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Biotechnological advances in Vitex species, and future perspectives

Vitex is a large genus consisting of 230 species of trees and shrubs with multiple (ornamental, ethnobotanic and pharmacological) uses. Despite this, micropropagation has only been used to effectively propagate and preserve germplasm a limited number (six) of Vitex species (V. agnus-castus, V. doniana, V. glabrata, V. negundo, V. rotundifolia, V. trifolia). This review on Vitex provides details of published micropropagation protocols and perspectives on their application to germplasm preservation and in vitro conservation. Such details serve as a practically useful user manual for Vitex researchers. The importance of micropropagation and its application to synthetic seed production, in vitro flowering, production of secondary metabolites, and the use of molecular markers to detect somaclonal variation in vitro, are also highlighted.

Depulped seeds of V. doniana Sweet germinate well after a hot water treatment [1] or after 21 days of hydrationdehydration cycles [31]. The seed germination of other Vitex species has not yet been studied. Thus, urgent attention is needed to conserve Vitex species. One biotechnological tool, in vitro propagation, provides a viable solution for the largescale propagation of medicinally important plants [53,92,65], including cryoconservation [94] and genetic transformation [96]. The micropropagation of three Vitex species (V. negundo, V. agnus-castus and V. trifolia) from 29 published reports of Vitex sp. has recently been reviewed [9]. However, that review lacks vital details about disinfection methods, temperature, light source and intensity, photoperiod, basal medium, plant growth regulator type and concentrations, medium pH, carbon source type and concentrations for culture initiation, multiplication and rooting, all of which are essential factors that influence the outcome of the in vitro protocol for Vitex species. Consequently, our review explores these fine-scale details of the different steps of the plant tissue culture protocols for Vitex spp. to allow plant biotechnologists to design new and detailed experiments. This review, which provides a detailed analysis of reports from 1986 to 2016 of the micropropagation of six Vitex species (V. agnus-castus L., V. doniana, V. glabrata R. Br., V. leucoxylon L., V. negundo L., V. trifolia L.) (Tables 1  and 2), provides a solid foundation for the sustainable social and economic use of valuable members of this genus. A brief background of the socio-economic importance of Vitex species for which micropropagation protocols exist is provided next.
V. agnus-castus (chast berry) is a deciduous shrub native of Mediterranean Europe and Central Asia. The fruit extract of V. agnus-castus is used to treat menstrual disorder (amenorrhoea, dysmenorrhoea), premenstrual syndrome, corpus luteum insufficiency, hyperprolactinaemia, infertility, acne, menopause and disrupted lactation [30]. The fruits and leaves of V. doniana (black plum) are either consumed raw or after processing while the leaves, fruits, roots, barks and seed of the plant are used in traditional medicinal in Africa to treat a wide range of ailments [28,29,32], and references therein]. V. glabrata is a tree commonly known as ''Kai Nano" in Thailand whose bark and roots are used as an astringent because the bark accumulates high levels of ecdysteroids, primarily 20-hydroxyecdysone or b-ecdysone [110]. The former compound, 20-hydroxyecdysone, can be synthesized in cell suspension cultures of V. glabrata [83,84,23]. V. leucoxylon is a large deciduous tree found in India and is commonly known in Marathi as Songarbhi. The crude alcoholic extract of its leaves possesses anti-psychotic, anti-depressant, analgesic, antiparkinsonian, anti-microbial, anti-inflammatory and woundhealing properties [100]. V. negundo is a woody, aromatic shrub used in Ayurveda, Unani, Chinese, and folk medicine [109,56], and has mainly anti-inflammatory, analgesic, antihyperglycaemic, hepato-protective, anti-microbial and snake venom neutralization activity [71]. V. trifolia is a component of a number of commercially available herbal formulations that employ its leaves, and which have antiseptic, aromatic, febrifuge, anodyne, diuretic, and emmenagogue activity, fruits, which have nervine, cephalic, emmenagogue, amenorrhoeatreating and anthelmintic activity, roots, which are used to treat febrifuge, painful inflammation, cough and fever, and flowers, which are used to treat fever [76].
This review highlights the advances made in the micropropagation (including synthetic seed technology), in vitro flowering, and production of secondary metabolites of Vitex species. Emphasis is also given to the use of molecular markers to detect variation arising from in vitro propagation. This review is useful for conservation biologists, plant physiologists and biotechnologists that aim to explore other unexplored Vitex species or to expand the repertoire of research existent for the currently investigated species.

Selection of suitable starting material and disinfection
The choice of explant often depends on the material that is available, and sometimes even on the season. The selection of explants in Vitex in vitro studies tends to be from young and actively growing shoots or branches, with shoot tips and nodal explants with dormant axillary buds being the first choice (Table 2) due to the presence of a predetermined meristem which allows for true-to-type clonal propagation.

Alternative explant sources
There are several studies available in which other explants were used. For instance, internodes or stem segments were used for callus induction and regeneration studies in V. negundo [103,77,26,79], V. leucoxylon [25], and V. trifolia [15]. Stem-induced callus of V. glabrata produced 20hydroxyecdysone (20-HES) in a liquid culture system [83,84,98]. V. negundo leaves were used for callus culture and  20-hydroxyecdysone (ecdysteroid) production was improved with the addition of precursors: 7dehydrocholesterol (10 mg/l; 1.31 mg/l/d; 1.36-fold higher than control), ergosterol (10 mg/l; 1.12-fold higher than control), and cholesterol (5 mg/l; 1.11fold higher than control [98]. [83,84,98,23] V.   [3] (continued on next page) Biotechnological advances in Vitex species, and future perspectives [15] (continued on next page) Biotechnological advances in Vitex species, and future perspectives  [13] to produce triterpenes [67]. Leaf segments of V. negundo with a midrib and adjacent stem segment were used to study callusmediated organogenesis, and their findings were compared with shoot tips and nodal explants [77]. Explants from in vitro germinated seedlings (hypocotyls, cotyledons, roots and shoot tips) can be employed [22], although seed-derived material is genetically dissimilar and is thus not suitable for clonal tissue culture experiments. There is one report on somatic embryogenesis but none on direct organogenesis (adventitious shoot regeneration) in the Vitex genus, although there are reports on callus-mediated organogenesis in V. trifolia [15,35,81], V. negundo [103,26,79], V. rotundifolia [72] and V. leucoxylon [25]. The chances of somaclonal variation are greater in the case of callus-mediated organogenesis or somatic embryogenesis [52,62], although the latter can provide a useful platform for genetic transformation studies. Somaclonal variation during in vitro cultures of Vitex species has not been reported yet, and this topic should be explored in future studies.

Influence of season
The impact of season for collecting nodal explants from V. negundo from Bhubaneswar, Orissa, in India was compared (1. March to May; 2. June to August; 3. September to November; 4. December to February), and collection between June and August showed higher shoot multiplication [80]. In other studies [10,11,12], nodes of V. trifolia collected from Aligarh, Uttar Pradesh, in India, from January to December, or shoots collected in March, September and October showed best bud break. None of these studies [80,10,11,12] reported the effects of season on explant contamination but such information is essential as explant disinfection is the most important step in the establishment of a tissue culture experiment [93].

Optimized disinfection procedures
Once the explant has been selected, surface disinfection is an important step to establish contamination-free cultures. Based on the Vitex tissue culture literature (Table 1), explants are generally washed under running tap water followed by a wash with a detergent used in a concentration range of 0.1-5%. LabolineÒ applied at 0.1% for 10 min was effective for V. trifolia [44,3,5], 5% LabolineÒ or TeepolÒ for 10 min for V. negundo [7] and V. trifolia [8], while 5% ExtranÒ for 10 min effectively disinfected V. negundo explants [107]. Explants are then surface disinfected with 0.01% (10 min)-0.1% mercury chloride (HgCl 2 ) (1-8 min; Table 1), although 0.1% HgCl 2 for 3 min was sufficient to obtain a 90% contamination-free culture for V. negundo nodes and shoot tip explants [102]. After treatment with HgCl 2 , explants were treated with sterile distilled water (SDW) or sterile double-distilled water (SDDW) three to seven times and then inoculated on basal medium supplemented with various plant growth regulators (PGRs). In some reports, HgCl 2 was omitted from the protocol. V. rotundifolia nodal explants were sterilized with 70% EtOH for 10 s followed by treatment with sodium hypochlorite (NaOCl; 2% active chlorine) for 15 min and rinsed three times with SDW [72]. Only 10% NaOCl for 10 min followed by 4-5 washes of SDW was effective for V. leucoxylon [25] (1) and (2), callus differentiated to 7-8 cm tall shoots after 50 d culture.
Little callus formed on media (3) and (4) surface disinfection and explant preparation can be divided into three stages: (1) removal of dirt and surface dust by washing under running tap water for 10-30 min followed by treatment with liquid detergent; (2) disinfection under a laminar air hood: treatment with EtOH, preferably 70%, which can penetrate bacterial cell walls (by disrupting hydrogen bonding) for at most 30 s otherwise explants can be damaged [70] and then 0.1% HgCl 2 for 1-3 min depending on the explant (juvenile explants can be treated for a maximum of 1 min and mature explants for as long as 3 min); (3) 2-3 washes with SDW. NaOCl is a much more environmental-friendly disinfectant then HgCl 2 [19]. The presence of endophytic microorganisms can cause problems during the establishment of in vitro cultures [57].
In our experiments, we observed contamination by bacteria in V. negundo nodal explants (Fig. 1) that were disinfected with (1) 0.1% HgCl 2 for 3 min followed by three rinses with SDW, (2) 70% ethanol for 2 min followed by treatment with 0.1% HgCl 2 for 3 min and three rinses with SDW, and (3) 0.1% HgCl 2 for 3 min followed by disinfection with 70% ethanol for 2 min and three rinses with SDW. Browning of explants was observed when HgCl 2 was used as the disinfectant (Fig. 1A-C) while surface disinfection with 70% ethanol for 2 min followed by three rinses with SDW allowed explants to remain green although contamination was still observed in more than 50% of cultures. However, 95% of contamination could be controlled by adding filter-sterilized antibiotics (41.28 lM kanamycin, 59.81 lM penicillin, or 34.39 lM streptomycin) to Murashige and Skoog (MS) medium [63] (Fig. 1E-H). Nanoparticles have been shown to control disinfection [17] and could be used in the future to disinfect explants from other Vitex species. Contamination by endophytes has not yet been reported in any Vitex species and could be controlled through meristem culture.

Light conditions
Most culture conditions for the in vitro growth of Vitex species require a 16-h photoperiod (more rarely 10, 12, or 14 h [77,15,107,108,50,75] under cool white fluorescent tubes at a photosynthetic photon flux density of 35-50 lmol m À2 s À1 (Table 2). However, a continuous supply of light was useful for the production of 20-HES from stem-induced callus of V. glabrata [83,84,98].
Light-emitting diodes (LEDs) and cold-cathode fluorescent lamps (CCFL) have seen expanded scientific applications [112], including in plant physiological studies [69,37]. To maximize plant production in vitro, or even to alter morphogenesis, changes in the intensity, type of light source, spectral range and photoperiod can be explored in the future for Vitex tissue culture and secondary metabolite production.

Medium composition
MS medium was most frequently used for tissue culture studies of Vitex species (Table 2)  For shoot tip and axillary shoot multiplication, the most frequently used cytokinins were 6-benzyladenine (BA), kinetin (Kin), and thidiazuron (TDZ) ( Table 2). In V. agnus-castus, Balaraju et al. Balaraju et al. [16] showed that BA or Kin, usually in the presence of an auxin, a-naphthaleneacetic acid (NAA), could induce shoots from shoot tips and nodal explants. In V. negundo, NAA induced callus formation when used alone, but organogenesis was observed when callus was transferred to medium containing BA and NAA [103]. Groach et al. Groach et al. [41] studied the effect of various carbon sources (sucrose, table sugar, sugarcane juice) and gelling agents (agar, sago powder) and found that sugar cane juice was a better carbon source than sucrose for axillary shoot multiplication of V. negundo.
Additives such as sodium sulphate, silver nitrate and phloroglucinol were used for tissue culture studies of V. negundo [24,85]. Silver nitrate is frequently used in in vitro plant propagation as it is a well-known ethylene inhibitor [55]. The addition of amino acids can improve the in vitro culture of many plants [105]. We conducted an experiment that employed nodal explants of V. negundo that were inoculated on MS medium supplemented with 4.44 lM BA, 41.28 lM kanamycin, 59.81 lM penicillin, and 34.39 lM streptomycin. When this medium, or medium containing 4.44 lM BA and 57.41 lM arginine, 68.43 lM glutamine or 86.86 lM proline, was used, shoot growth improved (Fig. 1A-D). Therefore, the effects of various additives on the morphogenic responses of different explants of Vitex species need to be explored. For example, Dadjo et al. Dadjo et al. [29] found that the number of somatic embryos induced from the leaves of V. doniana could be increased when tryptophan was added to MS medium.
Successful rooting of in vitro-raised shoots prior to their ex vitro establishment is an important aspect of plant tissue culture that ultimately renders a protocol efficient, or not. Rooting of shoots can be performed both under in vitro and ex vitro conditions, although the former is most common. In vitro rooting of most Vitex species was carried out using a basal medium (half-strength MS, Nitsch or WPM) with a low salt concentration in combination with an auxin [indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA, alone, or in combination ( Table 2)]. Phloroglucinol can induce a wide range of organogenic responses in in vitro plants, the most common of which is associated with improved rooting [91]. Ex vitro rooting of in vitro-raised shoots was performed by dipping the cut ends of V. negundo shoots in 1466-2442 lM IBA [7,8,85,78].
Several studies did not complete an acclimatization stage while others did not assess the success (plantlet survival) of acclimatization (Table 2). In general, well-rooted plantlets are washed under running tap water to remove adhering agar from the roots and transferred to a suitable substrate and plantlets in containers that are held under high humidity, followed by conditions [18,40] which differ widely depending on the species (Table 2).

Synthetic seeds
The use of synthetic seeds (synseeds) to transport or preserve important germplasm, or even to serve as a module for cryopreservation is well established in many plants [82,54]. Only two reports are available on synseed production in Vitex species.
Optimum synseeds were produced when V. negundo nodal explants were encapsulated in 3% sodium alginate (Naalginate) with 100 mM calcium chloride (CaCl 2 ). MS medium containing 2.5 lM kinetin (Kn) in combination with 1.0 lM a-naphthaleneacetic acid (NAA) resulted 92.6% conversion to plantlets in vitro [4]. More recently, Ahmed et al. Ahmed et al. [13] reported 92% survival of V. trifolia nodal segments encapsulated according to the Ahmad and Anis [4] method. This indicates that 3% Na-alginate with 100 mM CaCl 2 could be used to develop synthetic seeds of other Vitex species in the future. Encapsulated nodal segments could be stored at 4°C up to 8 weeks with 42.5% regeneration efficiency and plantlets, which rooted best on full-strength MS medium containing 0.5 lM NAA, were successfully acclimatized (92%) to field conditions [13].

In vitro flowering
One of the most captivating events in the lifecycle of a flowering plant is the shift from the vegetative phase to the reproductive phase, a complex developmental shift that can be determined by several factors. Under natural conditions, flowering duration varies widely, at least in V. rotundifolia and V. agnus-castus [43]. Flowers that are induced in vitro are an ideal source of explants for the production of haploids since the chance of contamination is very low and since flower induction can take place independent of the season [95,97]. In vitro flowering is also a useful strategy to investigate flowering physiology. Only two studies have examined in vitro flowering in V. negundo [107,104]: details of flowering conditions are described in Table 2). Data about in vitro flowering are not available in Thiruvengadam and Jayabalan [104]. Ahmad et al. Vadawale et al. [107] studied different C/N ratios (they increased the C/N ratio by increasing the sucrose concentration and maintaining the concentration of NH 4 NO 3 at 20.6 lM) and observed that the addition of 146.06 mM sucrose (C/N ratio = 7.08) resulted in in vitro flowering of 80% of cultures within 24 days on MS medium supplemented with 4.44 lM BA and 0.53 lM NAA. In that study, 90% of plants formed an inflorescence with an average of 5.1 flowers per plant [107]. Only one report is available on in vitro flowering, in V. trifolia [64], but details about in vitro flowering induction, specific culture conditions or data were not reported. This leaves a wide scope for plant physiologists and biotechnologist to explore this technique for other Vitex species.

Somatic embryogenesis
Somatic embryogenesis is the process by which somatic cells differentiate into somatic embryos. Somatic embryos, which morphologically resemble zygotic embryos, are used as a model system in embryological studies [58]. However, the greatest importance of somatic embryos, in medicinal plant biotechnology, is their practical application in large-scale vegetative propagation; in some cases, somatic embryogenesis is favoured over other methods of vegetative propagation because of the possibility of scaling up propagation by using bioreactors while somatic embryos or embryogenic cultures can be cryopreserved, allowing gene banks to be established while embryogenic cultures are also an attractive target for genetic modification [61]. Dadjo et al. Dadjo et al. [29] induced somatic embryos from leaf explants of V. doniana on ½ MS medium supplemented with 0.50 lM thidiazuron, 5.55 lM myo-inositol, and 49.74 lM silver nitrate or 6.25 lM tryptophan, but the conversion of somatic embryos to plantlets and field transfer and survival were not reported. Therefore, an efficient protocol should be developed for the propagation of V. doniana and other unexplored Vitex species using somatic embryogenesis.

Production of secondary metabolites
The medicinal properties of medicinal plants are derived from the presence of single or multiple secondary metabolites [39]. V. glabrata produces sterols like 7-dehydrocholesterol (7-DHC), a-ecdysone and 20-HES [110]. 20-HES has multiple uses, including as a growth stimulator for shrimp culture [21], an insecticide [33], an anabolic steroid in sports and bodybuilding, and as a tonic supplement for male and female reproductive systems [34]. Ten-year-old callus induced from stem explants (the exact explant source, i.e., node or internode, was not indicated) used [84] of V. glabrata (as suggested in Thavornnithi [99]; culture conditions described in Table 2) to study the effects of cholesterol, 7-DHC, and ergosterol fed to callus cultures for 0, 12, 24, 72, 96 and 120 h. They found that cholesterol did not increase 20-HES content but instead inhibited cell growth while 7-DHC and ergosterol increased 20-HES production 1.36-fold more than the control without affecting cell growth. Sinlaparaya et al. Sinlaparaya et al. [83], using the same callus as in two other studies [84,99], tested ½ MS, MS, ½ B5 and B 5 medium (syn. Gamborg medium [38] supplemented with 8.88 lM BA and 9.04 lM 2,4dichlorophenoxyacetic acid (2,4-D) for 1 to 5 weeks (detailed culture conditions in Tables 1 and 2). They found that highest cell dry weight (DW) (12.1 g/L) and maximum production of 20-HES (0.038% DW) was possible in B 5 medium in the third week. Thanonkeo et al. Thanonkeo et al. [98] used the same explant as that used in two other studies [83,84], namely callus from a 10-year-old V. glabrata culture, to test basal media, temperatures and sucrose concentrations (MS, ½ MS, B 5 and ½ B 5 ; 25 vs 30°C; 20, 30, 40 g/L sucrose) for 0-12 days at 2day intervals. Thanonkeo et al. Thanonkeo et al. [98] also observed that when cells were cultured in suspension cultures at 30°C on B 5 or ½ MS medium with 30 and 40 g/L sucrose, the production of 20-HES increased 1.09-fold more than the control. Feeding cholesterol at 5 mg/L as a precursor to biosynthesize 20-HES accumulated 1.11-fold more 20-HES than control cells. In another study [23], callus cultures were initiated using the same explants as in the three previous reports. Elicitation with chitosan at 50 mg/L resulted in 17.16 g/L biomass and 377.09 mg/100 g DW 20-HES, which were 1.62 and 8.33 times higher than the control cultures, respectively. Likewise, the addition of methyl jasmonate at 100 lM also enhanced growth and production of 20-HES. The highest growth and 20-HES production reached 14.44 g/ L and 621.76 mg/100 g DW, which were 1.35-and 14.54-fold higher than the control cultures [23]. Noel and Darit [67] isolated triterpenes from callus cultures derived from leaf explants of V. negundo but triterpenes were not quantified nor was the method used to induce callus described.

Molecular markers to detection somaclonal variation
Molecular markers play an important role in studies related to genomics, evolution, and phylogeny of medicinal plants [42].
Only three reports (Table 2) are available on the use of molecular markers in the Vitex genus, specifically in V. negundo and V. trifolia, primarily to verify the homogeneity of in vitroderived plant material. Ahmad and Anis [5] isolated DNA from micropropagated and field-grown plants (plant parts and other conditions were not reported) by the Doyle and Doyle [36] method and used random amplified polymorphic DNA (RAPD) markers (10 GC-rich decamer primers, OPA1-10) to verify the genetic fidelity of two-year-old micropropagated V. negundo plants versus the mother plant (control), and detected no genetic variation. Samantaray et al. Samantaray et al. [81] isolated DNA from fresh leaves of micropropagated and a field-grown mother plant by the cetyltrimethyl ammonium bromide (CTAB) method [20] with minor modifications; 1% polyvinylpyrrolidone (PVP) was added to remove polyphenols and RAPD and inter simple sequence repeats (ISSR) were used to test the genetic fidelity of V. trifolia; they found no genetic variation relative to the mother plant. Ahmad et al. Ahmad et al. [8] also carried out RAPD analysis of V. trifolia plantlets developed from axillary shoots and extracted DNA from young leaves using the [36] protocol. No phenotypic variation was observed in micropropagated plants of V. trifolia [81,8] and V. negundo [5]. A wider selection of molecular markers could assist in the discrimination of adulterants from pure sources, and to screen out somaclonal variants derived from bioreactors, synthetic seeds, or cryopreservation.

Conclusions and future perspectives
Even though there are an estimated 707 plant species known as Vitex sp. worldwide, 230 species have a taxonomically accepted name, 455 names are synonyms and 22 names are unresolved [74]. Protocols for the in vitro propagation of only six species have been established (Table 2), and most have been dedicated to V. negundo (27 articles from a total of 46 papers dedicated to the genus) and V. trifolia (9/46). The only four reports that exist for V. glabrata are related to the production of secondary metabolites [83,84,23,98] but a micropropagation protocol is not available for this species yet. Despite these studies, ironically, none of these studies was performed on the 15 Vitex species listed on the IUCN Red Data list [46]. Seven species are vulnerable (V. acunae, V. ajugaeflora, V. amamiensis, V. keniensis, V. parviflora, V. urceolata and V. zanzibarensis), five are endangered (V. cooperi, V. evoluta, V. gaumeri, V. kuylenii, V. lehmbachii), one is critically endangered (V. yaundensis), one is of low risk and least concern (V. longisepala) and data are deficient about V. heptaphylla IUCN Red Data list [46]. It is essential to provide useful, centralized and detailed information about protocols and in vitro experimental conditions that could urgently be applied to the remaining species, including the endangered ones, to establish in vitro propagation protocols suitable for clonal propagation, cryopreservation and genetic transformation. Photoautotrophic micropropagation, the use of bioreactors, or the use of thin cell layers [88], sonication and ultrasound [89], or magnetic fields [90] to alter or improve tissue growth in vitro are all aspects that merit investigation in Vitex species. Recent progress was made in the induction of V. agnus-castus polyploids using 0.05% colchicine while irradiation with 50 Gy of c-rays resulted in a single-stemmed plant type [14], emphasizing the importance of mutation technology as an applied breeding technique to induce variation.
This implies that the use of biotechnology for this genus is still at a nascent phase of development, and that there is still ample room for future exploration of the great majority of Vitex species. For example, the use of molecular techniques such as RFLP to authenticate V. glabrata [73], or the examination of explants for fungal or bacterial contaminants, e.g., in V. trifolia [113], prior to in vitro culture, indicate that biotechnology has pure and applied applications for Vitex research. Given the economic importance of these species, the in vitro protocols outlined in this review provide a platform for pure and applied studies to be conducted, including the use of bioreactors for mass production of important secondary metabolites or compounds of pharmaceutical and medicinal importance.