Smoke tree (Cotinus coggygria Scop.) propagation and biotechnology: A mini-review

South African Journal of Botany 114 (2018) 232–240

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Minireview

Smoke tree (Cotinus coggygria Scop.) propagation and biotechnology: A mini-review Jaime A. Teixeira da Silva a,⁎, Andrzej Pacholczak b,⁎, Agnieszka Ilczuk b,⁎ a b

P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken 761-0799, Japan Warsaw University of Life Sciences, Faculty of Horticulture, Biotechnology and Landscape Architecture, Department of Ornamental Plants, Nowoursynowska 159, 02-787 Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 15 May 2017 Received in revised form 8 November 2017 Accepted 10 November 2017 Available online xxxx Edited by AO Aremu Keywords: Auxin Biotechnology Biostimulants Cytokinins Ex vitro In vitro Ornamental

a b s t r a c t Cotinus coggygria Scop. (Anacardiaceae), commonly referred to as ‘smoke tree’ or ‘smoke bush’, is an attractive ornamental tree or large bush that has medicinal properties and multiple bioactivities. Seed germination can be high after treating seeds with H2SO4, but to avoid growing male trees that will not form attractive inflorescences, the rooting of cuttings from female trees is a preferred method of vegetative propagation. One method to intensify the rooting of stem cuttings is by shading mother plants and simultaneously applying an exogenous auxin or biostimulator. This method can increase the quality of rooted cuttings. In addition, shading causes anatomical changes in the stem structure and leads to the formation of adventitious root primordia. New EU regulations have forced member states to change or withdraw existing authorization for products used in plant protection that contain active substances like auxin (indole-3-butyric acid, IBA). Consequently, there is an active search for alternative measures to support the process of rooting woody plants. Examples of such preparations are biostimulators, including AlgaminoPlant, HumiPlant and Route®, which can increase the rooting of cuttings, affect processes associated with oxidative stress, and increase the intensity of gas exchange and the content of organic compounds. The most effective in vitro micropropagation protocol for C. coggygria involves the use of Murashige and Skoog basal solid or liquid media enriched with one of two cytokinins, 6N-benzyladenine or meta-metoxytopolins. The use of IBA has shown most success, both ex vitro and in vitro. All other aspects of Cotinus species biotechnology still need to be developed. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Cotinus coggygria Scop. (Anacardiaceae; The Plant List, 2017a) has nine synonymous varieties or subspecies and even though it has 21 species (accepted, synonymous, or contested; The Plant List, 2017b), published literature exists on the propagation biology and biotechnology of only one, C. coggygria. The common names ‘smoke tree’ or ‘smoke bush’ have been assigned to C. coggygria, which is a small tree or bush (Gilman and Watson, 1993) that is distributed primarily from southern Europe to western China, and is a hardy (e.g. ‘Nordine Red’) or purpleleaved (e.g. ‘Royal Purple’) ornamental (Fig. 1) (Pijut, 2000). Purple coloring of leaves results from the accumulation of anthocyanins in response to 300–400 nm of UV light and cold temperature (4–9 °C) (Oren-Shamir and Levi-Nissim, 1997). In the Northern hemisphere, Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, abscisic acid; BA, 6Nbenzyladenine; H2O2, hydrogen peroxide; IAA, indole-3-acetic acid; IAAO, oxidase indole-3-acetic acid; IBA, indole-3-butyric acid; LS, Linsmaier and Skoog medium; MemT, meta-metoxytopolin; MemTR, meta-metoxytopolin riboside; MS, Murashige and Skoog medium; NAA, 1-naphthaleneacetic acid; PPFD, photosynthetic photon flux density; POX, peroxidase; PPO, polyphenol oxidase; QL, Quoirin and Lepoivre medium. ⁎ Corresponding authors. E-mail addresses: [email protected] (J.A. Teixeira da Silva), [email protected] (A. Pacholczak), [email protected] (A. Ilczuk).

https://doi.org/10.1016/j.sajb.2017.11.009 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

smoke tree flowers in the summer and sets seed in autumn. This makes several C. coggygria cultivars an attractive option for landscaping, including in rooftop gardening where it has been shown to exhibit mild cold (−23 °C) tolerance (Fan and Wang, 2011). Plants grow well when potted in 70 peat:30 bark (v/v) (Cameron et al., 2005). There is a risk of fungal infection, especially by Verticillium dahliae, which causes plant stunting and early leaf senescence, thus negatively affecting its ornamental properties (Gilman and Watson, 1993; Xiong et al., 2014). A comprehensive review highlighted the pharmacological and phytochemical constituents of C. coggygria with a wide range of biological activities (“antioxidative, antibacterial, antifungal, antiviral, anticancer, antigenotoxic, hepatoprotective and anti-inflammatory”) derived from the plant's essential oils and extracts found in shoots, leaves, flowers and heartwood (Matić et al., 2016). Thus, in addition to its ornamental value, C. coggygria has important medicinal properties. This review explores the propagation of this ornamental, both via conventional seed or cutting propagation and by in vitro tissue culture.

2. Seed germination and seedling establishment Sexual or seed propagation of C. coggygria is a common method to produce many progenies but these are genetically heterogeneous.

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et al. (2014) evaluated the effect of H2SO4-based scarification, cold stratification and GA3 to break seed dormancy. Seeds that were only cold stratified for up to 120 days resulted in very low germination (0– 15.8%). The best results were obtained when seeds were acid-scarified for 60 min, treated for 24 h with 0.5–2.0 mg L−1 GA3 and cold stratified for 1–3 months. Germination was 65–73.3%, independent of GA3 concentration and the duration of chilling. The application of GA3 could not replace the stratification period but could stimulate seed germination (30.8–35.8% versus 5.0% in the control). Similar results (40.7%) were obtained by Deng et al. (2010) when seeds of C. coggygria var. cinerea were scarified and then immersed in 346.4 mg L−1 GA3 (Table 1). The differences between the results of those research groups can be attributed to the provenance of seeds. Seed used by Olmez et al. (2008, 2009) and by Guner and Tilki (2009) were from Turkey, those by Stilinovic and Grbic (1988) from Serbia and those by Pipinis et al. (2014) from Greece. It can be inferred that seeds from Turkey had lower dormancy than those from Serbia. It is also possible that seeds from Greece and Serbia had a similar hard testa that was impenetrable to water.

3. Vegetative propagation

Fig. 1. Five-year-old plants of Cotinus coggygria ‘Royal Purple’ grown in autumn.

Even though as many as 100,000–119,000 seeds/kg can be collected, some studies have indicated that C. coggygria seeds exhibit some dormancy and reduced (75%) germination ability, limiting the ability to propagate this plant (Hartmann et al., 1997; Olmez et al., 2008; Deng et al., 2010). Seeds have a hard testa and display internal dormancy (physical and physiological) (Stilinovic and Grbic, 1988; Olmez et al., 2009). Several methods are available to break seed dormancy, including scarification (mechanical, physical and chemical), cold stratification, soaking in gibberellic acid (GA3) or a combination of all of these (Stilinovic and Grbic, 1988; Pijut, 2000; Olmez et al., 2008; Guner and Tilki, 2009; Olmez et al., 2009; Deng et al., 2010; Pipinis et al., 2014). Seeds of C. coggygria can be harvested and sown in autumn without any treatment or in spring with a pre-treatment that can involve 30 min scarification in H2SO4 and subsequent stratification in moist sand for 45–60 days at 3 °C (Pijut, 2000). Olmez et al. (2008) soaked seeds in hot water (88–100 °C) for 24 h and cooled them immediately or soaked them for 24 h in tap water. To scarify seeds, they used 98% H2SO4 for 20, 50, or 80 min then placed seeds in the cold for 20, 40, or 60 days. Seeds were sown in pots in a greenhouse or in a field. Seeds immersed for 20–80 min in H2SO4 then cold stratified for 60 days and left for 22–25 days under greenhouse conditions resulted in 77.1–82.8% germination. In the control treatment, only 19.3% of seeds germinated after 52 days while under field conditions germination was even lower. Olmez et al. (2009), in ensuing experiments, immersed seeds in H2SO4 for 50 min and cold stratified (5 °C) them for 15 days, resulting in 88.1% germination in the laboratory, but in greenhouse conditions, 70.2% of seeds germinated after 10 min treatment with H2SO4 and 30day stratification. Guner and Tilki (2009) noted that the germination of C. coggygria seeds increased significantly when stratification period was extended (16.8% after 30 days, 72.3% after 120 days and 1.7% in the control). In addition, 10–40 min scarification in H2SO4 together with cold stratification for 30 or 60 days significantly improved germination percentage (95.8–99.0%) compared to other treatments. Pipinis

Seed germination tends to result in the production of male plants that are then not able to produce the attractive inflorescences typical of this ornamental. For this reason, vegetative propagation has received the greatest attention in terms of research output to date. Thus, it is common to propagate vegetative stem cuttings of female plants with indole-3-butyric acid (IBA) at 1.0–3.0 g L−1 and under misting in moist soil. Roots develop between 4 and 8 weeks, as was already shown by earlier studies, mainly with C. coggygria ‘Royal Purple’ (Kelley and Foret, 1977; Siftar, 1981; Blakesley et al., 1991, 1992; Pijut, 2000). Kelley and Foret (1977) claimed 33% rooting success of stems sampled in early June in the absence of any special treatments, increasing up to 95% when IBA was used. Blakesley et al. (1991) used young shoots 8– 10 cm long denuded of leaves in the basal 3–4 cm section. Even though these shoots were dipped in rooting medium, no roots formed during the summer growth period (July and August). In their study, high levels of rooting were associated with high levels of free indole-3-acetic acid (IAA) in plants. A subsequent study by the same authors (Blakesley et al., 1992) showed that etiolated shoots collected in May could induce higher levels of rooting using the same treatment. Cameron et al. (2001a) showed how removal of the shoot tip reduced rooting percentage from 95% to 40%, while fogging resulted in about 20% higher rooting than misting. In contrast to most of those studies where leaves were normally removed from shoots prior to rooting, Cameron et al. (2001b) dipped shoots in 1.26 g L−1 IBA for 5 s, and suggested leaving all leaves intact to allow for maximum leaf area. They obtained N80% rooting in contrast to 44% rooting when leaf area was reduced and b22% rooting when lateral branches were trimmed, perhaps explaining the 0% rooting observed by Blakesley et al. (1991). This may be

Table 1 Methods of breaking seeds dormancy of Cotinus coggygria Scop. Method

References

24 h soak in hot (88–100 °C) water and immediately cooling 24 h soak in tap water 98% H2SO4 scarification during 20–80 min

Olmez et al. (2008)

20–120 days stratification at 3–5 °C 24 h soak in GA3

Olmez et al. (2008) Stilinovic and Grbic (1988), Pijut (2000), Olmez et al. (2008), Guner and Tilki (2009), Olmez et al. (2009), Pipinis et al. (2014) Stilinovic and Grbic (1988), Pijut (2000), Olmez et al. (2008), Guner and Tilki (2009), Olmez et al. (2009), Pipinis et al. (2014) Deng et al. (2010), Pipinis et al. (2014)

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associated with the availability of carbon sinks for root formation (Aminah et al., 1997). Research on the effects of etiolation and shading of stock plants on the rooting of C. coggygria ‘Royal Purple’ stem cuttings was also conducted by Pacholczak et al. (2005). Etiolation of stock plant affects the stem anatomy of cuttings. Cell walls are poorly lignified, sclerenchyma layers are undeveloped, and root primordia grow easily outside the stem (Nicolini et al., 2001). In the Pacholczak et al. (2005) study, stock plants were first covered for 6 weeks with a double layer of white or black foil to etiolate plants or a nursery net to increase shading and limit photosynthetic photon flux density (PPFD) from 1850 μmol m−2 s−1 to 930 or 72 μmol m−2 s−1. Plants were then sprayed with an aqueous solution of IBA (200 mg L−1, 1 L of solution per shrub). Semi-lignified stem cuttings with four nodes and two leaves were prepared from the middle parts of shoots harvested from stock plants collected in mid-June and were treated with commercial rooting powders: Ukorzeniacz AB containing 0.3% NAA, or Rhizopon AA containing 2% IBA. Rooting powders were applied directly to the basal part of cuttings and 75–80% of cuttings rooted when material was collected from plants shaded by 50% or 96% and subsequently treated with Ukorzeniacz AB or Rhizopon AA, respectively. The additional application of auxin to the basal part of cuttings collected from shaded mother plants increased rooting to 85–90%. The application of Rhizopon AA, Ukorzeniacz AB or an aqueous solution of 200 mg L−1 IBA significantly stimulated the growth of roots (85% for Ukorzeniacz AB, 88% for Rhizopon AA and 90% for IBA). Since the first report by Frolich (1961), the etiolation of stock plants before cuttings are harvested is one of the simplest and cheapest methods to increase the effectiveness of rooting in stem cuttings, especially in woody plants. This is a useful method to increase the propagation efficiency of a number of plant species, including Pinus mugo, Quercus rubra, Acer saccharinum (Herman and Hess, 1963; Maynard and Bassuk, 1985) and Acer griseum (Maynard and Bassuk, 1999). In woody plants, the continuous layer of sclerenchymatic pericycle is the main anatomical obstacle to the development of roots from stem cuttings. Since the primordia of adventitious roots form endogenously in the vascular cylinder, this continuous layer of sclerenchymatic pericycle may create a mechanical barrier that hampers root emergence (Maynard and Bassuk, 1985; Caesar, 1990; Nicolini et al., 2001). On the other hand, the reduced ability of Quercus plants with a continuous sclerenchymatic pericycle to easily form adventitious root primordia may be related to the early cessation of meristematic activity in pericyclic cells. In contrast to other stem meristemoids, the meristematic activity of pericyclic cells ceases very early and its cells sclerify (Amissah et al., 2008). The poor formation of layers of sclerenchyma cells localized over the oil channels in shoots is the result of limited exposure to light (Maynard and Bassuk, 1996; Amissah et al., 2008). Stems of plants without a continuous layer of sclerified pericycle have a locally retained meristemoid potential in non-sclerified pericyclic cells and are known to root better and form many adventitious roots (Doud and Carlson, 1977). The reason may be the lack of anatomical obstacles that limit the emergence of adventitious roots and the formation of connections between the vascular systems of stems and roots (Nicolini et al., 2001; Pacholczak et al., 2005, 2006). Shaded stems also change morphologically and have smaller diameters and longer internodes relative to control plants (Pacholczak et al., 2005, 2006). During the acclimatization of shoots to ambient conditions, there is an increase in the level of total chlorophyll, free amino acids, polyphenolic acids and free IAA, and a drop in the content of soluble sugars, soluble protein and free ABA (Pacholczak et al., 2005, 2006). Following on from the Pacholczak et al. (2005) study, Pacholczak et al. (2012) evaluated the effect of several biostimulants on the rooting of C. coggygria ‘Young Lady’ stem cuttings (Fig. 2A, B): 0.2% AlgaminoPlant which consists of an algal extract (Sargassum sp., Laminaria sp., Ascophyllum sp., Fucus sp.) and α-amino acids, 0.2% HumiPlant which contains 12% humic acids, 6% fluvic acids and nitrogen, phosphorus, potassium, zinc and boron and 0.1% Route® which contains ammonium

Fig. 2. Cotinus coggygria ‘Young Lady’: A. stem cuttings, B. cuttings during rooting in greenhouse. C. Scale of root development in cuttings: 1 — no visible roots; 2 — a few (1– 3) short roots; 3 — 4–5 roots, some of them branched, no root ball formed; 4 — medium sized root system composed of 6–10 branched roots forming a root ball; 5 — well formed, branched root system forming a root ball (over 10 roots). Bars = 1.5 cm.

zinc acetate as the active substance. AlgaminoPlant together with HumiPlant and Route® alone were applied once, twice or three times in 7-day intervals. The effects of these three preparations were compared to a synthetic auxin and commercial rooting powder Rhizopon AA (2% IBA), an aqueous solution of IBA (200 mg L− 1), and cuttings sprayed with distilled water (control treatment). The biostimulants positively affected the rooting of cuttings. The best results were obtained after three rounds of application of Route® (65% of cuttings rooted, forming 5–6 roots that were 3–5 cm long) compared to the control (22% of cuttings rooted, forming few roots) (Table 2, Fig. 2C). When cuttings were sprayed once or twice with AlgaminoPlant containing HumiPlant, 45–50% of cuttings rooted and produced 3–4 short roots. Comparable rooting was observed when an aqueous solution of IBA was used, forming 5–6 branched roots. The application of Route® resulted in an increase in the level of polyphenolic acids in cuttings. These biostimulants affected the level of organic compounds such as chlorophyll, free amino acids and reducing sugars (Table 3). Pacholczak et al. (2013) next demonstrated the effect of 0.2% AlgaminoPlant and 0.1% Route® on the rooting of C. coggygria ‘Royal Purple’ stem cuttings (Fig. 3A). Similar to the experiments with ‘Young Lady’, cuttings were sprayed with these biostimulants. The efficiency of rooting induced by these stimulants was compared to the application of an aqueous solution of IBA (200 mg L−1) or Rhizopon AA. Cuttings rooted in a greenhouse (Fig. 3B) in 2 peat:1 perlite:1 sand (v/ v). The foliar application of biostimulants positively affected rooting:

↑ ↑ − ↑ ↑ − ↑ ↑ ↑ ↑ ↑ ↑

Organic compound

AlgaminoPlant + HumiPlant × 1

Route® × 1

Chlorophyll Reducing sugars Free amino acids Polyphenolic acids

↑ ↑ ↑ −

↑ − ↑ ↑

− − ↑ ↓ − ↑ ↑ × × − × ×

↑ × × − × ×

↑ × × ↓ × ×

41.6% after two applications of AlgaminoPlant, 42.3% after three applications of Route® and 30% in the control (Table 4). In another study, Pacholczak et al. (2015a) compared the effect of spraying with 0.2% AlgaminoPlant or 0.1% Route®, Rhizopon AA and an aqueous solution of IBA (200 mg L−1) on stem cuttings of C. coggygria ‘Royal Purple’ and ‘Young Lady’ (Tables 2 and 4). These experiments were carried out as described by Pacholczak et al. (2012). The rooting efficiency of stem cuttings of both cultivars was dependent on the year of the experiment. Root development was best in both cultivars in 2012: 20% of control cuttings of ‘Royal Purple’ rooted in the first year of the experiment and 60% in the second year whereas values for

Rooting degree

IBA, indole-3-butyric acid; ↑ — increase in level; ↓ — decrease in level; − — no effect; × — no data.

× − ↑ × − ↑ × ↑ ↑ × ↑ ↑ ↑ ↑ − ↑ ↑ ↑ 2010 2011 2012 2010 2011 2012 Rooting percentage

− − ↑ − − −

Season

IBA

× ↑ ↑ × ↑ ↑

AlgaminoPlant + HumiPlant × 1 AlgaminoPlant × 3 AlgaminoPlant × 2

Table 3 The effect of biostimulants on the contents of organic compounds in leaves of stem cuttings of Cotinus coggygria ‘Young Lady’ in 2010 relative to control treatment (Pacholczak et al., 2012).

↑ — increase in level; − — no effect.

Rhizopon AA

AlgaminoPlant × 1

235

Treatment

Analysis

Treatment

Table 2 The effect of treatments on rooting of Cotinus coggygria ‘Young Lady’ stem cuttings relative to the control (Pacholczak et al., 2012, 2015a).

AlgaminoPlant + HumiPlant × 2

AlgaminoPlant + HumiPlant × 3

Route® × 1

Route® × 2

Route® × 3

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Fig. 3. Cotinus coggygria ‘Royal Purple’: A. stem cuttings, B. cuttings during rooting in greenhouse. C. Scale of root development in cuttings: 1 — no visible roots; 2 — a few (1– 3) short roots; 3 — 4–5 roots, some of them branched, no root ball formed; 4 — medium sized root system composed of 6–10 branched roots forming a root ball; 5 — well formed, branched root system forming a root ball (over 10 roots). Bars = 1.5 cm. C from Pacholczak et al. (2015b), with kind permission.

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Table 4 The effect of treatments of Cotinus coggygria ‘Royal Purple’ stem cuttings on rooting and biochemical and physiological changes relative to the control (Pacholczak et al., 2013, 2015a, 2015b). Treatment Analysis

Season

Rhizopon AA

IBA

AlgaminoPlant × 1

AlgaminoPlant × 2

AlgaminoPlant × 3

Route® × 1

Route® × 2

Route® × 3

Rooting percentage

2010 2011 2012 2013 2010 2011 2012 2013 2012 2013 2012 2013 2012 2013 2012 2013 2012 2013 2012 2013 2012 2013

− ↑ ↑ ↑ ↑ ↑ − ↑ − ↑ − − × × × × × × × × × ×

↑ ↑ ↑ ↑ ↑ ↑ − ↑ − ↑ − − − − ↑ ↑ − ↑ − ↑ ↑ ↑

↑ − − − ↑ − − − ↑ ↑ ↑ ↑ − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑

↑ − − − ↑ − − ↑ ↑ ↑ ↑ − × × × × × × × × × ×

↑ − − − − − − ↑ ↑ ↑ ↑ − × × × × × × × × × ×

↑ ↑ − − − ↑ − ↑ − ↑ − ↑ − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑

↑ − − − − − − ↑ ↑ ↑ − ↑ × × × × × × × × × ×

↑ − − − ↑ − − − ↑ − ↑ − × × × × × × × × × ×

Rooting degree

Photosynthesis rate Respiration rate Chlorophyll Total sugars Reducing sugars Free amino acids Soluble proteins

IBA, indole-3-butyric acid; ↑ — increase in level/rate; − — no effect; × — no data.

‘Young Lady’ cuttings were less than 20% and 46%, respectively. The effect of biostimulants was significant in the second year of the experiment. The climatic conditions during the growth of stock plants before harvesting the cuttings, i.e. means of monthly temperatures and rainfall, are shown in Table 5, which demonstrates the impact of these factors on the physiological status of the field-grown stock plants from which cuttings were derived. In 2012 there was much less rainfall than in 2011 and this might have affected the condition of stock plants. In addition, night temperature during rooting was 15–20 °C while daily temperatures ranged between 25 and 35 °C. About 75% of ‘Royal Purple’ cuttings treated once with AlgaminoPlant and that rooted were comparable with rooting induced by a foliar application of IBA solution. In ‘Young Lady’, a single spray of cuttings with Route® or a triple spray with AlgaminoPlant resulted in the highest rooting percentage (75–85%). These experiments also show clearly that the application of biostimulants to cuttings, relative to the control, affected biochemical parameters, causing an increase in the level of polyphenolic acids and catalase activity but a decrease in the hydrogen peroxide (H2O2) content and peroxidase activity (Table 6). A follow-up experiment by the same research group (Pacholczak et al., 2015b) once again evaluated the influence of spraying aqueous solutions of the same biostimulants on the rooting of C. coggygria ‘Royal Purple’ cuttings (Table 4) using the methodology outlined by Pacholczak et al. (2015a). The aim of this study was to observe biochemical changes, respiration efficiency and photosynthetic rate in cuttings during the rooting period. Cuttings were sprayed once, twice or three times with one of two biostimulators, 0.2% AlgaminoPlant or 0.1% Route® at weekly intervals compared with Rhizopon AA and an aqueous solution of IBA

Table 5 Rainfall and mean temperatures in 2011–2012 at the Experimental Station: conditions of the Pacholczak et al. (2015a) study. Month

March

April

May

June

2011 rainfall (mm) 2012 rainfall (mm) 2011 mean monthly temp. (°C) 2012 mean monthly temp. (°C)

15.1 23.2 3.0 5.0

80.5 52.8 10.5 9.4

38.9 21.4 14.4 15.6

106.1 57.7 18.6 17.4

(200 mg L−1). Cuttings were rooted in new plastic tunnels equipped with automatic watering, a mist system and a shading device. Night temperature during rooting was 15–20 °C while daily temperatures ranged between 25 and 35 °C. Rooting percentage of untreated cuttings was dependent on the year of the experiments (73% rooting in 2012 vs 41% in 2013). The application of Rhizopon AA or the aqueous solution of IBA significantly enhanced rooting (61.7–86.7% and 61.0–76.7%, respectively), and the degree of rooting (2.5–3.0 and 2.6–2.9 on a 5-point scale), independent of the year of the experiment. When leaves were sprayed three times with AlgaminoPlant or once with Route®, best rooting resulted (2.6–2.7 on a 5-point scale) of stem cuttings in the second year of the experiment (Fig. 3C). An increase in the rate of photosynthesis and respiration and in the level of carbohydrates, free amino acids, soluble proteins (in both years of the experiment) and total chlorophyll (only in the second year) content of stem cuttings was observed in treatments that applied both biostimulants (Table 4).

Table 6 The contents of polyphenolic acid (mg g−1 d.m.), hydrogen peroxide (H2O2) (μg g−1 d.m.), catalase activity (CAT) (mkat g−1 d.m.) and peroxidase activity (POX) (nkat g−1 d.m.) in cuttings of two cultivars of Cotinus coggygria. (Modified from Pacholczak et al. (2015a).) Content

Cultivar

Season Control

Polyphenolic acid ‘Royal Purple’ 2011 2012 ‘Young Lady’ 2011 2012 H2O2 ‘Royal Purple’ 2011 2012 ‘Young Lady’ 2011 2012 CAT ‘Royal Purple’ 2011 2012 ‘Young Lady’ 2011 2012 POX ‘Royal Purple’ 2011 2012 ‘Young Lady’ 2011 2012

13.06 a 9.10 a 9.08 a 9.35 a 31.54 b 75.91 c 90.32 c 87.73 c 439.02 a 1004.59 a 430.49 a 860.29 a 0.836 b 1.207 c 0.724 b 1.020 c

AlgaminoPlant Route® 24.18 b 9.24 a 11.85 b 8.17 a 15.28 a 38.92 b 53.08 a 65.07 b 2226.78 c 1653.76 b 1126.54 c 1092.57 a 0.672 a 1.140 a 0.727 b 0.831 b

21.48 b 11.27 b 15.69 c 9.30 a 16.11 a 21.30 a 61.33 b 22.86 a 1679.49 b 1089.13 a 900.35 b 945.21 a 0.733 a 1.187 ab 0.608 a 0.694 a

Means in a row followed by the same letter do not differ significantly at p = 0.05.

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Table 7 Explant type and media composition for Cotinus coggygria shoot multiplication. Cultivar

Explant type

Medium composition

References

‘Royal Purple’ Genotype with green leaves and purple inflorescences (PAR) ‘Royal Purple’

Axillary buds, shoot tips, 2–3 nodal segments

MS + 1.0 mg L−1 BA + 30.0 g L−1 sucrose

Podwyszyńska et al. (2000)

Axillary shoots, few nodal segments Two nodal segments

LS + 0.23 mg L−1 BA + 30.0 g L−1 sucrose + 5.0 g L−1 Agargel™

‘Royal Purple’ ‘Royal Purple’; ‘Simfonia Verii’ ‘Royal Purple’

Axillary buds with leaf primordia A few nodal segments

‘Royal Purple’

A few nodal segments

‘Royal Purple’

Microshoots

−1

−1

−1

MS + 1.0 mg L BA + 30.0 g L sucrose + 4.0 g L Agargel™ + 0.001% activated charcoal QL + LS vitamin + 32.0 mg L−1 NaFeEDTA + 1 mg L−1 BA + 0.2 mg L−1 NAA + 0.1 mg L−1 GA3 + 40.0 g L−1 dextrose + 9.0 g L−1 Duchefa agar MS + 1.0 mg L−1 BA + 0.1 mg L−1 NAA + 30.0 g L−1 sucrose + 8.0 g L−1 Bacto agar

Cameron et al. (2005) Metivier et al. (2007)

Călinescu et al. (2009), Rovină et al. (2013) Jacygrad et al. (2012), Pacholczak et al. (2013) −1 −1 −1 Modified MS (550.0 mg L CaCl2·2H2O, 462.5 mg L MgSO4, 46.6 mg L Na2EDTA·H2O, Podwyszyńska et al. 34.8 mg L−1 FeSO4·7H2O) + vitamins (thiamine, pyridoxine, nicotinic acid) 1.0 mg L−1 + (2012) 1.0 mg L−1 MemT or 1.0–2.0 mg L−1 MemTR + 30.0 g L−1 sucrose + 5.5 g L−1 agar Liquid MS + 0.8 mg L−1 BA + 0.2 mg L−1 NAA + 20.0 g L−1 sucrose Shi and Zheng (2015)

BA, 6N-benzyladenine; GA3, gibberellic acid; LS, Linsmaier and Skoog (1965); MemT, meta-metoxytopolin; MemTR, meta-metoxytopolin riboside; MS, Murashige and Skoog (1962); NAA, 1-naphthaleneacetic acid.

4. In vitro propagation Cameron et al. (2005) tested the response of C. coggygria ‘Royal Purple’ to in vitro conditions and found that photoperiod had no effect on rooting percentage of cuttings harvested in August. However, cuttings derived from plants exposed to an 8-h photoperiod in September, as opposed to a 16-h photoperiod, showed greater rooting percentage, mirroring the etiolated nature of shoots indicated by Blakesley et al. (1992). Although Cameron et al. (2005) failed to disclose the disinfection procedure for treating stem cuttings used for the establishment of in vitro cultures, they did indicate that the basal medium should be Linsmaier and Skoog (1965) (LS) medium supplemented with 0.23 mg L−1 6Nbenzyladenine (BA; see notes in Teixeira da Silva, 2012), 30 g L−1 sucrose and 5 g L−1 Agargel (Table 7). Other culture conditions included a PPFD of 40 μmol m−2 s−1, culture temperature of 23 °C and a subculture every 6 weeks in which the apical growth was removed and 20mm long cuttings with multiple nodes were transplanted onto fresh medium. Rooting experiments used the same culture conditions but BA was substituted with IBA at 1 mg L−1 and PPFD was cut to half (Table 8). At this concentration of IBA, rooting was 68% when photoperiod was 8 h but 100% when photoperiod was 16 h. Metivier et al. (2007) found that 2.0 mg L−1 IBA could induce 100% rooting in ‘Royal Purple’ within 6 days. IAA at 20.0 mg L−1 induced 95% rooting (76% when 2.0 mg L− 1 was used), 1-naphthaleneacetic acid (NAA) induced 87% rooting at 0.2 mg L−1 (73% at 2.0 mg L−1)

while 2,4-dichlorophenoxyacetic acid (2,4-D) at any concentration, with or without other auxins, could not induce roots. In the Metivier et al. (2007) study, Murashige and Skoog (Murashige and Skoog, 1962) (MS) basal medium supplemented with 1.0 mg L− 1 BA and 0.001% activated charcoal induced roots from 2 to 3 cm microcuttings that were obtained from a commercial (Canadian) in vitro source (Tables 7 and 8). Other culture conditions included a 16-h photoperiod, a PPFD of 80–100 μmol m−2 s−1 and culture at 24 °C. Călinescu et al. (2009) found that ‘Royal Purple’ rooted better than ‘Simfonia Verii’ on Quoirin and Lepoivre (1977) basal medium supplemented with 40 g L−1 dextrose and 32 mg L−1 iron chelate and solidified with 9 g L−1 agar. In studies by Călinescu et al. (2009) and Rovină et al. (2013), axillary branches (containing 3–4 nodes) of one-year old trees growing wild in Arges were collected in March and July, treated with 0.03% Fundazol and placed at 4 °C until the explants were prepared for culture (Tables 7 and 8). Axillary buds with leaf primordia which served as the explants were first washed in running water, placed in 96% ethanol for 10 min, added to calcium hypochlorite (concentration not indicated) for 20 min and washed in sterile distilled water for 3– 15 min. Cultures were maintained at 22–24 °C, a 16-h photoperiod, with a PPFD of 35–45 μmol m− 2 s−1. Shoots of ‘Royal Purple’ and ‘Simfonia Verii’ rooted on medium with 0.25 mg L− 1 NAA (66% and 23% rooting, respectively). The addition of 0.25 mg L−1 IBA to the culture medium resulted in 50% and 12.5% rooting. Rooting on MS and LS basal media was lower than on Quoirin and Lepoivre basal medium. Rovină et al. (2013) noted callus induction at the base of shoots.

Table 8 Medium composition for rooting Cotinus coggygria microshoots. Cultivar

Explants type

‘Royal Purple’

Shoot tips cut under leaf ca. Modified MS (825.0 mg L−1 NH4NO3, 950.0 mg L−1 KNO3) + 1.0 mg L−1 IAA + Podwyszyńska et 3.0 mg L−1 IBA al. (2000) N1 cm long Modified MS (825.0 mg L−1 NH4NO3, 950.0 mg L−1 KNO3) + 1.0 mg L−1 IAA + −1 1.0 mg L IBA Shoot tips MS + 20.0 mg L−1 IBA + 30.0 g L−1 sucrose + 4.0 g L−1 Agargel™ Metivier et al. (2007) Shoot tips QL + LS vitamin + 32.0 mg L−1 NaFeEDTA + 0.25 mg L−1 NAA + 40.0 g L−1 Călinescu et al. dextrose + 9.0 g L−1 Duchefa agar (2009) −1 −1 −1 Shoot tips ca. 1–2 cm Modified MS (550.0 mg L CaCl2·2H2O, 462.5 mg L MgSO4, 46.6 mg L Podwyszyńska et al. (2012) Na2EDTA·H2O, 34.8 mg L−1 FeSO4·7H2O) + 1.0 mg L−1 vitamin (thiamine, −1 −1 −1 pyridoxine, nicotinic acid) + 3.0 mg L IBA + 30.0 g L sucrose + 5.5 g L agar Shoot tips MS + 20.0 mg L−1 IBA + 30.0 g L−1 sucrose + 8.0 g L−1 Bacto agar Pacholczak et al. MS + 0.2% AlgaminoPlant + 30.0 g L−1 sucrose + 8.0 g L−1 Bacto agar (2013) Shoot tips ca. 1.5 cm ½ liquid MS + 1.0 mg L−1 IBA + 20.0 g L−1 sucrose Shi and Zheng (2015)

Genotype with green leaves and purple inflorescences (PAR) ‘Royal Purple’ ‘Royal Purple’; ‘Simfonia Verii’ ‘Royal Purple’

‘Royal Purple’ ‘Royal Purple’

Medium compounds

IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog (1962); LS, Linsmaier and Skoog (1965); QL, Quoirin and Lepoivre (1977).

References

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Podwyszyńska et al. (2000) micropropagated two cultivars of C. coggygria: ‘Royal Purple’ and a genotype with green leaves and a purple inflorescence (PAR). The culture was initiated in July from axillary buds on MS medium with 1.0 mg L−1 BA (Table 7). Shoots were propagated on the same medium for one year and explants consisted of 2–3 nodal segments and apical shoot tips. The length of each subculture was 5– 6 weeks because shoots showed necrosis on apical parts and yellowing of leaves. Four types of microcuttings ca. 1 cm long with 2–3 leaves (A — shoot tips cut directly under leaf; B — shoot tips, cut under leaf, leaf was removed; C — shoot tips cut above leaf; D — 2–3 nodal segments) were rooted on modified MS medium with half the concentration of NH4NO3 and KNO3 and supplemented with 1.0 mg L− 1 IAA, 1.0, 2.0, or 3.0 mg L− 1 IBA, or 1.0 mg L−1 IAA with 0.1, 1.0, or 2.5 mg L− 1 NAA (Table 8). After 4 weeks, rooted microshoots were acclimatized in a greenhouse for 3 months in pots containing 4 peat:1 perlite (v/v; pH 6.0) with 1.5 mg L− 1 Azofoska fertilizer (1 (N):0.5 (P2O5):1.4 (K2O)). Type B microcuttings of PAR showed 96.6% rooting on MS medium supplemented with 1.0 mg L− 1 IAA and 3.0 mg L− 1 IBA. Type B microcuttings of ‘Royal Purple’ showed 100% rooting while types C and D showed 98.7% rooting on MS medium with 1.0 mg L−1 IAA and 1.0 mg L−1 IBA. The type of microcutting had no effect on the number of roots per shoot in both genotypes but longest roots were obtained in PAR type B microcuttings. All rooted in vitro shoots were acclimatized

in greenhouse conditions and achieved a height of 10–20 cm after 3 months. In a follow-up experiment, Podwyszyńska et al. (2012) improved the micropropagation efficiency of C. coggygria ‘Royal Purple’ on MS medium enriched by meta-metoxytopolin (MemT) and its riboside (MemTR). In vitro shoots were cultured on modified MS medium with 25% higher concentrations of Ca, Mg and Fe and enriched by 0.5 mg L−1 BA (Table 7). They were subcultured every 4–5 weeks for a total of 22 subcultures on the same medium. Cultures were placed in a growth room with a 16-h photoperiod and a PPFD of 40 μmol m−2 s−1 at 23 °C. The apical parts of microshoots ca. 3–4 cm long (i.e., shoot tips) were multiplied in the presence of 0.5, 1.0 and 2.0 mg L−1 MemT or MemTR. In the second and third subcultures, best multiplication was in medium with 1.0 mg L− 1 MemT (10.4 shoots explant− 1) or with 1.0–2.0 mg L−1 MemTR (9.5–10.7 shoots explant−1). More shoots elongated in the presence of 1.0 mg L−1 MemT in the second subculture (8 weeks) relative to medium with 0.5 mg L−1 BA (control) or 0.5– 2.0 mg L− 1 MemTR. A total of 51.9% of shoots regenerated on 0.5 mg L−1 MemT-supplemented medium rooted on MS medium with 3.0 mg L−1 IBA and formed 3.6 roots shoot− 1 (Table 8). In contrast, fewer shoots were obtained in medium containing 2.0 mg L− 1 BA (2.3% rooting and 0.7 roots shoot− 1). This latter BA concentration inhibited the rooting of shoots the most.

Fig. 4. Cotinus coggygria ‘Royal Purple’: A. in vitro shoot proliferation on MS medium with 1.0 mg L−1 6N-benzyladenine (BA), 0.1 mg L−1 1-naphtaleneacetic acid (NAA), and 30 g L−1 sucrose after 8 weeks of culture; B–C. rooting of shoots in vitro on MS medium with 20 mg L−1 indole-3-butyric acid (IBA) after 8 weeks of culture. Bars = 1 cm.

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Another group of researchers (Pacholczak et al., 2013) tested the effect of biostimulants on rooting of in vitro cuttings of C. coggygria ‘Royal Purple’. Microshoots were multiplied on MS basal medium supplemented with 1.0 mg L−1 BA and 0.1 mg L−1 NAA and placed in a 16-h photoperiod at 22 °C under a PPFD of 24 μmol m−2 s−1. For root induction, MS medium was supplemented with 0–70.0 mg L−1 IBA or enriched with one of two filter-sterilized biostimulants, 0.2% AlgaminoPlant or 0.1% Route®. MS medium without any additives was the control treatment. In the presence of 20.0 or 30.0 mg L− 1 IBA, 89% of cuttings rooted while only 19% of control shoots rooted (Table 8). Route® was unable to stimulate rooting in vitro whereas AlgaminoPlant induced rooting in only 20% of cuttings. Jacygrad et al. (2012) indicated that to achieve 96% shoot regeneration of C. coggygria ‘Royal Purple’ the best medium was MS (pH 5.8) when supplemented with 1.0 mg L− 1 BA, 0.1 mg L−1 IBA, and 30 g L−1 sucrose (Table 7). In this medium, the highest shoot number (4.6 explant−1 and about 18.1 cm long) was also observed when cultures were placed in a phytotron under a 16-h photoperiod at a PPFD of 100 μmol m−2 s−1 at 24 °C (Fig. 4A). A more recent study by Ilczuk and Jacygrad (2016) found a relationship between the rooting of microcuttings on MS medium with 20.0 mg L−1 IBA (Fig. 4B, C), the formation of root primordia, the activity of antioxidant enzymes and the content of organic compounds. To achieve this, anatomical and biochemical analyses were conducted. For anatomical analyses, plant material was collected daily from day 0 to day 20 from the start of the experiment while for biochemical analyses material was collected every 5 days. Pacholczak et al. (2005) indicated that in the basal part of the stem sclerenchyma may be found while Ilczuk and Jacygrad (2016) confirmed the presence of 2–3 layers of collenchyma under the periderm in microshoots. This could explain the improved rooting of in vitro shoots compared to stem cuttings. In this experiment, three phases of adventitious root development were defined. Induction took place from days 1 to 3, the growth of root primordia occurred from days 4 to 8 and root elongation was observed during days 9 to 15. Root primordia formed from the cells of medullary rays exactly between the oil channels in the parenchyma of primary bark. During the first phase of root primordia development (Table 9), the following trends were observed: an increase in oxidase indole-3-acetic acid (IAAO) activity and in the content of endogenous IAA and of H2O2 as well as a decrease in peroxidase (POX) and polyphenol oxidase (PPO) activity and the level of polyphenolic acids. The high activity of PPO and POX that accompanied the drop in activity of IAAO was very characteristic for the growth phase of root primordia and root elongation. In the last phase of root development, the content of endogenous auxin and H2O2 decreased.

Table 9 Changes in the activity and levels of oxidative enzymes and organic compounds during the development of root primordia in Cotinus coggygria ‘Royal Purple’ microshoots on MS medium with 20.0 mg L−1 IBA relative to MS without auxin (Ilczuk and Jacygrad, 2016). Enzyme/organic compounds

IAAO IAA and its derivatives PPO Polyphenolic acids CAT POX H2O2 Soluble proteins

Phases of adventitious root formation (days) Primordia Growth of roots Induction Primordia (1–3) development elongation outside the microshoot stem (9–15) (4–8) (15–30) ↑ ↑ ↓ ↓ − − ↑ −

↑ ↑ − ↓ − − ↑ ↓

↑ ↑ ↓ ↓ − ↑ ↓ ↑

− ↑ ↓ ↑ − ↑ ↑ ↓

CAT, catalase; H2O2, hydrogen peroxide; IAA, indole-3-acetic acid; IAAO, indole-3-acetic acid oxidase; POX, peroxidase; PPO, polyphenol oxidase; ↑ — increase in activity/level; ↓ — decrease in activity/level; − — no effect.

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Shi and Zheng (2015) were the first to use liquid MS medium to multiply and root C. coggygria ‘Royal Purple’ shoots (Tables 7 and 8). In their study, explants were clusters of shoots ≥15 mm long derived from solid medium culture. Shoots were placed in a bioreactor that consisted of a plant culture bottle and a liquid bottle connected to a number of 1-L plant growth units connected in parallel. MS medium was replaced to prevent contamination. The volume of liquid medium injected was 130 mL and shoots were immersed 12 times day− 1 for 5 min each time. In MS medium with 0.8 mg L−1 BA, 0.2 mg L−1 NAA and 20 g L−1 sucrose, 6 shoots explant−1 formed. At the rooting stage, in which immersion frequency was decreased to 6 times day−1, a maximum of 90% rooted shoots was possible in ½ MS medium with 1.0 mg L−1 IBA and 20 g L−1 sucrose. This method shortened the regeneration cycle by 2 weeks compared to solid culture. 5. Conclusions and recommendations for future research Most of the research conducted on Cotinus species has focused on basic propagation techniques and horticultural production, with limited research on biotechnology. This review focused almost exclusively on the economically important cultivar ‘Royal Purple’ of C. coggygria. All in vitro studies to date have focused on rooting since this step of the propagation protocol tends to be difficult in hardwood species and trees. Horticultural and biotechnological research in Cotinus species is at an infancy and has thus a lot to advance. The basic in vitro propagation of Cotinus species would benefit from the use of thin cell layer technology to increase productivity per explant (Teixeira da Silva and Dobránszki, 2013; Teixeira da Silva et al., 2015), in vitro flowering to obtain flowers all-year round to complete in vitro hybridization experiments (Teixeira da Silva et al., 2014), and organogenesis stimulated by ultrasound or sonication (Teixeira da Silva and Dobránszki, 2014) or by magnetic fields (Teixeira da Silva and Dobránszki, 2015) to try and stimulate the creation of somaclonal variants such as stable leaf chimeras, or by CO2 enrichment and alternative lighting sources to fortify the rooting of hardwood cuttings (Norikane et al., 2013). In Cotinus species, conventional propagation is by cuttings, and the use of biostimulants to enhance rooting is promising. The rooting action of biostimulants used to root C. coggygria stem cuttings is comparable to the auxin IBA. The ability to stimulate rhizogenesis in this difficult-toroot species is important. However, further research is needed to better understand the mechanism by which biostimulants impact root development to allow for sustainable production practices for applied ornamental horticulture. Authorship contribution statement All three authors contributed equally to the development of this review. Conflicts of interest The authors declare no conflicts of interest. References Aminah, H., Dic, J.M., Grace, J., 1997. Influence of irradiance on water relations and carbon flux during rooting of Shorea leprosula leafy stem cuttings. Tree Physiology 17, 445–452. Amissah, J.N., Paolillo, D.J., Bassuk, N., 2008. Adventitious root formation in stem cuttings of Quercus bicolor and Quercus macrocarpa and its relationship to stem anatomy. Journal of the American Society for Horticultural Science 13, 479–486. Blakesley, D., Weston, G.D., Elliott, M.C., 1991. Endogenous levels of indole-3-acetic acid and abscisic acid during the rooting of Cotinus coggygria cuttings taken at different times of the year. Plant Growth Regulation 10, 1–12. Blakesley, D., Weston, G.D., Elliott, M.C., 1992. Increased rooting and survival of Cotinus coggygria cuttings from etiolated stock plants. Journal of Horticultural Science 67, 33–37. Caesar, J.C., 1990. Effect of simulated shade-light quality on stem anatomy of Pinus contorta seedlings. IAWA Bulletin 11, 2–120.

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