5.1 Introduction
Water hyssop (Bacopa monnieri; family, Scrophulariaceae; genus, Bacopa) or brahmi (local name in India) is a semiaquatic herb and commonly grows in wetlands, damp, and marshy areas of warmer regions of the world (Al-Snafi 2013; Behera et al. 2016). There are more than 100 aquatic species found in the genus Bacopa across the globe (Russo and Borrelli 2005). It is native to India and Australia (Aguiar and Borowski 2013) and grown in East Asian countries like Arabian Peninsula, China, Sri Lanka, Nepal, Taiwan, and Vietnam and Florida (Khare 2003; Daniel 2005; Lansdown et al. 2013). The plant is found at 4400 ft altitude and can be cultivated easily depending on the availability of water (Bone 1996). In India, it ranked second among plants, based on uses as medicinal purposes along with advances in research and development and commercial value (Jain et al. 2013). B. monnieri is a small semiaquatic creeping, succulent herb having 10–30-cm-long stem and simple leaf and white or blue flowers with 5 mm fruit (capsule). The macroscopic studies revealed 5 mm cylindrical roots and cylindrical and glabrous stem with prominent nodes and internodes. Leaves are simple, sessile, glabrous, opposite, and obovate-oblong to spatulate in shape and generally long (0.6–2.5 cm) and wide (3–8 mm). The flowers are pale blue/pinkish white in color with five corollas, four stamens, two celled anthers, ovary with two chambers, and multiple ovules. Seeds are very small, irregular, and oblong in shape (Jain et al. 2016). Flowers and fruits are produced during summer (Bone 1996).
Uses of B. monnieri are known in the complementary and alternative medicines (CAM) since ancient times (Kean et al. 2017). It is an important constituent of Ayurvedic system of medicine and mainly as a nerve tonic for curing various neurological and neuropsychiatric diseases. The ancient Ayurvedic treatises like Charaka Samhita since the sixth century AD had mentioned the Bacopa formulations, against the mental conditions like anxiety, cognition, and diuretic, and an energizer for heart and nervous system. In the modern era, Bacopa is used for commercial mental tonic due to their bio-active compounds like alkaloids, bacosides, saponins, and sterols. The important phytochemicals of Bacopa are bacosides which are triterpenoid saponins of dammarane types (Sivaramakrishna et al. 2005) with 12 known analogs of bacoside family (Garai et al. 2009). Other saponins in Bacopa are novel bacopasides I–XII (Garai et al. 1996; Chakravarty et al. 2001, 2003) or alkaloids like brahmine, herpestine, nicotine, apigenin, cucurbitacins, d-mannitol, hersaponin, monnierasides I–III, or plantainoside (Kawai and Shibata 1978; Deepak et al. 2005; Bhandari et al. 2007; Kregel and Zhang 2007; Valko et al. 2007; Chakravarty et al. 2008; Phrompittayarat et al. 2007). Other chemical constituents contain glycoside, flavonoids, phytochemicals, amino acids, and esters (Behera et al. 2016). Among these, bacoside A is the most prominent saponin, and most of the research related to brahmi is based on bacoside A.
Based on containing highly important and bio-active metabolic compounds, they are used for curing illness and disorders like Alzheimer’s disease (Chaudhari et al. 2017), anti-amnesic activities (Saraf et al. 2008, 2010; Anand et al. 2010), antianxiety and antidepressant activities (Shader and Greenblatt 1995; Bhattacharya and Ghosal 1998), anti-arthritic activities (Viji et al. 2010; Vijayan et al. 2010), Antiepileptic (Dar and Channa 1999), antihyperglycemic activity (Ghosh et al. 2011), anti-inflammatory (Channa et al. 2006), antimicrobial effect (Joshi et al. 2013), antioxidant and adaptogenic properties (Bhakuni et al. 1969; Tripathi et al. 1996; Rao et al. 2000; Chowdhuri et al. 2002; Govindarajan et al. 2005), cardio-protective activities (Mohanty et al. 2010), central nervous system (Rao et al. 2000), DNA damage in humans and astrocytes (Elangovan et al. 1995; Kar et al. 2002), DNA replication in cancer cell lines (Channa et al. 2006), endocrine effects (Singh and Singh 1980), free radical scavenging effects (Yadav et al. 1989; Sivaranjan and Balachandran 1994), gastrointestinal effects (Sairam et al. 2001; Sumathy et al. 2002; Goel and Sairam 2002; Dharmani and Palit 2006), hepatoprotective (Sumathi and Nongbri 2008; Sumathi and Devaraj 2009), memoy enhancer (Abhang 1993), promoting hair growth (Jain et al. 2012, 2014; Jain 2016), sedative and tranquilizing properties (Bhakuni et al. 1969), stimulatory effect on thyroid function (Jain et al. 1994; Kar et al. 2002), withdrawal effects of morphine (Kar et al. 2002) or healings of wound (Sharath et al. (2010).
The application of combined traditional and modern biotechnological approaches accomplishes the genetic improvement of economic crops. Bacopa is mainly used as medicinal plant, and all efforts to conserve or its improvement to date are based solely on its bio-active bacoside A. Biotechnological techniques allow to develop desired traits in short time with elite characteristics. In recent years especially in the last decade or from the beginning of this millennium, Bacopa has gained attention of researchers, and studies related to its conservation and genetic improvement using different biotechnological and molecular biology like assessment of genetic diversity, artificial-induced mutation, application of in vitro plant tissue culture techniques for conservation, regeneration and bacoside production, synthetic seed production, use of nanoparticles (NPs) for callus induction or for biosynthesis of NPs, role of microbes for bacoside production, phytoremediation potential of Bacopa, genetic transformation studies in Bacopa, and genomics and transcriptomics of Bacopa have been reported. Thus, the aim of this chapter is to highlight the application of different biotechnological approaches used for the production, conservation, and secondary metabolite production of B. monnieri.
5.2 Genetic Diversity in B. monnieri
Exploitation of biodiversity of plants for improving the quantitative and qualitative characteristics with wide adaptation in different geographical regions is the basic of breeding programs in the modern scientific era. Rapid increase in human population demands to manipulate local cultivars with modern breeding programs to meet the demand of nutrition and other plant-based medicinal compounds used for the health of human beings. Contrarily, the erosion of genetic material due to overexploitation, use of elite cultivars, and lack of local genetic material bring such plants to endangered category. It is therefore the right demand to save these landraces with novel genes by applying modern biotechnological techniques for their conservation for future. In recent years, characterization of phenotype and genotype of different crops/species and modern techniques like gene mapping and sequencing enable researchers to exploit the functional genomics of desired species (Baloch et al. 2017). Molecular marker technique is used to measure direct genetic among species/genotypes/cultivars on the basis of morphological characteristics or geographical distribution. In Bacopa, different molecular markers have been reported to determine genetic diversity, and it is interesting to note that all works related to genetic diversity to date are based from India, where this plant is found in different parts and used commercially. Researchers collected plants from different geographical regions of India and used different molecular markers to find genetic diversity.
RAPD markers are widely used techniques for assessing the genetic diversity of Bacopa compared to other techniques employed (Karthikeyan et al. 2011; Kumar et al. 2013a, b; Srivastava et al. 2016; Anu et al. 2017). The first study of RAPD markers was reported by Darokar et al. (2001). They collected 25 accessions of Bacopa mainly from India and from Malaysia and tested with 40 RAPD primers. Out of these, 29 primers generated single or multiple polymorphic bands ranged from 2 to 8 per primer. The similarity of matrices was found between 0.8 and 1.0, which showed the medium polymorphism level. Thereafter, Karthikeyan et al. (2011) applied 10 RAPD markers on 25 of Bacopa accessions of different geographical regions of India and compared with in vitro shoot propagation. They got the band size ranged 200–870 bp with 113 amplified bands. Out of 113 bands, only 14 were polymorphic with range of 0–30.77%. A maximum number of 18 amplified bands were generated by OPD 08, but polymorphism was recorded only 1%. Cluster analysis indicated the two subgroups using similar coefficient. Similarly, Kumar et al. (2013a) collected eight accessions from Tamil Nadu region of India and applied six different OPL markers. They identified a total of 30 loci with 50% each of polymorphic and nonpolymorphic loci. The polymorphism ranged 0–83.33% as OPT-18 primers failed to generate any polymorphism (%). Contrarily, maximum polymorphism (%) was achieved when OPL-05 primers were applied.
Bacopa plants (18 accessions) collected from southern part of India were subjected to a total of 20 RAPD primers, out of which only 7 generated single or multiple bands. A total of 490 bands were recorded, and 328 and 162 were polymorphic and nonpolymorphic, respectively. Maximum genetic diversity (%) was recorded from OPT-1 and OPT-18 which was 69.2%, while minimum genetic diversity (%) was obtained from OPL-6 (17.8%). The similarity indices among all accession ranged from 0.8 to 1.0 (Kumar et al. 2013b). Anu et al. (2017) used 15 accessions (Western Ghats, South India), and 22 primers responded to all accession with a total of 197 bands with average polymorphic bands of 8.50. Sixteen out of 22 primers were 100% polymorphic with total of 187 polymorphic bands.
RAPD markers were also used for assessing the variation among in vitro regenerated plants in different studies. Ceasar et al. (2010) assessed the in vitro regenerated plantlets with five different RAPD markers, and all plants were monomorphic in nature and similar to mother plants. In vitro regenerated shoots of Bacopa were subjected to ten RAPD markers for assessing genetic fidelity. A total of 58 bands were amplified and only 8 were found polymorphic. The average polymorphism was recorded 13.19% (Pathak et al. 2013). Recently, Sharma et al. (2017a) achieved plant regeneration frequency of 0–20% from cryopreserved shoot tips and compared it with control plants. Their results revealed insignificant variation among shoots from both control and cryopreserved shoot tips using ten RAPD markers and HPLC (high-performance liquid chromatography) to quantify bacoside A contents. Their results revealed the genetic stability of in vitro regenerated shoots after cryopreservation treatment.
RAPD markers are also applied for assessing the genetic stability of Bacopa plants encapsulated with alginate. Randomly selected 19 plants after regeneration followed by regrowth of alginate-encapsulated uninodal cuttings were subjected to RAPD markers. A total of 334 bands were amplified with 72 (21.5%) polymorphic bands. The genetic distance of micropropagated plants ranged from 0.00 to 0.92, while encapsulated synthetic seeds showed 0.67–0.92 (Ramesh et al. 2011a). Muthiah et al. (2013) applied 20 ISSR (inter simple sequence repeats) and 25 RAPD primers to in vitro grown plantlets regenerated from encapsulated shoot tips for 6 months at 4 °C. A total of 130 bands with 125 monomorphic bands from ISSR primers were generated, whereas 25 RAPD primers generated 125 bands with 94% monomorphism. Their results from both studies revealed the use of more than one molecular marker for assessing genetic variability of Bacopa collected from nature or regenerated under in vitro conditions.
Two different markers RAPD and ISSR were used for amplification of 15 accessions collected from Central Indian States. RAPD markers generated a total of 197 bands with 8.95 bands per primer, and 187 bands were polymorphic, whereas 25 ISSR markers produced a total of 280 bands having 270 polymorphic bands with 10.8 bands/primer. The polymorphic information content (PIC) ranged 0.363 - 0.908 (RAPD) and 0.419 - 0.836 (ISSR). Whereas, similarity index ranged 0.16 - 0.95 (RAPD), 0.18-0.98 (ISSR) and 0.179 - 0.945 for ISSR and RAPD markers (Tripathi et al. 2012). Yadav et al. (2012) collected five different accessions of Bacopa from Central and southern and northern region of India. A total of 50 primers were tested, and only 14 produced a total of 515 DNA amplicon. On the basis of sequence of RAPD amplicon, they developed SCAR (sequence-characterized amplified region) primers and obtained single band (406 bp) of Bacopa in all five accessions. They concluded that RAPD and SCAR markers can be used for identification of fresh Bacopa plants. In the next step, they collected brahmi-based drugs from market, based on B. monnieri, and from other plants but sold as brahmi. Application of SCAR markers to these drugs revealed positive results to Bacopa-based drug samples, and drug samples from other plants were negative. They concluded that SCAR marker can be useful for the identification of Bacopa in fresh and in dry form.
There is a single report about the use of ISSR or amplified fragment length polymorphism (AFLP) markers for assessing the genetic diversity among Bacopa plants propagated under in vitro conditions or accession collected from nature (Krishna et al. 2013). They applied 15 ISSR markers to in vitro regenerated shoots of Bacopa up to 10 passages and achieved 57 bands with 56 monomorphic and 1 polymorphic band. The dendrogram analysis revealed the no or zero genetic inconsistency of plants cultured on standard or reduced culture conditions, whereas plants cultured on medium enriched with NAA (1-naphthalene acetic acid) and IBA (indolebutyric acid) revealed the minor variation. Impact of ecogeographical region on the quantification of bacoside A to check the chemodiversity was investigated on 75 accession of B. monnieri. Results revealed the clear impact of chemodiversity as bacoside A contents varied with region (Srivastava et al. 2016). They subjected 36 AFLP markers to 9 different samples of each location, and after initial screening, 2 best primer pairs were selected for final fingerprint generation from high- and low-yielding accessions of B. monnieri. They recorded 16 bands and 9 were found polymorphic with 56.25% polymorphism. There was no record of specific clustering using principal coordinate analysis (PCoA) or dendrogram.
5.3 Mutation Breeding of B. monnieri
The presence of important medicinal metabolites in Bacopa and their use as commercial drug create a new window for researchers to improve its characteristics. Researchers developed and are still developing the new protocol of in vitro regeneration with an objective to regenerate plants and to get higher concentrations of bacosides. But there is still a large gap, and researchers are trying to exploit the potential of Bacopa to develop new traits which can be grown in field conditions with superior agronomic and medicinal properties. Application of mutation breeding is an important and commonly practiced technique to create genetic variation among existing plant gene pool (Toker et al. 2007) which may help in selection process in a given environment (Yadav et al. 2007). As a result, there is a possibility of gaining large number of alleles (Chopra 2005) with a recessive or segregated (3:1) traits, and these traits must be controlled up to or beyond second generation (Micke and Donini 1993). In plant tissue culture, two types of mutagens, physical and chemical, are used for induced mutation. Limited studies highlight the use of physical mutagen like γ–rays (Varghese and Sathyanarayana 2007; Naik et al. 2012) or chemical mutagens like ethyl methanesulfonate (EMS) (Vajpaye et al. 2006; Naik et al. 2012), methyl methanesulfonate (MMS) (Vajpaye et al. 2006), or colchicine (Escandón et al. 2006; Kharde et al. 2017).
5.3.1 The Use of Physical Mutagens
In vitro nodal segments and leaf-induced calli (1 mg/l 2 4-dichlorophenoxyacetic acid (2,4-D)) of Bacopa were treated with γ-rays at the rate of 2.5 Gy/min; 0, 30, 40, 50, 60, 80, 90, and 100 Gy radiation treatments were used for nodal segment explant and 0, 30, 40, 50, 60, and 80 Gy for leaf-induced calli of two cultivars. Treatments of γ–rays induced morphological variability in both plants. They obtained 3.03% w/w bacoside A contents from nodal segment explant of Pragyashakthi cv. compared to 2.60% w/w (calli) and 1.60% w/w (control), whereas bacoside A contents were recorded as 2.61% w/w (calli), 1.75% w/w (control), and 1.17% w/w (nodal segment) from Calcutta Local cultivar (Varghese and Sathyanarayana 2007). Leaf explants of Bacopa were treated with 10, 20, 40, and 80 gray (Gy) for 0, 0.5, 1.0, 1.5, 2.0, and 2.5 hours (h) followed by culture on 2.0% sugar- and 2.0 mg/L KIN-containing medium. Eighty-four percent mortality rate of explant at 80 Gy and induced mutation at 10, 20, and 40 Gy (γ-rays) were reported, and five lines were produced which yielded more bacoside A content compared to control (Naik et al. 2012).
5.3.2 The Use of Chemical Mutagens
The plants of Bacopa were treated for 2 h with different concentrations of mutagens (0.001–5 mM EMS and 0.01–500 μM MMS) in order to evaluate the ecogenotoxicity by using comet assay to assess DNA damage. Acellular/in vitro exposed isolated nuclei or whole plants were exposed to these mutagens. The results indicated the dose-dependent DNA damage to both mutagens, and this damage was higher in root nuclei compared to leaf nuclei to both mutagens (Vajpaye et al. 2006). A study by Varghese and Sathyanarayana (2007) revealed the exposure of nodal segments and leaf-induced calli explants to 0.5% EMS for 0, 0.5, 1.0, 1.5, 2.0, and 2.5 h for two different cultivars (Pragyashakthi, Calcutta Local cultivar). They reported decreased bacoside A content with increase in exposure time to EM, whereas no increase in bacoside A content was recorded from leaf explant treated with EMS irrespective of exposure time.
Colchicine is another chemical mutagen applied for induced mutation or somaclonal variation in Bacopa plant. Colchicine treatment of nodal segments with 0.001% concentration for 24 or 48 h resulted in increased flower size (Escandón et al. 2006). They inoculated the explants on medium having 0.25 mg/l BAP (6-benzylaminopurine) and obtained two different plants (tetraploid) from control plants with difference in size and color of flower and leaf. Kharde et al. (2017) treated leaf explants with 0.1% and 0.2% colchicine for 1, 2, 3, 4, and 5 h and cultured on 1.1 μM IBA and 0.30 μM IBA. They observed changes or variations like leaf shape, number, and arrangements and enhanced bacoside contents which were higher when treated with 0.2% colchicine for 5 h, whereas treatment with 0.1% colchicine for 2 h yielded twofold bacoside contents that were recorded at 0.72%.
5.4 In Vitro Plant Tissue Culture of B. monnieri
B. monnieri is medicinal aquatic plant. It contains bio-active compounds, which have been used as medicine and attribute pharmacological activities (Ganjewala and Srivastava 2011). Zhou et al. (2009) reported at least 70 chemical constituents mainly saponins (Chillara et al. 2005), and bacoside A is the main saponin which attributes biological activities (Deepak and Amit 2004; Peng et al. 2010). Bacopa is a native plant of India which shows the narrow genetic diversity. The plant is used as memory enhancer (Charles et al. 2011), and a commercial drug is also available. The plant was reported threatened to extinction due to its wild collection and high demand (Tanvir et al. 2010; Tiwari and Singh 2010). Due to these factors, there is a need to develop strategies to conserve plant and also propagate to meet the demand of bacoside A. There are two ways to meet the objective: (a) the use of traditional vegetative propagation or seeds or (b) the application of in vitro plant tissue culture techniques.
Plant cell and tissue culture techniques include callus culture, cell suspension cultures, somatic embryogenesis, or organogenesis (Aasim et al. 2014) for the production of elite plants. These techniques can be used for isolation of economically important bio-active compounds. The results on different plants/crops show more advantageous for secondary metabolite isolation through in vitro culture compared to plant/seeds taken from field conditions. Furthermore, consistency, controlled conditions, and elite nature of cells/callus/plants taken from in vitro culture make it superior for metabolite production (Talukdar 2014). Furthermore, it is possible to alter the metabolite concentration with the aid of adding different chemicals/enzymes/organic compounds in the culture medium or controlled change in culture conditions like lights, temperature, etc. However, it is also significant to understand the variations in metabolite production or medicinal pathway (Al-Habori and Raman 2002). The in vitro techniques have two parts: (a) in vitro cell/callus/cell suspension and protoplast culture and (b) organogenesis or somatic embryogenesis based in vitro regeneration.
5.4.1 In Vitro Cell Suspension Culture/Callus Culture for Bacoside Production
In recent years, researchers reported work related to in vitro cell/callus/cell suspension of Bacopa with main focus on phytochemical production of bacoside. Cell suspension culture from callus is the most widely used technique used for secondary metabolites synthesis (Talukdar 2014). It also provides the facility to investigate the efficacy of variable organic and inorganic chemicals or biotic elicitors (Parale and Nikam 2009) or variable growth conditions on cell growth subsequently followed by secondary metabolite production of economic medicinal plants. Rahman et al. (2002) achieved friable green calli on leaf explant (0.5 mg/l KIN, 1 mg/l NAA, 1 mg/l casein hydrolysate, 30 g/l sucrose) and shifted to liquid medium with the same concentrations in complete darkness. They achieved bacoside A contents at the rate of 1 g/100 g dry cells. Leaf explants of Bacopa were used for callus induction by culturing it on 1 μM 2,4-D + 5 μM NAA-containing medium. The medium was also enriched with 0–125 μM glycine or 0–200 μM of phenylalanine, α-ketoglutaric acid, ferulic acid, or pyruvic acid singly. Application of 100 M pyruvic acid significantly enhanced the bacoside A from callus culture (Parale et al. 2010).
Successful callus induction from leaf explant using different combinations of BAP, IAA (Indole-3-acetic acid), KIN (1: 0.05: 0.05 or 1.5: 0.05: 0.05), and 2, 4-D: BAP (1:0.5; 1.5:0.5), was reported by Mendhulkar et al. (2011). They generated the cell suspension by shifting 1 g callus to liquid medium containing 1: 0.5 (2, 4-D: BAP). They treated the 21-day-old cell suspension with 0.2%, 0.6%, and 1.0% DMSO for 3 and 6 h and obtained maximum bacoside contents (4.6 ± 0.03 μg/mg) from suspension culture treated with 1% DMSO for 3 h. Bansal et al. (2014) optimized the KNO3, KH2PO4, glucose, and inoculum density for the growth of cell suspension and bacoside A contents, whereas application of RSM (response surface methodology), 5.67% glucose, 0.313% KNO3, and 0.29% KH2PO4 with inoculum density (0.66%) was optimized and revealed twofold biomass yield and 1.7-fold bacoside A.
Besides of use of callus derived cell suspension culture for Bacoside A production, callus culture using different explants, growth medium or adding different chemicals have been used also for Bacoside A synthesis. Showkat et al. (2010) induced callus using 0.5 mg/L 2,4-D from leaf explants. Bacoside and fingerprint profile of in vitro regenerated shoots using HPLC or HPLTC from callus revealed the similar phytochemical profile to that of mother plant or plants obtained from markets. Monica et al. (2013) obtained callus of Bacopa leaf by culturing it on medium with 0.5 mg/l 2,4 after 20 days and transferred it to a liquid medium similar to Rahman et al. (2002) containing 0.5 mg/l KIN, 1 mg/l NAA, 1 mg/l casein hydrolysate, and 30 g/l sucrose for 20 more days in darkness. They achieved 166% more saponin contents from cell suspension culture compared to plants taken from nature. Talukdar (2014) induced maximum callus culture from leaf and nodal segment explant and achieved highest callus weight on medium containing 0.2 mg/l NAA (24.67 g) or 2.0 mg/l 2,4-D (35.69 g) after 8 weeks of culture. They reported total bacoside content of 1.53% compared to 1.02% from field-grown Bacopa using HPLC. Recently, Hegazi et al. (2017a) reported the collection of B. monnieri plants from the Eastern Mediterranean coastal region of Egypt (North Sinai). They successful induced callus from leaf explant from medium enriched with 9 μM 2,4-D and 2.3 μM KIN. They also reported the effects of mevalonic acid (precursor) and chitosan and methyl jasmonate (elicitors) and got more biomass with 100 mg/L chitosan and highest bacoside A contents when 10 mM mevalonic acid was used. They also checked the efficacy of 100 mg/l chitosan (elicitors) and obtained 30.76-fold bacoside A contents compared to control plant.
5.4.2 In Vitro Regeneration/Organogenesis of B. monnieri
In recent years, large numbers of research work on in vitro regeneration of Bacopa have been published especially after in this millennium. The main objective in these studies was to develop or modify the existing protocols for the conservation of Bacopa as plant is considered as endangered in the literature. The demand of plant is increasing immensely and researchers are developing new protocols. This section presents the insight as regards in vitro regeneration techniques about growth medium, explants, plant growth regulators (PGRs), rooting, and acclimatization used by researchers. The information given in this section is based on the literature used and analyzed.
In vitro morphogenesis, shoot growth, and rooting vary with nutritional requirement of tissue used and plant type. The basic objective of adding basal medium is to meet the demand of macro- and micronutrients and vitamins, and their requirement also varies with the explant, tissue, or plant type (Saad and Elshahed 2012). The studies on plant tissue culture of Bacopa revealed the use of mainly MS medium at different concentrations like full MS (Gurnani et al. 2012; Asha et al. 2013; Jain et al. 2013; Kaur et al. 2013; Koul et al. 2014; Mohanta and Sahoo 2014; Subashri and Pillai 2014; Rency et al. 2016; Wangdi and Sarethy 2016; Haque et al. 2017; Srivastava et al. 2017; Zote et al. 2018), with some reports of using 0.5 MS (Haque et al. 2017) or one half MS (Jain et al. 2014), whereas B5 medium has also been reported in some studies (Mohapatra and Rath 2005; Monica et al. 2013; Koul et al. 2014).
The presence of reducing carbon or nonreducing carbon sources in plant tissue culture media is an important factor for providing energy and carbon source for photosynthesis or maintaining cell’s osmotic potential (Sumaryono et al. 2012) in the culture media, which in turn controls the morphogenetic potential (Yaseen et al. 2013). However, it depends mainly on concentration and type of carbon source and technique used for regeneration, callus induction, germination or rotting, etc. Most widely and recommended carbon source in tissue culture are sucrose, fructose, or glucose, but the most preferable carbon source is sucrose due to its effects and cost (Sumaryono et al. 2012). In vitro regeneration studies on Bacopa revealed the use of sucrose at different concentrations like 3% (Showkat et al. 2010; Vijayakumar et al. 2010; Asha et al. 2013; Kumari et al. 2014; Pandiyan and Selvaraj 2012; Begum and Mathur 2014; Behera et al. 2015; Nagarajan et al. 2015; Nandhini et al. 2015; Rency et al. 2016; Karataş et al. 2013, 2016, 2018; Narwal 2016; Wangdi and Sarethy 2016; Hegazi et al. 2017a, b; Srivastava et al. 2017; Ranjan and Kumar 2018) or reduced sucrose at the rate of 2.0% (Escandón et al. 2006; Kaur et al. 2013; Jain et al. 2014; Naik et al. 2014; Ranjan et al. 2018) in the culture medium for regeneration and rooting.
In vitro tissue culture of economic plants depends on culture medium composition like gelling agents which makes medium viscous (Jain 2006). There are several commercial gelling agents for plant tissue culture, but agar is the most preferable gelling agent compared to others like gelrite or phytagel or plant-based gums (Babbar et al. 2005). For Bacopa regeneration, solid medium gelled with agar or other gelling agents was preferred, but some studies also revealed the use of culture medium with reduced or no gelling agent (liquid medium) based on the need of the experiment. Agar at different concentrations has been successfully employed for in vitro regeneration with concentration of 0.65% (Showkat et al. 2010; Karataş et al. 2013, 2016, 2018, Karataş and Aasim 2014), 0.7% (Escandón et al. 2006; Kaur et al. 2013, Mohanta and Sahoo 2014; Behera et al. 2015; Mishra et al. 2015), 0.75% (Kaur et al. 2013), or 0.8% (Tiwari et al. 2001; Mohapatra and Rath 2005; Joshi et al. 2010; Prabha et al. 2010; Parale et al. 2010; Rout et al. 2011; Gurnani et al. 2012; Pandiyan and Selvaraj 2012; Rao et al. 2012; Asha et al. 2013; Begum and Mathur 2014; Kumari et al. 2014; Naik et al. 2014; Nagarajan et al. 2015; Narwal 2016; Rency et al. 2016; Wangdi and Sarethy 2016; Kashyap et al. 2017; Srivastava et al. 2017; Ranjan et al. 2018), whereas some studies revealed the use of phytagel (Hegazi 2016; Hegazi et al. 2017a, b) and gelrite (Nandhini et al. 2015) in the culture medium. A study by Yusuf et al. (2011) highlighted the comparison of different concentrations of isabgol (1.0%, 3.0%, and 5.0%) with agar (0.7, 1.0, or 1.5%) for in vitro regeneration of Bacopa.
Surface sterilization of plant seed or plant parts is the most important step toward plant tissue culture techniques which include the removal or minimizing the exogenous or in some cases endogenous microbial contamination (Buckley and Reed 1994). Micropropagation of aquatic plants usually involves the use of vegetative parts, directly subjected to surface sterilization without any substantial damage to explants during sterilization (Aasim et al. 2013). Selection of proper sterilizing agent and exposure time (Mihaljević et al. 2013) are of utmost importance and depend on physical or morphological characteristics of plant part like tissue’s hardness/softness (Srivastava et al. 2010). Like other aquatic plants, Bacopa is propagated through explants taken from vegetative parts and exposed to different sterilizing agents with different times of exposure. Studies on in vitro regeneration of Bacopa revealed the use of HgCl2 as major sterilizing agent at different concentrations like 0.01% (Showkat et al. 2010; Vijayakumar et al. 2010; Mohan et al. 2011; Jain et al. 2013; Kumari et al. 2014; Subashri and Pillai 2014), 0.05% (Zote et al. 2018), 0.2% (Koul et al. 2014), and 1% (Mohapatra and Rath 2005) with different exposure times. Thereafter, NaOCl is second most used sterilizing agent but at low concentrations like 0.5% (Soundararajan and Karrunakaran 2011) or 1% (Koul et al. 2014; Zote et al. 2018) and 2% (Naik et al. 2014; Umesh et al. 2014; Hegazi et al. 2017a, b).
Besides that, other detergents or antiseptic chemicals have also been reported for sterilization of Bacopa. It includes the use of Labolene detergent (Rout et al. 2011; Asha et al. 2013; Begum and Mathur 2014; Mohanta and Sahoo 2014; Nandhini et al. 2015); Teepol, a multipurpose detergent (Mohapatra and Rath 2005; Gurnani et al. 2012; Rao et al. 2012; Behera et al. 2015; Nagarajan et al. 2015; Kashyap et al. 2017; Ranjan et al. 2018); Savlon, antiseptic detergent (Vijayakumar et al. 2010; Mohan et al. 2011; Pandiyan and Selvaraj 2012; Ranjan and Kumar 2018); and Rankleen (Prabha et al. 2010) and Cetrimide, an antiseptic (Soundararajan and Karrunakaran 2011; Mishra et al. 2015; Srivastava et al. 2017). Sterilization process also involved the use of additive chemicals to enhance the sterilization efficiency. The chemicals used for sterilization with other major sterilizing agents are Tween (Escandón et al. 2006; Sharath et al. 2007; Yusuf et al. 2011; Kaur et al. 2013; Koul et al. 2014; Narwal 2016; Haque et al. 2017), alcohol (Showkat et al. 2010; Jain et al. 2014; Mohanta and Sahoo 2014; Subashri and Pillai 2014; Umesh et al. 2014; Nagarajan et al. 2015; Narwal 2016; Rency et al. 2016; Wangdi and Sarethy 2016; Kashyap et al. 2017; Srivastava et al. 2017; Ranjan et al. 2018), Bavistin fungicide (Kaur et al. 2013; Mohanta and Sahoo 2014; Haque et al. 2017), streptomycin + Bavistin (Mohan et al. 2011; Vijayakumar et al. 2010; Ranjan et al. 2018), and Bavistin + neomycin (Showkat et al. 2010).
The selection of proper explant is an important part of plant tissue culture protocol as it results in the development of adventitious or axillary shoots under in vitro conditions. Besides that, the presence or absence of meristematic cells in the explant also controls the regeneration process as organogenesis and somatic embryogenesis along with other factors like plant growth regulators, culture conditions, etc. For explant selection, different factors like explant age and size, plant quality, genotype, and objective of study (callus induction, somatic embryogenesis, organogenesis) must be taken in account (Smith 2012). For Bacopa micropropagation, different explants used can be classified as (a) explants regenerated adventitious shoots or (b) explants regenerated axillary shoots.
For adventitious shoot regeneration, leaf explant is the most widely used explant (Tiwari et al. 2001; Joshi et al. 2010; Parale et al. 2010; Rout et al. 2011; Vijayakumar et al. 2010; Yusuf et al. 2011; Rao et al. 2012; Jain et al. 2013; Koul et al. 2014, 2015; Naik et al. 2014; Umesh et al. 2014; Ayyappadas and Renugadevi 2015; Behera et al. 2015; Nandhini et al. 2015; Haque et al. 2017; Mehta 2017; Ranjan et al. 2018; Srivastava et al. 2017; Zote et al. 2018) followed by internode (Tiwari et al. 2001; Mohan et al. 2011; Yusuf et al. 2011; Rao et al. 2012; Kaur et al. 2013; Naik et al. 2014; Ayyappadas and Renugadevi 2015; Behera et al. 2015; Kashyap et al. 2017; Mehta 2017; Srivastava et al. 2017) and root explants (Vijayakumar et al. 2010).
Contrarily, different explants are also used for axillary shoot regeneration or callus induction like shoot apex/shoot meristem (Pandiyan and Selvaraj 2012; Jain et al. 2013; Kaur et al. 2013; Subashri and Pillai 2014; Ayyappadas and Renugadevi 2015; Hegazi 2016; Łojewski et al. 2016; Hegazi et al. 2017a, b), nodal segment from different parts of plants (Tiwari et al. 2001; Escandón et al. 2006; Prabha et al. 2010; Showkat et al. 2010; Vijayakumar et al. 2010; Yusuf et al. 2011; Gurnani et al. 2012; Pandiyan and Selvaraj 2012; Asha et al. 2013; Jain et al. 2013, 2014; Kaur et al. 2013; Kumari et al. 2014; Mohanta and Sahoo 2014; Naik et al. 2014; Subashri and Pillai 2014; Umesh et al. 2014; Ayyappadas and Renugadevi 2015; Behera et al. 2015; Mishra et al. 2015; Nagarajan et al. 2015; Narwal 2016; Wangdi and Sarethy 2016; Hegazi et al. 2017a, b; Kashyap et al. 2017; Mehta 2017; Srivastava et al. 2017; Ranjan et al. 2018), apical buds (Narwal 2016), and stem (Vijayakumar et al. 2010; Karataş et al. 2016; Zote et al. 2018).
The provision of PGR in the culture medium along with other factors like explant, basal medium, culture conditions, etc. controls the in vitro callogenesis and organogenesis. These PGRs in the culture medium are used at different concentrations based on the objective of the study. Cytokinins and auxins are used generally for in vitro regeneration. Cytokinins are used either singly or in combination with auxins, whereas auxins alone are used mainly for callus induction followed by organogenesis by transferring the calli to the medium enriched with cytokinins or auxins + cytokinins.
Bacopa is not a recalcitrant in nature and responds well enough to PGRs in the culture medium irrespective of explant even without meristematic regions like leaf or internodes. Different studies on Bacopa revealed the use of different cytokinins alone at variable concentrations for different explants. BAP is the most accepted and preferred PGR used for in vitro regeneration (Mohapatra and Rath 2005; Joshi et al. 2010; Prabha et al. 2010; Yusuf et al. 2011; Rao et al. 2012; Asha et al. 2013; Kaur et al. 2013; Jain et al. 2014; Kumari et al. 2014; Behera et al. 2015; Mishra et al. 2015; Nagarajan et al. 2015; Karataş et al. 2016; Srivastava et al. 2017; Haque et al. 2017). Other cytokinins used alone are KIN (Kumari et al. 2014; Naik et al. 2014; Wangdi and Sarethy 2016) and TDZ (Tiwari et al. 2001). A study by Begum and Mathur (2014) reported the use of BAP + KIN combination for in vitro regeneration of Bacopa, whereas Subashri and Pillai (2014) optimized different cytokinins (1.0 mg/l each of BAP and TDZ, 4.92 mg/l 2ip) for the regeneration of Bacopa in vitro. On the other hand, combination of cytokinin and auxins is also optimized for maximum shoot induction of Bacopa. These combinations include BAP + IAA (Gurnani et al. 2012; Narwal 2016; Ranjan and Kumar 2018), BAP + NAA (Rout et al. 2011; Jain et al. 2013; Rency et al. 2016; Ranjan et al. 2018), BAP + IBA (Zote et al. 2018), and KIN + IBA (Mehta 2017), whereas a combination of BAP + KIN + NAA has also been reported (Vijayakumar et al. 2010; Pandiyan and Selvaraj 2012; Ayyappadas and Renugadevi 2015). There are very few studies which reflected the use of TDZ alone or in combination with auxins. Karataş and Aasim (2014) reported the multiple shoot buds on TDZ, but these buds generated shoots when transferred to MS medium without PGRs. It is also interested to note that Bacopa can be propagated without any PGR in the culture medium (Koul et al. 2014) or shoot induction can be achieved by adding IAA or NAA in the culture medium (Mohanta and Sahoo 2014). In conclusion, all explants used for Bacopa regeneration respond well to PGRs irrespective of PGR type or concentration. It is also concluded that BAP solely and the combination of KIN and auxins (IAA, NAA, IBA) are most suitable for regeneration.
PGRs are generally used for callus or shoot induction in vitro. Researchers always tried nontraditional organic or inorganic chemicals or biological extracts for enhancing or inducing in vitro regeneration of economic plants. Being an economic plant, Bacopa is one of the plants subjected to different chemicals for exploiting the in vitro regeneration potential. Pothiaraj et al. (2016) used seaweed liquid extracts (SLEs) isolated from Gracilaria edulis and Sargassum wightii and compared with PGRs for B. monnieri. Application of 30% (S. Wightii) and 40% (G. Edulis) liquid extracts significantly enhanced the shoot and root proliferation with increased survivability of in vitro propagated plants. Kashyap et al. (2017) applied humin (a residue taken from acid-base treatment of vermicompost) alone or along with micronutrients, vitamins, or 3.0 mg/l BAP + 1 mg/l IAA in culture media and cultured nodal segment explants for shoot induction. They achieved higher shoot induction, leaf induction, plantlet weight, and survival rate on medium containing humins compared to humins with other supplements.
The rooting of in vitro regenerated shoots is linkage step between transfer of regenerated shoots/plantlets to external field conditions. Rooting followed by adaptation is an important part of successful plant tissue culture protocol. There are studies which skipped the rooting stage due to direct rooting of shoots (plantlets) in the culture medium due to the presence of auxins (Gurnani et al. 2012; Pandiyan and Selvaraj 2012) or even medium containing only KIN (Naik et al. 2014). Other studies even revealed the use of MSO (MS without any PGRs) for rooting and achieved high percentage of rooting (Asha et al. 2013; Mohanta and Sahoo 2014; Subashri and Pillai 2014; Ranjan and Kumar 2018). On the other hand, IBA was used most frequently auxin for rhizogenesis of Bacopa (Tiwari et al. 2001; Joshi et al. 2010; Rao et al. 2012; Kaur et al. 2013; Jain et al. 2013, 2014; Kumari et al. 2014; Behera et al. 2015; Karataş et al. 2016; Srivastava et al. 2017; Zote et al. 2018) followed by IAA (Rout et al. 2011; Narwal 2016; Rency et al. 2016). In all these studies, rooting response was high up to 100%, and these results reflected the easiness of rooting stage. Multiple shoot inductions with callogenesis during rooting medium containing IBA from the cut end of shoots were reported by Karataş et al. (2013). It shows that even auxins alone can also be used for direct plantlet regeneration in short time with longer shoots.
After rooting, the next stage is the adaptation/acclimatization of plantlets to external conditions. Bacopa is a semiaquatic plant which can survive in water and also in soil with high moisture. Soil as substrate for transferring plantlets for acclimatization has been reported in almost all of the studies. However, adaptation of in vitro regenerated plantlets in aquariums containing water was reported by Karataş et al. (2013). Furthermore, they also checked the plant growth in aquariums with different pH levels (4–10) and reported maximum plant growth at pH 8.0.
5.4.3 In Vitro Regeneration of B. monnieri for Bacoside and Other Metabolites
Although callus culture or cell suspension culture is the most accepted in vitro technique for isolation of secondary metabolites, in vitro regenerated shoots through organogenesis are also a good source of these metabolite isolations. The use of organogenesis for bacoside A and other secondary metabolites production is available. Praveen et al. (2009) regenerated Bacopa shoots in semisolid and liquid medium and gained more shoots from liquid medium. Analysis of bacoside A contents revealed more contents compared to semisolid medium, whereas more shoots were recorded from medium containing 2 mg/l KIN. Parale et al. (2010) used leaf explants for shoot induction in liquid medium containing 5 μM BAP. They also used organic supplements in the culture medium and noted enhanced bacoside A contents with 100 μM pyruvic acid. The bacoside contents were higher than control (4-fold) or naturally grown plantlets (1.2-fold). Sharma et al. (2013) applied methyl jasmonate to 1-month-old shoots and cultured it in liquid medium. They obtained maximum bacoside contents (1.8-fold higher than control) after 1 week.
Umesh et al. (2014) obtained plantlets by direct organogenesis (plantlets from leaf explant ın medium enriched with 2 mg/l KIN. However, bacoside contents varied with PGRs, and the highest concentration of bacopasides I and II was recorded on a medium with 1 mg/l BAP + 0.5 mg/l IAA or 2 mg/l KIN, respectively. Nandhini et al. (2015) achieved maximum shoot buds (162.33 ± 21.385) with 0.2 mg/L BAP. They checked the secondary metabolite contents of in vitro regenerated plantlets and found lower flavonoid contents compared to control plants, whereas minor differences in phenol and saponin contents were recorded when compared with control plants. Łojewski et al. (2016) used Mg and other metal-enriched media for shoot induction and Bacosides A contents. They achieved highest bacosides (37.3 mg/g dry weight) from cultures enriched with 1.0 mg/l BAP + 0.2 mg/l NAA + 0.25 g/l serine + 0.1 g/l Mg or 1.0 mg/l BAP + 0.2 mg/l NAA+0.5 g/l serine +0.5 g/l Mg. Hegazi et al. (2017b) obtained maximum shoot multiplication after six subculture (2.45 μM IBA+2.3 μM KIN). They also used precursor (mevalonic acid) and elicitors (chitosan and methyl jasmonate) for biomass and bacoside A production. One hundred micrometer methyl jasmonate enhanced the biomass, while 10 mM mevalonic acid resulted in 8.26-fold more bacoside A accumulation in shoots.
5.5 Regulation of Bacoside Biosynthesis by Beneficial Microbes
The application of biotic elicitors or chemical precursors/elicitors has also been used for increasing callus biomass and bacoside A production. Parale and Nikam (2009) inoculated callus derived from a liquid medium (5 μM NAA and 1 μM 2,4-D) with different strains of fungus used as elicitors. Only inoculation with Saccharomyces cerevisiae enhanced the bacoside contents up to 20%, whereas other biotic elicitors resulted in decreased bacoside contents from callus culture. Inoculation of plant beneficial microbes like Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 with Bacopa plant significantly enhanced resistance against nematode and also up-regulation of Bacoside biosynthetic genes. The genes in the pathway of bacoside biosynthesis were 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase. Further, the elicitation due to microbes enhanced the bacoside production significantly than the control treatments.
5.6 Cryopreservation of B. monnieri
Preservation of plant material for short time of few days to mid or long term upto few months is an important technique in germplasm conservation. These techniques are slow growth storage (in vitro techniques) or cryopreservation using liquid nitrogen (Ozudogru et al. 2010) or combination of different techniques for conservation. Slow growth conservation under in vitro conditions is based on slowing down the growth process of plant tissue without affecting its viability and regrowth under ambient conditions. The two most used techniques for short- to midterm preservation are encapsulation (up to 6 months) and vitrification (up to 12 months) for Bacopa. Sharma et al. (2011) used vitrification technique for cryopreservation of shoot tip explants. Their results revealed the significant increase in survival and regeneration frequency of cryopreserved explants when precultured with sucrose at 25 °C. Sharma et al. (2016) optimized the single-step protocol for regeneration, establishment, and medium-term conservation of Bacopa. Shoots were preserved for 12 months with relatively high survival rate and confirmed the genetic stability using molecular markers. In another study, Sharma et al. (2017a) cryopreserved the shoot tips of four different accessions with vitrification. They achieved 0–20% regeneration frequency from these cryopreserved explants. Comparison of these plants with non-vitrified plants using RAPD analysis or HPLC for bacosides revealed the genetic and biochemical stability.
5.7 Encapsulation (Synthetic Seed Production) of B. monnieri
Synthetic seed technology (SST) deals with the explant encapsulation regenerated in vitro/in vivo by applying alginate (Bukhari et al. 2014). It provides an alternative system for multiplication, storage, short-term preservation, and transportation of elite cloned traits (Gantait et al. 2015a). However, factors like explants, encapsulating agent, and matrix are significant for successful establishment of SST especially in medicinal plants. In recent years, SST has also been employed for Bacopa plants taken from in vitro regenerated plantlets. The first study was reported by Bansal and Pandey (2011), and they successfully regenerated the alginate-encapsulated shoot tip explants after storage. Similarly, shoot tip explants encapsulated with calcium alginate beads were stored at 24 ± 2 and 4 °C for 6 months and recorded 100% viability and regrowth of stored encapsulated shoot tips (Hegazi 2016), whereas shoot tips encapsulated with sodium alginate were also regenerated on medium fortified with cytokinins and auxins (Rency et al. 2016).
Besides shoot tip explant, nodal segment explant was also used for the encapsulation of Bacopa using different alginating agents. Sharma et al. (2012) assessed the encapsulated nodal segments of Bacopa and obtained 86.67% plantlet conversion after 6–8 weeks of storage. They also checked the efficacy of sodium alginate and CaCl2 on regeneration ability of encapsulated nodal segments. Nodal segments and shoot tips were encapsulated (3% sodium alginate, 80 mM NaCl) and stored at 4, 8, and 24 °C for 1 month. Thereafter, they were regenerated on medium having 0.44 μM BAP + 0.53 μM NAA, whereas storage for 6 months revealed the 100% regeneration from synthetic seeds derived from shoot tip at 4 °C (Muthiah et al. 2013). Gantait et al. (2015b) successfully encapsulated the nodal segment explants and obtained uniform beads when 2.5% sodium alginate + 75 mM NaCl was used and successfully obtained plantlets on 0.5 MS semisolid medium. These results clearly highlight the efficient use of SST for the conservation of Bacopa.
5.8 Phytoremediation Potential of B. monnieri
Bacopa is collected from nature as wild plant which is found in wet and marshy areas. These areas are generally polluted with industrial or pesticidal contaminants (Hussain et al. 2011) which pollute the water. The heavy metals contained in water are absorbed by Bacopa plants. Accumulation of Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn elements in Bacopa plants collected from nature was reported by Hussain et al. (2011). Similarly, Abdussalam et al. (2011) reported the bioaccumulation of Hg and Cd in Bacopa plants. Higher concentrations of Cd, Pb, Cu, and Zn (above threshold level) in Bacopa samples tested and reported inappropriate for human consumptions as herbal medicines by Srikanth Lavu et al. (2013). Mishra et al. (2016) collected the brahmi-based drugs from markets [Brahmi Ghrita (BG), Brahmi vati (BV), Saraswat Churna (SC)] and checked the heavy metal and pesticide residues in these samples. Their results highlighted the presence of heavy metals (Cd, Cr, Ni, Pb) or some pesticides like oxamyl, hexachlorocyclohexanes (α-HCH, β-HCH, and γ-HCH), dichlorodiphenyldichloroethylene, and dichlorodiphenyltrichloroethane. However, their concentration was below toxicity level. Keeping in view, collection of plant samples for herbal preparation is significant, and samples collected from the wild must be screened prior to making herbal medicines. Application of biotechnological techniques such as in vitro regeneration using plant tissue culture can be used to alleviate such types of risks.
5.9 Genetic Transformation Studies in B. monnieri
Advancement in genetic engineering techniques in recent years helps the researchers to incorporate elite genes of interest in plants in order to get traits with desired agronomic characteristics (Karami 2008). The development of reliable and repeatable in vitro regeneration protocol is prerequisite for successful genetic transformation. Besides that, other factors like genetic transformation technique used, type of genotype/cultivar and explant, PGR type and concentration, proper use of selection medium, Agrobacterium strains and cell density, etc. are also important to increase transformation efficiency. Furthermore, application of histochemical and molecular biology techniques (PCR, RT-PCR, hybridization) and bioassay is also vital for the confirmation of insertion, integration, and expression of genes in transgenes of different progenies. Genetic transformation of medicinal plants in recent years is gaining popularity in order to increase the economically important secondary metabolites.
Bacopa is also an important medicinal aquatic plant because it contains bacoside A which is a commercial brahmi-based drug used as brain tonic. Several techniques are in use for genetic transformation in plants, but interestingly, only Agrobacterium tumefaciens (Nisha et al. 2003; Ramesh et al. 2011b; Aggarwal et al. 2013; Yadav et al. 2014; Kumari et al. 2015; Paul et al. 2015; Croom et al. 2016; Sharma et al. 2017b)- and Agrobacterium rhizogenes (Majumdar et al. 2011; Bansal et al. 2014; Paul et al. 2015; Largia et al. 2016)-mediated genetic transformation have been reported for Bacopa to date. Agrobacterium-mediated genetic transformation is now most widely used genetic transformation technique for both monocots and dicots (Karami 2008). The advantages of Agrobacterium-mediated transformation are stable DNA integration into genome but with low copy numbers (Shou et al. 2004) and stable transgene expression in progeny (Hu et al. 2003).
5.9.1 A. tumefaciens-Mediated Genetic Transformation in B. monnieri
The first report on Agrobacterium-mediated genetic transformation of Bacopa was reported by Nisha et al. (2003). After that, the number of other studies by different researchers were published in current decade (Ramesh et al. 2011b; Aggarwal et al. 2013; Yadav et al. 2014; Kumari et al. 2015; Paul et al. 2015; Croom et al. 2016; Sharma et al. 2017b). In these studies, researchers used different Agrobacterium strains with plasmid-containing genes, selection medium, explant type, and different techniques for confirmation of transgenes. Leaf explant was the most preferable explant for genetic transformation studies (Aggarwal et al. 2013; Yadav et al. 2014; Kumari et al. 2015; Paul et al. 2015; Sharma et al. 2017b), whereas other explants like node (Ramesh et al. 2011b) and TCL from leaf or stem (Croom et al. 2016) were also used successfully for genetic transformation. For these explants, different types and concentrations of PGRs were used like 1.5 mg/l BA + 0.1 mg/l NAA + 0.1 mg/l GA3 (Nisha et al. 2003) and 1.0 mg/l BAP + 0.1 mg/l NAA + 0.1 mg/l GA3 (Ramesh et al. 2011b). On the other hand, 1–2 mg/l BA and 0–0.2 mg/l IAA (Kumari et al. 2015) and 2.0 mg/l BAP and 2.5 mg/l KIN (Sharma et al. 2017b) were added in the culture medium for putative transgenic shoot induction. Contrarily, Paul et al. (2015) cultured leaf explants on MSO for transgenic shoot induction of Bacopa.
Incorporation of specific gene of interest is the basic aim of any genetic transformation study that is driven by specific constitutive or non-constitutive promotors. The genetic transformation studies of Bacopa revealed the use of reporter or selectable marker genes driven by constitutive promotor. In these studies, uid was the most widely used gene (Nisha et al. 2003; Ramesh et al. 2011b; Aggarwal et al. 2013; Yadav et al. 2014; Kumari et al. 2015; Croom et al. 2016; Sharma et al. 2017b), whereas genes like neomycin phosphotransferase (nptII) (Nisha et al. 2003; Aggarwal et al. 2013; Yadav et al. 2014; Paul et al. 2015), hpt (Ramesh et al. 2011b; Kumari et al. 2015) and GFP (Croom et al. 2016), cryptogein gene (Paul et al. 2015), and tryptophan decarboxylase (tdc) or strictosidine synthase (str) (Sharma et al. 2017b) were also reported. It was also interested to note that CAMV 35S was the most used promotor in these studies. NOS promotor was also used for nptII gene in some studies (Nisha et al. 2003; Aggarwal et al. 2013). Based on the presence of reporter or selectable marker genes along with explant type, provision of proper selective agent and its concentration are also important for the enhancement of genetic transformation efficiency. The studies reflected the use of single antibiotic at the rate of 50 mg/l hygromycin (Sharma et al. 2017b) or two selective agents (antibiotics) like 15 mg/l kanamycin (kan) and 300 mg/l cefotaxime (cef) (Nisha et al. 2003; Yadav et al. 2014), 10 mg/l hygromycin (hyg) and 250 mg/l cef (Ramesh et al. 2011b), 50 μg/ml kan and 500 μg/ml carbenicillin (Aggarwal et al. 2013), 200 mg/l cef and 10 mg/l hyg (Kumari et al. 2015), or 500 mg/l cef and 100 mg/l kan (Paul et al. 2015).
After successful development of transgenes, the confirmation of gene integration and expression in different progenies is an important factor to obtain transgenic plants or lines. Techniques like GUS (β-glucuronidase) activity, polymerase chain reaction (PCR) analysis, and reverse transcription polymerase chain reaction (RT-PCR) were used by Nisha et al. (2003) and Kumari et al. (2015), whereas Ramesh et al. (2011b) confirmed transgenes by GUS and PCR analysis. Aggarwal et al. (2013) used different techniques like GUS, PCR, and RT-PCR analysis of nptII gene for confirmation. Yadav et al. (2014) applied techniques like histochemical GUS analysis, PCR analysis of nptII and GUS gene, and fluorometric GUS assay for the confirmation of transgenes, whereas RT-PCR analysis of GFP transcript and GUS analysis of putative transgenes were reported by Croom et al. (2016). Sharma et al. (2017b) used series of techniques like GUS, PCR, Southern blot hybridization, and RT-PCR. They also used metabolite profiling and quantification by HPLC.
5.9.2 A. rhizogenes-Mediated Genetic Transformation in B. monnieri
Hairy root (HR) culture is an important technique used for developing adventitious root induction using Agrobacterium rhizogenes for obtaining secondary metabolites. The inoculation of explants taken from medicinal plants with A. rhizogenes helps to produce bio-active compounds. There are few reports available which highlight the successful use of A. rhizogenes for hairy root production to increase bacoside production. Different A. rhizogenes strains were inoculated with explants like leaf (Majumdar et al. 2011; Bansal et al. 2014; Paul et al. 2015; Largia et al. 2016) or internode (Bansal et al. 2014) followed by culture on MS medium without any PGR (MSO). For selection, different antibiotics like 500 mg/L ampicillin (Majumdar et al. 2011; Largia et al. 2016) or 500 mg/l cef and 100 mg/l kan (Paul et al. 2015) were added in the selection medium. After genetic transformation, putative transgenes (HR) were confirmed by PCR and RT-PCR of rol AB or rol A, TR, and ags genes (Majumdar et al. 2011). Paul et al. (2015) used PCR analysis for the detection of the rol genes (rolA, rolB, rolC, rolD) and TR DNA (aux1, aux2, ags, mas1, mas2) and also used semi-qRT-PCR technique. They also checked the bacoside contents of transgenes by HPLC, whereas Largia et al. (2016) confirmed the transgenes with PCR for rol A gene, Southern blot hybridization, and elicitation of transformed plants with chitosan and also performed HPLC analysis to confirm the bacoside contents.
5.10 Application of Nanoparticles (NPs) in B. monnieri
Application of nanoparticle (NP) in vitro studies is gaining popularity among researchers in recent years. These NPs are in use for various purposes like antimicrobial activities (Klaine et al. 2008) or toxicological studies (Krishnaraj et al. 2012). These studies are majorly on microorganism or model organism. In recent years, NPs are also applied on plants for different objectives like germination or plant growth (Monica and Cremonini 2009). Furthermore, these NPs have clear impact on biological and pharmacological activities of some plants (Gandhare et al. 2016). In vitro plant regeneration techniques provide an alternative and efficient way of using NPs for different plant species of economic importance. Krishnaraj et al. (2012) exposed the Bacopa seeds to AgNPs and AgNO3 at different concentrations (10 ppb, 100 ppb, 10 ppm and 100 ppm) or cultured the Bacopa seedlings in hydroponic system containing 10 ppm AgNPs, 10 ppm AgNO3 and control without any NPs. Results revealed no effects of AgNPs, while AgNO3 hindered the seed germination with 45% at 10 ppm and zero at 100 ppm. Scanning electron microscopy (SEM) studies revealed the no severe toxic effects on plant morphological characteristics subjected to AgNPs. A couple of studies revealed the use of two different NPs in the culture medium at the rate of 16 × 1010, 16 × 105, and 16 × 103. In first study, silver nanoparticles (Kalsaitkar et al. 2014) and, in second study, copper nanoparticles (Gandhare et al. 2016) were applied for callus induction. In both studies, callus were induced at first and then transferred to medium with respective NPs resulting in almost similar results for both NPs. Minimum to medium callus growth was recorded on medium containing 16 × 05 and 16 × 103 AgNPs or CuNPs. Callus color was changed from green to light brown (16 × 105) or dark brown (16 × 105) but with no change in color when cultured on medium with 16 × 103 AgNPs or CuNPs, whereas complete callus inhibition was recorded at higher concentration of AgNPs or CuNPs. Besides using NPs on plant growth or callus induction, B. monnieri have been reported for biosynthesis of different NPs like gold (Babu et al. 2013; Bommavaram et al. 2013; Bindhu and Umadevi 2014) or platinum (Nellore et al. 2013).
5.11 Transcriptomics and Genomics Resources of B. monnieri
B. monnieri is a diploid plant species with chromosome number 2n = 64. Only up to recent its transcriptomic and genomics studies have been studied. Comparative transcriptomic studies revealed high-quality reads of 22.48 million and 22.0 million in shoot and root samples, respectively. Overall, 26,412 and 18,500 genes were annotated in root and shoot samples, respectively. Lastly, the 43 transcripts related to secondary metabolism were selected after mapping to 133 KEGG pathways (Jeena et al. 2017). The bacoside biosynthesis-related transcripts such as CYP450 monooxygenases, GTs, and β-amyrin synthase were in excess in root tissues; however, their expression was dominating in shoot tissues indicating the site of biosynthesis (Jeena et al. 2017). The identified genes would be useful for bacosides and other secondary metabolites by metabolic engineering either in homologous or heterologous expression system. Another study of de novo assembly of transcriptome of the plant revealed 10,556 simple sequence repeat (SSR) out of 8892 transcripts (Prabhudas and Natarajan 2017).
5.12 Conclusion and Future Prospects
B. monnieri is widely distributed in different geographic regions, but genetic variability studies are very limited. Similarly, use of physical and chemical mutagens resulted in increased Bacoside A contents. Due to its high bacoside A contents, the plant is widely collected from field conditions, and studies revealed the presence of certain heavy metals and pesticidal residues collected from the wild. Therefore, screening of these plants to heavy metals prior to use for making herbal medicines is important. Furthermore, the plant is also considered as threatened endangered plant by some researchers. This problem can be overcome by employing plant tissue culture techniques for its conservation and mass production to enhance bacoside A contents by using callus or cell suspension culture or directly from regenerated shoots. Other important advancements in recent years are the encapsulation technique to make synthetic seeds, the use of biotic elicitors for bacoside production, and the use of NPs for callus induction and also biosynthesis of NPs. Studies in Bacopa revealed the A. tumefaciens- and A. rhizogenes-mediated genetic transformation. However, in these studies, reporter or marker genes were used, and there is a need to incorporate genes related to bacoside A contents. During the last two decades, Bacopa is the most important aquatic plant due to its commercial value, but one major area in which the plant needs more research work is the functional genomics, genome sequencing, gene expression, and plant omics. Application of biological tools like QTL or MAS for identifying the potential genes to exploit the full potential of Bacopa plants.