Introduction

Thidiazuron (TDZ; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, molecular weight 220.25, molecular formula C9H8N4OS) is a synthetic plant growth regulator (PGR) that was originally registered as a cotton defoliant by Schering AG (Berlin, Germany) (Arndt et al. 1976). TDZ possesses cytokinin-like activity and does not contain the purine ring seen in other adenine-type cytokinins such as benzylaminopurine (BA), kinetin (Kin), and zeatin. The exploitation of TDZ in many aspects of plant cell, tissue, and organ culture studies, such as callus induction, somatic embryogenesis, and shoot organogenesis and proliferation has proved that TDZ is a potent regulator of these morphogenic processes. Lu (1993) reviewed the use of TDZ for adventitious shoot regeneration, axillary shoot induction, and somatic embryogenesis in many plant species, and indicated that TDZ induces as many or more adventitious shoots than adenine-type cytokinins for most of the species in which it had been tested. In some plant species, such as Dianthus caryophyllus (Lu et al. 1991; Nugent et al. 1991) and Rosa sp. (Lu 1993), TDZ was even more effective than BA, Kin, and zeatin at inducing shoot regeneration. The potency of TDZ has been demonstrated for the in vitro propagation of many recalcitrant, woody, and legume species (reviewed by Huetteman and Preece 1993; Lakshmanan and Taji 2000). A review by Schulze (2007) highlighted the role of TDZ in improving cereal tissue culture, minimising the recalcitrant nature of the Poaceae, and extending the application of transformation protocols to elite genotypes and more readily available explants. The morphoregulatory role of TDZ and the characterisation of TDZ-induced morphogenic effects was reviewed by Murthy et al. (1998), who indicated that TDZ stimulates endogenous plant-growth-regulating compounds in excised and intact tissues, and acts by modulating endogenous PGRs, either directly or as a result of induced stress. Guo et al. (2011) reviewed TDZ as a multi-dimensional PGR, and summarised the biochemical and biophysical responses of plant cells to TDZ and related mechanisms. TDZ was effective for flower induction in vitro in many plant species including Bambusa edulis (Lin and Chang 1998), Dendrobium nobile (Wang et al. 2009), and Rauvolfia tetraphylla (Faisal et al. 2005). Moreover, TDZ can significantly enhance transformation frequencies by improving the vigour of transgenic shoots (Joersbo et al. 1999). Recently, several authors contributed to a book edited by Ahmad and Faisal (2018) on TDZ and its applications to several aspects of plant tissue culture including morphogenesis, somatic embryogenesis, and micropropagation of herbaceous and woody plant species, highlighting the use of TDZ for the tissue culture of medicinal plants (Ahmad and Shahzad 2018; Deepa et al. 2018) and its potential use as an elicitor for the production of secondary metabolites (Unal 2018).

Unlike other cytokinins, TDZ is resistant to endogenous cytokinin oxidase, which makes it fairly stable in tissue culture (Mok et al. 1982). The metabolism of TDZ is extremely slow while that of zeatin is completely metabolised by plant tissues within hours of its application (Mok and Mok 1985). TDZ suppresses the activity of cytokinin oxidase (Horgan et al. 1988; Hare et al. 1994), which can result in the accumulation of purine cytokinins in plant tissues. When Hordeum vulgare leaves were treated with 10−8 to 10−5 M TDZ for 24 h, transcription was substantially accelerated in an in vitro system containing chromatin and RNA polymerase I from TDZ-treated leaves (Karavaiko et al. 2004). Nisler et al. (2016) reported two new TDZ derivatives (1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl) phenyl]-3-(1,2,3-thiadiazol-5-yl)urea) as being very potent inhibitors of cytokinin oxidase/dehydrogenase, an enzyme that catalyzes the degradation of cytokinins. A possible modulation of endogenous gibberellin by TDZ has also been proposed (Hutchinson et al. 1997a). Treatment with TDZ enhanced stress-related genes (Zhang et al. 2006) leading to the accumulation of ethylene (Yip and Yang 1986; Hutchinson et al. 1997b; Murthy 1997). The accumulated ethylene in turn inhibits auxin transport (Radhakrishnan et al. 2009). The accumulation of stress signaling molecules such as abscisic acid and proline was also associated with TDZ treatment (Murch et al. 1999; Jones et al. 2007). Moreover, TDZ purportedly modulates the influx/efflux of calcium, which is an important signaling molecule that initiates a cascade of metabolic events (White and Broadley 2003; Jones et al. 2007). Several developmental patterns in plant tissue culture were attributed to the regulatory role of TDZ in the biosynthesis and accumulation of endogenous hormones.

Despite numerous studies characterising TDZ as a potent PGR across a wide range of plant species, disturbance of normal plant development is associated with its application. Early reports by Huetteman and Preece (1993) and Lu (1993) highlighted that the formation of fasciated and compact shoots, hyperhydricity and difficulty in rooting were the main undesirable effects of prolonged exposure to TDZ while several other negative effects were subsequently reported in literature. The focus of this review is to summarise and discuss the abnormalities associated with the use and application of TDZ, with a focus on different strategies used to avoid or overcome the occurrence of such abnormalities in in vitro cultures, with details described in Table 1.

Table 1 Studies on TDZ-induced abnormalities in plant tissue culture
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Morphological, anatomical abnormalities and loss of morphogenic ability

TDZ (> 2.0 µM) induced undesirable changes in plant morphology, such as abnormal leaf morphology, fasciated shoots, and swollen shoot bases in many plant species (Table 1), including Spathiphyllum cannifolium (Fig. 1a, b; 2.27–8.08 µM TDZ; Dewir et al. 2006a), Cordyline fruticosa (Fig. 1c, d; 13.6–18.2 µM TDZ; Dewir et al. 2015), the Aglaonema hybrid ‘Valentine’ (9.1 µM TDZ; El-Mahrouk et al. 2016), Prunus armeniaca (20 µM TDZ; Goffreda et al. 1995), and Musa spp. (> 2.0 µM TDZ; Shirani et al. 2009). The differentiation of tetrafoliate single leaves and stalk-like structures without a shoot apex were reported in Arachis hypogaea in response to 4.5 µM TDZ (Akasaka et al. 2000). Histological observations revealed that the malformation most often obtained in A. hypogaea was a shoot-like structure that lacked a shoot apical meristem and had disorganised vascular bundles (Akasaka et al. 2000). Abnormal bulblets with small bulb scales and swollen basal plates from bulb scales of the Lilium oriental hybrid ‘Casablanca’ formed in media containing 4.5 µM TDZ (Han et al. 2005). Some of these abnormalities may appear during the first culture, or after continuous cultures. Continuous culture in TDZ-containing media induced abnormal bud primordia in D. caryophyllus (Ahmad et al. 2006) and A. hypogaea (Akasaka et al. 2000) which failed to develop into plantlets. Additionally, a TDZ-induced shoot culture of Aloe polyphylla developed swollen buds that did not develop into shoots (Ivanova and van Staden 2008).

Fig. 1
figure 1

Detrimental side effects of thidiazuron (TDZ) during shoot proliferation of several plants in vitro. Spathiphyllum cannifolium (a swollen shoot base and abnormal morphology at 2.27–8.08 µM TDZ; b normal proliferation at13.9 µM kinetin—in vitro shoots were cultured on MS medium solidified with 0.2% gelrite and incubated in a 16-h photoperiod (PP), 35 µmol m−2 s−1, and 25 °C), Cordyline fruticosa (c stunted and swollen shoots at 13.6 µM TDZ; d normal proliferation at 0.5 µM TDZ—in vitro shoots were cultured on MS medium solidified with 0.2% gelrite and incubated at 16-h PP, 55 µmol m−2 s−1, and 25 ± 2 °C), Conocarpus erectus (e callus formation at the shoot base with 2.3 µM TDZ; f normal proliferation with 8.9 µM benzyl adenine [BA]—in vitro shoots were cultured on MS medium solidified with 0.8% agar and incubated at 16-h PP, 35 µmol m−2 s−1, and 25 °C), Arbutus unedo (g hyperhydric shoots at 18.2 µM TDZ; h normal shoots at ≤ 13.6 µM TDZ—in vitro shoots were cultured on MS medium solidified with 0.8% agar and incubated at 16-h PP, 25 µmol m−2 s−1, and 25 ± 2 °C), and Cissus rhombifolia (I, shoot tip necrosis with 4.5 µM TDZ; J, normal shoot in response to 4.4 µM BA—in vitro shoots were cultured on MS medium solidified with 0.8% agar and incubated at 16-h PP, 25 µmol m−2 s−1, and 25 ± 2 °C). Unpublished photographs (Y.H. Dewir)

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Loss of morphogenic ability was also reported during somatic embryogenesis. Embryogenic masses of A. hypogaea were less responsive towards differentiation at high TDZ concentrations (13.62–45.41 µM) and several of these structures dedifferentiated and became necrotic (Joshi et al. 2008). TDZ-induced somatic embryos of Oncidium sp. failed to develop into plantlets, and instead developed into green abnormal structures that eventually turned brown and deteriorated (Chen and Chang 2000). In Rosa chinensis, TDZ-induced somatic embryos showed abnormal morphology, and turned brown when TDZ was applied at 11.25 µM (Chen et al. 2014).

Physiological abnormalities in shoots and stems

Inhibition of shoot proliferation

Despite the high cytokinin-like activity of TDZ during shoot proliferation, the inhibition of this process may also occur due to its application. The negative effects of TDZ on shoot proliferation have been reported in many plant species, including S. cannifolium (Dewir et al. 2006a) and Cotoneaster wilsonii (Sivanesan et al. 2011). In Bacopa monniera, the frequency and number of adventitious shoot buds (initiated at 6.8 µM TDZ) declined from 85 to 50% and from 53.5 to 16.9, respectively, after three subcultures (7 weeks in each passage) in media containing 2.2 µM TDZ (Tiwari et al. 2001).

Explants in plant tissue experiments tend to be continuously exposed to TDZ for a long duration, similar to other cytokinins, until organogenesis. Thus, explants are subjected to an overdose, resulting in the inhibition of shoot proliferation and other abnormalities. The optimal duration of TDZ exposure was less than 7 days in Phaseolus vulgaris (Malik and Saxena 1992), Pelargonium × hortorum (Hutchinson and Saxena 1996), and Manihot esculenta (Bhagwat et al. 1996). Malik and Saxena (1992) reported that a TDZ pulse treatment (10 µM) of P. vulgaris seeds for just 1 day was sufficient to induce direct shoot organogenesis (ten shoots/seedling), and the number of shoots after 7 days (35 shoots/seedling) was comparable to numbers after a continuous 4-week treatment (30 shoots/seedling). However, the optimal TDZ concentration and duration of exposure are species-dependent due to genotypic differences. In M. esculenta, exposure for 6–8 days to 0.22 µM TDZ was optimal for shoot proliferation (12.4 and 11/nodal explant, respectively), while longer or shorter durations (4 and 10–26 days) reduced shoot proliferation to 6–8 shoots (Bhagwat et al. 1996).

To avoid TDZ-induced inhibition of shoot proliferation, the TDZ concentration, exposure duration, plant genotype, and subculture of TDZ-exposed cultures to a secondary medium should be considered. A secondary medium lacking TDZ was used to improve Cassia angustifolia shoot proliferation and elongation by exposing shoot tips to 0.5 µM TDZ for 4 weeks (Siddique et al. 2015), while a secondary medium for 12 weeks containing 1 µM BA and 0.5 µM NAA effectively enhanced shoot multiplication from 4.5 to 20.3 shoots and shoot length from 1.4 to 4.7 cm 4-week-old cultures of Salix tetrasperma exposed to 2.5 µM TDZ (Khan and Anis 2012). The efficiency of a secondary medium lacking PGRs or containing 1.0 µM BA singly or in combination with 0.5 µM NAA on explants exposed for 7 days to TDZ (5 µM) was also evaluated for 4 and 8 weeks in Vitex trifolia (Ahmed and Anis 2012). BA (1.0 µM), when applied with NAA (0.5 µM), resulted in highest shoot number (22.3) and shoot length (5.2 cm) after 8 weeks of culture (Ahmed and Anis 2012).

Inhibition of shoot growth and elongation

Although TDZ has been shown to be superior to other cytokinins for shoot proliferation (reviewed by Huetteman and Preece 1993; Lu 1993; Murthy et al. 1998), problems with short and compact shoots have been reported in many plant species, including Juglans nigra (Van Sambeek et al. 1997), Rollinia mucosa (Figueiredo et al. 2001), and Alpinia zerumbet (Victório et al. 2011). Despite the high efficiency of TDZ at 2.5 µM, compared to zeatin, Kin, BA and 2-isopentenyladenine (2iP), in the regeneration of Saccharum officinarum embryogenic callus, it produced a high percentage (84%) of stunted shoots shorter than 1 cm (Chengalrayan and Gallo-Meagher 2001). Huetteman and Preece (1993) indicated that TDZ-inhibited shoot elongation may be due to its high cytokinin activity while the presence of a phenyl group in TDZ may be a possible cause of shoot bud fasciation. A study by Hutchinson et al. (1997a), which used gibberellin-synthesis inhibitors (triazoles and ancymidol) to improve TDZ-induced somatic embryogenesis in Pelargonium × hortorum, indicated the possible modulation of endogenous gibberellin by TDZ. TDZ-induced stunted growth may be related to gibberellin metabolism or proline biosynthesis, but this remains unclear. In general, stunted growth is not desired for micropropagation due to the difficulty in isolating individual shoots from multiple shoots, which increases the labour and time taken, and the cultivation of extra subcultures for their elongation and growth. On the other hand, TDZ-stunted shoots facilitates excision due to thick stems, for example, in Dendranthema (Teixeira da Silva 2003).

Previous studies showed different strategies to overcome the TDZ-induced inhibition of shoot elongation, such as sub-culturing onto PGR-free medium or medium containing another cytokinin, or the inclusion of other cytokinins with TDZ. Shoot elongation was favoured in the absence of PGRs in Lavandula stoechas (Nobre 1996) while inhibited elongation in Vaccinium vitis-idaea was overcome by transferring cultures inhibited by TDZ (1 µM for 8 weeks) to media containing 1 µM zeatin for 4 weeks (Debnath 2005). A two-step regeneration strategy, using TDZ to induce bud/shoot formation, followed by the use of zeatin to promote shoot elongation, was also reported for northern highbush blueberry cultivars and Vaccinium angustifolium (Song and Sink 2004; Debnath 2009; Liu et al. 2010). In Picea glauca, adventitious shoots elongated more when TDZ was combined with BA or zeatin (Ellis et al. 1991). In Vaccinium macrocarpon, the inclusion of 5 µM 2iP in the TDZ (10 µM)-containing culture initiation medium for 15 days and transfer of explants to a secondary medium containing a low TDZ concentration (1 µM) and 10 µM 2iP for 7 days followed by transfer to PGR-free medium resulted in enhanced shoot elongation in 2 weeks while no elongation was observed on initiation medium (5 µM 2iP and 10 µM TDZ) even after 4 months (Qu et al. 2000). A secondary medium containing 25 µM 2iP alone was used to elongate TDZ (10 µM)-regenerated Pisum sativum adventitious shoots within 4–10 weeks while shoots remained stunted when secondary medium with auxins or free of PGRs was used (Bohmer et al. 1995).

Hyperhydricity

Hyperhydricity is a disorder of tissue-cultured plants in which leaves become translucent and stems become swollen, distorted, and brittle. The environment inside a tissue culture vessel affects the normal growth and physiology of plants, and ultimately gives rise to morphological and physiological malformation and malfunctions (Park et al. 2004; Chakrabarty et al. 2006; Dewir et al. 2006b, 2015). Cytokinins are among the various factors that cause hyperhydricity in vitro (reviewed by Vieira de Vasconcelos 2012; Dewir et al. 2014). Kadota and Niimi (2003) indicated that phenylurea derivatives (11 µM N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) or 44 µM TDZ) promoted nearly a tenfold increase in the number of hyperhydric shoots in Pyrus pyrifolia than adenine derivatives (44 µM BA or 0.44 µM Kin). Different cytokinins (BA, Kin, 2iP, and TDZ) induced hyperhydricity in Hypericum hirsutum and H. maculatum, and TDZ had the greatest negative effect at 1.8 µM. These anomalies could explain the reduced shoot biomass production and inhibition of pseudohypericin biosynthesis (Coste et al. 2011). Hypericin (R=CH3) and pseudohypericin (R=CH2OH) are dianthrone derivatives with antiviral and anticancer activities (Prince et al. 2000; Kirakosyan et al. 2008). In Hypericum species, Liu et al. (2007) reported that TDZ at 0.23 µM resulted in malformed leaves and poor shoot growth and decreased hypericin content while 2.27 µM increased the formation of clustered shoots and hypericin content in H. perforatum. There was a 25-fold increase in the number of dark glands between H. sampsonii and H. perforatum, but only a 4.21-fold increase in the production of hypericin, suggesting that there was no direct correlation between gland number and hypericin production. Although hypericin metabolism occurred in black glands and the number of black glands increased following TDZ treatment, the production of hypericin was dependent on the metabolic efficiency of black glands on leaves. Thus, TDZ concentration and its induced anomalies affect the accumulation of secondary metabolites in tissue culture.

TDZ-induced hyperhydricity of regenerated shoots was reported in several plant species (Table 1), including Arbutus unedo (18.2 µM TDZ; Fig. 1g, h; El-Mahrouk et al. 2010), Pluchea lanceolata (> 2.3 µM TDZ; Kher et al. 2014), A. polyphylla (2.5 µM TDZ; Ivanova and van Staden 2011), and J. nigra (0.05–0.1 µM TDZ; Bosela and Michler 2008). High concentrations of TDZ (6.8–9.1 µM) and repeated subcultures resulted in hyperhydric C. wilsonii shoots (Sivanesan et al. 2011). Among different cytokinins (2iP, TDZ and zeatin; 2.25–22.5 µM) tested for shoot multiplication of Hymenocallis littoralis, TDZ showed the lowest total chlorophyll content at all tested concentrations (Yew et al. 2010). Ultrastructural analysis of Annona glabra in vitro leaves from regenerated axillary shoots on WPM medium (McCown and Lloyd 1981) supplemented with cytokinins (4.5 µM TDZ, 4.4 µM BA, 4.6 µM Kin, or 4.6 µM zeatin) revealed that TDZ, compared to other cytokinins, resulted in a significant reduction of chlorophyll a content and formation of abnormally shaped chloroplasts rich in plastoglobules, which is associated with a marked disorganization of the chloroplast endomembrane system (Oliveira et al. 2008). In D. caryophyllus, TDZ at 0.04–0.4 µM induced hyperhydricity in regenerated axillary buds and decreased the stability of the photosynthetic membrane, and the autolytic destruction of photosynthetic pigments of isolated chloroplasts was very strong in TDZ-treated material but weak in material treated with 0.4 µM BA (Genkov et al. 1997). TDZ (0.93 µM) induced the expression of stress-related genes in Medicago sativa callus, including the trehalose-6-phosphate phosphatase (TPP), 1-aminocyclopropane-1-carboxylate synthase (ACS) and proline dehydrogenase genes (Zhang et al. 2006) while TDZ-induced expression of ACS resulted in enhanced ethylene accumulation. Several studies indicated increased ethylene in response to TDZ treatment (Yip and Yang 1986; Hutchinson et al. 1997b; Murthy 1997). In wilted Triticum aestivum leaves, 10 µM TDZ exerted stress-induced ethylene production equal to that exerted by 1 mM BA, indicating that TDZ is more active than BA by two orders of magnitude (Yip and Yang 1986). During somatic embryogenesis of Pelargonium × hortorum, hypocotyl explants treated with 10 µM TDZ resulted in elevated levels of ethylene within 6 h in the headspace of culture vessels (Hutchinson et al. 1997b). Ethylene has been proposed as a negative by-product of the TDZ-mediated metabolic cascade (Hutchinson et al. 1997b), so it is likely that TDZ-induced hyperhydricity could be an indirect response to increased ethylene production.

Shoot tip, shoot and tissue necrosis

Shoot tip necrosis (STN), a common physiological deformity in plant tissue culture, is associated with the type and concentration of cytokinin in the culture medium (Bairu et al. 2009). In STN, the necrotic shoot tip initially becomes brown and eventually dies (Fig. 1i, j—unpublished results on Cissus rhombifolia; Lakshmi and Raghava 1993; Bairu et al. 2009). Baskaran et al. (2014) noted that although 13.6 µM TDZ produced a significantly higher number of shoots (37 per explant) of Coleonema pulchellum after 10 weeks of culture than BA (4.4–22.2 µM) or meta-topolin (4.2–20.7 µM), those shoots exhibited symptoms of STN. TDZ at 20 µM induced tissue necrosis in Hypericum erectum (Kim et al. 2006). In H. hirsutum and H. maculatum, the addition of 1.8 µM TDZ to liquid culture medium led to malformation and STN (Coste et al. 2011). Murch et al. (1999) noted the accumulation of abscisic acid, proline, and ions in TDZ-induced regeneration in peanut. Axillary shoots of Cercis canadensis proliferated using TDZ (0.2–22.7 µM) had leaves that were chlorotic and at a high concentration of TDZ (22.7 µM) had excessive necrosis and phenolic accumulation (Mackay et al. 1995). Prolonged exposure to TDZ may also result in the accumulation of phenolic compounds, as was reported during the induction of Pelargonium × hortorum somatic embryogenesis (Hutchinson and Saxena 1996). The exudation of phenolic compounds was enhanced by TDZ (Shirani et al. 2009). The accumulation of these stress-related substances might be responsible for TDZ-induced STN. However, several other factors including aeration, medium pH, nutrients and salt strength were reported to affect STN (reviewed by Bairu et al. 2009).

Callus formation

The use of TDZ may result in the formation of callus during axillary shoot proliferation, which is technically difficult and laborious to dissect from single shoot buds. Moreover, it could inhibit axillary shoot proliferation, as the regeneration pathway is directed toward callogenesis and indirect organogenesis. At 10 µM TDZ, axillary shoot proliferation of Cassia alata was coupled with callus formation and the number of shoots was reduced from 17.9 to 7.2 when nodal segments were exposed for 4 weeks to TDZ at 5 and 10 µM, respectively (Ahmed and Anis 2014). The formation of callus at basal cut ends of node explants on cytokinin-enriched medium is frequent in species with strong apical dominance (Preece et al. 1991). Marks and Simpson (1994) attributed the formation of basal callus to the action of accumulated auxins at the basal ends, stimulating cell proliferation. TDZ also exhibits auxin-like activity by modulating the biosynthesis and accumulation of endogenous auxins (Mok and Mok 1985; Murthy et al. 1995; Murch and Saxena 2001) thus, enhancing callus formation. In B. monniera, treatment with TDZ (> 6.8–22.7 µM for 7 weeks) produced excessive callus, and the shoot buds remained stunted (Tiwari et al. 2001). Callus type in Chirita swinglei could be altered depending on the use of TDZ at 1 µM or other cytokinins (Zhang et al. 2016). Callus formation has been reported as an undesirable side-effect of TDZ in a number of plants (Table 1), including Conocarpus erectus (2.3 µM TDZ for 6 weeks; Fig. 1e, f; Dewir et al. 2018), Albizia lebbeck (> 1 µM TDZ for 4 weeks; Perveen and Anis 2015), Leucaena leucocephala (0.35–1.5 µM TDZ for 3 weeks; Pal et al. 2012), Vigna subterranea (0.11–45 µM TDZ for 4 weeks; Silué et al. 2016), and Ericaceous species (Cao and Hammerschlag 2000; Qu et al. 2000; Debnath 2005; Guo et al. 2011).

Cytogenetic variation

In vitro plants are usually proliferated for several subculture cycles prior to rooting and acclimatisation and cytogenetic variations may occur in this long process. TDZ-induced somaclonal variations can be a valuable source for new genetic material. All off-type plants of Tulipa gesneriana ‘Blue Parrot’ micropropagated through long term cyclic subcultures (4 years) on TDZ (4.5–9.1 µM)-supplemented medium and grown in an insect-proof tunnel for four growing cycles, had a red–purple flower color instead of purple–violet flowers (Podwyszyńska 2005; Podwyszyńska et al. 2006). However, high genetic fidelity and true-to-type clones are critical for commercial micropropagation to maintain the essential characteristics of the mother plant. Several indicators, including morphological characteristics, chromosome numbers, isozyme profiles, and PCR-based molecular markers [randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism] can be used to assess genetic fidelity (Gostimsky et al. 2005; Podwyszyńska et al. 2006), including of tissue-cultured plants (Kacar et al. 2006). Khoddamzadeh et al. (2010) reported DNA polymorphism with 17% dissimilarity among the TDZ (13.6 µM) regenerated plants to the mother plant Phalaenopsis bellina using RAPD. Less variability (5.95%) was detected within TDZ (6.8 µM)-propagated D. nobile using RAPD and SCoT (Bhattacharyya et al. 2014) and TDZ (0.909 µM)-propagated Cymbidium giganteum (5.81%) plants using RAPD (Roy et al. 2012). RAPD and ISSR analysis of micropropagated T. gesneriana ‘Blue Parrot’ through cyclic subcultures (4 years) on TDZ (4.5–9.1 µM) medium revealed 45 and 55% polymorphism, respectively, while no polymorphism was detected in progeny lines derived from 2-year-old cultures (Podwyszyńska et al. 2006). DNA polymorphism (20%) was also reported in protocorm like-bodies (PLBs) of Phalaenopsis gigantea after 20 weeks of culture on medium containing 4.5 µM TDZ and 65.5 µM chitosan while reducing the culture period to 16 weeks resulted in no variations (Samarfard et al. 2014). Therefore, limiting the number of subculture cycles could maintain clonal characteristics, but the amenability to a TDZ-induced mutagenic effect is species-dependent.

In plants, albinism is a phenomenon in which the partial or complete loss of chlorophyll may result from differences in genotype, environmental conditions, or genome-based modifications that reduce chlorophyll biosynthesis and eventually damage the photosynthetic apparatus, causing photo-bleaching (Kumari et al. 2009). Albinism has been reported to be a cytogenetic variation associated with TDZ treatment in vitro (Table 1). Albino plants are difficult to root in vitro, and do not survive ex vitro, therefore, albinism is clearly undesirable for plant mass propagation. Lin and Chang (1998) reported that 30% of TDZ-regenerated (4.5 µM for 3 weeks) B. edulis axillary shoots were albino, albinism occurred by the second subculture and all albino plants did not survive ex vitro conditions. Lin et al. (2007) induced flowers from adventitious albino shoots of Dendrocalamus latiflorus after an 8-month subculture on medium with 0.45 µM TDZ, and shoots were multiplied on medium containing 0.45 µM TDZ, but no seeds were produced. Thus, albinism is also considered as a biotechnological limitation to in vitro hybridization. Plant regeneration from mature zygotic embryo-derived callus in several rice varieties on medium containing 2.3–9.1 µM TDZ and 5.4 µM NAA resulted in the regeneration of some albino shoots in every variety tested compared to 8.9–17.8 µM BA with 5.4 µM NAA (Azria and Bhalla 2000). Albinism is affected by the genotype, physiological state of the donor plants, culture temperature, and medium composition including sucrose concentration, and PGRs (reviewed in Kumari et al. 2009).

Plastid development requires compatibility between nuclear and chloroplast genomes which encode proteins essential for chloroplast development and function. Genetic studies in albino plants indicated that it is a recessive trait governed by many loci (Kumari et al. 2009). A large deletion of plastid DNA may be responsible for the regeneration of many albino rice plants (Harda et al. 1992). Recently, Zeng et al. (2017) observed that an albino rice plant had a mutation in the OsABCI8 gene which is localized in the chloroplast and plays a major role in chloroplast development and biosynthesis of chlorophyll precursor. Albino plants accumulated significantly higher levels of iron and nickel than wild type plants. In vitro albino Agave angustifolia plantlets regenerated on MS medium supplemented with 0.11 µM 2,4-d and 22.2 µM BA showed low expression levels of genes involved in photosynthesis and carotenoid biosynthesis, suggesting a disruption of these enzymes and processes in albino plants (Us-Camas et al. 2017). In in vitro plant cells, TDZ promotes the passage and storage of endogenous plant signals and iron, and modifies endogenous PGRs as well as the activities of antioxidant enzymes such as catalases and peroxidases (reviewed by Murthy et al. 1998; Guo et al. 2011). However, the mutagenic effect of TDZ and associated molecular mechanisms remain unknown, calling for further investigations.

Altered rooting or loss of rooting ability

TDZ influences the morphogenesis and rooting efficiency of shoots in culture when used at concentrations above the threshold levels and/or for long durations (Dewir et al. 2016). The loss of rooting ability is due to carryover effects of TDZ when used at the shoot multiplication stage which is dependent on TDZ concentration. In Solanum melongena, adventitious shoots regenerated by 0.2 µM TDZ failed to develop roots in several rooting induction media while at 0.01 µM TDZ, only 6% rooting was obtained in ½ MS medium supplemented with 0.6 µM IAA (Magioli et al. 1998). Interestingly, rooting reached 70% when callus was maintained on PGR-free medium before shoot excision for 2 weeks after bud induction by TDZ (Magioli et al. 1998). In L. leucocephala, rooting was 0% for axillary shoots regenerated on 0.45–2.27 µM TDZ for 30 days and there was a gradual increase in rooting up to 76.6% by decreasing TDZ concentration to 0.05 µM (Shaik et al. 2009). Moreover, the authors indicated that a TDZ (0.45 µM) pulse for 24 h for shoot multiplication caused high rooting (82–87%) of the regenerated shoots. TDZ-related inhibitory effects on rooting were also reported for Cicer arientinum (Murthy et al. 1996), Tamarindus indica (Mehta et al. 2004), and T. aestivum (Li et al. 2003). Inhibited root initiation caused by an extended period of exposure to cytokinins could be induced by modified metabolic enzyme activity or an edited receptor site ultimately reducing the effectiveness of auxins (Javed et al. 2013). The period of exposure to TDZ can also impact the timing of root initiation, with longer rooting times observed as a side effect of incubation with high concentrations of TDZ. In cultures of S. melongena initiated from root explants on root induction medium supplemented with auxin and TDZ, only shoots formed, but not roots (Franklin et al. 2004). The carryover effects of TDZ on the loss of rooting ability in the Fabaceae family can be overcome or minimised through several techniques and alternative methods to promote rooting (reviewed by Dewir et al. 2016), including subcultures on a PGR-free medium, using a reduced concentration of salt, adjusting the auxin type, concentration and timing of application, use of liquid rather than semi-solid medium, or exposing unrooted shoots to a high concentration of auxin as a pulse. Although grafting is a skill-based and tedious technique, it can be an alternative to in vitro rooting in difficult-to-root or recalcitrant species. Rooting was impossible for adventitious shoots of P. sativum regenerated with TDZ (1–50 µM) and so in vitro grafting was used resulting in a 100% grafting rate (Bohmer et al. 1995).

Species-specific effects of TDZ concentration

Some plant species are receptive to a wide range of TDZ concentrations and subculture cycles than others as confirmed by numerous successful protocols for tissue culture using TDZ, in which these plant species exhibited normal growth. In contrast, several other plants (Table 1) such as Cassia alata (Ahmed and Anis 2014), Glycine max (Mante et al. 1989), Leucaena leucocephala (Pal et al. 2012), Juglans nigra (Bosela and Michler 2008), and Musa sp. (Shirani et al. 2009), exhibited abnormal growth and showed sensitivity to very low TDZ concentrations (> 0.1–10 µM) while other species such as Aglaonema ‘Valentine’ (El-Mahrouk et al. 2016), Bambusa edulis (Lin and Chang 1998), and Cordyline fruticosa (Dewir et al. 2015) showed abnormalities at relatively high concentrations (> 13.6–27.2 µM). A slight increase in TDZ concentration reduced shoot proliferation in Cimicifuga racemosa, but increasing the TDZ concentration from 2 to 18 µM reduced the number of shoots from 14.8 to 3.5 per explant (Lata et al. 2002). Additionally, increasing TDZ concentration switched the regeneration pathway in Saintpaulia ionantha, where a low concentration (˂ 2.5 µM) induced shoot organogenesis while 5–10 µM induced somatic embryogenesis (Mithila et al. 2003). A similar switch from shoot organogenesis to somatic embryogenesis was observed in Ochna integerrima when TDZ concentration was increased from 5 to 10–15 µM (Ma et al. 2011) or in Metabriggsia ovalifolia when 2.5 µM TDZ was increased to 25 µM (Yao et al. 2016). Tremendous improvement in the tissue culture of woody and recalcitrant plant species has been achieved using TDZ. While a low concentration of TDZ in the range of nM to a few µM has shown the ability to induce axillary shoots, higher concentrations favor callus formation and proliferation of adventitious shoots (Huetteman and Preece 1993; Debnath 2018; Vinoth and Ravindhran 2018).

Conclusions

The efficiency of TDZ has been proved in tissue culture applications in several plant species, but it can lead to deformities if not used properly. To understand and avoid these deformities, we reviewed the detrimental side effects of TDZ in 40 plant species belonging to 23 families (Table 1), nine of which belong to the Fabaceae. These detrimental effects included morphological abnormality, loss of morphogenic ability, inhibition of shoot proliferation and shoot elongation, STN, hyperhydricity, cytogenetic variations such as albinism and DNA polymorphism, and altered rooting or loss of rooting ability, all of which hinder the in vitro propagation of plant species. These detrimental effects occur due to the inappropriate use of TDZ. TDZ is highly stable due to its slow metabolism and resistance to cytokinin oxidase. It enhances the synthesis of adenine-type cytokinins, modulates endogenous hormones, and exhibits both auxin and cytokinin-like activities. Moreover, it promotes stress genes and leads to the production of ethylene and stress signaling molecules. TDZ can thus be used solely to achieve different morphogenic and regeneration pathways. The appropriate and optimal concentration of TDZ is species-specific. However, the use of TDZ at low concentrations, pulse treatment and short periods of exposure are effective strategies to avoid TDZ-induced abnormalities. In Bactris gasipaes, a pulse treatment with 0.36 µM TDZ for 14 days minimized the negative effects caused by prolonged exposure to TDZ (Graner et al. 2013). Recently, Kumari et al. (2018) reported that overnight soaking of C. arietinum seeds in a 20 µM TDZ solution prior to culture in PGR-free medium reduced the detrimental effects of TDZ in proliferated axillary shoots. The inclusion of other cytokinins with TDZ in the primary medium and the use of a secondary medium lacking PGRs or containing adenine-derived cytokinins is an efficient two-step regeneration strategy to overcome growth deformities, enhance shoot elongation and minimize the carry-over effects of TDZ during the rooting stage. Other culture conditions including medium type and strength, gelling agent, ventilation and light intensity are important factors to maintain normal growth characteristics. Limiting the number of subculture cycles on TDZ-supplemented medium needs to be considered to maintain the clonal characteristics of regenerants. Further studies are required to improve our understanding of the physiological and metabolic mechanisms associated with using TDZ. Additionally, studies on the molecular mechanisms of TDZ-induced cytogenetic variations are necessary since genetic fidelity is a serious concern, particularly for commercial propagation. Finally, many plant research-based journals have historically placed greater emphasis on positive results than on negative results, so it is expected that the negative impact of TDZ on plant morphogenesis in vitro may be much higher than that reported in this review, simply because negative results tend to be deemphasized (Teixeira da Silva 2015).

Author contribution statement

All four authors equally contributed to and are responsible for conceiving the manuscript, developing its drafts and all revisions.