Biotechnology for propagation and secondary metabolite production in Bacopa monnieri

Abstract

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as “Brahmi” is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity.

Key points
• Critical and up to date records on in vitro propagation of Bacopa monnieri
• In vitro propagation and elicitation of secondary metabolites from B. monnieri
• Molecular markers and transgenic studies in B. monnieri

Full text

Vol.:(0123456789)
1 3
Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-022-11820-6
MINI-REVIEW
Biotechnology forpropagation andsecondary metabolite production
inBacopa monnieri
RupaSanyal1· SaheliNandi1· SharmilaPandey1· UjaniChatterjee1· TulikaMishra2· SutapaDatta3·
DorairajArvindPrasanth4· UttpalAnand5· AbhijitBhagwanMane6· NishiKant7· NirajKumarJha8·
SaurabhKumarJha8· MahipalS.Shekhawat9· DevendraKumarPandey10· AbhijitDey11
Received: 22 December 2021 / Revised: 3 February 2022 / Accepted: 4 February 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Bacopa monnieri (L.) Wettst. or water hyssop commonly known as “Brahmi” is a small, creeping, succulent herb from the Plantagi-
naceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant
has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids,
herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect
of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses
many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its
multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which
compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated
medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the
plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well
as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production
of different types of secondary metabolites. Diverse invitro methods such as organogenesis, cell suspension, and callus cultures have
been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised
leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exog-
enous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation
and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the
natural and invitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated
transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The pre-
sent review aims at depicting the recent research outcomes of invitro studies performed on B. monnieri which include root and shoot
organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by invitro propagation, acclimatization
of the invitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity.
Key points
• Critical and up to date records on invitro propagation of Bacopa monnieri
• Invitro propagation and elicitation of secondary metabolites from B. monnieri
• Molecular markers and transgenic studies in B. monnieri
Keywords Micropropagation· Biotechnology· Cell suspension cultures· Elicitation· Saponins· Bacosides· In vitro
propagation· Pharmacological activity
Introduction
Bacopa monnieri (L.) Wettst. from the Plantaginaceae fam-
ily is an amphibian plant of the tropical regions that usually
grow on the banks of rivers and lakes (Binita etal. 2005). It
* Devendra Kumar Pandey
dkpandey1974@gmail.com
* Abhijit Dey
abhijit.dbs@presiuniv.ac.in
Extended author information available on the last page of the article

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is found in the tropical and subtropical regions worldwide
which include India, Sri Lanka, Nepal, Taiwan, China, Paki-
stan, and Vietnam. It is also reported from Florida, Hawaii,
southern states of the USA, and the Mediterranean Basin.
In India, it occurs in Assam, Delhi, Manipur, Goa, Andhra
Pradesh, Gujarat, Bihar, Kerala, Karnataka, Tamil Nadu,
Punjab, Andaman, Rajasthan, Orissa, and West Bengal. It
is also found throughout the Western and Southern Peninsula
(Lansdown etal. 2013). Figure1 presents the global distri-
bution of B. monnieri (source: https:// www. gbif. or g/ speci es/
31711 69). Popularly referred to as Jalanimba, Brahmi, or the
thinking man’s herb, B. monnieri is a major ancient Medhya
Rasayana drug in the Ayurveda (Abdul Manap etal. 2019). It
is employed as a valuable component in a number of Ayur-
vedic preparations, such as Brahmi rasayana, Brahmivati,
Brahmighrit, and Sarasvatarisht (Sharma etal. 2016). In
Ayurveda, it has been used as a brain tonic to improve mem-
ory, concentration, and learning capacity and also to cure
mental illness (Brimson etal. 2021; Lopresti etal. 2021).
The plant improved cognitive and behavioral parameters in
children as well as in adolescents (Kean etal. 2017). B. mon-
nieri has been suggested in the Indian Materia Medica and
Traditional Chinese Medicine for the remedy of a variety of
mental medical conditions such as insomnia, poor cogni-
tion, anxiety, psychosis, a deficit of concentration, epilepsy,
insanity, Alzheimer’s disease, and depression (Moskwa etal.
2020; Halder etal. 2021; Anand etal. 2022). The plant was
found to improve respiratory function during bronchocon-
striction and also is utilized as a cardiac tonic and digestive
aid in India and Pakistan (Saha etal. 2020). Additionally, the
plant possesses neuroprotective, anti-neuro-inflammatory,
pro-cognitive, neuropsychiatric, antinociceptive, analgesic,
anticancer, antioxidant, antipyretic, and anticonvulsant prop-
erties (Nemetchek etal. 2017; Ranjan etal. 2018; Abdul
Manap etal. 2019; Brimson etal. 2020; Castelli etal. 2020;
Jeyasri etal. 2020; Kiani etal. 2020; Micheli etal. 2020;
Cheema etal. 2021; Datta etal. 2021; Dutta etal. 2021;
Paul etal. 2021; Sharma etal. 2022). The plant extract also
offered protection against tacrolimus-mediated kidney toxic-
ity and opioid induced toxicity (Shahid etal. 2016; Oyouni
etal. 2019). The plant has also exhibited anti-anhedonia
(Micheli etal 2020), vasodilatory (Kamkaew etal. 2019),
hippocampus-strengthening (Promsuban etal. 2017), and
anti-cytotoxic-genotoxic (Dogan and Emsen 2018) proper-
ties. The plant also did not exhibit any acute and chronic tox-
icities in a rat model (Sireeratawong etal. 2016). The active
phytochemicals of B. monnieri include saponins, alkaloids,
and sterols. The alkaloid brahmine was first reported from
the plant. Eventually, several other alkaloids such as nicotine
and herpestine were also reported. The plant houses other
major phytochemicals viz. des-saponin glycosides-triterpe-
noid and saponins (bacosides A and B). It also possesses
other minor constituents such as betulic acid, bacosides A1
and A3, hersaponin, monnierin, herpestin and flavonoids,
glucuronyl-7-luteolin, luteolin-7-glucoside, and glucuronyl-
7-apigenin. The pharmacological attributes of B. monnieri
for enhancing memory and cognition have been credited to
the presence of various triterpenoid saponins like bacosides
A, B, C, and D also referred as “memory chemicals” (Dey
etal. 2019; Banerjee etal. 2021; Nandy etal. 2022).
Fig. 1 Worldwide distribution of B. monnieri (source: https:// www. gbif. org/ speci es/ 31711 69)

Applied Microbiology and Biotechnology
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The seeds of B. monnieri are considered as poor prop-
agules because of their short viability (two months), and
the seedlings often die at the two-leaved stage, making the
growth difficult from seeds. Vegetative propagation is slow
which is also hampered by particular habitat conditions
and inferior performance of the propagules. Further, ris-
ing demand for the plant materials due to over-exploitation
put pressure on the supply of this medicinal species caus-
ing adulteration of plant materials (Tiwari etal. 2001). An
effective and most suitable alternative is the development
of invitro techniques for the conservation and sustainable
yield of medico-botanicals and their phytochemicals. True-
to-type, infection-free and compatible plants for identifica-
tion, characterization, and quantification of phytochemicals
can be provided by the invitro propagation. In the past two
decades, invitro technology has been progressively applied
for rapid clonal propagation and conservation of threatened
and valuable plant germplasm. Therefore, the implementa-
tion of invitro techniques might be a promising alternative
for B. monnieri multiplication and conservation.
Taxonomic description
B. monnieri also known as Herpestis monniera or water
hyssop, locally called Jalanimba or Brahmi in India, is a
much used Ayurvedic herb belonging to the Plantaginaceae
family.
Taxonomic classication
Kingdom: Plantae
Division: Angiosperms
Class: Eudicots
Order: Lamiales
Family: Plantaginaceae
Genus: Bacopa
Species: monnieri
B. monnieri is a small, annual, succulent, creeping, multi-
branched herb with numerous prostrate branches and roots
arising from the nodes. The plant grows to a height of about
2–3 feet (60–90cm), and the branches are 10–35cm long.
Roots developing from the nodes are small, thin, wiry, much
branched, and creamish-yellow in color. The stem is soft,
green or purplish green, thin measuring about 1–2mm in
thickness, and slightly bitter in taste. The nodes and inter-
nodes are prominent and glabrous. Each flower is small,
solitary, and axillary in position having four to five petals.
The shades of the flower range from white, purple, pink to
pale violet. The pedicels are 6–30mm long and bracteoles
shorter than pedicels. The leaves of B. monnieri are succu-
lent or fleshy and relatively thick. They are oblanceolate in
shape, sessile, stalkless measuring about 0.6–2.5cm long
and 3–8mm broad, and oppositely arranged on the stem
(Rameshwari etal. 2013). The fruits are ovoid, two celled
and two valved capsules, acute apex, and tipped with style
base. It is slightly bitter in taste with no distinct odor. Fig-
ure2A represents the habit of B. monnieri (source: Wikime-
dia commons; Creative Commons Attribution 3.0 Unported
license; Attribution: Forest & Kim Starr), and Fig.2B rep-
resents the flower of B. monnieri (source: Wikimedia com-
mons; Creative Commons Attribution 2.0 Generic license).
Species, as well as family names, are verified from www.
thepl antli st. com.
Phytochemistry
Phytochemicals are classified into two categories viz., pri-
mary and secondary components. The primary components
in B. monnieri are proteins, amino acids, sugar, and chloro-
phyll. B. monnieri contains alkaloids, saponins, and sterols
(secondary components). The alkaloids, “brahmine” was
isolated at first, and other alkaloids such as nicotine and
herpestine were also isolated later. Subsequently, saponins
like D-mannitol, hersaponin, and sterols like β-sitosterol
and stigmasterol were obtained. Besides, monnierin,
wogonin, betulic acids, and oxindin were also reported
(Al-Snafi2013). The major compounds were recorded as
tritetrapenoid saponins such as bacosides and bacopasapo-
nins. Besides, two saponins were found as aglycones, viz.
jujubogenin and pseudojujubogenin (Dey etal. 2019) which
mainly differ from each other in the nature of sugar units,
because the position of the glycosidic chain and the olefinic
side chain which is different in the aglycone (Rajan etal.
2015). Few other major active components recorded were
bacopaside I, bacopaside II, bacopaside X, bacoside A3,
bacopaside N2, and bacopasaponin C. Some minor active
components were bacopaside III, bacopaside IV, bacopaside
V, bacopasaponin E, and bacopasaponin F (Murthy etal.
2006). The most common and major phytochemical, baco-
side A, was found to be levorotatory which is the mixture of
four triglycosidic saponins like bacoside A3, bacopasaponin
C, bacopaside II, and jujubogenin; this jujubogenin is an
isomer of bacopasaponin C (Dey etal. 2020). Besides, baco-
genins A1, A2, A3, and A4 were identified from bacoside A,
with the help of hydrolysis process. Another common phy-
tochemical, bacoside B, which is the mixture of four digly-
cosidic saponins like bacopaside N1, bacopaside N2, and
bacopasides IV and V (Dey etal 2020). The dammarane-
type triterpenoid saponins viz. bacopasaponins A, B, and C,
which were isolated with different names, such as 3-O-a-L-
arabinopyranosyl-20-O-a-Larabinopyranosyl-jujubogenin,
3-O-[a-L-arabinofuranosyl (I → 2) a-Larabinopyranosyl]
pseudojujubogenin and 3-O-[β-D-glucopyranosyl (1 → 3)

Applied Microbiology and Biotechnology
1 3
{a-L-arabinofuranosyl (1 → 2)}a-Larabinopyranosyl]. Baco-
pasaponin D is also isolated as 3-O-[α-Larabinofuranosyl
(I → 2) β-D-glucopiranosyl] pseudojujubogenin by the
spectroscopic and chemical transformation method. The
saponin, bacoside A1, and triterpenoid saponin A3 were
also identified (Dey etal. 2020). A minor saponin, baco-
side A1 which is known as 3-O-[a-L-arabinofuranoyl
(1 → 3)-β-L-arabinopyranosyl] jujubogenin and triterpenoid
saponin, bacoside A3 were also isolated by chemical and
spectral analyses and known as 3-b-[O-b-Dglucopyranosyl
(1–3)-O-[aL-arabinofuranosy (1 → 2)]-Ob-D-glucopyrano-
syl)oxy] jujubogenin. Along with this bacogenin A4 was
characterized as ebelin lactone pseudojujubogenin. Le etal.
(2015) recognized novel saponins like bacopasides I–XII.
Bacopasides N1-N2 were also recorded from the plant.
(3alpha)-3-Hydroxylup-20(29)-en-27-oic acid which is trit-
erpene bacosine was isolated and identified from the aerial
parts of B. monnieri (Kishore etal. 2017; Ghosh etal. 2011).
From the aerial four cucurbitacins, bacobitacins A-D (1–4)
and cucurbitacin E(5), a known cytotoxin along with other
three known phenylethanoid glycosides, monnieraside l,
lll, and plantioside B, were also isolated (Bhandari etal.,
2007). Besides, stalks and leaves of B. monnieri were found
to contain 88.4% moisture. Carbohydrates (5.9g), fat (0.6g),
protein (2.1g), crude fiber (1.05g), ash material (1.9g),
phosphorus (16.0mg), calcium (202mg), iron (7.8mg),
Fig. 2 (A) Habit of B. monnieri
(source: Wikimedia commons;
Creative Commons Attribution
3.0 Unported license; Attribu-
tion: Forest & Kim Starr).
(B) Flower of B. monnieri
(source: Wikimedia commons;
Creative Commons Attribu-
tion 2.0 Generic license). (C)
Microprpagation in B. monnieri
(unpublished photograph of
Dr. Dey). (D) 2D structures of
the major secondary metabo-
lites found in B. monnieri (a:
bacoside A, b: bacoside B, c:
bacopaside I, d: bacopaside II,
e: bacopaside N2, f: bacopaside
X, g: bacoside A3, h: baco-
pasaponin C) (structure source:
http:// www. chems pider. com/)
A B C
D
ab
cd
e
f
gh

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1 3
nicotinic acids (0.3mg), ascorbic acids (63mg), and some
amount of energy (38cal) were recorded along with this
moisture (Devendra etal. 2018). Figure2D represents the
2D structures of the major secondary metabolites found in
B. monnieri (a: bacoside A, b: bacoside B, c: bacopaside
I, d: bacopaside II, e: bacopaside N2, f: bacopaside X, g:
bacoside A3, h: bacopasaponin C) (structure source: http://
www. ChemS pider. com).
Biotechnological aspects ofB. monnieri
Source ofexplants
The factors on which the regeneration potential of an explant
depends are genotype, size, age, source of explant, physi-
ological and developmental stage, presence of meristematic
region, proper sterilization, etc. (Dey etal. 2020; Tandon
etal. 2021). Almost all healthy plant parts can be employed
as the explant-source like apical or nodal meristem, root,
shoot, leaf, bud, seed, etc. Leaf, internodes, and nodes are
mostly used as explants for plant regeneration, especially
for shoot formation. Direct somatic embryos were also
found using the leaf explants derived from the microshoots
(Khilwani etal. 2016). The use of microshoots, leaf, or inter-
nodal explants excised from the basal region of the plant B.
monnieri was found to be very effective for direct organo-
genesis of shoots (Sarkar and Jha 2017). Position, type,
and orientation of explants in the medium were recorded
to influence the direct and indirect organogenesis of shoots
(Saha etal. 2020). For successful synthetic seed production
in B. monnieri, shoot tips were used as explants, cultured in
the MS medium, and supplemented with 75mM calcium
chloride and 2.5% sodium alginate (Pramanik etal. 2021).
Nodal segments and leaves, as explants, were also reported
for bacoside production (Praveen etal. 2009). The uses of
axillary bud, younger nodes, shoot tips, and young leaves
excised from the young shoots have been used for the estab-
lishment of callus cultures (Bhusari etal. 2013; Showkat
etal. 2010).
Culture conditions
For invitro propagation, plants are cultured in a suitable
culture vessel containing various nutrient media under
aseptic and controlled parameters. Culture conditions like
temperature, light intensity, pH, CO2 concentration, etc.
are needed to be optimized for the best growth and mor-
phogenic response of the plant materials invitro. The light
intensity used was 50µmol/m2/s PPFD (photosynthetic pho-
ton flux density) by Dey etal. 2019 which has been reported
as 80–100µmol/m2/s PPFD by Banerjee and Srhivastava
(2007). In another study, a fluorescent lamp of 40W was
used as a light source at night (Naik etal. 2017). The tem-
perature was reported to be 23 ± 2°C (Samanta etal. 2019).
The relative humidity provided for the incubation was found
to be 55–60% (Ranjan etal. 2018). Some sources have
shown to adjust the pH at 5.8 of the media with 1N HCl
or 1N NaOH solutions, and then, the culture media were
autoclaved for 15–20min at 121°C temperature and 15lb
pressure (Binita etal. 2005). In some experiments, 4.5 pH
has also been provided for the accumulation of biomass and
production of bacoside (Naik etal. 2010). If culture vessels
were used, after autoclaving, they were transferred to the
media room under controlled aseptic conditions for further
experiments. After this, all the subcultures were generally
conducted in intervals of four weeks (Srivastava etal. 2017).
Surface sterilization
Different plant parts/organs collected from the field or
greenhouse are surface sterilized before setting up the
invitro cultures. Surface disinfection of the explants is a
significant step prior to the establishment of invitro cul-
ture since microorganisms develop quicker in tissue culture
medium than the explants do (Kim etal. 2017). Contamina-
tion with microorganisms such as viruses, bacteria, yeast,
fungi, etc. is counted to be one of the most important hin-
drances during invitro culture of plants. These microbes
compete unfavorably with plant tissues for nutrients and
elevate the culture mortality rate. They can also result in
tissue necrosis, invariable growth, decreased shoot prolifera-
tion, and reduced rooting. The very first step in surface steri-
lization of the cut explant of B. monnieri was performed by
a thorough washing of the explant under running tap water
for 20min to 2h to remove the superficial mud and dust
particles adhering to the surface (Banerjee and Modi 2010;
Sharma etal. 2016). Labolene (5% v/v for 15min) (Banerjee
and Modi 2010), teepol (5% for 30min) (Mohapatra and
Rath 2005), Cetavlon (1—2% for 10min) (Sharma etal.
2010), etc. have also been used as mild liquid detergents
for initial microorganism elimination from the surface of
the explants. Kalita etal. (2018) in their experiment used
70% ethanol for 30s to disinfect nodal segments of B. mon-
nieri followed by a solution with 25% sodium hypochlorite:
0.01% Tween-20 for about 25min. Afterwards, the explants
were washed thrice with sterile distilled water. Few research-
ers have also recommended the use of a systemic fungicide
called Bavistin®. Ceasar etal. (2010) soaked excised shoot
buds in 0.1% (w/v) Bavistin® containing carbendazim for
20min and then washed thrice with sterile distilled water. As
reported by most of the researchers, the final step of washing
was performed with 0.1% (w/v) mercuric chloride (HgCl2)
solution for 3 to 10min followed by washing with sterile
distilled water to remove traces of HgCl2 (Haque etal. 2017;
Parale etal. 2010; Sharma etal. 2010). The final wash was

Applied Microbiology and Biotechnology
1 3
performed carefully several times (4–5) with autoclaved
distilled water prior to inoculation of the explants in the
culture medium. In place of HgCl2, 40% diluted H2O2 (v/v)
for 10min was used as a surface sterilant by Karataş and
Aasim (2014). Then, they were washed with double distilled
water for 5min by continuous stirring. Some researchers
also used 70% alcohol for surface decontamination (Ceasar
etal. 2010; Kalita etal. 2018).
Media andplant growth regulators (PGRs)
Plant tissue culture medium is most important for plant
growth invitro and basal medium, in the same way, is forti-
fied with some necessary nutrients like carbohydrates, vita-
mins, minerals, and many additives for the proper growth of
the plant. Murashige and Skoog (MS) medium, B5 (Green-
way etal. 2012), Linsamaier and Skoog (LS), and Schenk &
Hildebrandt (SH) are the most used media for invitro culture
of the plants. For B. monnieri, MS medium of full strength
has been proven as the most suitable culture medium but
several works like shoot multiplication and bacoside produc-
tion have been observed in Gamborg’s B5 medium (Koul
and Mallubhotla 2020). Plant growth regulators (PGRs) are
some chemical compounds that regulate plant’s growth and
development in many ways by promoting or inhibiting them.
In plant tissue cultures, many PGRs are supplemented to the
medium for seed germination, promoting elongation or dif-
ferentiation of cells etc. MS medium fortified with different
concentrations of PGRs like auxins, cytokinins, etc. in dif-
ferent combinations has shown to produce multiple shoots
and buds. Using nodes of B. monnieri as explants, Sanputa-
wong etal. (2021) conducted a study where MS medium was
supplemented with 0–1mg/L napthalene acetic acid (NAA)
and 0–2mg/L 6-benzyladenine (BA). These combinations
of PGRs have produced good callus growth and shoot
amplification. BA and thidiazuron (TDZ) (0.5mg/L and
0.25mg/L, respectively) have also been shown to enhance
the bacoside production in the cell suspension cultures
(Kharde etal. 2018). MS basal medium of half strength,
supplemented with 0.5mg/L benzyl amino purine (BAP)
and 1mg/L indole-3-butyric acid (IBA), was found to pro-
vide better results in terms of induction of longer shoots and
roots, respectively, in greater numbers (Ceasar etal. 2010).
For micropropagation of B. monnieri, MS medium supple-
mented with gibberellin A3 was found as one of the most
suited PGRs with different combinations of NAA, 2–4-D,
kinetin (kin), BAP, etc. for callus induction, multiple shoot
formation, and root growth (Murthy etal. 2019). In addition,
100ml/L of banana extract and 100ml/L of coconut water
were added as the sources of PGRs in the MS basal medium
which showed maximum numbers of rooting from the regen-
erated shoots (Soundararajan and Karrunakaran 2011). In
another study, Gracilaria salicornia extracts were used as a
source of PGR for B. monnieri in vitro propagation as 25%
of this extract had shown 82.2% root induction and 60% of
this extract had shown 85.9% shoot induction after induction
to the medium (Rency etal. 2017). Figure2C presents the
micropropagation in B. monnieri (unpublished photograph
of Dr. Dey).
Carbon source
Carbohydrates are one of the main sources of carbon for
plants as it controls the developmental patterns, absorption
of energy, etc. (Dey etal. 2020). Sucrose is usually found
to be the best carbon source in the culture media among all
the carbohydrates which can be substituted by galactose,
lactose, mannose, melibiose, and cellobiose in the culture
(Srivastava etal. 2017). However, sucrose is the main car-
bohydrate to help in the translocation of phloem sap (Fink
etal. 2018). Even 2% sucrose supplementation in the MS
medium increased the shoot-biomass and enhanced baco-
side A content in the regenerated shoots (Naik etal. 2010).
Besides, 20g/L sucrose with 7g/L agar, supplemented to
MS medium, was found to be best suited for induction of
roots (Ranjan etal. 2018). Supplementation of 250mM
sucrose enhanced the somatic embryogenesis up to 70%
probably via supplying the necessary energy needed to
form somatic embryos (Saha etal. 2020). Glucose as well
increased the biomass as well as bacoside A accumulation in
hairy roots (Bansal etal. 2015). Sucrose, sorbitol, maltose,
fructose, and glucose in different combinations in differ-
ent media were also supplemented for shoot regeneration,
bacoside, and biomass production. In one study, it was sug-
gested to check every sugar combination for their suability in
obtaining the best results in the propagation of plants invitro
(Naik etal. 2017).
Nitrogen source
Nitrogen has a plethora of roles in plant growth and develop-
ment in invitro conditions. Nitrogen has been reported to
influence cell differentiation and to enhance the totipotency
of the cell. A nitrogen source is not always required to be
added exogenously to the culture medium but for some spe-
cial advantage, it may be applied (Kovalchuk etal. 2018).
Double strength of NH4NO3 added to the MS medium pro-
duced maximum and efficient yield of bacoside A. Even this
supplemented media produced biomass and shoots in greater
numbers (Naik etal. 2011). Potassium nitrate (KNO3) was
also found to be effective as a nitrogen source for bacoside
A production and for obtaining increased biomass (Bansal
etal. 2015). Nitrogen is the main component of amino acids,
and sometimes amino acids like L-asparagine, L-glutamate,
adenine, etc. are added to the culture media as organic nitro-
gen sources. In an experiment on biomass production in B.

Applied Microbiology and Biotechnology
1 3
monnieri, they added 0.1g/L l-tryptophan; 0.5g/L and
0.25g/L serine were added in the MS medium for better
response (Muszyńska etal. 2016).
Additives
The beneficial nutritive chemicals, those act as chelators
or pH controlling buffer systems to improve the produc-
tion rate, micro-salts availability to the plant part used as
explant by providing sufficient nutrients, are known as addi-
tives (Dey etal. 2020). Most of the time, agar is used as
an additive to solidify the medium for better rooting and
shoot regeneration (Showkat etal. 2010). In many studies,
casein hydrolysate was used to supplement the liquid MS
medium for suspension culture to produce maximum baco-
side (Kharde etal. 2018). Methyl jasmonate was also found
to be effective in bacoside A production, as an additive in
invitro raised shoots of B. monnieri. In addition, salicylic
acid (SA) and pyruvic acid (PA) were applied as additives
for bacoside production (Koul and Mallbhotla 2020; Parale
etal. 2010). Zinc oxide nano-particles were also added in
the suspension cultures for better plant growth (Bhardwaj
etal. 2018). Magnesium sulfate, zinc hydroaspartate, and
anthranilic acid have produced increased biomass follow-
ing being supplemented in the media for B. monnieri shoot
cultures (Lojewski etal. 2014).
In vitro propagation
In vitro propagation or micropropagation is a technique used
for the vegetative growth and multiplication of plants from
tissues or seeds in aseptic and controlled conditions on arti-
ficial growth media. Micropropagation is usually executed in
two ways: direct and indirect. The indirect process involves
callus development from explant followed by shoot and
root formation, while the direct process involves shooting
and rooting on the explant. With its rapidity and limitless
potential, plant tissue culture offers novel opportunities to
deal with various shortcomings in the areas of medicinal
plant cultivation, conservation, and exploitation. Some of
the exciting applications are exploiting genetic engineering
and somaclonal variation for crop improvement, fast micro-
propagation to produce quality plants, invitro conserva-
tion and germplasm exchange and production of secondary
metabolites, invitro selection for resistance to biotic and
abiotic stress, etc. A great deal of medicinal plants are not
the producer of seeds, or the seeds are too minute to be
germinated in soil. Thus, mass-scale propagation of disease-
free plants is a common problem. In this situation, the plant
tissue culture technique presents a remarkable potential for
fast and true-to-type mass scale propagation of the plants in
disease-free conditions.
The ability of rapid multiplication into true-to-type
plants and efficiency in B. monnieri transplantation can
be employed in conservation strategies and mass propaga-
tion of the plant for commercial use. B. monnieri has an
upright market demand owing to the medicinal attributes it
possesses. In India, the estimated consumption of this plant
is approximately 1000 tons/year (Kharde etal. 2017). The
annual demand of B. monnieri during the years 2004 to 2005
was 6621.8 tons with an annual growth rate of 7% accord-
ing to the National Medicinal Plants Board (NMPB). This
demand is growing rapidly with the growing popularity of
drugs consist B. monnieri (Sharma etal. 2010). Recently,
NMPB and Technology Information Forecasting and Assess-
ment Council (TIFAC) have identified this plant among the
seven prime medico-botanicals suggested for prompt rec-
ognition to be provided and are indexed in the list of highly
endangered Indian medicinal plants (Tripathi etal. 2012).
Micropropagated plants grow rapidly and mature early com-
pared to the progenies propagated via seeds. Plants gener-
ated from tissue culture can multiply through the increased
production of axillary and/or adventitious shoots either by
direct or indirect organogenesis followed by generation of
roots and also by somatic embryogenesis.
Callus induction
A callus is an undifferentiated and unorganized mass of cells
produced from plant tissue (explant) cultured on an appro-
priate medium supplemented with PGRs. Callus cultures
are a source of tissues for plant regeneration, chromosomal
variation (somaclonal variation), secondary metabolite pro-
duction, and cell suspension culture. Apical shoot tip, leaf,
embryo, stem, nucellus, germinating grains, stamen, root,
basal plate meristem, etc. can be used as explants for the
inception of callus cultures. The cells of explants divide
continuously to give rise to a soft, irregular shaped callus.
In most cases, the explant produces callus within 3 to 8days
of incubation (Jat etal. 2016). Callus formation and its sub-
sequent regeneration are the main steps in in vitro propaga-
tionof plants. From the shoot tip explant of B. monnieri,
callus was induced in MS media fortified individually with
various auxins (NAA, IAA, and 2,4-D). Callus development
was recorded the highest in media with 2,4-D (2mg/L) and
moderate in media treated with IAA and NAA (Talukdar
2014). Soft, yellowish green to brownish callus tissues were
obtained from the leaf explants on MS media supplemented
with 0.5mg/L 2,4-D (Showkat etal. 2010). MS media com-
posed of various concentrations and combinations of PGRs
were used for callus formation of which combination of 6
BA (2ppm) and IAA (1ppm) were the most useful for the
formation of soft, yellowish-green calli in 15days (Ahmed
etal. 2014). In callus initiation from leaf explant, the best
result was obtained when 0.5mg/L NAA and 0.5mg/LBAP

Applied Microbiology and Biotechnology
1 3
were added to the MS basal media resulting in 75% callus
formation. In addition, for the nodal segment as explants,
0.5mg/L IAA and 4.0mg/L BAP added to MS media sup-
ported 85% callus formation (Ranjan etal. 2018). Hegazi
etal. (2017) recorded that 9µM 2,4-D and 2.3µM kin added
to MS medium produced the best callus initiation in B. mon-
nieri. In addition, it generated an increased percentage of
fresh weight and yellowish, white friable callus. Ali etal.
(2021) obtained maximum callus generation from the leaf
explants with NAA (2.5mg − 1) showing 94.22% generation
rate accompanied via 2,4-D (2.5mg − 1) showing 82.43%
generation rate; in the case of nodal explants, the highest cal-
lus formation was detected with 2,4-D (2.5mg − 1) showing
71.14% generation rate followed by NAA (2.5mg − 1) show-
ing 62.15%. In intermodal explants, most of the calli forma-
tion was noted in the presence of 2,4-D (2.5mg − 1) show-
ing 65.21% generation rate followed by NAA (2.5mg − 1)
showing 52.14% generation rate. Samanta etal. (2019) in
their experiment observed that when BAP (5mg/L) was sup-
plemented in MS media following 60days of culture, callus
formation and potent growth were achieved. Hence, they
concluded that BAP (5mg/L) needed more time to initiate
callus formation. During invitro propagation, leaf segments
were chosen mostly over the internodal segments as explant
sources for callus formation because of the presence of soft
tissue, no woody structure, and broad surface area (Dey etal.
2020). High concentrations of phenolic compounds are often
correlated with the superior antioxidant capacity of the plant
(Aras etal. 2018; Silinsin and Bursal 2018). Many pheno-
lics such as quinic acid, p-coumaric acid, and malic acid
were found to be present in the plant extracts (Bursal etal.
2019). Owing to the presence of high phenolics in B. mon-
nieri, the callus culture often became brown and to prevent
this browning; different PGRs (2,4-D, NAA, IBA, and BAP)
at different concentrations were used by Meenashree etal.
(2017), out of which NAA produced healthy callus without
any browning. Lowering the amount of nitrate source and
incorporation of ascorbic acid (100mg/L) in media also
helped in attenuating the browning of callus. Dogan (2020)
reported that the percentage of callus induction and inten-
sities of callus growth from nodal explants reduced with
an increment in NaCl concentrations (salt stress). In addi-
tion, browning, yellowing, or even deaths of the callus were
observed due to salt toxicity.
Shoot organogenesis
Shoot multiplication from a single explant is one of the
prime highlights for micropropagation, germplasm conser-
vation, and biomass production. Many researchers recom-
mended the potency of nodal explant to produce multiple
shoots (Dey etal. 2020). Shoot organogenesis can directly
take place on the isolated explant such as leaf and stem via
direct organogenesis or can be found only following callus
generation by indirect organogenesis. The concentration of
cytokinins and auxins in the culture medium is a significant
factor affecting the degree of multiplication. The cytokinin
signaling pathway exhibits a potential target for controlling
de novo shoot organogenesis and invitro plant regeneration.
The two fundamental types of cytokinins utilized in plant
tissue culture are BAP and kin. Binita etal. (2005) reported
solid media with more potential for bud proliferation from
the leaf whereas the liquid medium was suggested to be
more effective for bud proliferation from axillary nodes and
internodes. Saha etal. (2020) reported enhanced direct shoot
organogenesis from the leaf and internodal explants without
the supply of exogenous PGRs, and the induction rate was
increased when exogenous cytokinins and some additives
were used. Direct shoot organogenesis was obtained in a cul-
ture medium containing a combination of BAP (17.80μM)
and IAA (2.28μM) producing maximum shoot initiation
(85.2) with larger shoot production (Mahender etal. 2012).
MS medium fortified with 0.25mg/L BA + 0.25mg/L NAA
showed the maximum number of shoots per explant com-
pared to the medium with other combinations of BA + NAA.
Shoot regeneration is inhibited with a higher concentration
of NAA in combination with all concentrations of BA. The
shoots developed from the leaf explants were comparatively
longer than those from the other explants (Karatas etal.
2013). When the swollen nodes were sub-cultured either on
MS medium or MS media supplemented with 1.0mg L-1
GA3, the highest shoot proliferation (114.2 shoots/ node)
with an average shoot length of 6.4cm was noted. Chauhan
and Shirkot (2020) in their experiment observed that MS
medium supplemented with 1.0mg/L BAP and 0.5mg/L
kin showed the best invitro shoot multiplication. The shoots
produced from MS medium containing 0.5mg/L BAP were
reported long and healthy and on increasing the BAP con-
centration to 2.0mg/L, the rate of shoot multiplication
declined. Various concentrations of different carbon sources
(glucose, sucrose, and mannitol) were evaluated to find out
the best response in regeneration events, among which 5%
sucrose in MS media was recorded to be the most useful for
shoot generation (22.6 shoots/explant), and in case of leaf
explant, 3% sucrose was found more effective (20.6 shoots/
explant) (Srivastava etal. 2017). Similarly, it was noted
that the medium with 2% sucrose and 4.5 pH demonstrated
increased shoot biomass up to 150.50 shoots/explant, fresh
wt. 6.31g, and dry wt. 250mg (Naik etal. 2010). In a study
on the effect of different concentrations of NaCl on B. mon-
nieri, it was observed that in shoot, the Na+ content was
enhanced with a rise in NaCl level in the medium, and both
K+ and Ca2+ levels decreased in the shoot, and as a result, a
remarkable reduction was recorded in shoot number/culture,
fresh and dry weights, shoot length, and water content in the
tissues (Ahire etal. 2013).

Applied Microbiology and Biotechnology
1 3
Root organogenesis
In plant tissue culture, root organogenesis is primarily influ-
enced by different types and concentrations of sucrose, min-
erals, PGRs, etc. in the media and other factors in a culture
like pH, temperature, etc. Rooting is especially essential and
is considered as the ultimate achievement for micropropaga-
tion (Chen etal. 2014). Best root induction (almost 80%),
reported by Ranjan etal. (2018), was achieved on the MS
media enriched with 7g/L agar and 20g/L sucrose. Com-
binations of 20g/L sucrose, 30g/L fructose, and 40g/L
glucose (all as carbohydrate sources), supplemented in liquid
and solid MS medium, using a stem of B. monnieri with
three nodes as an explant have also been recorded to produce
a maximum number (23) of roots using a horizontal culture
method (Ahmed etal. 2014). In addition to this, selective
shoot rooting medium (SSRM, MS medium fortified with
4.9μM IBA + 25mg/L hygromycin) induced rooting from
the shoots, resistant to hygromycin (Mahender etal. 2012).
In another study, 0.5mg/L phloroglucinol on MS medium
of half strength with 1mg/L IBA gave the best outcome for
root organogenesis, in terms of length (8.7cm) and numbers
(16.5) (Ceasar etal. 2010). Invitro regeneration of B. mon-
nieri has also shown the best combination of 1mg/L of BAP
and 3mg/L of IAA for root induction (Gurnani etal. 2012).
Some experiments have shown better rooting of regenerated
shoots from the 1/2 MS media with 0.5mg/L NAA, 1%
sucrose, and 2% jiggery (Bhusari etal. 2013). An evaluation
on the impact of some polyamines (PAs) inin vitro propaga-
tion of B. monnieri was conducted where the MS media with
1mg/L IBA and 1mM spermine has produced the high-
est number of roots from the regenerated shoots (Dey etal.
2019). Croom etal. (2016) found transverse thin cell layers
(tTcl) of leaf and stem as explants producing 100% roots in
the MS medium fortified with 5µM IBA, using a liquid lab
rocker (LLR) box. An investigation on the impact of abiotic
stress using NaCl and polyethylene glycol (PEG) in invitro
raised B. monnieri has shown a good response in root for-
mation on the solid/liquid MS medium supplemented with
0.8mg/L NaCl whereas 26g/L PEG and enhanced concen-
trations of NaCl have shown to decrease the development of
roots (Hussien etal. 2017). In another study, 60ml extracts
of Cyanobacteria, Aulosira fertillissima, with the MS liquid
medium of 40ml have produced superior rooting response
(Banerjee and Modi 2010).
Somatic embryogenesis
Somatic embryogenesis is a tool of regeneration or
developmental pathway which forms the non-zygotic
cell devoid of vascular connection with the original tis-
sue. These non-zygotic embryos are formed from a sin-
gle or grouped somatic cell. The rate of germination in
somatic embryogenesisis very high (80%-85%). This
process goes through different stages which are globular
stage (small globose or spherical structure), heart shape
stage (three-lobed structure with pale yellow color), and
torpedo stage (elongated heart shape with pale yellow
color) (Samanta etal. 2019). Many researchers reported
various types of culture media for growing somatic
embryos of B. monnieri. Other researchers recorded that
the somatic embryos were developed in high frequency
in B5 medium fortified with 2,4-D (0.25 and 0.5mg/L)
alone or combined with BAP (0.5mg/L). The concentra-
tion of 2,4-D (1.0mg/L) in the B5 medium was found to
be important for the growth of somatic embryos. When
2,4-D was present at low concentration in B5 medium,
the embryos grew in high frequency, and absence of
2,4-D in medium, no embryos were formed. However,
somatic embryos failed to germinate in the specific media
where only 2,4-D was present. BAP (0.5mg/L) was found
to be essential for the maturation of somatic embryos.
PGR-fortified MS medium was reported ineffective for
embryo growth (Jain etal. 2010). Parale and Sangle
(2020) reported that with the decreasing concentration
of 2,4-D or kin in the media, the number of embryos also
decreased. BA and 2,4-D influenced somatic embryo-
genesis from leaf explants of B. monnieri. The develop-
mental frequency of somatic embryos depended on the
concentration of PGRs. MS media supplemented with BA
(12.5µM) and 2,4-D (1.0µM) produced somatic embryos
in a maximum frequency of 47.1%. When the 2,4-D con-
centration was increased in the medium, the frequency of
explant producing somatic embryo decreased. Somatic
embryogenesis was also influenced by the concentration
of sucrose. Sucrose (250mM) containing media helped to
produce somatic embryos in the highest frequency (77%)
as observed by Khilwani etal. (2016). Parale and Sangle
(2020) cultured calli on the full strength of MS medium
with 1.5%, 3%, or 4% sucrose and half strength of MS
media with 1.5%, 3%, or 4% sucrose without PGRs. The
full strength of MS media with a low concentration of
sucrose produced the embryo, but when the concentra-
tion of sucrose was high (4%), the somatic embryos were
not found. Ali etal. (2021) stated that a mixture of 2,4-D
(2.0mg/L) and kin (1.5mg/L) was useful for somatic
embryogenesis in maximum frequency (84%). Embryoid
differentiation took place in MS media with 0.5mg/L 2,4-
D. MS media with or without BA were also used for fur-
ther development of these embryoids (Saha etal. 2020).
Hardening andacclimatization
In vitro raised plantlets when pass through the process
of acclimatization (hardening) show a higher rate of sur-
vival and vigorous growth when transferred to the soil

Applied Microbiology and Biotechnology
1 3
(Castañeda-Méndez etal. 2017). The in vitro generated
plants are directly hardened in the greenhouse stage. Ini-
tially, water was needed to be sprayed five times a day at
four-hour intervals to maintain high humid conditions
(Ranjan etal. 2018). After root development from plantlets
(3cm in length) in culture vessels, the roots were washed
properly, and a sticky semi-solid agar medium was removed
carefully from roots under running tap water (Mehta 2017).
Most of the reports indicated that the plantlets were treated
with 0.1% Bavistin® for 10min during hardening to protect
the fungal attack. In addition, it was recommended to trans-
fer these treated plantlets directly to the plastic pot which
contained different mixtures of sterilized soil with various
combinations such as soil mixed with either vascular arbus-
cular mycorrhizae (VAM), or farmyard manure, or fly ash,
or vermicompost, or agropeat (Sharma etal. 2018). Vari-
ous kinds of soil mixtures with different ratios were found
to be used such as soil and organic manure (2:1); soil and
vermicompost (3:1) (Haque and Ghosh 2013); soil and ver-
miculite (50:50) (Chaudhry etal. 2019); soil and soilrite
(1:1) (Showkat etal. 2010); sand, soil, and farmyard manure
(1:1:1) (Binita etal. 2005); and sand, soil, coco peat, and
farmyard manure (1:1:1:1) (Ranjan etal. 2018). In a few
studies, the plantlets were kept for 2weeks (or 10–15days)
in the culture room following transformation from invitro to
plastic pots (Sharma etal. 2010). In a few reports, plantlets
were shifted to 25°C–30°C temperature and 80%-90% rela-
tive humidity and under a photoperiod of 16h for acclimati-
zation (Sharma etal. 2010). According to an experiment by
Chauhan and Shirkot (2020), the survival rate was found to
be 80% when the plantlets were kept in coco peat following
six weeks of hardening. In sand and soil, the survival rate
was recorded to be 42% or 68% after six weeks of plant-
lets transfer. Moreover, 48% survival rate of the plantlets
was noted in the mixture of coco peat and sand after six
weeks of transfer to this mixture. The hardened plantlets
were also transferred to a mixture of soil, sand, and farm
yard manure (FYM) in 1:1:1 ratio, in a small pot covered
with a glass jar for one week. After removing this glass jar,
the pot was transferred to the glass house, and the growth
rate of plantlets was observed for 2, 4, 6, and 8weeks.
The height of the hardened plantlets after the second week
was recorded to be 2.96cm with an average growth of the
leaves that was recorded as 19.50 per plant. Following the
4th week, the plantlets reached a height of 4.22cm, and
the number of leaves were recorded as 25.50 per plant; the
height of the plantlets increased to 5.18cm as well as the
number of leaves was increased to 31.00 per plant after the
6th week; after 8th week, the survived plantlets reached to
6.08cm of height with 39.00 leaves per plant (Chauhan
and Shirkot 2020). Finally, the plantlets were successfully
transferred to the net house (under shade) following accli-
matization for further growth (Srivastava etal. 2017). After
acclimatization, 93% of B. monnieri plantlets survived in the
natural environment (Hegazi 2016). Sharma etal. (2017a)
reported 90% survival rate whereas Mehta (2017) recorded
a 100% survival rate for the B. monnieri plantlets.
In vitro production ofsecondary metabolites
B. monnieri houses its prime secondary metabolites as
the dammarane type triterpenoid saponins which showed
anti-oxidant, anti-amnestic, and nootropic effects (Dey
etal. 2020). Among these, the most profound saponin is
bacoside, and the other saponins recorded are bacopaside
I-XII, including bacopasaponin F, bacopasaponin D, baco-
pasaponin C, bacopaside V, bacopaside II, etc. (Majum-
dar etal. 2011). Besides saponins, alkaloids like brahmin,
herpestine, and nicotine (Lala. 2020) and flavonoids like
luteolin, luteolin-7-glucoside, glucoronyl-7-lutoelin, gluc-
oronyl-7-epigenin, etc. have also been recorded from the
plant (Jat etal., 2016). Glucose (5.67%), KNO3 (0.313%),
and KH2PO4 (0.29%) with 0.66% inoculum density in cells
cultured in MS medium demonstrated an enhanced rate of
bacoside A production (Bansal etal. 2017). Another invitro
study assessed the bacoside A concentration in the shoots
of B. monnieri cultured in MS medium supplemented with
2mg/L kin. An addition of cytokinin resulted in a higher
accumulation of bacoside A in the regenerated shoots in
the liquid medium (Praveen etal. 2009). In another study,
regenerated adventitious shoots produced the maximum
amount of bacoside A in the medium fortified with 0.20mM
copper (Cu) and 2% sucrose at 4.5 pH (Naik etal. 2010,
2015). In addition, organic supplements like SA and PA
added to the shoot cultures have shown an increase in the
bacoside A accumulation in the shoots (Saha etal. 2020).
Methyl jasmonate (MJ) has also been noted to promote the
production of bacoside A in the shoot cultures of B. mon-
nieri (Sharma etal. 2013). MJ (50µM) combined with SA
(50µM) enhanced the accumulation of bacoside A besides
producing bacoside A3, bacopaside II, and bacopasaponon C
(Largia etal. 2015). Colchicine (0.1%) treatment only for 2h
also enhanced the bacoside concentration in the regenerated
plants (Kharde etal. 2017). In an Agrobacterium rhizogenes-
transformed B. monnieri plant, the regenerated hairy shoots
produced a higher amount of bacoside A (Largia etal. 2016).
Combinations of some rhizospheric microorganisms like
Glomus intraradices, Trichoderma harzianum, and Bacil-
lus megaterium, using a method called Fourier Transform
Near-Infrared (FTNIR), have shown to enhance the produc-
tion of bacoside A in B. monnieri (Gupta etal. 2015). The
bacoside A content was recorded higher during February
to May under stress conditions which was implicated in
the invitro production of the compound under stress. Bal-
loon type bubble bioreactor and glass bottle bioreactor have
also been employed, and the former was found to be more

Applied Microbiology and Biotechnology
1 3
preferred for bacoside production (Sharma etal. 2019).
Dey etal. (2020) recorded High Performance Thin Layer
Chromatography (HPTLC) as a potent technique to assess
bacoside A content and also compared the compound among
invitro and nature grown plantlets.
Use ofmolecular markers
High survival rate and genetic stability were found using
molecular markers (random amplified polymorphic DNA
(RAPD) (OPC-15) and inter simple sequence repeat (ISSR)
(UBC-808)) on B. monnieri shoots conserved for 12months
(Sharma etal. 2016). RAPD studies also revealed no nota-
ble reproducible variation among the control sets and the
invitro-cryopreserved B. monnieri plants (Sharma etal.
2017b). In another study, 20 ISSR primers generated 130
clear and reproducible amplicons, with 125 bands show-
ing monomorphism. In addition, 25 RAPD markers showed
115 bands with 94% of these being monomorphic following
6months of storage (Muthiah etal. 2013). Clonal fidelity
of the regenerated B. monnieri was investigated using ISSR
and RAPD markers that also revealed high monomorphism
(90%) between the mother plant and invitro regenerated
plantlets (Dey etal. 2019).
In order to determine the diversity in the wild popula-
tions, about 35% variations were found using RAPD and
ISSR. However, ISSR markers showed higher variation
(44.9%) than the RAPD markers (23%) (Bansal etal. 2014a).
RAPD and ISSR analysis also revealed genetic diversity in
15 B. monnieri accessions from central India which was
reported necessary to be used for their conservation as well
as for breeding (Tripathi etal. 2012).
Table 1 In vitro shoot and root formation in B. monnieri via direct organogenesis
Explants Culture conditions Basal medium Additives for root
induction
Additives for shoot
formation
Reference
Stem with three nodes -Liquid and solid MS 20g/L sucrose, 30g/L
fructose, 40g/L
glucose
20g/L glucose Ahmed etal. (2016)
Nodal and leaf -MS 20g/L sucrose and
7g/L agar
1.0mg/L BAP and
0.5mg/L NAA
Ranjan etal. (2017)
Leaf -MS - 3% sucrose, 4mg/L
BAP, 0.1mg/L
NAA, and 4g/L
Gelrite®
Thi etal. (2012)
Leaf and internode -½ MS 1.0mg/L IBA and
0.5mg/L phloroglu-
cinol
- Ceasar etal. (2010)
Axillary node -Liquid MS 60ml Aulosira
extracts, 1mg/L kin
(15 roots of 4cm)
60ml Aulosira
extracts, 1mg/L kin
Banerjee and Modi
(2011)
Shoot tips -MS 1.5mg/L IAA,
0.5mg/L IBA
1mg/L meta-topolin
(mT)
Pramanik etal. (2021)
Leaf -MS 17.80μM BAP and
2.28μM IAA
25mg/L hygromycin
and 4.9μM IBA
Mahender etal. (2012)
Axillary nodes and
internodes
-Liquid MS - 0.2µM IAA, 1.1µM
BA
Binita etal. (2005)
Microshoots/leaf or
internode
50–60% humidity;
25 ± 1°C; 16h PP;
PFD 40μmol/S/m2
MS 3% sucrose, 0.75%
agar
- Sarkar and Jha (2017)
Axillary nodes, young
leaves, shoot tips
-50% MS 1% sucrose, 2% jag-
gery, 0.5mg/L NAA
20g/L jaggery, 5g/L
agar, 10g/L sucrose,
3mg/L BAP, and
0.5mg/L IBA
Bhusari etal. (2013)
Leaf -MS - 500μg/mL of carbeni-
cillin or cefotaxime
Aggarwal etal. (2013)
Young shoots -MS 3% sucrose, 0.8% agar,
1mg/L IBA, 1mM
spermine
2mg/L BAP, 0.5mg/L
TDZ, 0.5mg/L kin,
0.5 and 1mM sper-
mine, spermidine,
putrescine
Dey etal. (2019)

Applied Microbiology and Biotechnology
1 3
Application oftransgenics
Earlier, Agrobacterium tumefaciens strain EHA105 harbor-
ing the binary vector pBE2113 with the genes for GUS and
neomycin phosphotransferase was used to transform B. mon-
nieri plants. It showed 60% transformation frequency and
took two months for regeneration of the transgenics from the
leaf explants (Nisha etal. 2003). In another study, A. tumefa-
ciens LBA4404 harboring pCAMBIA1301 containing ß-glu-
curonidase (uidA) and hygromycin phosphotransferase (hpt)
genes demonstrated fast and efficacious shoot organogen-
esis invitro and transgenic plant production in B. monnieri
(Mahender etal. 2012). B. monnieri when transformed using
three strains of A. tumefaciens such as LBA4404, EHA105,
and GV3101 containing expression vector pCAMBIA2301
with β-glucuronidase (GUS), no remarkable variation in the
transformation efficiency among the three strains was noted
(Yadav etal. 2014). Effective shoot regeneration (87.5%)
and genetic transformation (82.5%) of B. monnieri were
achieved by A. tumefaciens-mediated transformation con-
firmed via GUS assay and PCR mediated detection of hptII
gene (Kumari etal. 2015). In another report, factors influ-
encing genetic-transformation and shoot-organogenesis in B.
monnieri were also standardized (Aggarwal etal. 2013). In
another study, a quick regenerating A. tumefaciens-mediated
transformation method for B. monnieri (L.) was achieved as
a heterologous expression model of Catharanthus roseus
derived terpenoid indole alkaloid producing genes (tryp-
tophan decarboxylase and strictosidine synthase) normally
absent in B. monnieri (Sharma etal. 2017c). B. monnieri
transformed with Agrobacterium rhizogenes strains LBA
9402 and A4 stimulated bacopa saponins synthesis in trans-
formed calli and as well as in plants which was attributed
to the endogenous elicitation mediated by A. rhizogenes
Ri T-DNA (Majumdar etal 2011). Further, Ri crypt-trans-
formed (encoding proteinaceous elicitor cryptogein) B. mon-
nieri showed a notably higher accumulation of bacoside A3
(Majumdar etal. 2012). The crypt-transformed B. monnieri
kept long-term, exhibited notably higher bacoside content
invitro (1.66- to 2.05-fold higher than the non-transformed
ones) (Paul etal. 2015). B. monnieri, genetically transformed
with using different A. rhizogenes strains (viz. A4, R1000,
SA79, MTCC 532, and MTCC 2364), displayed higher pro-
duction of hairy root biomass and higher accumulation of
bacoside A (except A4 strain) compared to the non-trans-
formed lines (Bansal etal. 2014b). A. rhizogenes (A4 and
MTCC 532 strains)-derived hairy roots exhibited the highest
regrowth frequency. Moreover, a high biomass producing
Table 2 In vitro callus induction and somatic embryogenesis in B. monnieri
Explants Basal medium Additives Response Reference
Shoot tip MS 2mg/L 2,4-D Callus development in higher
percentage
Talukdar (2014)
Leaf from microshoots MS 12.5µM BA, 1µM 2,4-D Formation of somatic embryos Khilwani etal. (2016)
Leaf MS 2ppm BA, 1ppm IAA Successful induction of soft
yellowish-green callus
Ahmed etal. (2014)
Leaf petiole MS 0.5mg/L kin and 0.25mg/L
2,4-D; 0.25mg/L 2,4-D; and
0.1mg/L BAP
Best callus induction Mehta etal. (2012)
Leaf and stem MS 2µM BA, 0.2% Gelrite®,
30g/L sucrose, 0.7% agar, 6
and 8µM NAA
Callus induction Shrivasatava and Rajani (1999)
Apical or axillary bud,
internode, and leaves
MS 1mg/L 2,4-D; 0.2mg/L
kinetin
Embryo maturation and pro-
duction of plantlet from it
Samanta etal. (2019)
Leaf MS 16 × 103 of silver nanoparticles Medium callus growth Priya etal. (2014)
Internode and leaf MS 3% sucrose; 0.65% agar; 0.25,
0.50, and 1mg/L of BA and
NAA
Callus induction Karatas etal. (2013)
Leaf B5 medium 0.8% agar; 0.25 and 0.5mg/L
2,4-D; and 0.5mg/L BAP
Somatic embryogenesis at
high frequency
Jain etal. (2010)
Leaf ½ or full strength MS 20µM 2,4-D and 20µM kin,
3% sucrose, 0.8% agar
Somatic embryo induction Parale and Sangle (2020)
Young nodes MS 1mg/L IBA and 1mg/L IAA,
3% sucrose, 0.65% agar
Callus formation Showkat etal. (2010)
Nodes and leaves MS 2mg/L 2,4-D; 1.5mg/L kin Maximum (84%) induction
and formation of somatic
embryogenic calli
Ali etal. (2021)

Applied Microbiology and Biotechnology
1 3
line, upon elicitation, produced 5.83% of bacoside A five and
three-times more than the untransformed and transformed
non-elicited control sets, respectively (Largia etal. 2016). In
addition, the overexpressing Sorghum bicolor–derived vacu-
olar proton pyrophosphatase gene (SbVPPase) attenuated
salt stress in transgenic B. monnieri transgenics produced by
A. tumefaciens-mediated transformation (Ahire etal. 2018).
In another report, insertion of rol genes modulated the mor-
phogenic potential in transgenic B. monnieri derived from A.
tumefaciens-mediated transformation (Sarkar and Jha 2021).
Conclusions
B. monnieri is considered as a potent medicinal plant con-
taining an array of phytochemicals viz. alkaloids, flavonoids,
saponins, and glycosides. The plant possesses several phar-
macological activities attributed to its bioactive compounds
especially bacoside A. B. monnieri is commercially impor-
tant due to its presence in various herbal formulations used
against neurological disorders. This plant has always been a
subject of interest to a myriad of researchers. A substantial
number of studies have been conducted on this plant focus-
ing on its tissue culture and biotechnology to propagate the
plant invitro and also for sustainable and stable produc-
tion of its phyto-constituents. The present review provides
a comprehensive account of its invitro propagation stud-
ies such as callus induction, root and shoot organogenesis,
somatic embryogenesis, and secondary metabolite produc-
tion (Tables1, 2, and 3). Besides, insights in the molecular
marker-based studies revealed the clonal fidelity among the
natural and invitro generated plantlets. Agrobacterium-
mediated transformation system was mostly used to opti-
mize bacoside production, biomass yield, and heterologous
expression of secondary metabolite producing genes. The
regenerated plants can be used as a continual provision for
Table 3 In vitro production and enhancement of bacosides using elicitors in B. monnieri
Strategies Explants Media Elicitors Reference
Cell suspension culture Shoot MS + 2.5μM BA Glucose (5.67%), KNO3
(0.313%), and KH2PO4
(0.29%)
Bansal etal. (2017)
In vitro regeneration Leaf Semi-solid and liquid MS 2mg/L kin Praveen etal. (2009)
Plant cell or organ culture Leaf Liquid MS + 5μM BA 100µM pyruvic acid Parale etal. (2010)
In vitro propagation Stem (internodes) MS + 0.1mg/L TDZ 150mg/L chitosan, 2mg/
mL yeast extracts
Kamonwannasit etal. (2008)
In vitro propagation Leaf MS + 2mg/L kin 0.20mM copper, 2%
sucrose
Naik etal. (2015)
Hairy root cultures Leaf Liquid MS + 1mg/L BAP,
0.1mg/L NAA, 3%
sucrose, and 0.8% agar
10mg/L chitosan Largia etal. (2016)
In vitro shoot cultures Shoot MS 45mg/L CuSO4Roy etal. (2017); Sharma
etal. (2015)
Suspension cultures Leaf B5 + 1mg/L 2,4-D 1mg/L salicylic acid Koul and Mallubhotla (2020)
In vitro liquid shoot
cultures
Shoot with 5–6 nodes Liquid MS + 4.44μM BAP,
0.54μM NAA
50µM methyl jasmonate,
50µM salicylic acid
Largia etal. (2015)
In vitro propagation Leaf MS + 3% sucrose, 1.1µM
BA, 0.30µM IBA
1% colchicine Kharde etal. (2017)
Cell suspension culture Leaf Liquid MS + 0.4mg/L
2,4-D
0.5mg/L BA and kin,
0.25mg/L TDZ
Kharde etal. (2018)
In vitro propagation Stem with leaves Liquid MS + myo- inositol,
nicotinic acid, 4mL/L
vitamin B1, 1mg/L BAP,
0.2mg/L NAA
0.5mg/L anthranilic acid Lojewski etal. (2014)
In vitro propagation Leaf and internode MS basal salt + 3% sucrose NaCl, CuSO4Roy etal. (2017)
In vitro propagation Leaf MS + 9µM 2,4-D; 2.3µM
kin
100mg/L chitosan and
10mM mevanolic acid
Hegazi etal. (2017)
In vitro propagation Stem segments and leaf Liquid MS + 5µM BA 750mg/L Saccharomyces
cerevisiae or Mucor sp.
derived biotic elicitors
Prakash and Dayaram (2009)
Cell suspension culture Leaf, node, internode MS + 1mg/L different
auxins
0.75 and 1ppm zinc oxide
nanoparticles
Bharadwaj etal. (2018)
In vitro shoot regeneration Adventitious shoots MS + 2mg/L kin 2 × NH4NO3Naik etal. (2011)

Applied Microbiology and Biotechnology
1 3
uniform raw materials for commercial production of second-
ary metabolites which in turn will minimize the pressure on
the natural populations and hence can be indirectly useful
for conservation.
Acknowledgements The corresponding author thankfully acknowl-
edges “Faculty Research and Professional Development Fund”
(FRPDF) for financial assistance from Presidency University.
Author contribution RS prepared the primary draft. SN, SP, and UC
revised it and designed the figures. RS, SN, and NK prepared the tables
and contributed to the discussion part. TM, UA, NKJ, MSS, and DKP
edited the primary draft. SD, SKJ, and ABM revised the manuscript,
and AD designed, conceptualized, edited, and supervised the entire
work. All authors read and approved the manuscript.
Declarations
Ethics approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Conflict of interest The authors declare no competing interests.
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Authors and Aliations
RupaSanyal1· SaheliNandi1· SharmilaPandey1· UjaniChatterjee1· TulikaMishra2· SutapaDatta3·
DorairajArvindPrasanth4· UttpalAnand5· AbhijitBhagwanMane6· NishiKant7· NirajKumarJha8·
SaurabhKumarJha8· MahipalS.Shekhawat9· DevendraKumarPandey10· AbhijitDey11
1 Department ofBotany, Bhairab Ganguly College (affiliated
toWest Bengal State University), Feeder Road, Belghoria,
Kolkata700056, WestBengal, India
2 Department ofBotany, Deen Dayal Upadhyay Gorakhpur
University, Gorakhpur, UttarPradesh273009, India
3 Department ofZoology, Bethune College, Kolkata (affiliated
toUniversity ofCalcutta), Kolkata, WestBengal700006,
India
4 Department ofMicrobiology, School ofBiosciences, Periyar
University, Salem636011, Tamilnadu, India
5 Department ofLife Sciences, Ben-Gurion University
oftheNegev, 84105Beer-Sheva, Israel
6 Department ofZoology, Dr. Patangrao Kadam
Mahavidyalaya, Sangli (affiliated toShivaji University
ofKolhapur), Sangli, Maharashtra416308, India
7 Department ofBiotechnology, School ofHealth andAllied
Science, ARKA Jain University, Jamshedpur832108,
Jharkhand, India
8 Department ofBiotechnology, School ofEngineering
& Technology (SET), Sharda University, 201310,
GreaterNoida, UttarPradesh, India
9 Plant Biotechnology Unit, Kanchi Mamunivar Government
Institute forPostgraduate Studies andResearch,
Puducherry605008, India
10 Department ofBiotechnology, Lovely Professional
University, Phagwara144402, Punjab, India
11 Department ofLife Sciences, Presidency University, 86/1
College Street, Kolkata700073, WestBengal, India