Appropriate selection and collection of explants is the first and foremost step for a successful in vitro regeneration study. Even though A. vasica is a perennial shrub and collection of explants can be done round the year; the most active growth stage was considered to retain the regeneration ability of collected explants. Preferable time of explant collection for in vitro regeneration is considered to be between November and March [8], [31], based on certain aspects like ontogenetic or physiological age and position (certain part of the plant, from where explants are collected) or size of explants. A number of explants, such as whole leaf, leaf disc, petiole, shoot tip, nodal segment, axillary meristems and root have been utilized for initiation of in vitro direct or indirect regeneration of A. vasica that has been summarized in Table 1. Among these explants, the sole use of nodal segments from field-grown plants was the most prevalent in majority of the reports [8], [9], [10], [32], [33], [34]. Additionally, when the regeneration efficiency of nodal segments was compared with other explants like shoot tip [35], [36], [37], the nodal segment explants displayed better response based on multiple shoot initiation and subsequent proliferation. Similar trend was also observed in case of indirect regeneration, where the nodal segment explants induced higher frequency of callogenesis in comparison to shoot tip, petioles, and leaf disc explants [38]. To induce cell culture and to obtain maximum cell biomass, Singh et al. [39], unconventionally used root segment explants and attained significant results. On the other hand, Madhukar et al. [40] used leaf explants to develop cell suspension culture via friable callus induction from leaf explants. Even after considering the superior morphogenetic competence of nodal segment explants, leaf explants was preferred for induction and subsequent regeneration of callus [7], [31], [41], [42], [43], [44]. In couple of the instances, the specific age of the explant source (mother plant) was mentioned either as 2–3 years old plant [35] or 6–7 year old flowering plant [8]; however in majority of the reports the age and stage of mother plants were not mentioned, which is considered to be a major factor during explant selection.

Following the collection, surface sterilization and preparation, the explants undergo processing for optimization of in vitro regeneration protocol via standardization of type and formulation of basal media, vitamins, carbohydrates, levels of solidifying agent, pH and plant growth regulators (PGRs). Influence of these factors on micropropagation of A. vasica has been summarized in Table 1. For multiple shoot initiation and subsequent proliferation (Fig. 1a and b), full strength Murashige and Skoog [48] (MS) medium was the only choice as found in all the published reports on A. vasica. Supplementation of PGRs in MS medium significantly varied, as displayed by the reports on shoot multiplication. In several reports, combination of cytokinin and auxin were preferred. For instance, 0.5–2 mg/l N6-benzyladenine (BA) was used as cytokinin in combination with 0.05–0.2 mg/l α-napthalene acetic acid (NAA), which was used as auxin [9], [32], [35], [49]. As an additional cytokinin source, 1 mg/l 6-furfurylaminopurine (Kinetin or Kn) was used along with equal concentration of BA for shoot multiplication [8]. Similarly, Roja et al. [36] used 1 mg/l supplementation of gibberellin A3 (GA3) with 1 mg/l BA to enhance the shoot multiplication frequency of A. vasica. Later on, Lone et al. [37] added 0.5 mg/l NAA and 0.5 mg/l thidiazuron (TDZ) with 2 mg/l BA to improve the regeneration efficiency of BA; wherein, 100% of explants produced the maximum (23.3) shoots/explant in 28 days. However, in contrast, there are several reports on the sole use of BA for initiation of high frequency multiple shoots, wherein very high concentrations of 10 mg/l [8], [34] or 22.20 µm [50]BA were used. As an outcome, 93.33% explants produced ∼11 shoots/explant, each 5.2 cm in length. Exceptionally, a very low level (1.1 mg/l) of BA alone induced multiple shoots in 6 days of culture and eventually produced 7.4 shoots of 7.2 cm length with 2.8 of leaves/shoot [10].

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Fig. 1. Micropropagation of Adhatoda vasica: (a) Direct shoot initiation (arrows) from nodal segment explant after one week of inoculation in MS medium with 1.1 mg/l BA, (b) elongation and proliferation of initiated shoots at 10 days of culture, (c) multiple shoot proliferation and root initiation (arrows) in MS medium with 1 mg/l IBA and 0.25 mg/l NAA, (d) in vitro rooting in clump after 14 days of inoculation (arrows), (e) induction of friable calli in MS medium with 3 mg/l 2,4-D, (f) induction of organogenic calli in MS medium with 0.5 mg/l 2,4-D, (g) indirect rooting and shoot bud (arrows) initiation after 21 days. [Photographs are not in scale].

Source: Original and unpublished photographs from the experiments, carried out by the authors.