Plant Cell Rep (2016) 35:2207–2225

DOI 10.1007/s00299-016-2041-8

REVIEW

Micropropagation and genetic transformation of Tylophora indica

(Burm. f.) Merr.: a review
Jaime A. Teixeira da Silva1 • Sumita Jha2

Received: 19 June 2016 / Accepted: 15 August 2016 / Published online: 23 August 2016
Ó Springer-Verlag Berlin Heidelberg 2016

Abstract been shown to accumulate tylophorine suggesting that

Key message This review provides an in-depth and in vitro biology and transgenic methods are viable ways of
comprehensive overview of the in vitro culture of Ty- clonally producing valuable germplasm and mass produc-
lophora species, which have medicinal properties. ing compounds of commercial value. Further studies that
Abstract Tylophora indica (Burm. f.) Merr. is a climbing investigate the factors affecting the biosynthesis of Ty-
perennial vine with medicinal properties. The tissue culture lophora alkaloids and other secondary metabolites need to
and genetic transformation of T. indica, which has been be conducted using non-transformed as well as transformed
extensively studied, is reviewed. Micropropagation using cell and organ cultures.
nodal explants has been reported in 25 % of all publica-
tions. Leaf explants from field-grown plants has been the Keywords Asclepiadaceae
In vitro
Micropropagation

explant of choice of independent research groups, which Morphogenesis
Somatic embryogenesis
Tissue culture

reported direct and callus-mediated organogenesis as well Transformed cultures
as callus-mediated somatic embryogenesis. Protoplast-
mediated regeneration and callus-mediated shoot organo-
genesis has also been reported from stem explants, and to a Introduction
lesser degree from root explants of micropropagated plants
in vitro. Recent studies that used HPLC confirmed the There are currently 90 Tylophora species with accepted
potential of micropropagated plants to synthesize the major names, and a multitude of synonyms and species whose
T. indica alkaloid tylophorine prior to and after transfer to nomenclature is being revised (The Plant List 2016). Ty-
field conditions. The genetic integrity of callus-regenerated lophora indica (Burm. f.) Merr. (syn Tylophora indica var.
plants was confirmed by RAPD in a few reports. Tissue glabra (Decne.) H. Huber) (The Plant List 2016), of the
culture is an essential base for genetic transformation Asclepiadaceae family and commonly known as Indian
studies. Hairy roots and transgenic T. indica plants have ipecacuanha in English, or as Antamul in India (see list of
vernacular names in Table 1), is a climbing perennial vine.
Communicated by N. Stewart. Kirtikar and Basu (1991) and Schmelzer and Gurib-Fakim
(2013) offer a botanical description of this medicinal plant,
& Jaime A. Teixeira da Silva and indicate that T. indica is a perennial climber that can
[email protected] reach up to 1.5–3 m in length, forms short stocky rhizomes
& Sumita Jha (3–4 mm thick) and fibrous roots. The simple opposite
[email protected]; [email protected] leaves, which can be 2–10 cm long, have margins that can
1 be entire, ovate or orbicular. Many green-yellow flowers
P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Miki-cho,
Kagawa-ken 761-0799, Japan (outside) with a purple inside form on an axillary umbel-
2 like cyme, which is the inflorescence. The fruit is 5–10 cm
Department of Botany, Centre of Advanced Study, University
of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, long with many 2–2.5 cm long seeds. The plant is usually
India propagated by seed collected from plants in the wild and

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2208 Plant Cell Rep (2016) 35:2207–2225

Table 1 Vernacular names for Tylophora indica genetic transformation (Bajaj and Ishimaru 1999; Roy-
chowdhury et al. 2013b), synthetic seed production (Sharma
Language/dialect Common/vernacular name
et al. 2013), or bioreactor production of secondary metabo-
Bengali Antamul lites (Baque et al. 2012). This review highlights the advances
Gujarati Damnivel that have been made thus far in the tissue culture of Ty-
Hindi Antamul, jangli pikvam lophora species, although the analysis in Table 2 reveals that
Kannada Adumutadhagida, nipaladaberu the majority (63/65 studies, or 97 %) of studies have
Marathi Khodiki, pittakhadi, rasna focused on T. indica with only a single study on T. ovata
Oriya Mendi, mulini (Lindl.) Hook. ex Steud. (syn. T. ovata var. balansae
Sanskrit Arkaparni, shwasaghni (Costantin) Tsiang, T. ovata var. brownii (Hayata) Tsiang
Tamil Asthma kodi, nanjaruppan, nancaruppan, & P.T. Li, and T. ovata var. ovata), by Jeyachandran and
naippalai, mirkkurinja, tellavidavela, Bastin (2014) and one study on T. subramanii Henry
kondachani (Murukan et al. 2015).
Telugu Kakapala, verripala
Modifed from Shahzad et al. (2016), and also based on Dhandapani
and Balu (2002) Morphogenesis and propagation of Tylophora
indica in vitro
vegetative propagation is poorly explored (Dhandapani and
Balu 2002). However, more recently, Mehandru et al. The explant is the central unit of plant tissue culture, and its
(2014) used stem cuttings of T. indica from field-grown choice depends on seasonal availability or quality of
plants for clonal propagation using an aeroponic system mother plant material, on the experimental objective, and
and found that 100 % of stem cuttings rooted with 2 g/l on its responsiveness in vitro. The most popular explant
indole-3-butyric acid (IBA), performing better than cut- used for the tissue culture of Tylophora species has been
tings rooted directly in soil (77 % of cuttings while only leaves (Fig. 1a) from field-grown and micropropagated
6.65 % of control cuttings rooted in the absence of an plants.
auxin). T. indica has been extensively studied since 1970 when
Tylophora indica has numerous medicinal properties, Rao et al. (1970) reported the induction of callus from stem
including antioxidant, antiallergic, anti-angiogenic, explants which differentiated into roots, shoots and bipolar
antibacterial, anticancer, antifeedant, anti-inflammatory, somatic embryos (SEs). Rao et al. (1970) also demon-
antimicrobial, antitumor, antiasthmatic, cardioprotective, strated SE development in a cell suspension culture while
diuretic, hepatoprotective, and displaying immunomodu- the histological basis of morphogenesis was described by
latory activity, all of which have been recently reviewed by Rao and Narayanswami (1972). The morphogenetic
Shahzad et al. (2016) and will thus not be included in this potential of dedifferentiated cells of T. indica in vitro was
review. Overexploitation and the lack of organized culti- demonstrated using various types of explants, namely
vation strategies underscore the importance of developing leaves (50 % of reports, Table 2; Fig. 1a), internodes or
biotechnological approaches for the rapid and reproducible stems, petioles and roots. Micropropagation based on the
in vitro propagation of this medicinal plant species and the use of shoot tips or nodal explants involving apical or
stable and improved in vitro and in planta production of its axillary bud proliferation has been the method of choice in
valuable pharmaceuticals that endow it with these widely 25 % of published reports (Table 2).
reported medicinal properties.
Two Tylophora species are on the Red List of the
International Union of Conservation of Nature, Tylophora Use of explants from ex situ plants
cameroonica N.E.Br., listed as near threatened, and Ty-
lophora urceolata Meve, listed as vulnerable International Leaves from ex situ plants have been the primary source
Union for Conservation of Nature and Natural Resources for callus induction and indirect shoot regeneration from
(IUCN 2015). Other than these two species, currently the dedifferentiated callus, with only few reports on somatic
use of biotechnology is not a tool for preservation of rare or embryogenesis, most of which have not been substantiated
endangered material, but rather a tool for mass propagation by suitable histological analyses.
of clonal material, or as a stable base for creating a sterile The earliest report of direct shoot organogenesis from
in vitro milieu to engage in other applied biotechnologies mature leaves of field-grown plants was by Bera and Roy
for improvement of medicinal plants such as tissue culture (1993), inducing as many as 304 shoot buds/explant in
(Yoshimatsu 2008), somatic hybridization (Murch and Saxena optimized medium but no histology was performed. Man-
2001), germplasm cryopreservation (Dixit et al. 2004), jula et al. (2000) induced embryogenic callus from mature

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 Table 2 Micropropagation and tissue culture of Tylophora indica, except for Tylophora ovata and Tylophora subramanii (chronological listing)
Explant conditions and disinfection Culture medium, PGRs and Culture conditions*** Remarks, experimental outcome and References
procedures additives*,** maximum productivity, acclimatization
and variation

Young vines ? 10 % NaOCl ? SW ? 5 mm MS ? 1 mg/l 2,4-D ? 1 mg/l 2-BTOA Continuous light. CWFT. LI NR. Shoots and somatic embryos formed Rao et al. (1970)
explants (CIM). MS ? 2 mg/l 2,4-D ? 10 % 25 ± 2 °C. 50–60 % RH
CM ? 200 mg/l CH ? 100 mg/l myo-
inositol (CPM). 2 % sucrose. pH, agar
conc. NR
Young vines ? 10 % NaOCl ? SW ? 5 mm White’s ? 1 mg/l 2,4-D ? 1 mg/l Continuous light. CWFT. LI NR. Callus developed within 4 w. Histology of Rao and
internodes = explants 2-BTOA (CIM). pH 5.8. 2 % sucrose. 25 ± 2 °C. 50–60 % RH shoots and somatic embryos described Nayaranaswami
Plant Cell Rep (2016) 35:2207–2225

Agar conc. NR (1972)

Stem from stem cuttings and roots from MS ? 2 mg/l 2,4-D ? 10 % Continuous light. Light source In the 1973 study, the addition of Benjamin and
germinated seedlings CM ? 200 mg/l CH ? 5 mg/l AdS and LI NR. 25 ± 2 °C. phenylalanine—an alkaloid precursor—to Mulchandani
(CIM). Other conditions NR 50–60 % RH CIM did not change the level of steroids (1973, 1976)
and alkaloids. In the 1976 study, the
irradiation of callus with gamma rays
decreased the content of b-amyrin, b-
sitosterol, stigmasterol and campesterol
Leaf, stem and root segments of field-grown MS ? 2 mg/l 2,4-D ? 0.2 mg/l Kin (CIM PP and LI NR. CWFT. Callus was induced within 2 w, shoot buds Benjamin et al. 1979
plants (age and disinfection procedures NR) for 3 explants). MS ? 2 mg/l 25 ± 2 °C after another 3–4 w, and complete
NAA ? 0.2 mg/l Kin (SIM for stem and plantlets 4–6 w thereafter. Alkaloids
roots). MS ? 2 mg/l IAA (RIM). pH, could not be synthesized in in vitro callus
carbohydrate source, agar conc. NR cultures, even in the presence of alkaloid
precursors. Tissue cultured plants showed
a similar alkaloid profile as normal field-
grown plants
Stems (ca. 5 mm long) of field-grown MS ? 2 mg/l 2,4-D ? 0.2 mg/l Kin Continuous light. Light source Stem segments produced callus in 4 w. Mhatre et al. (1984)
plants ? 0.1 % HgCl2 10 min ? 49 SDW (CIM). MS ? low auxin ? high NR. 1000 lx. 25 ± 2 °C Protoplasts isolated from callus when
cytokinin (shoot buds). MS ? 10 % incubated for 7 h in light 2 %
CM ? 1 mg/l 2,4-D (SEIM). pH 5.5–5.8 cellulose ? 1 % macerozyme ? 1 %
hemicellulose ? 0.6 M mannitol.
Embyoids and shoot buds developed from
callus. Regeneration of whole plants was
possible
Nodes (1–1.5 cm) of ex situ MS ? 5 mg/l BA ? 0.5 mg/l 16-h PP. CWFL. 2800–3000 lx. Axillary shoots did not develop in the Sharma and Chandel
plants ? TeepolTM 15 min while NAA ? 100 mg/l AA (SIM, SMM; 4–6 25 ± 2 °C. 60 % RH presence of BA, Kin, 2iP, NAA, IAA, (1992)
stirring ? RTW 2 h ? 0.1 % HgCl2 w subcultures). MS ? 1 mg/l IAA 2,4-D or GA3 alone. Callus formed when
7 min ? SDW (RIM). pH 5.8. 3 % sucrose. 0.8 % agar 2,4-D (1-5 mg/l) and NAA (1–2 mg/l)
were used in combination. In SIM,
axillary buds sprouted within 10 d from
60 % of explants. Subculture on SIM
formed 5.38 shoots/explant and 92 % of
new shoots formed roots on RIM within 4
w. 90-100 % survival in SoilriteTM
(Keltech Energies Ltd., Bangalore, India)
2209

123
 Table 2 continued
2210

Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and

123
variation

Leaves (2.5-3 cm long) of ex situ plants (age MS ? 5 mg/l BA ? 0.5 mg/l AdS (SIM, 16-h PP. CWFL. 35.7 lE cm-2 Direct shoot bud induction at apical and Bera and Roy (1993)
NR) ? 70 % EtOH 1 min ? 0.1 % SMM). MS ? 3 mg/l IAA (RIM). pH 5.8. s-1. 25 ± 1 °C. 55–60 % RH basal part of whole leaf or cut surfaces.
HgCl2 ? 5 % Teepol 10–12 min (continuous 3 % sucrose. 0.6 % agar Prolific shoot bud induction
shaking) ? SW ? whole or cut (304/explant). Shoot multiplication every
leaves = explants 4-6 w. Plantlets transferred to soil in pots
with 60-72 % survival
Mature leaves (6-m-old greenhouse MS ? 1-2 mg/l BA or 3–5 mg/l Kin or Continuous light. CWFT. 30 embryoids/100 mg callus formed, with Manjula et al. (2000)
plants) ? RTW ? 1 % 2 mg/l Kin ? 0.1 mg/l IAA (CIM; 30-d 40 lmol m-2 s-1. 25 ± 2 °C 27 % conversion into plantlets, which
LaboleneTM ? DW ? 0.1 % HgCl2 subcultures). MS ? 2 mg/l could be acclimatized in sand: soil (1:1)
5 min ? SDW ? 1 cm2 explants Kin ? 0.1 mg/l IAA (SIM/SEM/SEIM). with 90 % survival
pH 5.8. 3 % sucrose. 0.8 % agar
Mature leaves (greenhouse plant) ? 1 % MS ? 2 mg/l 2,4-D ? 0.01 mg/l Kin Continuous dark (CIM) or 10-h 25 SEs/initial callus cluster (0.5 g) in 85 % Jayanthi and Mandal
TeepolTM ? RTW ? 0.1 % HgCl2 (CIM; 14-d subcultures). MS ? 2 mg/l PP ? CWFT ? 50 lmol m-2 of callus. No variation claimed using (2001)
5 min ? 59 SDDW ? 0.5 cm2 explants 2iP (SEIM). PGR-free MS (SEGM). pH s-1 (SEIM). 25 ± 1 °C RAPD
5.8. 3 % sucrose. 0.2 % phytagel
First and second expanded leaves from terminal MS ? 10 lM 2,4,5-T or 10 lM 2,4-D 16-h PP. Light source NR. 96 % (2,4-D) or 100 % (2,4,5-T) of leaf Faisal and Anis (2003)
1 cm ? TW 30 min ? 5 % TeepolTM (CIM). MS ? 5 lM Kin (SIM). ‘ 50 lmol m-2 s-1. 25 ± 2 °C explants formed callus. 64.8 shoots/callus
5 min ? 0.1 % HgCl2 MS ? 0.5 lM IBA (RIM). pH 5.8. 3 % clump formed on SIM, with 85 % of
3 min ? 49 SDW ? 1 cm2 explants sucrose. 0.8 % agar callus explants being responsive. In RIM,
90 % of shoots rooted, forming 4.3 roots/
shoot. Plantlets acclimatized in sterilized
vermiculite, watered with ‘ MS every
3 days for 2 w, then transferred to pots
with garden soil (survival % NR)
Young shoots (May–June growth of 15-y-old MS (initial shoot induction). 16-h PP. CWFL. 48 lmol m-2 Initial shoots formed plantlets that were Chaudhuri et al.
trees) ? 5 % TeepolTM 10 min ? RTW MS ? 26.8 lM BA (SIM). s-1. 24 ± 1 °C. 50–60 % RH then used to source two types of root (2004, 2005, 2006),
30 min ? DW ? 0.1 % HgCl2 MS ? 28.54 lM IAA (RIM). pH 5.8. explants: WRS and GRS. 78 % of GRS Roychowdhury et al.
18-20 min ? 4–59 SDW ? shoot tips 3 % sucrose. 0.8 % agar formed shoots in 24 weeks but 75 % (2013a, 2015a, b)****
(10 mm) = explants. Roots of could form shoots in response to 5.36 lM
micropropagated plants used as explants BA within 6 w. 92 % of WRS formed
shoots in 12 w. 24.61 lM 2iP in SIM,
rather than BA, could also induce shoots
in 61 % of GRS in 6 w or in 89 % of
WRS in 12 w. 100 % of GRS or WRS
(pooled) shoots could form roots on RIM,
with 10 roots/shoot after 4 w. 88 % (from
SEs) or 96 % (from shoots) survival in
soil ? sand (1:1)
Young shoots ? RTW 30 min ? 5 % MS ? 10 lM 2,4,5-T (CIM). MS ? 5 lM 16-h PP. Light source NR. 100 % of explants formed callus. 45 Faisal and Anis (2005)
TeepolTM 5 min ? DW ? 0.1 % HgCl2 Kin (SIM). ‘ MS ? 0.5 lM IBA (RIM). 50 lmol m-2 s-1. 25 ± 2 °C. shoots/callus clump formed on SIM, with
3 min ? SDW several times ? 0.5–1 cm pH 5.8. 3 % sucrose. 0.8 % agar 60 % RH 80 % of callus explants being responsive.
long stem explants In RIM, 90 % of shoots rooted, forming
4.3 roots/shoot. Plantlets acclimatized in
sterilized vermiculite, watered with ‘ MS
every 3 days for 2 w, then transferred to
pots with garden soil (survival % NR)
Plant Cell Rep (2016) 35:2207–2225
 Table 2 continued
Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and
variation

Petioles (3–5 mm long) (age of mother MS ? 10 lM 2,4-D ? 2.5 lM TDZ (CIM; 16-h PP. CWFL. 50 lmol m-2 100 % of explants formed callus. 56 Faisal et al. (2005)
plants NR): same protocol as Faisal 21-d subcultures). MS ? 2.5 lM TDZ s-1. 24 ± 2 °C. 60 % RH shoots/callus clump formed on SIM, with
and Anis (2005) (SIM). ‘ MS ? 0.5 lM IBA (RIM). 90 % of callus explants being responsive.
pH 5.8. 3 % sucrose. 0.8 % agar In RIM, 90 % of shoots rooted, forming
4.3 roots/shoot. Plantlets acclimatized in
sterilized vermiculite after dipping cut
ends of shoots in 150 lM IBA for
30 min, watered with ‘ MS every 3 days
Plant Cell Rep (2016) 35:2207–2225

for 2 w, then transferred to pots with

garden soil (100 % survival).
Photosynthetic parameters between
in vitro plantlets and ex vitro acclimatized
plants were comparable
15-d-old leaves (outdoor plants) ? 70 % MS ? 7 lM 2,4-D ? 1.5 lM BA (CIM). 16-h PP. CWFT. 50 lmol m-2 92 % of explants formed callus. 100 % of Thomas and Philip
EtOH 30 s ? 0.1 % HgCl2 ? 2 drops MS ? 8 lM TDZ (SIM).‘ MS ? 3 lM s-1. 24 °C callus cultures formed shoots, with 66.7 (2005)
Tween-20 5 min ? 39 SDDW ? 1 cm2 IBA (RIM). pH 5.8. 0.8 % (w/v) agar. shoots/callus culture. 92 % of
explants Carbohydrate NR acclimatized plants survived in soil.
Leaves (1-y-old plants) ? DW 29 ? 1 % MS ? 0.5 lM TDZ ? 1.5 lM 2,4-D 16-h PP. CWFT. 50 lmol m-2 18.2 globular SEs/explant. 65 % of cultures Chandrasekhar et al.
TeepolTM 5 min ? 0.1 % HgCl2 (SEIM). PGR-free MS (SEGM). 2 % Na- s-1. 25 ± 2 °C. formed plantlets. 90 % of SEs (2006)
5 min ? 4–59 SDDW ? 8 9 20 mm alginate ? 5 % Ca-alginate (SE germinated. 65-70 % survival after
explants synseed). pH 5.7. 3 % sucrose. 0.8 % acclimatization in garden soil ? sand
agar (1:1). No morphological variation
claimed
Internodes (outdoor plants, age NR) ? 1 % MS ? 4 lM 2,4-D (SEIM). MS ? 6 lM 16-h PP. CWFT. LI NR. 71 % of callus formed SEs (49 SEs/g Thomas (2006)
Savlon (detergent) Kin, 10 lM GA3 or lM ABA (SEGM). 24 °C callus). 94 % of acclimatized plants
10 min ? SDDW ? 0.1 % HgCl2 pH 5.8. 200 mM sucrose. 0.8 % (w/v) survived (substrate NR)
5 min ? 39 SDDW ? 10 mm explants agar
Young shoots from 2-y-old plant ? RTW MS ? 2.5 lM BA ? 0.5 lM NAA (SIM). 16-h PP. CWFL. 50 lmol m-2 Synthetic seeds (encapsulated nodes) were Faisal and Anis (2007)
30 min ? 5 % LaboleneTM ‘ MS ? 0.5 lM IBA (RIM). pH 5.8. s-1. 24 ± 2 °C. 60 % RH produced with 3 % N alginate and
5 min ? 39 SDW ? 0.1 % HgCl2 3 % sucrose. 0.8 % agar complexed with 100 mM CaCl2
2H2O for
3 min ? 49 SDW ? stem segments with 30 min. 72 % of beads converted into
nodes (3-5 mm long) = explants plantlets when plated immediately, or
only 50.3 % when stored for 8 w at 4 °C.
43 % of synseeds germinated directly on
sterilized SoilriteTM moistened with ‘
MS after 6 w. After an initial transfer for
1 m to SoilriteTM, 90 % of germinated
plantlets survived in soil
Same explants and protocol as Faisal MS ? 2.5 lM BA ? 0.5 lM 16-h PP. CWFL. 50 lmol m-2 On SIM, 93 % of nodal segments formed Faisal et al. (2007),
and Anis (2007) NAA ? 100 mg/l AA (SIM). ‘ s-1. 24 ± 2 °C. 60 % RH shoots (8.6/explant). explants being Faisal and Anis (2010)
MS ? 0.5 lM IBA (RIM). Subcultures responsive. In RIM, 90 % of shoots
on all media every 2 w. pH 5.8. 3 % rooted, forming 4.5 roots/shoot. 90 % of
sucrose. 0.8 % agar (0.25 % gelrite for plantlets acclimatized in sterilized
RIM) vermiculite survived. Acclimatized plants
showed higher SOD (and other
antioxidant) levels when placed at a
higher light irradiance
2211

123
(300 lmol m-2 s-1)
 Table 2 continued
2212

Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and

123
variation

Leaves (10-y-old plant) ? 5 % TeepolTM MS ? 2 mg/l 2,4-D ? 0.5 mg/l 16-h PP. Light source NR. 100 % of explants formed callus in 7 d and Singh et al. (2009, 2010)
10 min ? RTW ? 70 % EtOH Kin ? 200 mg/l CH (CIM, CPM). 2000 lx. 26 ± 2 °C 35 shoots/callus cluster in 15–20 d. The
2 min ? 0.1 % HgCl2 MS ? 1 mg/l Kin ? 0.5 mg/l IAA level of amino acids increased in callus
2 min ? SDDW ? 1 cm2 explants (SIM). 4-w subcultures. pH 5.8. 3 % between day 10 and 20 (assessed by SDS-
sucrose. 0.8 % agar PAGE)
Leaves from 2nd node (greenhouse plants, Liquid MS ? 0.4 M mannitol ? 3 % Darkness ? 25 °C (callus 10.3 9 105 protoplasts yielded from 1 g of Thomas (2009)
age NR) ? 20 % NaOCl ? 2 drops sucrose ? 2–6 lM 2,4-D (PCM). 0.4 ml induction from protoplasts). leaf tissue. Protoplasts showed 84 %
Tween-20 20 min ? 59 SDW ? of PCM with 0.2 M mannitol added each All other cultures: viability after 4 h, but only 65 % after
leaf tissue without lower epidermis week. PCM – mannitol (SIM). 200 lmol m-2 s-1. Temp., 24 h. 44.2 shoots formed per callus
and midrib ? protoplast isolation MS ? 5 lM TDZ ? 0.4 lM NAA light source, PP NR cluster. 80 % of acclimatized plants
(SELM). ‘ MS ? 3 lM IBA (RIM). pH survived in soil
5.8. 0.8 % (w/v) agar
Nodal explants (size, age NR) ? TW ? MS ? 1 mg/l BA (SIM; subcultures NR). 18-h PP. 25 ± 1 °C. Light Shoots formed from 64 % of nodes and Gami and Parmar (2010)
0.5 % NaOCl 20 min ? 0.01 % HgCl2 MS ? 0.05 mg/l IAA (RIM). pH 5.6–5.8. source, LI, RH NR 70.5 % of shoots rooted. 99 % survival of
5 min ? SDW 3 % sucrose. 0.8 % agar acclimatized plants in cocopeat, soil and
sand (ratio NR). Different extracts from
in vitro vs in vivo leaves and roots
showed weak antimicrobial activity
relative to 0.01 g/l tetracycline, the
positive control
Nodal explants (size, age NR) ? 5 % MS ? 3 mg/l 2,4-D (CIM). MS ? 3 mg/l 12-h PP. CWFT. 3000 lx. Explant swelling after 6–8 d on CIM, and Kaushik et al. (2010)
TeepolTM 20 min ? 0.1 % HgCl2 BA (SIM; 3-w subcultures). MS ? 2 mg/ 25 ± 2 °C. 70 % RH callus formation from cut ends. 80 % of
5–10 min ? 3–59 SDW l IBA ? 4 mg/l NAA (RIM). pH, explants formed callus. 80 % of explants
carbohydrate source, agar conc. NR formed shoots directly and 90 % of shoots
induced roots in RIM. Chlorophyll,
carbohydrate, protein and lipid content
not different between in vivo and in vitro
regenerants. Acclimatization not
performed.
Nodes (5-y-old plant) collected from Sept. to MS ? 2 mg/l BA ? 0.2 mg/l 16-h PP. CWFL. 48 lmol m-2 95 % of nodes formed an average of 4.86 Rani and Rana (2010)
Nov. ? 5 % TeepolTM 10 min ? RTW GA3 ? 100 mg/l myo-inositol (SIM; 4-w s-1. 24 ± 1 °C. 60–65 % RH shoots/explant. 90 % of shoots rooted,
30 min ? DW ? 0.1 % HgCl2 subcultures). MS ? 0.5 mg/l IBA (RIM). forming 4.55 roots/shoot. 96 % of
10 min ? 4–59 SDW (explant size NR) pH 5.8. 3 % sucrose. 0.8 % agar plantlets could be acclimatized in VC
Leaves (age of mother plant MS ? 2.5 lM BA ? 3.5 lM 2,4-D (CIM). 16-h PP. CWFL. 60 lmol m-2 80–90 % of explants callused. 4.6 Rathinavel and
NR) ? RTW ? 5 % TeepolTM MS ? 1 lM Kin (SIM). PGR-free ‘ MS s-1. 25 ± 2 °C shoots/explant on SIM. 90 % of shoots Sellathurai (2010)
16–20 min ? SDW ? 70 % EtOH (RIM). Subcultures every 35 d. pH 5.8. rooted. 62 % acclimatization in sterilized
30 s ? 0.1 % HgCl2 3 % sucrose. 0.8 % agar peat soil and sand (1:1)
7 min ? 3–49 SDDW ? 0.5 cm2 explants
Plant Cell Rep (2016) 35:2207–2225
 Table 2 continued
Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and
variation

Leaves (5-y-old plant) ? RTW 30 min ? 5 % MS ? 5 lM 2,4-D (CIM; Khatoon et al. 16-h PP. CWFL. 40 lmol m-2 Number of shoot buds/culture: 23.4 (BA) or Sahai et al. (2010a, b),
TeepolTM 15 min ? RTW ? 0.1 % HgCl2 2013). MS ? 5 lM BA (CIM; all other 3 s-1. 25 ± 2 °C. 70 % RH 26.8 (TDZ) (2010a), 46.8 (2010b). Jahan et al. (2013),
3 min ? 3–49 SDW ? 1 cm2 explants (or studies). MS ? 2.5 lM TDZ (CIM; Number of roots/shoot: 5.8 (2010a), 6.4 Khatoon et al. (2013)
1.5 cm long green root segments: 2010b) 2010a). MS ? 5 lM BA (SIM). ‘ (2010b). 5–10 mg/l NAA also effective
MS ? 0.5 mg/l IBA (RIM). pH 5.8. 3 % for root formation (2010a, 2010b). 90 %
sucrose. 0.8 % agar survival of acclimatized plants in
soil ? garden manure (1:1) (2010a), or
vermiculite (2010b). Antibacterial (Jahan
Plant Cell Rep (2016) 35:2207–2225

et al. 2013) and antifungal activity

(Khatoon et al. 2013) claimed from 70 %
ethanolic extract of leaves and callus.
Leaves (1-y-old plant) ? RTW MS ? 2 mg/l BA ? 0.5 mg/l IBA (CIM). 16-h PP. CWFL. 2500 ± 500 lx. 95 % of leaves formed callus and 15.22 Verma et al. (2010),
15–20 min ? 1 % TeepolTM MS ? 0.1 mg/l TDZ (SIM). ‘ 26 ± 2 °C. 55 ± 5 % RH shoots/explant. 85 % of shoots rooted Sharma et al. (2014)
2–4 min ? 3–49 SDW ? 0.1 % HgCl2 MS ? 0.5 mg/l IBA (RIM). pH 5.8. with 7.75 roots/shoot within 8 d.
2–3 min ? rinses and explant size NR Subcultures, carbon source, and gelling Internodes and petioles could also induce
agent NR callus, but less than leaves. 75 %
acclimatization in VC and autoclaved soil
(1:3). 93 % genetic similarity shown with
ISSR markers
1 cm leaf segments from in vitro plants MS ? 2 mg/l Kin ? 1 mg/l NAA (SEIM, 16-h PP. CWFL. 74.3 % of explants formed SEs on SEIM Davendra et al. (2011)
SEGM). pH 5.6. 3 % sucrose 50–60 lmol m-2 s-1. 25 °C. and 77.2 % germinated after subculture
70 % RH every 2 w. Synthetic seeds (encapsulated
nodes) were produced with 3 % N
alginate and complexed with 50 mM
CaCl2
2H2O for 30 min. 20.2 % of beads
germinated and 5.2 % of those converted
into plantlets when 30-d-old SEs were
used and plated on ‘ MS.
Acclimatization not performed
Leaves and stems (3-y-old plant) ? RTW MS ? 4.65 lM Kin ? 29.4 lM NAA 16-h PP. CWFL. 50 lmol m-2 Green callus formed within 7–8 d and Kaur et al.
30 min ? 0.1 % TeepolTM (CIM). MS ? 8.88 lM BA (SIM). ‘ MS s-1. 25 ± 2 °C shoots formed within 6–8 w in about (2011a, b, 2014),
5 min ? TW ? 0.1 % Bavistin or ‘ MS ? 9.84 lM IBA (RIM). pH 5.8. 90 % of cultures (2011a; 85 % from Anand et al. (2012)
10–12 min ? 0.1 % HgCl2 2 % sucrose. 0.8–1 % agar leaves in 2011b). 92 % acclimatization in
2–4 min ? 3–49 SDW ? 5–6 cm stem or soil ? VC ? Azobacter (N2
intact leaves (explants) fixer) ? Pseudomonas (phosphate
solubilizer) (1:1:1:1) (2011b), or
88–90 % acclimatization in soil ? VC
(1:1) (2011a, 2011b). HPTCL detected
80, 28.3 and 24.5 lg/ml tylophorine in
the leaves of in vitro plants, suspension
cultures and callus, respectively
Internodes (age of mother plant NR) ? RTW MS ? 3.25 lM BA ? 1.5 lM IAA (CIM; 16-h PP. CWFL. 60 lmol m-2 Callus was induced within 8 w. 100 % of Mahesh et al. (2011)
30 min ? 70 % EtOH SIM). MS ? 12.25 lM Kin ? 3.75 lM s-1. 26 ± 2 °C. 55–60 % RH explants formed callus, shoots, and roots
50 s ? SDDW ? 0.1 % HgCl2 NAA (SIM). MS ? 1.25 lM (organogenesis not quantified).
2 min ? 29 SDDW (5 min each) Zea ? 2.75 lM IAA (RIM). pH 5.7. 3 % Acclimatization claimed, but not shown.
sucrose. 0.8 % agar
2213

123
 Table 2 continued
2214

Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and

123
variation

Young leaves (plant age MS ? 0.2 mg/l BA ? 2.0 mg/l NAA Callus 7 d in darkness ? light. Callus formed after 15 d. 37.5 % band Chaturvedi and
NR) ? RTW ? TeepolTM (CIM). MS ? 1 mg/l BA ? 1 mg/l Kin PP, light source, LI, temp, RH polymorphism in regenerants using Chowdhary (2012)
10–15 min ? 0.1 % HgCl2 (SIM). 20-d subcultures (CIM, SIM). pH NR RAPD (46 % using ISSR). Rooting and
2–3 min ? 39 DW ? 1 cm2 explants 5.8. 3 % sucrose. 0.8 % agar acclimatization not performed
Young leaves (plant age NR) ? TW MS ? 2.5 mg/l BA (CIM; SIM; SMM). 16-h PP. CWFL. 2000 lx. Callus formed in 7 d on 90 % of explants. Kalimuthu and
29 ? 5 % TeepolTM 10 min ? TW ? 1 % MS ? 0.5 mg/l IBA (RIM). pH 5.8–5.9. 25 ± 2 °C Shoots developed from callus within 20 d. Jeyaraman (2012)
Bavistin (fungicide) 20 min ? TW ? 70 % 3 % sucrose. 1 % agar 95 % survival after acclimatized in
alcohol 30 s ? 0.12 % HgCl2 soil ? sand ? compost (1:1:1). Alkaloids
3 min ? 3–49 DDSW tested by TLC
Shoot tips, leaves, nodes (plant age MS ? 1 mg/l 2,4-D ? 1 mg/l Kin (CIM; No conditions reported 80 % of shoot tips and leaves formed callus Patel and Patel (2012)
NR) ? Tween-20 ? NaOCl shoot tips). MS ? 1 mg/l 2,4-D ? 1 mg/l (60 % in nodes). 100 % of callus formed
2 min ? 0.5 % HgCl2 3–4 min ? water BA (CIM; leaves, nodes). MS ? 3 mg/l shoots and all shots rooted.
BA ? 0.5 mg/l IBA (SIM). MS ? 1 mg/l Acclimatization not performed
IBA (RIM). pH, carbon source, gelling
agent NR
Leaves of 1-y-old plant ? 29 DW ? 1 % MS ? 1 mg/l IAA (RIM). pH 5.8. 3 % Constant darkness. 25 ± 1 °C Adventitious roots formed within 2 w and Rashmi et al. (2012)
TeepolTM 5 min ? 0.1 % HgCl2 sucrose. 0.2 % gelrite profuse roots by 6 w (24.33 roots/explant;
5 min ? 4–59 SDW ? 5 mm2 explants 2 mg/l IBA induced 15.33 roots/explant)
Aged leaves (base of plants) and young leaves MS ? 2 mg/l BA (SIM; old leaves). 16-h PP. CWFL. 50 lmol m-2 Shoot primordia formed from cut edge of Haque and Ghosh
(from terminal 5th – 8th nodes) of 2-y-old MS ? 2 mg/l BA ? 0.2 mg/l IAA (SIM; s-1. 23 ± 2 °C old leaves within 26–30 d but within (2013)
plants ? 2 % Bavistin 10 min ? 5 % young leaves). ‘ MS ? 0.2 mg/l IBA 40–45 d from youg leaves. Young leaves
Tween-20 3 min ? 0.1 % HgCl2 (RIM). 30-d subcultures. pH NR. 3 % formed 25.2 shoots/explant (18.6
8 min ? 39 DW ? cut in half sucrose. 0.7 % agar shoots/explant in old leaves). 100 % of
shoots rooted on RIM, forming 8.3 roots/
shoot. 93.3 % of acclimatized plantlets
survived in soil ? VC (3:1). No variation
between micropropagated plants and
mother plant shown by RAPD.
Acclimatized plants flowered, formed
fruit and R1 seed
Leaves from 1-y-old plant ? cut into small MS ? 1 mg/l BA ? 1 mg/l 2,4-D (CIM). 12-h (CIM) or 16-h (SIM) PP. Callus induced after 16 d. Shoot buds Sadguna et al. (2013)
pieces (size NR) ? RTW 10 min ? 0.1 % MS ? 1 mg/l BA ? 2 mg/l glutamic Light source NR. 2000 lx. formed after 4 w (18 shoots/explant) in
HgCl2 6 min ? 4–59 SDW acid (SIM). ‘ MS ? 4 mg/l IBA (RIM). 25 ± 2 °C 90 % of explants. Callus and rooting not
Subculture every 20 d. 3.5 % sucrose. pH quantified. Acclimatization not performed
and gelling agent NR
Shoots from 1-y-old plant ? 39 TW ? leaves CIM (Chandrasekhar et al. 2006). 16-h PP. CWFL. 36 lmol m-2 Wide antimicrobial activity (growth Sellathurai et al. (2013)
in SDW 30 min ? 5 % TeepolTM Subculture every 3 w. pH 5.8. 0.8 % agar. s-1. 26 ± 2 °C. 55–60 % RH inhibition) shown by callus extracts
5 min ? DW ? 70 % alcohol ? 0.1 % Carbon source NR
HgCl2 5 min ? 39 SDW
Leaves, nodal segments (size NR) from 1-y-old MS ? IAA ? Kin (SIM from nodal PP, light source, LI NR. Shoot formation after 4 w, plantlet Anjum et al. (2014)
plant ? RTW ? cetrimide ? streptomycin segments; CIM from leaf segments; conc. 25 ± 2 °C. 55 ± 5 % RH formation after 8 w
sulfate ? bavistin ? HgCl2 ? 70 % NR). MS ? 2 mg/l BA ? 0.5 mg/l NAA
alcohol ? SDDW (in all cases, exact (SIM from leaf segments)
concentrations and exposure times NR)
Plant Cell Rep (2016) 35:2207–2225
 Table 2 continued
Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and
variation

1-m-old leaves ? TeepolTM (conc. time Beads ? Zenk et al. (1975) basal Shaking at 90 rpm. 25 ± 2 °C. Relative to the control (0.16 %), Chaturvedi et al. (2014)
NR) ? RTW ? 0.1 % HgCl2 medium ? 5 % sucrose ? 5 mg/100 ml Light conditions NR kaempferol content increased to 2.07 %
2 min ? 39 DW ? homogenized (protocol cinnamic acid and 3.31 % after 2 and 3 w, respectively,
NR) ? alginate beads (2 % Na- when beads with agarized suspension
alginate ? 50 mM CaCl2 30 min) culture were cultured in liquid medium
Shoots (age of mother plants NR) ? RTW MS ? 2.5 mg/l BA ? 3.5 mg/l NAA 16-h PP. Light source, LI NR. 73 % of explants formed shoots. 100 % Jeyachandran and Bastin
30 min ? nodes 109 TW ? 0.2–0.5 % (SIM). ‘ MS ? 2.5 mg/l IBA ? 2 mg/l 25 ± 2 °C acclimatization in sterile soil, sand, and (2014)à
Bavistin ? 0.03 % streptomycin IAA (RIM). Subcultures NR. pH 5.8. organic manure (1:1:1)
Plant Cell Rep (2016) 35:2207–2225

10 min ? SDW 29 ? savlon (1.5 % Carbon source and gelling agent NR

chlorohexidine gluconate ? 3 % cetrimide)
10 min ? SDDW ? 0.01 % HgCl2
1 min ? SDW
Nodes ? RTW 20 min ? 0.1 % Tween-20 MS ? 1 mg/l AdS ? 0.5 mg/l 2,4-D 16-h PP. Light source NR. 75 % of explants formed shoots and 65 % Mohan et al. (2014)
5 min ? TW ? 70 % EtOH (SIM). MS ? 1 mg/l IAA (RIM). pH 5.8. 2000 lx. 22 ± 1 °C. 75 % RH of shoots rooted on RIM. 70 %
5 min ? water ? 0.1 % HgCl2 2 % sucrose. 0.8 % agar acclimatization
3–5 min ? SDW
Adventitious roots (size NR) deried from MS ? 2 mg/l IBA ? 0.1 mg/l BA (SIM). 16-h PP. CWFL. 50 lmol m-2 25 shoots/root explant within 4 w. Nayeem et al. (2014)
in vitro plant leaves produced according to ‘ MS ? 0.2 mg/l IAA (RIM). pH 5.8. s-1. 26 ± 2 °C Acclimatization in sterile cocopeat, sand,
Rashmi et al. (2012) (RIM = MS ? 1 mg/l 3 % sucrose. 0.8 % agar and soil (1:1:1), with 80 % survival.
IAA).
Nodes ? RTW 30 min ? 1 % TeepolTM MS ? 2 mg/l 2,4-D ? 0/1 mg/l Kin 16-h PP. Light source, LI NR. 85 % of explants formed callus within 25 d. Patel and Nadgauda
10 min ? wash ? 0.1 % HgCl2 (CIM). MS ? 0.4 mg/l BA ? 0.1 mg/l 25 ± 2 °C 98 % of explants formed shoots (2014)
2–3 min ? 4–59 DDW Kin (SIM, RIM). 30-d subcultures. pH (2.6/explant) within 7 d. 97 %
5.8. 2 % sucrose. Gelling agent NR acclimatization in soil ? SoilriteTM (ratio
NR) after a 5 min dip in Bavistin
Nodes ? 29 DW ? 1 % TeepolTM MS ? 1.5 mg/l BA ? 0.5 mg/l NAA 16-h PP. CWFL. LI NR. Different alternative carbon sources were Rajavel and Stephan
5 min ? 0.1 % HgCl2 5 min ? 4–59 SDW (SIM). pH 5.8. 3 % sucrose. 0.8 % agar 25 ± 2 °C tested on the effect on shoot formation. (2014)
95.2 % of explants formed shoots with
AR grade sucrose (94.8 % with white,
refined sugar; 76.8 % with sugarcane
juice; 73.8 % with unrefined, brown
sugar; 67.6 % with jaggery), forming
16.5, 15.5, 12.4, 11.5 and 9.3
shoots/explant, respectively, when added
at 3 % in all cases
Leaves from mature plant (age NR) ? RTW MS ? 1 mg/l 2,4-D ? 1 mg/l Kin (CIM). PP and light source NR. 2000 lx. 80 % of explants formed callus. 80 % of Rathod et al. (2014)
30 min ? detergent 5 min ? TW ? 0.1 % MS ? 2 mg/l BA ? 0.2 mg/l IAA 25 ± 2 °C. 50–60 % RH callus formed shoots (8.1 shoots/callus
HgCl2 2 min ? 4–59 SDW ? 1 cm2 (SIM). pH 5.8. 3 % sucrose. 0.8 % agar cluster). Protein, reducing sugar and total
explants amino acids, as well as peroxidase and
IAA oxidase were highest in the initial
callus phase (among 4 in vitro stages
tested)
Leaves (age NR) ? 70 % EtOH MS ? 2 mg/l BA ? 2 mg/l 2,4-D (CIM). 16-h PP. Light source and LI Callus formed after 20 d in 80 % of Dhokrat et al. (2015),
1 min ? TW ? 0.2 % HgCl2 time MS ? 2 mg/l BA ? 0.2 mg/l IAA NR. 25 ± 2 °C explants. Shoot tips induced callus and Jogdand et al. (2016),
NR ? 39 SDW (SIM). pH 5.8. Carbon source NR. 0.3 % shoots simultaneously. Acclimatization Shimple and Pandhure
clerigelas NR (2016)
2215

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 Table 2 continued
2216

Explant conditions and disinfection procedures Culture medium, PGRs and additives*,** Culture conditions*** Remarks, experimental outcome and References
maximum productivity, acclimatization and

123
variation

Nodes from young shoots ? RTW MS ? 1 mg/l Kin ? 4 mg/l NAA (CIM). 16-h PP. CWFL. 50 lmol m-2 Callus was induced within 6–8 d, and Murukan et al. (2015)
30 min ? 5 % labolene ‘ MS ? 2 mg/l BA (SIM). ‘ s-1. 25 ± 2 °C covered the explant after 3 w. 40–50
5 min ? 39 SDW ? 0.1 % HgCl2 MS ? 4 mg/l IBA (RIM). pH 5.8. 3 % shoots/node formed. Leaves also formed
3 min ? 39 SDW sucrose. 0.8 % agar callus and shoots, but in lower amounts
and took longer. 80 % of shoots rooted in
RIM
Leaf and nodal segments ? RTW (time MS ? 2 mg/l Kin ? 0.5 mg/l IAA (SIM: 16-h PP. Light source and LI 100 % of explants formed callus in 12 w. A Soni et al. (2015a)
NR) ? 0.2 % Cetrimide (leaves: 5 min; nodes). MS (liquid) ? 1 mg/l NR. 25 ± 2 °C. 55 ± 5 % RH maximum of 3.59 shoots formed/explant.
nodes: 7 min) ? 0.25 % Streptomycin BA ? 0.5 mg/l IAA (CIM, SIM: leaves; 95.1 % of shoots rooted. Acclimatization
sulphate ? 0.5 % Bavistin (leaves: 15 min; precursor feeding). MS ? 1 mg/l IBA in autoclaved SoilriteTM ? soil (1:1).
nodes: 20 min) ? 0.1 % HgCl2 (leaves: (RIM). pH 5.7. Carbon source NR. Increased levels of tylophorine were
3 min; nodes: 5 min) ? 70 % EtOH 0.63 % agar induced after feeding tyrosine in culture
1 min ? 69 SW
Nodes ? RTW 30 min ? 5 % Teepol MS ? 0.5 mg/l BA (SIM). ‘ MS ? 1 mg/ 16-h PP. Light source NR. 100 % shoot formation and 95 % root Soni et al. (2015b)
8–10 min ? SDW several times ? Bavistin l IAA (RIM). pH 5.8. Carbon source and 3000 lx. 25 ± 2 °C. 65–70 % induction (organogenesis not quantified).
1 h ? 39 SDW ? 0.1 % HgCl2 gelling agent NR RH Acclimatization in sand ? garden soil
4–6 min ? SDW (1:1)

2-BTOA 2-benzothiazoleacetic acid; 2,4-D 2,4-dichlorophenoxyacetic acid; 2,4,5-T 2,4,5-trichlorophenoxy acetic acid; 2iP N6-(2-isopentenyl) adenine; AA ascorbic acid; ABA abscisic acid;
AdS adenine sulphate; BA N6-benzyladenine (BA is used throughout even though BAP (6-benzylamino purine) may have been used in the original, according to Teixeira da Silva 2012a; CH
casein hydrolysate; CIM callus induction medium; CM coconut milk; CPM callus proliferation medium; CWFT white fluorescent tube(s); d day(s); DDW double distilled water; DW distilled
water; EtOH ethyl alcohol (ethanol); GA3 gibberellic acid; GRS gree-root-segment = green region of roots that is not WRS; HgCl2 mercury chloride; HPTLC high performance thin layer
chromatography; IAA indole-3-acetic acid; IBA indole-3-butyric acid; ISSR inter simple sequence repeat; Kin kinetin (6-furfuryl aminopurine); LaboleneTM a detergent (Qualigens, Mumbai,
India); LI light intensity; m month(s); MS Murashige and Skoog (1962) medium; NAA a-naphthaleneacetic acid; NaOCl sodium hypochlorite; NR not reported in the study, PCM protoplast
culture medium; PGR(s) plant growth regulator(s); PP photoperiod; RAPD random amplified polymorphic DNA; RH relative humidity; RIM root induction medium; rpm revolutions per
minute; RTW running tap water; SDW sterilized (by autoclaving) distilled water; SDDW sterilized (by autoclaving) double distilled water; SDS-PAGE sodium dodecyl sulfate polyacrylamide
gel electrophoresis; SE somatic embryo; SEGM somatic embryo germination medium; SEIM somatic embryo induction medium; SELM shoot elongation medium; SIM shoot induction medium;
SMM shoot multiplication medium; SOD superoxide dismutase (EC 1.15.1.1); SW sterile water; TeepolTM a detergent (Reckitt and Colman, Slough, UK); TDZ thidiazuron (N-phenyl-N’- 1,2,3-
thiadiazol-5-ylurea); temp temperature; TLC thin layer chromatography; VC vermicompost; w week(s); White’s White’s nutrient solution (White 1943); WRS white-root-segment = growing
white apical region of the roots (15–20 mm); Zea trans zeatin
* Even though the term ‘‘calli’’ was used in the original, the term callus has been used here based on the recommendation of Teixeira da Silva 2012b
** All percentage values represent w/v (weight/volume) for solids or v/v (volume/volume) for liquids, unless otherwise specified
*** The original light intensity reported in each study has been represented since the conversion of lux to lmol m-2 s-1 is different for different illumination (main ones represented): for
fluorescent lamps, 1 lmol m-2 s-1 = 80 lx; the sun, 1 lmol m-2 s-1 = 55.6 lx; high voltage sodium lamp, 1 lmol m-2 s-1 = 71.4 lx (Thimijan and Heins 1983)
**** For the 2005, 2006, 2013 and 2015 studies, the basic protocol to establish the initial cultures and to obtain the in vitro aseptic explants used for genetic transformation experiments, and
eventually transgenic plants, used the Chaudhuri et al. (2004) protocol. Claims of somatic embryogenesis without sufficient proof (cytological, histological, genetic), i.e., only photos of
macromorphology
à
Tylophora ovata
Tylophora subramanii
Plant Cell Rep (2016) 35:2207–2225
Plant Cell Rep (2016) 35:2207–2225 2217

Fig. 1 The in vitro culture of Tylophora indica. a Direct shoot supplemented with 0.5 mg/l IBA, after 8 weeks of culture at
organogenesis from mature (thick, leathery, and dark green) leaves 24 ± 1 °C with a 16-h photoperiod. c In vitro root induction from
(from first 10 leaf pairs from the base) after 50 days of culture in 12-day-old shoot (2.5 cm) derived from (a) on half-strength MS
Murashige and Skoog (MS) medium supplemented with 2 mg/l BA. medium fortified with 0.1 mg/l IBA. d Three-month-old hardened
Leaf explants were collected from a four-year-old plant maintained plants kept in earthen pots containing a mixture of soil and
inside a shade-net house to avoid direct sunlight and exposed to vermicompost (3:1, v/v) and maintained inside a greenhouse
60–65 % relative humidity using a misting system. In vitro cultures (30 ± 2 °C, 14-h photoperiod, 60–65 % relative humidity). e Ex
were incubated inside a growth chamber maintained at 23 ± 2 °C vitro plant (22 months old) maintained in the field under full sunlight
with a 16-h photoperiod and at a photosynthetic photon flux density of and natural conditions without any additional care. f Flower of ex
approximately 50 lmol m-2 s-1 emitted by cool fluorescent tubes vitro plant. All photos (unpublished) provided with kind permission
(Philips India Ltd.). b Direct shoot organogenesis from green root of Dr. B. Ghosh (a, c–e), and Dr. D. Roychowdhury (b)
segments, excised from 4 to 6-week-old in vitro plant, on MS medium

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2218 Plant Cell Rep (2016) 35:2207–2225

Leaves from field-grown plant (1-2 years old)

Sterilization: TeepolTM (10-15 min) HgCl2 (0.1% for 10-15 min) 5 washes in dH 2O

Explant size: ~ 2 cm2

Shoot induction media*: MS + BA/Kin (2-5 mg/L) + IAA (0.2-1.5 mg/L)

Culture conditions: 24±1 C;16-h photoperiod; 3000±500 lux; 60±5% RH

3-4 cycles of 4-week 6-8 weeks

subcultures:
organogenic 6-8 weeks
Induction of nodular meristemoids with SAM through organogenesis
potential retained;
>25 shoots / explant
6-8 weeks
Media:
Development of 2-3 cm long microshoots MS + BA (1-4 mg/L) ± NAA
(0.02-0.05 mg/L)*

Axillary bud multiplication and proliferation Root induction media:

MS/2 + IBA (0.1-0.2 mg/L) ~1-1.5 cm green root segments
6-8 weeks
excised from in vitro mother plant
MS + BA/KIN (0.5-2 mg/L) + NAA (0.1-0.5 mg/L) + AA (0-100 mg/L)* 4-6 weeks

Maintained for >1 year in vitro

Nodal explant Rooted plants Root plants

Hardening: soil: compost/vermiculite (3:1, v/v); 30±2 C; 14-h photoperiod; 60±5% RH

2-4 weeks

*depends on genotype/population Transfer to soil

Fig. 2 In vitro plant regeneration in and mass propagation of Tylophora indica can be achieved in several ways, based on Table 2

leaves and subsequent development of SEs, but the con- Phillip (2005) were the first to provide histological evi-
version frequency to SEs depended on the concentration dence of indirect shoot formation from immature leaves of
and combination of indole-3-acetic acid (IAA), 6-benzy- field-grown plants, noting 100 % regeneration potential by
ladenine (BA) and kinetin. Jayanthi and Mandal (2001) long-term (up to 180 days) callus cultures. Haque and
induced SEs from embryogenic callus induced on mature Ghosh (2013) showed a different morphogenic response by
leaf explants from ex situ plants, obtaining 50 plantlets/g of young and mature leaves of field-grown plants grown
callus in 5 months. Somatic embryogenesis has also been in vitro: while aged leaves formed shoots directly, young
reported from leaf explants by Chandrasekhar et al. (2006) leaves first formed nodular meristemoids. The Haque and
and Sahai et al. (2010a), and from internodes (Thomas Ghosh (2013) study is the only publication in which
2006). micropropagated plants transferred to field flowered, and
Faisal and Anis (2003) induced shoots indirectly from 28.5 % of plants produced fruit. The development of a
callus in 85 % of leaves from field-grown plants, from stem successful protocol for the micropropagation of Tylophora
explants (Faisal and Anis 2005) and from petiole explants plants (Fig. 1c–e) is a prerequisite for more advanced
(Faisal et al. 2005), a similar result being obtained by studies such as genetic transformation.
Verma et al. (2010) leaf, petiole and internode explants. To induce callus using leaf and stem explants, MS
Shoot induction was possible from mature leaf explants medium supplemented with 2.5–7.5 lM 2,4-D or 2,4,5-T is
(Rathinavel and Sellathurai 2010; Anjum et al. 2014) of optimal while shoot organogenesis from this callus can be
young leaves (Kalimuthu and Jeyaraman, 2012). Kaur et al. induced in MS medium supplemented with 5 lM kinetin or
(2011a) developed a protocol for the induction of shoots BA, or 8 lM thidiazuron (Faisal and Anis 2003, 2005;
from the stems of field-grown plants, and a subsequent Thomas and Philip 2005; Fig. 2). Microshoots can be
protocol for the acclimatization and ex situ establishment rooted in half-strength MS medium containing 0.5–0.1 lM
of tissue cultured plants (Kaur et al. 2011b). Thomas and IBA.

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Plant Cell Rep (2016) 35:2207–2225 2219

Use of explants from in vitro plants followed by acclimatization in vermiculite in a growth

chamber under a 16-h photoperiod for 4 weeks (Fig. 2).
All studies that have used in vitro tissue to initiate in vitro
cultures have employed leaf and root explants for whole
plant regeneration. There are three reports on the use of root Protoplast culture and morphogenesis
explants excised from in vitro plants to develop whole plants.
Chaudhury et al. (2004) induced nodular shoot buds from Mhatre et al. (1984) were the first to report plant regen-
green root segments in the presence of a cytokinin, and eration from protoplasts in T. indica from callus induced on
embryogenic callus from the same explants in the presence stem explants. The yield of viable protoplasts, and induc-
of BA and 2iP (N6-(2-isopentenyl) adenine), with 42 % of tion of shoots, was higher when freshly induced callus was
explants converting to SEs. Sahai et al. (2010b) reported used than from five year old callus that had been regularly
direct shoot organogenesis and callus-mediated somatic subcultured. Thomas (2009) regenerated T. indica plants
embryogenesis in green root segments, and provided a from mesophyll-protoplast-derived callus that had been
detailed histological assessment of shoot development. induced from the leaves of field-grown plants. Protoplasts
Nayeem et al. (2014) induced shoots from adventitious roots have not yet been used to generate hybrids in Tylophora.
that developed from leaf explants, either directly or via callus
formation. Devendra et al. (2011) claimed to induce SEs
from leaf explants but provided no histological evidence. Optimal in vitro protocol

Based on the protocols described in Table 2, it is evident

Use of axillary buds, nodes and shoot tips that T. indica is an interesting example of a plant species
showing morphogenic potential from almost all vegetative
Sharma and Chandel (1992) first reported axillary bud parts. However, for the purpose of mass clonal propaga-
proliferation and propagation from nodal stem segments in tion, the most commonly used explant, namely shoot tips or
T. indica in response to cytokinins and auxin in basal nodes, is not very suitable due to a low rate of multipli-
medium supplemented with ascorbic acid. Faisal et al. cation. To maintain the fidelity of the genetic and chemical
(2007) used 100 mg/l ascorbic acid in addition to auxins profile of the parent plant, it is necessary to avoid propa-
and cytokinins to improve shoot number and length from gation protocols that involve dedifferentiation of explant
nodes. Axillary bud multiplication and micropropagation cells to friable callus and indirect morphogenesis requiring
were subsequently reported by Gami and Parmar (2010) auxin and/or cytokinin supplementation in the basal med-
and Kaushik et al. (2010). Rani and Rana (2010) collected ium, which is very prevalent in T. indica. To induce de
nodal explants during different seasons and found that the novo shoots from leaf explants of field-grown plants via the
frequency of bud break and shoot number/explant were formation of nodular meristemoids, MS medium supple-
maximum when collected in September–November. mented with 2–3 mg/l BA or kinetin, together with low
Micropropagation from nodal explants was also reported levels of IAA (0.2–0.5 mg/l) can be used with subcultures
by Mohan et al. (2014) and Patel and Nadgauda (2014). every 4–6 weeks. Similarly, shoot organogenesis can be
While shoot multiplication using a pre-existing meristem induced via the formation of nodular meristemoids in green
has been the method of choice for clonal propagation of root segments excised from 6 to 8 week old rooted in vitro
plants in many plant species, in T. indica, the fewest plants. Thus, induction of nodular meristemoids from leaf
reports of shoot organogenesis involve nodal explants. The explants is a suggested method of choice to establish and
rate of multiplication using nodal explants is not mentioned in vitro culture and to propagate plants for commercial
in most publications and needs to be improved. It is also purposes (Fig. 2).
unclear whether a range of endophytic fungi isolated from
leaves and stems (Kumar et al. 2011) may impact the
effectiveness of organogenesis in vitro. Secondary metabolite production in vitro
An optimum number of shoots per nodal explant with an
average length of 3–4 cm can be obtained in MS medium Benjamin and Mulchandani (1973) first reported secondary
supplemented with 2.5 lM BA or kinetin, 0.1 lM NAA, metabolite production in T. indica in vitro callus induced
and 50–100 mg/l ascorbic acid after 6 weeks of culture from stem segments and roots from in vitro germinated
(Faisal et al. 2007). Rooting of these shoots is optimum in seedlings. The callus did not form any phenanthroin-
half-strength MS medium supplemented with 0.5 lM IBA dolizidine alkaloids even after feeding precursors, but

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phytosterols were detected. Benjamin et al. (1979) further roots in excised leaf and stem explants infected with
investigated alkaloid synthesis in callus cultures and Agrobacterium rhizogenes strain A4. In that study, as many
in vitro regenerated plants: while alkaloids were not as 60 % of inoculated shoots formed hairy roots with dif-
detected in callus, the alkaloid profile of tissue culture- ferent transformed root clones (Fig. 3a) that accumulated
derived flowering plants were similar to field-grown plants. tylophorine at 0.16–0.29 mg in roots/Petri dish and
Jha et al. (2005) investigated the potential of differentiated 1.03–1.29 mg/g root dry weight. The transformed roots
or morphogenic root-derived callus cultures and plants could be successfully cultured in liquid medium, forming
regenerated from root segments of T. indica. The level of higher biomass, yield and tylophorine content than in solid
tylophorine—a phenanthroindolizidine alkaloid and the medium. This is a prerequisite for scale-up studies. The
main alkaloid in T. indica—in nodular meristemoid and secretion of tylophorine in liquid root culture medium was
friable embryogenic cultures and in plantlets regenerated a significant finding for large-scale production using
in vitro prior to and after transfer to the field was assessed bioreactors.
by HPLC. Tylophorine was detected in all in vitro cultures, Spontaneous regeneration of plants from Ri (root-in-
in the shoots and roots of in vitro plantlets as well as in the ducing)-transformed roots in plant growth regulator-free
leaves, stems and roots of one-year-old plants after transfer basal medium (Fig. 3b, c) was reported by Chaudhuri
to the field. Friable embryogenic cultures had double the et al. (2006). The Ri-transformed T. indica plants had
tylophorine content when the culture period was extended 160–280 % higher tylophorine content than untrans-
from 4 to 12 weeks, and 12-week-old tissue cultured formed plants, and an equivalent 350–510 % higher
plantlets had a 21-fold higher tylophorine content than biomass (Chaudhuri et al. 2006). The same group
4-week-old plants. The tylophorine content of one-year-old (Roychowdhury et al. 2013a, 2015a, b) then assessed the
micropropagated plants growing in the field and wild plants morphological and genetic stability of long-term
was comparable. Kaur et al. (2011a) detected 71–80 lg/ml (4–6 years) in vitro hairy root cultures and plants
of tylophorine in tissue culture-derived plantlets, confirmed derived from transgenic hairy roots (Fig. 3d). Among the
by Kaur et al. (2011b) study (80 lg/ml). Kaur et al. most notable morphological variations observed were
(2011b) also found that suspension cultures and callus shorter shoots with more nodes and leaves/plant, both in
produced 28.3 and 24.5 lg/ml of tylophorine, respectively. in vitro plantlets and in one-year-old greenhouse-grown
Soni et al. (2015a), using precursor feeding of 2 mg/l tyr- plants (Roychowdhury et al. 2013a). Despite this varia-
osine, induced 27.7, *12.5, *9.5, and *4.5 lg/ml of tion, no genetic variation (RAPD profiles) was detected
tylophorine in in vitro-derived plantlets, shoots, callus and (Roychowdhury et al. 2015a). The rolA, rolB, rolC, and
mother plants, respectively. rolD genes were stably inserted (Fig. 4), as confirmed by
RT-PCR, in all clones and tylophorine content, as
confirmed by HPTLC, was almost two-fold higher than
Molecular verification of somaclonal variation in non-transformed plants (Roychowdhury et al. 2013a,
2015b).
Molecular markers have not been extensively used in Ty- The morphogenic potential of transformed hairy root
lophora biotechnology. While two studies (Jayanthi and cultures was not affected by the presence of rol genes of
Mandal 2001; Haque and Ghosh 2013) showed no variation the Ri plasmid and plants regenerated both via direct (less
(i.e., polymorphism) in random amplified polymorphic common) and indirect (more common) organogenesis as
DNA (RAPD) banding between mother plants and in vitro well as via callus-mediated somatic embryogenesis
regenerants, Chaturvedi and Chowdhary (2012) reported (Chaudhuri et al. 2006; Roychowdhuri et al. 2015b). In
37.5 % polymorphism while Pathak et al. (2013) showed these studies, since Ri-transformed plants showed
62.1 % polymorphism. enhanced tylophorine production, and since such high
tylophorine-containing plantlets could be stably micro-
propagated as non-transformed plants, this technique can
Genetic transformation, hairy root production, be commercialized for the production of T. indica sec-
secondary metabolites and bioreactors ondary metabolites. A protocol for the induction of Ri-
transformed roots, regeneration of plants via direct
Tissue culture forms an important structural basis for organogenesis and indirect somatic embryogenesis in T.
genetic transformation studies. Several studies reporting on indica, does not require exogenous supplementation of
the development of transgenic T. indica exist. The first phytohormones at any stage thereby ensuring the genetic
(Chaudhuri et al. 2005) documents the induction of hairy stability of Ri-transformed plants.

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C D

Fig. 3 Spontaneous regeneration of Ri-plant from hairy root culture cultured under 16-h photoperiod on MS medium showing somatic
of Tylophora indica. a Ri-transformed root culture on MS medium embryogenesis (bar 0.5 cm). c Single germinated somatic embryo on
(bar 1.3 cm). b Spontaneously regenerating Ri-transformed callus MS medium (bar 0.3 cm). d Ri-transformed plant

Conclusions and future perspectives factors affecting the biosynthesis of T. indica alkaloids and
other secondary metabolites need to be conducted using
The development of an economically viable scale-up cul- transgenic cultures via exogenous and endogenous elicita-
ture system using transformed root cultures is a pre-re- tion (Chaudhuri et al. 2009; Ramirej-Estrada et al. 2016)
quired for the large-scale production of tylophorine and biotransformation (Banerjee et al. 2012).
(Roychowdhury et al. 2013b), although there are several The following topics still need to be explored in Ty-
other means of establishing in vitro cultures (Fig. 2). Hairy lophora in vitro biotechnology: anther culture (e.g., Teix-
roots serve as a continuous source of target metabolites of eira da Silva et al. 2015), the use of thin cell layers
parent plants due to their genetic stability, and ability for technology (Teixeira da Silva and Dobránszki 2013, 2015),
rapid growth in plant growth regulator-free liquid medium in vitro flower induction (e.g., Teixeira da Silva et al.
in bioreactors (Stiles and Liu 2013; Mehrotra et al. 2015), 2014), or CO2 enrichment for increasing biomass (e.g.,
allowing for hairy root-mediated biotransformation (Ban- Norikane et al. 2013). More recently, an aqueous extract of
erjee et al. 2012). These technologies would allow for the T. indica leaves was used to synthesize silver nanoparticles
use of T. indica hairy roots to further improve the (Oke et al. 2015), but that procedure would need to be
biosynthetic potential of superior clones. Studies on the improved to make it more economically viable than for

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1 2 3 4 5 6 7 8 9 10 1112 13 Compliance with ethical standards

Conflict of interest The authors have no conflicts of interest to

declare.

300bp rolA
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