Keywords

12.1 Introduction

Cytokinins are a major class of plant growth hormones that induce cell division in tissues (Koshimizu and Iwamura 1986). Its major properties include impeding the senescence process mainly by preventing protein degradation that further increases the activity of RNase (Hall 1973). The primary function of cytokinins is suppressing the apical dominance and thus culminating a way for the development of buds at lateral positions (Leopold and Kriedemann 1975). Considering the extensive uses of cytokinins, there has been an increase in demand on the usage of synthetic cytokinins having analogous effects to naturally derived cytokinins. A novel class of cytokinins, i.e., topolins, is recently being used in plant tissue culture. Chemically, they are regarded as aromatic cytokinins (ARCKs), and structural studies showed that topolins consist of a hydroxylated benzyl that is attached at N6-position of adenine (Aremu et al. 2012a). mT was first isolated from the leaves of Populus × canadensis. The nomenclature of the hormone has been derived from the Czech word ‘topol’ that signifies the plant poplar from where it was first isolated (Strnad et al. 1997). Furthermore, the cytokinin activity depends on the N1 position which should remain free since in the case of hydroxyl at the ortho-position, hydrogen bonding occurs between this group and the N1 atom, which in turn makes the meta-substituents highly potent (Holub et al. 1998). Henceforth, the name meta-topolin [6-(3-hydroxybenzylamino)purine)] is clearly justified. Generally, mT serves as substrate for O-glucosyltransferase (ZOG1 enzyme found in Zea mays and Phaseolus lunatus). Additionally, from the reports on correlation between the activity of mT and their ability to serve as substrate for ZOG1 enzyme, it can be concluded that there is a similarity between the receptors and the binding sites of mT (Mok et al. 2005). The physical attributes of mT are, namely, ‘solid form’ and ‘off white’ to ‘white’ colour. During preparation of stock solution, mT is readily soluble in water, but other solvents like KOH and dimethyl sulfoxide (DMSO) are also equivalently effective. mT also has many multifaceted properties like evading the ill effects of shoot-tip necrosis and hyperhydricity and causing delay in senescence that eventually facilitates multiple shoot regeneration and proliferation (Malá et al. 2013).

12.2 Principles of Regeneration via In Vitro Shoots

The development and regeneration of new organs from different explants are based on the phenomena known as ‘totipotency’, which means that a single cell can develop into a functional organ. The entire process of shoot organogenesis spans entirely over three broad aspects, which are competence, determination, and morphogenesis (Sugiyama 1999). Competence is regarded as the initial step of shoot organogenesis where cell signalling induced by hormones leads to dedifferentiation of cells (Howell et al. 2003). The next step is determining the identity of the organ fixed by the proportion of plant growth hormone; in case of shoot organogenesis, it is possible through cytokinins (Gahan and George 2008). Morphogenesis is the ultimate step where shoot induction finally occurs (Sugiyama 1999).

Furthermore, the phenomena of shoot induction can be categorized into two pathways, namely, direct and indirect pathway. The direct pathway involves the formation of shoot bud, and there is no formation of callus (Yancheva et al. 2003), whereas indirect pathway involves an intermittent step where formation of callus occurs and from adventitious shoots induces from the respective callus (Gahan and George 2008).

12.3 In Vitro Shoot Regeneration in Plant System

The regeneration of shoots from desired explants, mediated via direct or indirect organogenesis approach, serves as a steadfast methodology for micropropagation (Gahan and George 2008). The regeneration of shoots in multiple numbers depends on the type of explant utilized and also on an array of factors like the nutrients utilized in basal media, the environmental factors, and the type and dosage of plant growth hormone used (Gantait et al. 2014).

12.3.1 Influence of Explant

The fundamental criteria before establishment of in vitro culture is the proper selection of explant that should be free from contamination and can be imparted by physical factors like dust or dirt and biological factors like bacteria, fungi, and other microorganisms (Gantait and Kundu 2017). There are innumerable types of explants utilized in micropropagation for inducing shoot directly. Shoot tips and axillary buds are predominantly used explants in any micropropagation experiment since it is easily available and it maintains the genetic integrity in the in vitro-derived plantlets from the mother plant (Rout et al. 2006). Histological studies showed that the shoot-tip region contains a zone comprising of meristematic cells that exhibit the phenomena called ‘totipotency’ and possess the ability of accumulating plant growth hormones at an anticipated level (Akin-Idowu et al. 2009). Another desirable explant utilized is nodal segment, which has the capability to generate multiple axillary buds, since bud break can be easily induced in a short period of time with the application of desired level of plant hormones (Gantait et al. 2009). Some other explants apart from shoot tip and nodal segment such as leaf, corm, bulb, epicotyl, hypocotyledon, cotyledon, root, zygotic embryos, and tillers exhibited promising results and displayed a positive correlation with shoot regeneration (Meyer et al. 2009; Nas et al. 2010; Niedz and Evens 2010; Valero-Aracama et al. 2010; De Diego et al. 2011, Moyo et al. 2011; Niedz and Evens 2011a; Baskaran et al. 2012; Aremu et al. 2014; Moyo et al. 2014; Masondo et al. 2014; Ncube et al. 2015; Aremu et al. 2016; Chiancone et al. 2017; Baskaran et al. 2018a, b; Behera et al. 2018; Chauhan and Taylor 2018; Ahmad and Anis 2019).

12.3.2 Influence of Basal Media

In any micropropagation setup, the basal media enriched with macro- and micronutrients is essential for proper regeneration of explant. The popularly used basal media is Murashige and Skoog (MS) (Murashige and Skoog 1962) medium for shoot regeneration (Table 12.1). There are also other types of media apart from MS media, where the results achieved were quite promising, such as Le Poivre (LP) basal medium (Cortizo et al. 2009); Woody Plant (WP) medium (De Diego et al. 2010; Lattier et al. 2014; Mirabbasi and Hosseinpour 2014; Wen et al. 2016; Rakrawee et al. 2018; Tongsad et al. 2018; Nowakowska et al. 2019), comprising low concentration of nitrate and ammonium ions (Bosela and Michler 2008); Nas and Read medium (NMR) (Nas et al. 2010); Murashige and Tucker (MT) medium (Niedz and Evens 2011b); Quoirin and Lepoivre (QL) (Quoirin and Lepoivre 1977) medium where calcium nitrate is the sole nitrogen source (Lattier et al. 2013); and Driver and Kuniyuki (DKW) medium (Driver and Kuniyuki 1984) that possess minimum proportions of ammonium ions with calcium nitrate serving as nitrogen sources (Gentile et al. 2017; Nacheva et al. 2015; Stevens and Pijut 2018).

Table 12.1 Influence of meta-topolin (mT) on induction, proliferation, and elongation of in vitro multiple shoots
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12.3.3 Influence of Carbon Source and Concentration

The explants that require regeneration under in vitro conditions possess partial autotrophic condition; hence, carbon source is mandatory (Van Huylenbroeck and Debergh 1996). Carbon source, mainly sugars, is required for fine-tuning the osmotic regulation (Hazarika 2003). Majority of research reports indicated the use of optimum amount of carbon source (2–3% sucrose), which is prevalently used (Table 12.1). However, as low as 0.57% sucrose was used to assess its effect on multiple shoot induction from shoot tip of Asparagus officinalis that eventually resulted in elongated multiple shoots (Hudák et al. 2013). It is noteworthy to mention that there is a single instance wherein glucose was used as a carbon source instead of sucrose (Nacheva et al. 2015).

12.3.4 Influence of Physical Environment

Light and temperature are the indispensable parameters for successful establishment of any in vitro culture. Light is dependent upon two factors, namely, intensity and duration. The source of light utilized in any in vitro culture room is fluorescent lamp that can be ‘cool’ or ‘warm’ (Gantait and Kundu 2017). Based on the research reports of past one decade, it was observed that the optimum light intensity ranging between 40 and 300 μmol/m2/s was maintained for successful in vitro regeneration (Table 12.1). Additionally, temperature also plays an obligatory role for normal growth of in vitro culture, and it is mainly dependent on the basic design and structural organization of laboratory. The temperature regime adopted during establishment of in vitro multiple shoot culture protocols is summarized specifically in Table 12.1. Relative humidity (RH) also serves as a regulatory factor in any commercial tissue culture laboratory. Lower levels of RH result in instilling a positive growth in explants, since it strikes a perfect balance with the transpiration rate, whereas higher levels of humidity cause degenerative disorders in explants (Ghashghaie et al. 1992). Generally, the optimum level of RH recurrent in all the relevant literatures scrutinized is around 60%.

12.3.5 Influence of Meta-topolin

There are ~100 reports on the usage of meta-topolin as a plant growth hormone for shoot regeneration that have been documented and illustrated precisely in this chapter. For shoot induction and multiplication, proper dosage of cytokinins is essential (Gahan and George 2008). mT proved to be an effective cytokinin for multiple shoot culture in a number of medicinal, ornamental, and aromatic plants (Fig. 12.1). In majority of the cases, mT alone was sufficient to induce multiple shoots and also aid in shoot proliferation in many species summarized in Table 12.1. A range of 5–10 μM mT was employed to induce multiple shoot proliferation in various plant species through direct and indirect regeneration systems (Bairu et al. 2009a; Vinayak et al. 2009; Nas et al. 2010; Swart et al. 2012; Clapa et al. 2014; Al et al. 2014; Mukherjee et al. 2020). It was observed that even at a concentration lower than 5 μM, mT resulted in successful shoot induction (Wojtania and Węgrzynowicz-Lesiak 2012; Hudák et al. 2013; Aremu et al. 2014; Al et al. 2015; Lata et al. 2016).

Fig. 12.1
figure 1

Influence of meta-topolin (10 μM in semi-solid MS medium with 3% sucrose incubated at 25 ± 1 °C temperature, 50 μmol/m2/s PPFD light intensity, 16 h photoperiod with 60% RH) on in vitro multiple shoot proliferation of several plant species. Multiple shoot cultures of (a) Anthurium andreanum, (b) Musa sp. cv. Grande Nine, (c) Coleus forskohlii Briq., (d) Gerbera jamesonii Bolus, (e) Rauwolfia serpentina (L.) Benth. ex Kurz., (f) Sphagneticola sp., (g) Fragaria × ananassa Dutch, (h) Vanilla planifolia Andrews (figures are not in scale). (Source: Unpublished photographs of Saikat Gantait)

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12.3.6 Influence of mT in Combination with Other PGRs or Additives

mT was also coupled with various classes of plant hormones and also growth additives that gave fruitful results (Table 12.1). mT when used in combination with auxins provided positive results towards shoot induction; however, the formation of regenerative callus was observed when it was exogenously used with auxins like indole-3-acetic acid (IAA) (Meyer et al. 2009). Alternatively, multiple shoot was observed in Coleonema album when combined with indole-3-butyric acid (IBA), where myo-inositol (MI) was used as an additive (Fajinmi et al. 2014). Surprisingly, in combination with N6-benzyladenine (BA), shoot induction occurred along with the formation of callus in the basal end, when shoot tips of Coleonema pulchellum were cultured (Baskaran et al. 2014). In combination with other cytokinins, a high synergistic effect was observed with mT. A high frequency of multiple shoot formation was observed when mT was used in combination with BA (Baskaran and van Staden 2013). Even the use of abscisic acid (ABA) along with mT gave positive results in multiple shoot regeneration in Pelargonium hortorum (Wojtania and Skrzypek 2014). Combination of mT with gibberellic acid (GA3) resulted in shoot multiplication with increased shoot length (Engelmann-Sylvestre and Engelmann 2014; Wen et al. 2016). Similarly, with the application of additives like MI and casein hydrolysate along with mT resulted in longer shoot length in Pistacia vera (Benmahioul 2017). Additives like trimethoprim, yeast extract, and yeast malt broth when used in the medium together with mT also resulted in an increase in nodal length of multiple shoots in Merwilla plumbea (Baskaran et al. 2012). Synthetic cytokinins like thidiazuron (TDZ) when used in combination with mT and an additive AgNO3 exhibited better results during in vitro culture of nodal segments of Dendrobium aphyllum (Bhattacharyya et al. 2018a). Activated charcoal serves as a potent additive for in vitro culture since it has the capability of absorbing hazardous phenolic compounds that are generated during in vitro regeneration (Gantait et al. 2009). Activated charcoal gave promising results with high frequency of shoot multiplication when used in combination with mT (De Diego et al. 2011; Engelmann-Sylvestre and Engelmann 2014; Grulichova et al. 2017).

12.4 Conclusion and Future Prospect of mT Use

In this chapter, the extensive use of mT in various plant species along with their appropriate doses has been scrutinized and elaborately described. The usage of mT in various in vitro cultures has shown promising results when used alone or in combination with other plant growth hormones or additives. The other facets of mT apart from effective shoot induction and multiplication include increase in shoot length, fresh weight, and increase in photosynthetic capacity of in vitro-derived plantlets (Ahmad and Anis 2019). It also has an additional capability of inducing regenerative callus when coupled with some auxins, thus highlighting the efficiency of mT. Considering these positive attributes, we can consider mT as an upcoming plant growth regulator that can be utilized commercially. The need for a comprehensive and reproducible protocol on in vitro culture of plantlets using mT is an utmost necessity for commercialization of plant tissue culture system globally. However, the utility of this compound needs to be harnessed in plants having medicinal and aesthetic value.