Keywords

15.1 Horticulture, Propagation Systems, and the Importance of Commercial Micropropagation

Horticulture, the production of fruit, vegetables, and ornamental and medicinal plants, is a sector of great socioeconomic importance in world agriculture. The color, texture, size, and flavor of horticultural products are the predominant qualities needed for successful commercial activity in this area. Product nutraceutical value is also a quality that plays an important role.

Fruit, vegetables, and ornamental and medicinal plants can be propagated sexually, conventional vegetatively, and through micropropagation. In those propagated by seeds, the seedlings are produced commercially and used for direct (field) or indirect seeding, by transplanting pre-germinated seedlings under substrate and greenhouse conditions. In the seed propagated group, we can divide the cultivated species into two. The first group is self-propagating, for example, in lettuce cultivars (Lactuca sativa). In this case, after the development of the new cultivar, seed production is naturally obtained by natural self-fertilization. The other group is equivalent to F1 hybrid seeds, where heterozygous plants are used to obtain homozygous ones, naturally (tomato) or induced by controlled manual pollination, in order to obtain pure lineages (highly homozygous). Specific selected lines are crossed with each other to obtain the F1 hybrids, highly heterozygous, genetically uniform, and with characteristics of commercial interest (RHS 2019). In both cases, seed propagation reduces phytosanitary problems, when due care is taken to produce high-quality seeds.

The other group which uses conventional vegetative propagation refers to those species propagated by stem cutting (guava, chrysanthemum, and sweet potato), grafting (apple, citrus, mango, and grape), air layering (litchi), and the use of stolons (strawberries), rhizomes (banana, ginger, turmeric), divisions (banana, pineapple), and tubers (potatoes), among others. Although vegetative propagation maintains the genetic characteristics of new plantlets obtained, the methods of vegetative propagation have resulted in the dissemination and increase in the frequency of serious crop-specific diseases, especially those of viral or bacterial origin, resulting in a significant reduction in horticultural productivity.

A third group is micropropagated plants obtained under in vitro conditions, using plant tissue culture techniques. The need for micropropagated plants was based on issues found especially in vegetatively propagated plants. These included inefficient multiplication rate, failure to obtain a large number of shoots from a mother plant throughout the year, and the occurrence of diseases that were serious in these species. The latter resulted in the accumulation of contamination in plants and significant and gradate reduction in productivity along cycles of propagation (Cardoso et al. 2018), for example, garlic (Allium sativum) crop cultivation in Brazil, where conventional vegetative propagation through bulbils leads to the accumulation of viruses of the genera Allexivirus, Carlavirus, and Potyvirus, and significant reduction in bulb productivity with the advancement of propagation cycles and production of bulbs (Fernandes 2012). In different garlic cultivars that have undergone micropropagation, including clonal virus-free programs using shoot tip/meristem culture, the increased productivity can reach up to 141% in the first cycle of bulb production, compared to plants without clonal virus-free programs, with productivity up to 49% higher until the fifth cycle of bulb production, even after virus re-infection in this period (Melo Filho et al. 2006). This demonstrated a marked difference in micropropagation methods, in relation to those conventionally used, especially when micropropagation was the only method producing commercially virus-free clonal plantlets.

15.2 The Success of Commercial Micropropagation with the Discovery of Cytokinins

The historical success of plant tissue culture came about with advances in knowledge of plant hormones or plant growth regulators (PGRs), especially cytokinins. One of the oldest trials demonstrating the key role of cytokinins in the success of in vitro development was reported with tomato plants, where it was possible to obtain unlimited growth of roots in a culture medium containing minerals, sucrose, and vitamins, while the shoot tips maintained limited growth under the same conditions, without the addition of PGRs or even with the addition of only auxins to the culture media. Only when the adventitious root was induced, the restarting of growth on in vitro shoots was reported, demonstrating that an unknown diffusible factor from roots was capable of sustaining cell division (White 1934). Before that, Haberlandt (1913) had reported the presence of a diffusible substance in vascular tissue capable of stimulating cell division. However, the discovery was made of the first cytokinin, kinetin (6-furfurylaminopurine), a synthetic molecule found in autoclaved herring sperm DNA, which enabled cell proliferation of tobacco pulp cells (Miller et al. 1955). By means of this molecule and in search of similar molecules, the first natural cytokinin, zeatin, found in immature endosperms of maize (Zea mays) seeds was reported (Miller 1961; Letham 1963). The characterization of a cytokinin was due to its physiological effects in promoting cell division and other regulatory functions similar to kinetin (Skoog and Armstrong 1970), associated with molecules with similar molecular structure (Strnad 1997; Frébort et al. 2011).

Other synthetic molecules with cytokinin effect were also obtained with the ability to promote effects very similar to zeatin in vegetables. Of these, N6-benzyladenine (BA) is one of the most used and most effective synthetic PGRs for use in plant micropropagation, especially for the large-scale production of micropropagated horticultural plantlets (Oliveira et al. 2009; Silva et al. 2012; Ahmadian et al. 2017). It promotes cell division, dedifferentiation, adventitious regeneration, and the induction of multiple sprouts in individualized explants, by stimulating the cellular division of meristems located in the axillary buds of these shoots and breaking the apical dominance exerted by auxins (Müller and Leyser 2011).

In vitro cytokinins have made plant micropropagation a highly profitable commercial activity, in particular by promoting the formation of a large number of shoots from individual explants in a short period of time, making large-scale micropropagation an alternative for many species which have limited propagation by seed or even conventional vegetative propagation, such as low yield of new plantlets and important phytosanitary problems.

In addition to the isoprenoid-based cytokinins (ISCK) (Strnad 1997), some phenylureas also have cytokinin activity (Bruce and Zwar 1966), such as forchlorofenuron (CPPU; Kapchina-Toteva et al. 2000) and thidiazuron (TDZ; Sunagawa et al. 2007).

The discovery of the natural meta-hydroxylated analogues of BA called topolins (Strnad 1997), such as 6-(2-hydroxybenzylamino)purine and 6-(3-hydroxybenzylamino)purine also called ortho- and meta-topolin (oT, mT), was made more recently. These aromatic cytokinins were proven as efficient as isoprenoid (ISCK) and phenylurea types. Topolins have since been used in the multiplication/sprouting/shoot proliferation of different species, such as sugarcane (Vinayak et al. 2009), Pelargonium × hortorum and P. × hederaefolium (Wojtania 2010), the hybrid C35 of Citrus sinensis × Poncirus trifoliata (Chiancone et al. 2016), and Prunus spp. (Monticelli et al. 2017), among others.

15.3 The Importance of N6-Benzyladenine in Commercial Micropropagation

The most widely used cytokinins for micropropagation are BA and TDZ, especially in the in vitro establishment phases, helping to maintain and stimulate cell division of shoot tips taken from donor plants and inoculated in vitro as reported in Aloe vera (Lavakumaran and Seran 2014) and even promoting adventitious organogenesis in non-meristem tissues, in order to obtain adventitious shoots from leaf segments of different commercial cultivars of Anthurium andraeanum (Cardoso and Habermann 2014). Cytokinins have also been reported to induce somatic embryogenesis, such as in Dendrobium ‘Chengmai Pink’ (Chung et al. 2005); however, the embryogenic response of somatic tissues is controlled by a complex mechanism involving hormones including cytokinins, auxins, abscisic acid, and ethylene and stress-related responses (Fehér 2015).

In in vitro establishment of culture medium, cytokinins, such as BA and TDZ, are used for the regeneration and multiplication phase, stimulating the morphogenetic response and development of multiple axillary or adventitious shoots in explants from different plant species, making micropropagation an efficient tool for clonal plant production.

In conclusion, especially for the cytokinin BA, its use in micropropagation protocols was a trigger for the development of commercial techniques, allowing regulation of in vitro regeneration processes, as well as the development of practically all existing commercial micropropagation protocols for different species used in horticulture and other agricultural applications. BA is the most widely used cytokinin in the micropropagation plant industry because of its efficiency, availability (Bairu et al. 2007), and effective response in different plant species, including herbaceous and woody plants used in horticulture.

For example, in Prunus avium cv. Lapins, BA was the only cytokinin capable of inducing shoot multiplication, resulting in a multiplication rate of up to 2.83 in culture medium containing 5 μM of this cytokinin and 0.5 μM indole-3-butyric acid (IBA), compared to other cytokinins such as TDZ {maximum multiplication index (MMI)—1.4)}, N6-isopentenyladenine (iP) (MMI—1.17), and kinetin (KIN) (MMI—1.61) (Ruzic and Vujovic 2008).

Using the Prunus ‘Flordaguard’ rootstock, the addition of BA to the culture medium increased the percentage of explants with shoots (80%), and up to four shoots per explants were obtained using concentrations close to 4.0 mg L−1 (Radmann et al. 2011).

Similar results were obtained in a comparative study using BA and KIN cytokinins in the in vitro cultivation of gerbera, Philodendron, Spathiphyllum, and Musa cultivars (Vardja and Vardja 2001).

15.4 BA Is Not Effective for All Micropropagated Species

The question about testing these new cytokinins, the topolins, was needed. This was due to the fact that BA also leads to undesirable effects in micropropagated plantlets. Vardja and Vardja (2001) micropropagated different ornamental species and noted that for Cordyline, Dracaena, and Dieffenbachia genera, although the cytokinin BA increased the multiplication rate, there were undesirable effects, such as vitrification (hyperhydricity) and reduction in rooting and elongation, in the subsequent phases of in vitro cultivation. At the Laboratory of Plant Physiology and Tissue Culture (UFSCar), our research group observed different symptoms of BA in plant tissues of different horticultural species: in tea tree (Melaleuca alternifolia), we found marked reduction in size of shoots (Fig. 15.1a, b, 1.0–3.0 mg L−1) and early leaf necrosis in explants (Fig. 15.1b, 3.0 mg L−1) compared with normal development in BA-free culture medium (study of Carla Midori Iiyama); in strawberry (Fragaria × ananassa), some cultivars showed as symptoms severe reduction of petiole size, yellowing of leaves, and callus production (instead of shoots) using 0.5 mg L−1 BA, and reduction of concentration to 0.25 mg L−1 resulted in good shoot proliferation with no symptomatic shoots (Fig. 15.1c; study of Camila Y. Nishimura Saziki); in the medicinal plant Phyllanthus amarus, the main symptoms observed using 0.5 mg L−1 BA were callus development in the basal region of shoots and hyperhydricity in leaves and stems (Fig. 15.1d, study of Maria Eduarda Barboza Souza de Oliveira).

Fig. 15.1
figure 1

Different symptoms observed in horticultural species micropropagated in culture medium containing N6-benzyladenine (BA): (a) multiple shoot induction (arrows) in explants of tea tree (Melaleuca alternifolia); (b) symptoms of reduction of size and leaf necrosis (ln) caused by BA in high concentration; (c) symptoms of size reduction and leaf yellowing in strawberry (Fragaria × ananassa); (d) hyperhydricity in shoots and callus development (cd) in basal region of shoots of the medicinal plant Phyllanthus amarus

Full size image

There are published reports for different micropropagated species in which BA is a potent inhibitor of adventitious rooting on in vitro shoots, such as those observed in the medicinal plant Achyrocline satureioides, in which concentrations of BA above 2.5 μM completely inhibited the formation of roots in in vitro shoots cultivated in Woody Plant Medium (Guariniello et al. 2018). Vitrification or hyperhydricity is another physiological abnormality observed in plants cultured in vitro normally caused by the addition of cytokinins, such as BA and TDZ, into the culture medium. The type and concentration of these cytokinins as well as the type of gelling agent and the physical state of the culture medium affect this undesirable response in some micropropagated plants (Ivanova and Van Staden 2011).

For these reasons, in the micropropagation industry, studies using the alternative cytokinins, topolins, for micropropagation of horticultural plant species are mostly concentrated on comparisons of efficiency with the cytokinin BA, e.g., mT, compared to BA added to the culture medium for in vitro evaluations of multiplication or shoot proliferation efficiency and shoot/plantlet development (Table 15.1).

Table 15.1 The use of topolins on micropropagation of horticultural species and its effects on shoot proliferation, in vitro development and quality of plantlets
Full size table

In this sense, an advantage in the use of topolins, compared to BA, would be in maintaining or increasing the positive effects of BA (Werbrouck et al. 1996), especially for efficient induction of multiple shoots that enable the commercial micropropagation of different horticultural species (Cardoso 2018; Cardoso et al. 2018). mT application is usually associated with a reduction of undesirable effects reported in plants cultivated with BA or TDZ cytokinins in culture medium, such as hyperhydricity and inhibition of rooting (Bairu et al. 2007).

For this reason, and due to the similar effects to BA, topolins are used especially in the induction of multiple shoots in the multiplication phase of micropropagation (Table 15.1).

15.5 Topolins Have Effects Not Only on the Multiplication Phase

The use of topolins may also have other effects on later phases of multiplication and micropropagation, such as rooting and acclimatization (Werbrouck et al. 1996). In the micropropagation of potato (Solanum tuberosum) cv. Jaerla, an increase in the dry mass and survival percentage of acclimatized plantlets was observed (91.8–94.7% survival rate) by adding 0.005–0.01 mg L−1 of the meta-topolin riboside (mTR) compared to untreated plants (70.9% survival). This response was attributed to an increase in endogenous cytokinin content in shoots under acclimatization conditions, promoted by the application of mTR, which resulted in lower plant losses in this phase (Baroja-Fernandéz et al. 2002).

In banana ‘Williams’, the application of specific topolins, such as meta-methoxytopolin (MemT) and meta-methoxytopolin-9-tetrahydropyran-2-yl (MemTTHP), promoted an increase of in vitro shoots and root length under acclimatization, while other more common topolins such as mT or mTR did not have the same effect (Aremu et al. 2012). The studies done demonstrated the diversity of responses to different types of topolins, according to the species used. Interestingly, Aremu et al. (2012) observed a reduction in the levels of chlorophylls a, b, and a + b with the use of most of the topolins, compared to the control.

The use of mT improves the late phases of somatic embryogenesis, for example, in hybrid papaya, mT applied in the concentration of 10 μM stimulated sprouting in 40–45% of somatic embryos, with similar effects to BA at 1.8 μM (Solórzano-Cascante et al. 2018). Similar results were obtained by Bhattacharyya et al. (2018a) who reported increases in percentage of shoot emergence of encapsulated PLBs of Ansellia africana orchid by using 5.0–10.0 μM of MemTTHP topolin. In Dendrobium aphyllum orchids, the use of mT up to a concentration of 15 μM increases the percentage of transverse-Thin Cell Layers (tTCLs) that proliferated shoots (79.43±0.12) and number of shoots per tTCL (11.22±0.39) (Bhattacharyya et al. 2018b).  

Another positive effect of the use of mT was reported for Corylus avellana micrografting on Corylus colurna rootstocks, in which up to 70% survival of micrografts was reported with the shoots obtained using mT treatment and only 30% in the treatment with BA (Gentile et al. 2017).

The different types of topolins may also result in effects on metabolism, affecting, for example, in vitro secondary metabolite production. In Aloe arborescens, Amoo et al. (2012) observed significant effects of different types and concentrations of topolins on the production of in vitro secondary metabolites, such as iridoids, total phenolics, flavonoids, and condensed tannins. Similar results demonstrated that the use of mT at 20 μM alone in the culture medium promoted increase in total phenolic and flavonoid content in the medicinal plant Huernia hystrix, whereas the combination of this topolin with 10 μM NAA resulted in increase in shoot proliferation (3×) but had negative effects on the levels of these same secondary metabolites (Amoo and van Staden 2013).

15.6 Topolins Show Similar Negative Effects to BA Depending on Species, But Could Be an Important Tool for Commercial Micropropagation

Despite the positive responses cited in most studies using topolins as cytokinins, some studies have also reported non-significant or even negative effects of topolins, compared with BA used in culture medium. As examples, in ‘Williams’ and ‘Grand Naine’ bananas, the rate of abnormalities obtained with mT was similar to that obtained with the use of BA, both up to 15 μM, but treatments with topolins mT (3.3 roots) and mTR (0.4 roots) at 22.2 μM resulted in a drastic reduction in root numbers, whereas cytokinin BA maintained the number of roots (10.4 roots) compared to control (11.8 roots) (Bairu et al. 2008). In the same study, the authors also observed a dwarf mutant plant in the seventh cycle of in vitro multiplication, identified in the treatment with topolins and using a specific molecular marker. Drastic reductions in rooting percentage of micropropagated shoots were also observed in the medicinal plant Huernia hystrix using 20–25 μM mT (<10% rooting) compared to the use of BA at the same concentration (>90% rooting).

Thus, as observed in previous studies with different classes of plant hormones or different molecules of the same class, the cytokinins show different responses according to species and genotypes (genotype-dependent responses). Thus, it is expected that the responses to topolin-type cytokinins will also be genotype-dependent. This means that topolins have advantages in the micropropagation of some species over other cytokinins, such as BA and TDZ, while in other species BA, the most widely used cytokinin in micropropagation, remains the mainstay of efficient micropropagation protocols.

In this case, the most reasonable proposal is that topolin-type cytokinins are an important and additional tool in the micropropagation of species used in horticulture, especially those with in vitro cultivation issues (Werbrouck 2010; Amoo et al. 2011; Bandaralaje et al. 2015) and for which the most available cytokinins on the market do not result in efficient protocols for commercial micropropagation.

For example, in the micropropagation of the medicinal species Harpagophytum procumbens, excessive callus production at the base of the explants and shoot apical necrosis were reported as physiological disorders caused by BA addition to the culture medium (Bairu et al. 2011). These same symptoms were reported as a problem in the micropropagation of other species, such as in walnut Corylus colurna (Gentile et al. 2017). In both cases, the replacement of BA with mT resulted in resolution of the problem, and in the latter case, the use of mT also led to a significant increase in the percentage of acclimatized plants (78–84% survival in acclimatization), compared to symptomatic plants micropropagated using BA (24–30% survival only).

Similarly, in vitro micropropagation issues, such as early leaf senescence and loss of bud regeneration capacity in different Pelargonium cultivars, were overcome with mT at 0.5–1.0 mg L−1. This resulted in increased multiplication rates of six different cultivars, including those showing symptoms of vitrification, shoot deformities, excessive callus formation, and death (67% of all cultivars) in the presence of cytokinin BA at 0.5 mg L−1 in the culture medium. Using mT as a cytokinin in the culture medium, the authors reported shoot proliferation during five subcultures in all six Pelargonium cultivars tested, with higher multiplication rates and none of the negative symptoms reported for BA treatments (Wojtania 2010).

15.7 Some Remarks About Differential Responses to Topolins and BA

The mechanisms underlying species peculiar responses to the various types of cytokinins are not yet elucidated. Possibly, the differences in species or cultivar responses to treatment with different types of cytokinins are associated with the fact that they represent a different stimulus for the natural biosynthesis of individual cytokinin types in the plants in question (Aremu et al. 2014) and thus lead to different responses regarding proliferation, undesirable symptoms, and subsequent effects on rooting and acclimatization of the sprouts and plantlets obtained.

In many studies, the lower toxicity of topolins, compared to BA and TDZ, is mentioned because the external application of cytokinins leads to natural CK biosynthesis in plants. The final metabolites generated by topolins are more easily degradable, especially O-glucosides, a readily storable form of cytokinins which are rapidly converted into plant-active cytokinin bases (Bairu et al. 2009; Strnad 1997). However, this explanation cannot be applied to all species, since undesirable effects such as inhibition of in vitro sprouting, callus production, and rooting inhibition have also been reported in plant species using some topolins, similar to those reported for the cytokinins BA and TDZ (Table 15.1).

One hypothesis that could explain the heterogeneity of responses to cytokinins among species, especially the differences between BA and topolins, may be related to the type of proliferation of new shoots under in vitro conditions in the production of commercially micropropagated plantlets. Although most species used in horticulture are micropropagated using the multiple shoot induction technique (Cardoso 2018; Cardoso et al. 2019), in some species this is precluded for a variety of reasons, such as negative effects of cytokinins on the in vitro culture environment (Duarte et al. 2019). Micropropagation using the micro-cutting technique, in which the nodes and internodes are segmented and induced to sprout new axillary shoots has been demonstrated is a viable and efficient alternative to multiplying shoots of some horticultural crops (Cardoso and Teixeira da Silva 2013; Duarte et al. 2019).

For example, in the propagation of Krymsk®5 rootstock (Prunus fruticosa × Prunus lannesiana), a higher number of shoots/explants (2.23) using BA instead of mT (0.57) are reported, but the use of mT resulted in a higher number of nodes per explant and shoot length than those cultivated with other cytokinins, including BA (Tsafouros and Roussos 2019). In this case, if each node were segmented and considered as a source of explants (microcuttings) to maintain sprout proliferation, it is possible that the multiplication rate using mT would increase significantly.

15.8 Conclusions

In conclusion, the cytokinins topolins discovered recently are being used as an important tool in the micropropagation of species of great importance for horticulture. In particular, the use of topolins in many cases has addressed the micropropagation and proliferation of high-quality shoots of species or genotypes in which other cytokinins were not efficient for shoot proliferation or resulted in undesirable effects, commonly caused by cytokinins BA and TDZ. However, it is important to note that the use of topolins is subject to the same symptoms caused by other cytokinins at high concentrations or even in species in which BA is still the most efficient for micropropagation. For this reason, studies that consider in particular evaluation of the genetic stability of micropropagated plants with these cytokinins are necessary because they should increase the use of topolins on a commercial scale, aiming at the production of horticultural species. In addition, topolins could increase efficiency of micropropagation in some species. The high costs of are among the major current challenges in large-scale micropropagation (Cardoso et al. 2018), and the increased efficiency of micropropagation is a preponderant factor for effective cost reduction (Chen 2016). The combination of techniques such as temporary immersion bioreactors, light-emitting diodes (LEDs), and photoautotrophic cultivation, among others, commercially emergent with the use of topolins added to the culture medium as well as the effects of topolins in tissue culture applied to plant breeding could also be considered and studied. As example, the use of topolins in the substitution of other cytokinins was also used for inducing gametic embryogenesis in microspores isolated from recalcitrant Citrus genotypes (Chiancone et al. 2015).