. (2024). Cutting propagation of *Bornmuellera tymphaea*, a nickel

hyperaccumulator for use in agromining: Effects of rooting media and auxins on stem cuttings. *Ecological Research*, 1-8. https://doi.org/10.1111/1440-1703.12459