In Vitro Cellular & Developmental Biology - Plant

https://doi.org/10.1007/s11627-023-10375-z

PLANT TISSUE CULTURE

Development of an improved and simple shoot tip cryoconservation

protocol for cryobanking of Bacopa monnieri (L.) Wettst. germplasm
Ravi Gowthami1 · Neelam Sharma1 · Ramesh Chandra1 · Jinsa Sara Kurian2 · Era Vaidya Malhotra1 ·
Anuradha Agrawal1,3

Received: 9 June 2023 / Accepted: 4 August 2023 / Editor: Barbara Reed

© The Society for In Vitro Biology 2023

Abstract
Bacopa monnieri (L.) Wettst. is a high value medicinally important plant used as a memory enhancer, the germplasm of which
requires to be conserved long-term due to its over-exploitation. Access to an efficient shoot tips cryoconservation protocol that
results in high post-thaw regrowth and is applicable to many genotypes is a prerequisite for long-term conservation of valuable
germplasm. Experiments were conducted to assess the effect of shoot tip size and cryoconservation techniques, including vitrifi-
cation (V) and droplet-vitrification (DV), to improve post-thaw regrowth in two accessions of B. monnieri. Optimal results after
cryopreservation were obtained when small size (SS – 0.5 × 0.5 mm) shoot tips were excised from cultures pretreated on MS basal
medium supplemented with 0.3 M sucrose (SM) for 4 to 16 wk, precultured on SM medium for 2 d and dehydrated with Plant
Vitrification Solution-2 (PVS2) at 0 °C for 35 min. High post-thaw regrowth of > 50% was obtained using V technique, which was
further enhanced by using DV technique (approximately 80%). Genetic integrity of regenerated plantlets after cryoconservation
using V and DV showed no significant variation among the control and cryoconserved plants using 39 Inter Simple Sequence
Repeats (ISSR) markers. Further, the developed protocol was evaluated in other four accessions and four-fold enhanced post-thaw
regrowth (approximately 80%) and reduction of preparation time by approximately 20 wk was achieved. These results demon-
strated that this improved protocol has potential to be implemented for cryobanking of in vitro germplasm of Bacopa monnieri.

Keywords Cryopreservation · Droplet-vitrification · Genetic stability · Medicinal plant · Probabilistic viability assessment ·
Vitrification

Introduction protocols have primarily been developed for a restricted set

Cryoconservation protocols have been established in various
plant species worldwide, especially using in vitro derived
explants (Panis 2019). It is worth noting that most of these
Ravi Gowthami and Neelam Sharma equally contributed and share
first authorship.
* Anuradha Agrawal
anuagrawal1@yahoo.co.in
1
Tissue Culture & Cryopreservation Unit, ICAR-National
Bureau of Plant Genetic Resources (NBPGR), Pusa Campus,
New Delhi, India
2
Mar Athanasios College for Advanced Studies Tiruvalla
(MACFAST), Pathanamthitta, Kerala, India
3
Present Address: Indian Council of Agricultural Research
(ICAR), National Agricultural Higher Education Project
(NAHEP), Krishi Anusandhan Bhawan-II, Pusa Campus,
New Delhi 110012, India
of genotypes, typically employing vitrification-based tech-
niques. However, for a protocol to be applicable for long-
term conservation of germplasm, it should be applicable to
a wide-range of species and genotypes (Benson 2008; Panis
2019; Panis et al. 2020; Zhang et al. 2023). Such generic
protocols are being used in cryobanking for very few crops,
namely banana, sweet potato, mints, alliums, taro, and tem-
perate fruits (Panis 2019; Wilms et al. 2020; Agrawal et
al. 2022a). Regarding medicinal plants, the research is at
a preliminary stage (Sharma et al. 2020a; Agrawal et al.
2022b), and there are scanty reports on successful cryo-
conservation of medicinal plants of India using shoot tip
explants, including B. monnieri (Sharma et al. 2011, 2017),
Dioscorea deltoidea (Mandal and Dixit 2000; Dixit-Sharma
et al. 2005; Mandal and Dixit-Sharma 2007; Sharma et al.
2022), Gentiana kurroo (Sharma et al. 2021), Holostemma
annulare (Decruse et al. 2004), Picrorhiza kurroa (Sharma
and Sharma 2003), and Rauvolfia serpentina (Ray and

1Vol.:(0123456789)
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 Gowthami et al.
Bhattacharya 2008). The genetic stability aspect of cryo-
conserved shoot tips has been addressed only in a few plants,
including B. monnieri (Sharma et al. 2017), D. deltoidea
(Dixit-Sharma et al. 2005; Mandal and Dixit 2000; Sharma
et al. 2022), and G. kurroo (Sharma et al. 2021).
Bacopa monnieri (L.) Wettst. is an endangered, vegetatively-
propagated medicinal plant, popularly used as a nootropic, or
brain tonic and memory enhancer, in epilepsy and Alzheimer’s
disease, as an anticancer in Ayurvedic Materia Medica, and
used for asthma, ulcers, tumors, gastroenteritis, ascites, enlarged
spleen, anemia, inflammations, and leprosy (Sharma et al.
2017). Recent research focused primarily on cognitive enhanc-
ing effects, specifically mental functions affirm the traditional
Ayurvedic claims about the plants medicinal properties (Saha
et al. 2020; Jeyasri et al. 2020). Besides being used for multiple
ailments, the herb is utilized in phytoremediation (Jauhari et al.
2017). According to the International Union for Conservation
of Nature (IUCN), this species is assessed as Least Concern
(LC), and it is also listed as a threatened species in a Conser-
vation Assessment and Management Prioritization (CAMP)
workshop held at Madhya Pradesh during 2006 (Gowthami et
al. 2021). To ensure its availability for subsequent generations
and pharmaceutical industry, conservation of germplasm of this
precious Indian medicinal herb is essential.
Ex situ conservation of B. monnieri seeds in seed gene-
banks is difficult due to insufficient seed availability and poor
germination (Dubey 2015). Further, propagation through
seeds is low due to short viability (2 mo with frequent seed-
ling death (Volluri et al. 2011). Conventionally, the crop is
propagated vegetatively. Hence, to safeguard this valuable
species, a total of 32 accessions of B. monnieri collected from
different agro-ecological regions of the country have been
conserved in vitro using slow-growth conservation strategies
in In Vitro Active Genebank (IVAG) of ICAR-NBPGR, New
Delhi, India (Sharma et al. 2007, 2012, 2016, 2020b). For
long-term storage, cryoconservation experiments conducted
in the present authors’ laboratory, so far, resulted in low
post-thaw regrowth (0 to 20%) of shoot tips in four acces-
sions (Sharma et al. 2011, 2017). Despite the low levels of
post-thaw regrowth, an important aspect of these results was
the stability of recovered plants as assessed by biochemi-
cal (bacoside) and molecular (RAPD) analyses. However,
the post-thaw regrowth level was low to apply the protocol
for cryobanking of germplasm in genebanks in accordance
with the probabilistic viability assessment for cryobanking
of vegetatively propagated crops (Panis et al. 2005; Volk et
al. 2017). Several factors of pre-, during- and post-cryocon-
servation influences the post-thaw regrowth rates, including
the pretreatment period, the conditions and medium, the type
and size of explants, the preculture media, the duration and
temperature, the osmoprotection duration, the vitrification
solution type, the composition, the exposure duration and
temperature, the regrowth media, and the cryoconservation
13
technique (Gowthami et al. 2023; Zhang et al. 2023). Hence,
the current study was aimed to develop an efficient, easy, and
widely applicable protocol for long-term conservation of B.
monnieri germplasm held in the IVAG of ICAR-NBPGR,
New Delhi, India. The experiments were performed to
assess various parameters, such as (1) size of explants, (2)
pre-growth conditions, (3) cryoprotective dehydration con-
ditions, and (4) cryoconservation technique (comparison of
vitrification, and droplet-vitrification techniques) with the
aim of achieving high-regrowth and genetic stability.
Material and Methods
Plant Material The present study utilized in vitro grown cul-
tures of six accessions of B. monneri (IC353204, IC375976,
IC249250, IC426442, IC342108, and IC468878; hereinafter
denoted as BC4, BCJ, BC1, BCM, BC5, and BC7, respectively)
that were maintained in the IVAG at ICAR-NBPGR, New
Delhi, India. These accessions have been conserved for more
than 20 yr under various normal- and slow-growth conditions
with frequent subculturing every 12 to 24 mo, respectively.
In Vitro Stock Cultures Multiplication
For generation of explants for experiments, in vitro stock
cultures were multiplied on shoot multiplication medium
(MM) composed of Murashige and Skoog (MS; Murashige
and Skoog 1962) basal medium with 0.2 mg ­L−1 6-benzylad-
enine (BA) (Sigma-Aldrich®, St. Louis, MO) and 3% (w/v,
0.08 M) sucrose (Hi-Media®, Mumbai, India) and gelled
using 0.8% (w/v) agar (Hi-Media®) at pH 5.8. Nodal seg-
ments (0.8 to 1.0 cm) were subcultured onto MM medium
every 6 wk and maintained under standard culture room
conditions (SCC) of 25 ± 2 °C and a 16-hr photoperiod
(40 μmol ­m−2 ­s−1) supplied by white, fluorescent tubes
(Philips, Mumbai, India) (Sharma et al. 2007, 2016).
Pretreatment of Donor Plants (DPs) To produce physiolog-
ically suitable stock cultures of DPs for cryopreservation
experiments, nodal segments (about 5 mm in length) from
healthy, green, proliferating shoot cultures on MM medium
were transferred to a pretreatment medium, including (1)
MM medium or (2) MS medium with 0.3 M sucrose at
pH 5.8 (hereafter called SM medium) in glass test tubes
(Borosil, New Delhi, India) for a period of 4 to 24 wk
depending on the experiment. These cultures were main-
tained under SCC.
Shoot Tip Excision and Preculture Shoot tips of two differ-
ent sizes, namely large size (LS) and small size (SS), were
excised from 4- to 24-wk-old (depending on the experiment)
healthy in vitro cultures using a stereobinocular microscope
(Olympus, Haryana, India) in aseptic conditions (Fig. 1A-B).
 SHOOT TIP CRYOCONSERVATION BACOPA MONNIERI
Figure 1.  Effect of size of shoot tips on post-thaw regrowth (%) of
genotype BC4 of Bacopa monnieri (L.) Wettst. by vitrifictaion (V)
and droplet-vitrification (DV) techniques. Shoot tips of large size
(1.5 mm × 1 mm with 4 to 5 leaf primordia) and small size (0.5 mm
X 0.5 mm with 2 leaf primordia) excised from 24-wk-old stock shoots
pretreated on SM medium, precultured on SM medium for 2 d and
The LS shoot tips were excised by removing the overlap-
ping leaves until 1.5 mm × 1 mm size (length × breadth) was
attained such that the meristem was covered with 4 to 6
leaf primordia (Fig. 1B). The SS shoot tips (Fig. 1A) were
excised by removing the overlapping leaves until the glassy
apical dome (0.5 mm × 0.5 mm) with two leaf primordia
remained and the apical domes could be easily observed
under a microscope. Excised shoot tips were inoculated on
a Petri dish (60 mm diameter) (Hi-Media®) with 10 mL of
preculture medium (SM) and precultured for 2 d under SCC.
Vitrification Protocol (V) The protocol by Sharma et al.
(2017) was followed. Precultured shoot tips (SS and LS)
were placed in a sterile 1.8 mL cryovials (Nunc™, Thermo
Fisher Scientific® Roskilde, Denmark) containing 1.0 mL
of cryoprotectant Plant Vitrification Solution 2 (PVS2) com-
prising 0.4 M sucrose (Hi-Media®, Mumbai, India), 30.0%
(w/v) glycerol, 15.0% (w/v) ethylene glycol, and 15.0% (w/v)
dimethyl sulfoxide (Sigma-Aldrich) in liquid MS medium
adjusted to pH 5.8 (Sakai et al. 1990) and depending on the
experiment incubated for 35 min at 0 or 25 ºC. For PVS2
incubation at 0 ºC, cryovials with shoot tips and PVS2 were
placed in a pre-cooled labtop cooler (0 °C) (Nalgene®,
Thermo Scientific, Waltham, MA), and for 25 ºC PVS2
incubation, cryovials with shoot tips and PVS2 were kept in
a cryovial holder in a laminar air flow cabinet (LAF, Klen-
zaids, Mumbai, India). After PVS2 dehydration, cryovials
were placed in a cryostick and quickly placed into liquid
nitrogen (LN) in a Dewar flask for a minimum of 1 h fol-
lowed by rapid thawing in water held at 45 °C for 2 min
and 25 °C for 1 min with constant slow stirring (Sharma
et al. 2011, 2017). After rapid thawing, using a sterile Pas-
teur pipette, the PVS2 solution was removed, and shoot tips
dehydrated with PVS2 for 35 min at 0 ºC prior to cryoconservation.
Vertical bars and error bars represent mean ± SE, respectively. Sig-
nificant differences (P ≤ 0.01) among techniques (lower case) and
between size of shoot tip within the technique (upper case) are repre-
sented by different alphabets by Tukey HSD test.
were rinsed twice with an unloading solution (US; 1.2 M
sucrose in liquid MS medium; pH 5.8) and incubated for
20 min at room temperature. After rewarming, shoot tips
were placed on a sterile dry filter paper (55 mm diameter,
qualitative filter paper, Ambay Biotech, India) held in Petri
dishes (Hi-Media®) (60 mm diameter), and shoot tips were
then plated on 10.0 ml of SM medium in Petri dishes (Hi-
Media®) (60 mm diameter) for regrowth.
Droplet‑Vitrification Protocol (DV) Precultured shoot tips
were placed in a sterile 1.8 mL cryovials containing 1.0 mL
of PVS2 and incubated for 35 min at 0 or 25 ºC (depending
on the experiment) as mentioned in the vitrification proto-
col section. Sterile aluminum foil strips (20 × 5 mm), which
were slightly folded (approximately 2 mm) at one end (for
holding), were placed in a sterile Petri dish (60 mm diam-
eter, (Hi-Media®) and two PVS2 droplets (5.0 µL each) were
placed on each strip using a sterile Pasteur pipette (Thermo
Fischer), and 7 to 8 explants were accommodated in each
drop 2 min prior to the completion of the PVS2 incuba-
tion time. After PVS2 incubation, aluminum foil strips with
PVS2 droplets that were embedded with shoot tips were
immersed into LN directly, and after attaining freezing, the
strips were placed into sterile cryovials (1.8 mL) that were
filled with LN; the filled cryovials were placed in a cryos-
torage box that was then placed in a thermocol packing box
for 1 h. Thereafter, the shoot tips were rewarmed by taking
out the aluminum foil strips from the cryovials and imme-
diately transferring them into 10 mL of US in sterile Petri
dishes (35 mm diameter, Hi-Media, Mumbai) and incubating
for 20 min at 25 ± 2 °C. After 20 min, the shoot tips were
processed as given in V technique. In both the techniques,
13
 Gowthami et al.
controls were represented by the shoot tips dehydrated with
PVS2 followed by US without LN freezing.
Regrowth and Ex Vitro Establishment Initially for 5-d, the
Petri plates with shoot tips were incubated in the dark at
25 ± 2 °C then later moved to SCC. Regrowth was assessed
by recording the total number of shoot tips regrown and
formed normal shoots after 1 to 24 wk of plating (depend-
ing on the experiment). After a wk of sprouting (1.5 mm in
length with 4 leaf stage) regrown shoot tips were inoculated
onto the MM medium for multiplication and root formation.
To evaluate the efficiency of ex vitro establishment, 8 (DV)
to 12- to 18-wk-old (V) plantlets on MM with well-formed
roots (10 to 15 roots with 2 to 3 cm) regenerated after cryo-
preservation were planted in pots as reported by Sharma et
al. (2017).
Standardization of Parameters for Cryoconservation of B.
monnieri Germplasm This study was formulated into three
experiments, including (i) assessment of size of shoot tips,
(ii) effect of donor plant age, pretreatment media, PVS2
dehydration and the cryopreservation technique and (iii)
applicability of protocol to other genotypes. Details are
enumerated below.
Effect of Size of Shoot Tip Shoot tips of two different sizes,
LS and SS, as described above, were used to assess the effect
of shoot tip size on post-thaw regrowth in BC4. LS and SS
shoot tips were dissected from 6-mo-old stock cultures pre-
treated on SM medium and precultured on SM medium for
2 d at SCC followed by PVS2 dehydration for 35 min at 0 ºC
using V and DV techniques. Based on the results of experi-
ment 1, SS shoot tips were used in subsequent experiments.
Effect of Donor Plant Age (Pretreatment Duration), Pretreat‑
ment Media, PVS2 Dehydration, Temperature, and Cryocon‑
servation Technique For this set of experiments, selected
parameters include donor plant age (pretreatment duration):
4 to 24 wk; pretreatment media: MM and MS with 0.3 M
sucrose (SM); PVS2 dehydration temperature: 0 or 25 ºC;
and cryopreservation techniques: V and DV. Two accessions,
BC4 and BCJ, were used to assess effects of these param-
eters on post-thaw regrowth. SS shoot tips excised from 4- to
24-wk-old cultures pretreated on MM or SM media, precul-
tured on SM medium for 2-d on SCC, and subjected to PVS2
incubation for 35 min at 0 or 25 ºC were cryopreserved using
V or DV technique.
Applicability of the Protocol to Other Genotypes Based on
the results of the above experiments on two accessions, the
standardized protocol was tested in the other four acces-
sions of B. monnieri: BCM, BC1, BC5, and BC7. For this,
SS shoot tips dissected from 4- to 16-wk-old cultures were
13
pretreated on SM medium and precultured on SM medium
for 2 d on SCC and subjected to PVS2 dehydration for
35 min at 0 ºC using V and DV technique.
Assessment of Genetic Stability Cryoconserved plants
were analyzed for their genetic stability using Inter Sim-
ple Sequence Repeats (ISSR) markers. Total genomic DNA
was isolated using the CTAB method (Doyle and Doyle
1987) from young leaves of three replicates of stock plants
(4-wk-old) (controls), plants conserved by V and DV tech-
niques. PCR amplification reactions using 39 ISSR primers
(Table 1; Eurofins Genomics India Pvt. Ltd., India) were set
up such that each 10.0 μL reaction volume contained 40 ng
template DNA, 1.0 μL 10X PCR buffer, 250.0 μM of each
dNTP, 1.5 mM M ­ gCl2, 0.8 μM primer, and 1U Taq DNA
Polymerase (Vivantis Technologies, Malaysia) in a thermal
cycler (Gene Pro, Hangzhou Bioer Technology Co., China).
The thermal profile was programmed as: initial denaturation
at 94 °C for 2 min, 37 cycles of denaturation at 94 °C (10 s),
primer annealing temperature depending on primer (30 s),
primer extension at 72 °C (65 s) and final extension at 72 °C
for 10 min. The amplified products were mixed thoroughly
with 6X loading dye and resolved on 2.0% agarose gels in
1X TAE buffer. DNA profiles were photographed on a Gel
Documentation System (GenoSens 2100, Clinx Science
Instruments Co., China). Sharp bands were scored for their
presence (1) or absence (0) and size of the amplified product
was determined by co-electrophoresis of standard molecu-
lar weight marker. Pairwise similarity among the analyzed
samples was estimated using Jaccard’s similarity coeffi-
cient (Jaccard 1908) and cluster analyses were performed
by the unweighted pair group method with arithmetic mean
(UPGMA) using the NTSYS-PC version 2.0 (Rohlf 1998).
Statistical Analysis Each experiment was conducted in a
completely randomized design replicated three times with
15 shoot tips for each treatment in each replication. The data
in percentage was arcsine transformed before analysis. The
data provided represents mean ± standard error (SE). Data
obtained from experiment 2 was analyzed using the General
Linear Model (GLM). Wherever applicable, data was sub-
jected to ANOVA and means were separated using Tukey
HSD test at 95% and 99% confidence interval (CI), to test the
significance of each treatment. Data was analyzed in SPSS
software version 22. The graphical figures were generated
using Excel 2010.
Results and Discussion
Effects of Size of Shoot Tips on Post‑Thaw Regrowth Size
of shoot tips has been found to be a crucial parameter for
obtaining high survival and regrowth after cryopreservation
 SHOOT TIP CRYOCONSERVATION BACOPA MONNIERI

Table 1.  Details of ISSR Serial no Primer Name Primer Sequence (5'—3') Annealing No. of frag- Size of
primers used in the study Temp. (ºC) ments ampli- amplified
fied fragments

1 IS-6 GAG AGA GAG AGA GAG AC 51.0 8 100–900

2 IS-8 AGA GAG AGA GAG AGA GC 51.0 6 200–900
3 IS-9 TGT GTG TGT GTG TGT A 52.5 5 400–1000
4 IS-10 CGA GAG AGA GAG AGA GA 55.8 4 200–600
5 IS-11 CAC ACA CAC ACA CAG​ 52.5 4 300–700
6 IS-61 GAG AGA GAG AGA GAG AT 54.0 3 200–400
7 IS-53 AGA GAG AGA GAG AGA GC 54.0 5 200–800
8 IS-65 AGA GAG AGA GAG AGA GT 52.5 1 600
9 UBC 814 CTC TCT CTC TCT CTC TA 50.0 1 900
10 UBC 820 GTG TGT GTG TGT GTG TC 50.0 1 400
11 UBC 825 ACA CAC ACA CAC ACA CT 52.7 8 200–800
12 UBC 826 ACA CAC ACA CAC ACA CC 52.7 3 200–900
13 UBC 828 TGT GTG TGT GTG TGT GA 50.0 3 300–600
14 UBC 834 AGA GAG AGA GAG AGA GTT​ 50.6 7 200–800
15 UBC 835 AGA GAG AGA GAG AGA GTC​ 50.6 4 200–1000
16 UBC 836 AGA GAG AGA GAG AGA GTA​ 50.0 5 300–900
17 UBC 840 GAG AGA GAG AGA GAG ATT​ 50.6 7 150–1000
18 UBC 841 GAG AGA GAG AGA GAG ATC​ 53.8 8 200–900
19 UBC 842 GAG AGA GAG AGA GAG ATG​ 53.8 6 200–700
20 UBC 843 CTC TCT CTC TCT CTC TGA​ 47.8 2 400–800
21 UBC 858 TGT GTG TGT GTG TGT GGT​ 50.8 4 500–1000
22 UBC 859 TGT GTG TGT GTG TGT GGC​ 51.2 5 250–800
23 UBC 860 TGT GTG TGT GTG TGT GGA​ 52.4 3 600–900
24 UBC 861 ACC ACC ACC ACC ACC ACC​ 56.6 5 300–800
25 UBC 873 GAC AGA CAG ACA GAC A 53.4 4 400–900

(Bettoni et al. 2019a, b; Normah et al. 2019; Pathirana et al.
2021). Preliminary experiments were conducted to know the
post-thaw regrowth pattern of SS and LS exposed to differ-
ent duration of PVS2 dehydration (0, 10, 15, 20, 25, 30, 35,
40, 45, 50 min). A gradual increase in post-thaw regrowth
of cryopreserved shoot tips (SS and LS), as PVS2 dehydra-
tion duration increased from 0 to 35 min was observed and
maximum pos-thaw regrowth was 42.5% (SS) and 30% (LS)
after 35 min of PVS2 exposure using V technique. Further
increase in PVS2 dehydration duration to 40 and 45 min.
resulted in decrease in post-thaw regrowth and 50 min. expo-
sure was lethal to both the sizes. Overall post-thaw regrowth
was higher in SS shoot tips than LS shoot tips across all the
PVS2 exposure durations. Hence, 35 min. of PVS2 exposure
was used in all the experiments in the present study (data
not provided).
Results of experiments conducted to determine optimum
shoot tip size for cryopreservation in BC4 using V and DV
techniques following the previously standardized procedure
with few modifications (Sharma et al. 2017) were presented
in Fig. 1. In the control (+ PVS2,—LN,), both large size (LS)
and small size (SS) shoot tips exhibited near 100% regrowth.
Size of shoot tips significantly affected the post-thaw
regrowth of shoot tips when exposed to PVS2 followed by
LN treatment (+ PVS2, + LN) (Fig. 1). Using V technique,
shoot tips exhibited 30% and 42.5% post-thaw regrowth,
while by DV higher regrowth of 40.8% and 63.2% was
recorded with LS and SS shoot tips, respectively (P ≤ 0.01).
In the previous report, we used approximately 1 mm shoot
tips from 24-wk-old donor plantlets on MM medium that
were precultured on MS medium with 0.3 M sucrose and
achieved 20% post-thaw regrowth. However, in present study
significantly improved the post-thaw regrowth was obtained
by using 0.5 mm shoot tips excised from 24-wk-old cultures
pretreated on MS medium with 0.3 M sucrose and precul-
tured on MS medium with 0.3 M sucrose for 2-d. Also, the
size of the shoot tips was found to significantly affect the
post-thaw regrowth irrespective of the technique used. Simi-
lar increased regrowth after cryopreservation using smaller
shoot tips (1 mm) (compared to 1.5 to 2 mm) has also been
reported in grapes and taro (Marković et al. 2013; Bettoni
et al. 2019a, b). Lower post-thaw regrowth with larger shoot
tips may be ascribed to uneven and improper dehydration
of larger shoot tips leading to death of the tissues due to

13
 Gowthami et al.

formation of ice crystals, less efficient heat exchange dur- Table 2.  Analysis of variance for post-thaw regrowth of cryocon-
ing cooling/thawing (Harding et al. 2009; Jiang et al. 2019; served shoot tips of Bacopa monnieri (L.) Wettst. (BC4)
Panis et al. 2005; Zhang et al. 2015). Small sized shoot Source of variation DF Mean Square
tips avoid the occurrence of temperature gradients between
Donor plant age (DPG) 5 689.22**
external and more internal cell layers, which is detrimental
Pretreatment media (PM) 1 10,563.21**
during cryopreservation. However, shoot tips must be of a
PVS2 temperature (PVS2) 1 14,972.58**
sufficiently large size for rapid and direct regrowth to take
Technique (T) 1 4153.91**
place after cryopreservation (Engelmann 2014; Volk and
DPG x PM 5 32.91**
Caspersen 2007). Generally apical shoot tips, with apical
DPG x PVS2 5 85.74**
dome partially covered or covered with one or two shoot leaf
PM x PVS2T 1 3825.73**
primordial, measuring 1 to 3 mm in length are the preferred
DPG x T 5 9.42*
explants for cryoconservation though the actual size should
PM x T 1 681.60**
be determined for each plant group/species/genotype (Bet-
PVS2 x T 1 347.54**
toni et al. 2021; Normah et al. 2019). The outcome of exper-
DPG x PM x PVS2 5 50.94**
iment clearly indicates SS shoot tips are the better explants
DPG x PM x T 5 34.23**
over LS shoot tips for cryopreservation. Therefore, all the
DPG x PVS2 x T 5 24.68**
preceding experiments were performed using SS shoot tips.
PTM x PVS2 x T 1 1289.47**
DPG x PM x PVS2 x T 5 36.021**
Effect of Donor Plant (DPs) Age, Pretreatment Medium, and
Error 96 3.36
PVS2 Dehydration Temperature The success of cryopreser-
vation is governed by many factors, including physiological **
P ≤ 0.01 * P ≤ 0.05
state of the stock plants, overall plant quality and uniform-
ity, age of the plants in culture, and preconditioning (Bet-
toni et al. 2021; Panis 2019; Wang et al. 2021; Wilms et al. reduced water content of explants by high sucrose thereby
2020). ANOVA analysis showed that post-thaw regrowth of enhancing freeze tolerance (González-Arnao et al. 1998).
shoot tips was significantly influenced by donor plant age, High sucrose concentration in the shoot tips is known to
pretreatment medium, PVS2 dehydration temperature and be helpful to prevent damage caused by dehydration and
cryopreservation techniques (Table 2). In addition, significant freezing (Vandenbussche et al. 1999). Preculture of explants
interactions were observed between all the factors except the with high sucrose reported to increase total soluble protein
interaction of donor plant age and PVS2 dehydration tem- (Suzuki et al. 2006; Wang et al. 2004), total soluble sugar
perature, which was non-significant. Effect of donor plant age (Wang et al. 2004), abscisic acid (Suzuki et al. 2006), and
(4 to 24 wk), pretreatment medium (MM and SM) and PVS2 proline (Suzuki et al. 2006), which may eventually improve
dehydration temperature (0 and 25 ºC) on post-thaw regrowth the post-thaw regrowth of explants (Reed 2008; Engelmann
using V and DV techniques in two accessions (BC4 and BCJ) 2014).
are presented in Table 3. Irrespective of age of DPs, pretreat- Irrespective of the technique, pretreatment media, and
ment medium and PVS2 dehydration temperature, the PVS2 PVS2 dehydration temperature, the regrowth percent-
treated non-frozen controls (-LN) exhibited regrowth of 93.3 age decreased significantly as the age of the shoot tip DPs
to 96.7% in BC4 and 90.1 to 100% in BCJ. In the current increased from 4 to 24 wk using V (60 to 10%, respectively)
study, we found that PVS2 dehydration at low temperature and DV (80 to 20%, respectively) (Table 3). The highest
(0 ºC) was more favorable for cryopreservation compared regrowth of about 60 to 56% occurred when shoot tips were
at higher temperature (25 ºC). These results conform to the isolated from 4 to 12-wk-old cultures using V technique and
earlier reports on medicinal plant Dioscorea deltoidea (Man- 86 to 80% regrowth occurred when shoot tips were excised
dal and Dixit-Sharma 2007), and some tropical plants like from 4 to 16-wk-old cultures using DV technique. Between
banana (Panis et al. 2005) and taro (Sant et al. 2008). The the two techniques tested, in the conditions of this experi-
reduced toxicity of vitrification solution and increased post- ment, DV technique was found to be more efficacious than
thaw survival/regrowth at lower temperatures (0 ºC) may be V in both the accessions with approximately 26% enhanced
due to slower penetration of PVS2 at 0 ºC compared to that post-thaw regrowth. In the present study, the highest post-
at 25 ºC leading to reduced toxic effects, or tissue damage of thaw regrowth was obtained in shoot tips dissected from 4 to
the shoot tips (Reed 2008; Bettoni et al. 2021). 12 wk (V) and 4 to 16 wk (DV) of DPs; and approximately
Pretreatment of DPs on MS medium supplemented with 20% decrease in post-thaw regrowth was observed when
0.3 M sucrose for 4 to 24 wk resulted in high post-thaw shoot tips were excised from older age plant (16 to 24 wk).
regrowth in comparison to the pretreatment on multipli- Similar studies on the impact of donor plant age on post-
cation medium (MM; 0.08 M sucrose). It may be due to thaw regrowth were also published in Dahlia (Gowthami

13
 SHOOT TIP CRYOCONSERVATION BACOPA MONNIERI

Table 3.  Effect of donor plant age, pregrowth media and Plant Vitrification Solution 2 (PVS2) incubation temperature on post-thaw regrowth of
two accessions of Bacopa monnieri (L.) Wettst. (BC4 and BCJ) using vitrification (V) and droplet vitrification (DV) techniques
Donor plant age Preg. media PVS2 Incu. temp BC4 BCJ
Control (PVS2 V (LN) DV (LN) Control (PVS2 V (LN) DV (LN)
treated) treated)

4 wk SM 0 ºC 96.7 ± 0.8a 60.1 ± 1.2a 86.7 ± 1.7a 100 ± ­0a 60.5 ± 0.7a 86.5 ± 1.0a
25 ºC 93.5 ± 1.2ab 30.5 ± 0.7e 40.5 ± 1.5e 96.7 ± 2.4abc 40.1 ± 1.0 cd 59.4 ± 0.8d
MM 0 ºC 93.3 ± 2.0ab 42.7 ± 1.3c 50.3 ± 0.7d 93.3 ± 1.3abc 46.7 ± 0.9b 54.7 ± 1.2e
25 ºC 93.3 ± 1.2ab 30.5 ± 1.4e 33.3 ± 1.2fg 90.5 ± 1.7cd 36.2 ± 1.0e 43.5 ± 2.0gh
8 wk SM 0 ºC 96.7 ± 0.6a 56.4 ± 1.8a 80.1 ± 2.6b 100 ± ­0a 60.1 ± 0.1a 83.6 ± 1.3ab
25 ºC 93.3 ± 1.0ab 30.5 ± 0.4e 40.5 ± 1.0e 96.5 ± 1.5abc 40.1 ± 1.3cd 52.8 ± 1.8f
MM 0 ºC 96.7 ± 2.1a 36.3 ± 2.5d 44.0 ± 0.7d 90.7 ± 1.9cd 46.7 ± 0.6b 48.7 ± 2.1fg
25 ºC 93.3 ± 0.6ab 30.0 ± 0.6e 33.3 ± 1.5fg 90.0 ± 1.5cd 30.6 ± 0.7ef 40.8 ± 1.4hi
12 wk SM 0 ºC 96.7 ± 1.1a 56.4 ± 0.8a 80.1 ± 2.0b 98.5 ± 1.2ab 58.1 ± 1.2a 80.8 ± 3.2b
25 ºC 93.3 ± 0.3ab 30.0 ± 0.4e 40.0 ± 1.0e 96.5 ± 0.4abc 30.3 ± 0.6f 51.5 ± 2.3ef
MM 0 ºC 96.7 ± 0.1a 36.6 ± 0.3d 40.7 ± 2.1e 90.0 ± 1.0 cd 37.6 ± 2.4d 40.5 ± 1.0hi
25 ºC 93.3 ± 1.5ab 20.3 ± 2.4f 30.3 ± 3.0gh 90.0 ± 1.5 cd 23.3 ± 1.0gh 36.7 ± 1.2ij
16 wk SM 0 ºC 96.7 ± 1.0a 50.5 ± 0.5b 80.1 ± 1.8b 96.5 ± 2.1abc 46.7 ± 1.8b 79.5 ± 2.5b
25 ºC 93.3 ± 0.7ab 29.5 ± 1.6e 33.4 ± 2.0 fg 92.8 ± 1.8abc 30.1 ± 0.8f 46.7 ± 2.2fg
MM 0 ºC 93.3 ± 1.8ab 30.2 ± 1.0e 36.5 ± 0.4ef 90.0 ± 1.0cd 37.6 ± 1.5d 34.6 ± 0.8jk
25 ºC 96.7 ± 0.1a 20.2 ± 1.2f 30.1 ± 2.2gh 90.0 ± 1.0cd 20.0 ± 0.5hi 20.6 ± 0.2m
20 wk SM 0 ºC 96.4 ± 0.2a 42.8 ± 2.3c 63.5 ± 3.4c 96.2 ± 1.5abc 43.5 ± 1.5bc 66.7 ± 1.0c
25 ºC 96.7 ± 0.8a 29.3 ± 2.1e 30.4 ± 2.5gh 90.7 ± 3.1bcd 24.4 ± 0.9gh 30.3 ± 1.0kl
MM 0 ºC 93.5 ± 1.2ab 30.1 ± 0.7e 32.6 ± 1.5fgh 90.7 ± 2.2bcd 26.7 ± 2.6fg 33.3 ± 1.3jk
25 ºC 90.1 ± 1.9b 16.6 ± 0.4f 25.1 ± 1.4j 90.5 ± 1.9 cd 13.5 ± 1.1jk 20.5 ± 2.4 m
24 wk SM 0 ºC 96.5 ± 1.2a 42.5 ± 1.1c 63.2 ± 1.6c 96.1 ± 1.3abc 43.3 ± 1.4bc 65.1 ± 0.6c
25 ºC 96.7 ± 0.6a 29.1 ± 1.2e 28.1 ± 1.0i 90.5 ± 1.3 cd 16.7 ± 1.2ij 26.7 ± 0.3 l
MM 0 ºC 93.3 ± 1.0ab 30.1 ± 1.7e 30.0 ± 1.3gh 90.8 ± 1.6bcd 20.8 ± 2.9hi 30.9 ± 1.1kl
25 ºC 93.3 ± 2.1ab 10.6 ± 3.2g 22.7 ± 0.5k 90.1 ± 1.4cd 10.0 ± 1.7k 20.8 ± 0.5m

Values are mean ± SE

Values superscripted with same letter in each column are not significantly different (P ≤ 0.01)

et al. 2023), where shoot tips from donor plants that were 6
and 8 wk old showed higher regrowth rates (23.33%) than
those from cultures that were 4 and 12 wk old and 13% and
6.67%, respectively. This confirms the importance of physi-
ological stage of the stock plants (DPs) for increasing the
tolerance to cryogenic procedures (Reed 2008). Although
there is no consensus on the culture age or pretreatment
duration for successful cryoconservation due to the wide
variety of techniques and culture systems used, it is apparent
that physiological differences between explants in terms of
pretreatment conditions have an impact on cryoconservation
success; however, such differences may be species-specific.
Applicability of the Protocol to Other Genotypes Results
of experiments to assess applicability of above standard-
ized protocol to other accessions and optimizing pretreat-
ment duration for best post-thaw regrowth were presented
in Table 4. Non- frozen shoot tips (-LN; controls) exhib-
ited approximately 90% regrowth in all the accessions
with normal growth (Fig. 2C, F, G). For cryoconserved
shoot tips, high post-thaw regowth of 76 to 86% using DV
and ≥ 45% using V technique was observed in all the six
accessions tested using SS from 4 to 16-wk-old cultures
pretreated on SM medium depending on the genotype and
age of the donor plants (DPs). Genotypic dependent success
of cryopreservation protocol is reported in several species
(Sharma et al. 2017, 2022; Vollmer et al. 2019; Wilms et al.
2020). Though statistically significant difference in regrowth
levels were observed in few accessions, the difference was
very low, including 3 to 10% using V and 3 to 4% using DV
(P ≤ 0.01). This indicates the feasibility of its application in
long-term conservation of germplasm, hence the protocol
being generic and widely applicable.
Comparison of Efficacy of Vitrification (V) and Droplet‑Vitrifi‑
cation (DV) Protocols Improved regrowth of 76 to 86% (DV)
and 40 to 60% (V) in all the six accessions in comparison to
0 to 20% regrowth following previously developed protocols

13
 Gowthami et al.

Table 4.  Mean regrowth of six accessions of Bacopa monnieri (L.) Skoog medium supplemented with 0.3 M sucrose (SM medium) and
Wettst. (BC4, BCJ, BCM, BC1, BC5, BC7) using vitrification (V) precultured on SM medium for 2 d at 25 °C and dehydrated with
and droplet vitrification (DV) techniques. Shoot tips (0.5 X 0.5 mm) PVS2 for 35 min at 0 °C prior to cryopreservation
excised from 4 to 16 wk donor plants pretreated on Murashige and

Accessions Donor plant age and technique

4 wk 8 wk 12 wk 16 wk
V DV V DV V DV V DV

BC4 60.1 ± 1.2a 86.7 ± 1.7a 56.4 ± 1.8ab 80.1 ± 2.6ab 56.4 ± 0.8ab 80.1 ± 2.0a 50.5 ± 0.5a 80.1 ± 1.8a
BCJ 60.5 ± 0.7a 86.5 ± 1.0a 60.1 ± 0.1a 83.6 ± 1.3a 58.1 ± 1.2a 80.8 ± 3.2a 50.7 ± 1.8a 79.5 ± 2.5a
BCM 57.5 ± 0.5ab 85.0 ± 2.8a 57.5 ± 1.1ab 80.0 ± 0.4ab 55.0 ± 1.5b 79.8 ± 1.7a 46.0 ± 1.5b 79.0 ± 1.0a
BC1 60.0 ± 0.6a 84.6 ± 2.5a 56.5 ± 1.0ab 82.1 ± 0.2a 56.4 ± 1.0ab 79.9 ± 0.8a 50.0 ± 1.1a 76.4 ± 1.1b
BC5 60.0 ± 0.2a 82.0 ± 2.0ab 57.8 ± 0.5ab 81.0 ± 0.3ab 52.6 ± 0.7c 80.0 ± 0.4a 46.4 ± 1.2b 77.7 ± 1.5b
BC7 60.0 ± 1.1a 84.6 ± 0.6ab 56.8 ± 1.3ab 81.8 ± 1.2ab 55.5 ± 0.9b 79.5 ± 0.9a 47.0 ± 1.1ab 79.5 ± 1.4a

Values are mean ± SE

Values superscripted with same letter in each column are not significantly different (P ≤ 0.01)

Figure 2.  Comparison of efficacy of vitrification (V) and droplet-
vitrification (DV) techniques in genotype BCJ of Bacopa monnieri
(L.) Wettst. (A-C) regrowth of shoot tips after cryopreservation (A)
vitrification (B) droplet-vitrification (C) control (D-E) Proliferation
of regenerated shoot tips (D) vitrification (E) droplet-vitrification
(F) control (G) Shoot multiplication on MM medium comrprising
Murashige and Skoog basal medium with 0.2 m ­ gL−1 6-benzyladenine
(H-J) Ex vitro establishment of cryoconserved shoot tips (H) Plantlets
for ex vitro transfer (I) Hardening in protrays (J) Ex vitro regeneration
in pots. Scale bar = 1 cm.

using V technique and LS shoot tips was obtained (Sharma
et al. 2011, 2017) (Table 4). Besides enhanced regrowth,
the advantage of DV protocol standardized in present study
was consistency and quick response (2 to 3 wk) compared
to present and previous V technique wherein sometimes the
regrowth occurred very late (up to 8 to 24 wk). Importantly
in all the treatments, irrespective of the technique, initially
one sprout with shoot tip was obtained without any callus
formation which is desirable as it indicates growth from pre-
existing meristem in the shoot tip (Fig. 2A to F). Plantlets
with multiple shoots and roots amenable for ex vitro transfer
were formed after 8 wk (DV) and 12 to 18 wk (V) of shift-
ing to MM medium (Fig. 2G, H). Irrespective of the cryo-
conservation technique and treatments, all the regenerated
plantlets transferred ex vitro were successfully established
with 100% survival in portrays (Fig. 2I) and pots (Fig. 2J),
this justifies the maintenance of regeneration capacity after
cryoconservation.
Based on the present results of the six accessions, it is
evident that the highest regrowth of approximately 80%
was obtained when shoot tips were excised from 4 to
12-wk-old cultures pretreated on SM medium, exposed to
35 min of PVS2 dehydration at 0 ºC using DV and ≥ 50%
using V in all the six accessions. Significant improvement
in post-thaw regrowth using DV, as compared to V con-
forms to the earlier similar reports in a number of species
including endangered medicinal plant (Panis 2019; Sharma
et al. 2021).

13
 SHOOT TIP CRYOCONSERVATION BACOPA MONNIERI

Probabilistic Viability Assessment for Cryobanking Based
on high post-thaw regrowth (≥ 80%) using DV in above
experiments and according to probability equation (Volk
et al. 2017) the number of shoot tips calculated to be cry-
oconserved in each vial to obtain at least one plant after
cryoconservation was presented in Table 5. Considering
all the accessions 5 to 7 shoot tips are sufficient to obtain
one viable growing plant at higher confidence level of 99%
and 95% using DV while with V technique 14 to 24 shoot
tips at 99% confidence level and 11 to 18 shoot tips at 95%
confidence level are required. Thus, the standardized proto-
col has the ability for long-term conservation of germplasm
of B monnieri. Cryobanking of these accessions, therefore,
is being done with 10 shoot tips per vial and with target
to cryobank at least 200 shoot tips in 3 to 4 independent
experiments.
Genetic Stability of Cryoconserved Plants Genetic stabil-
ity is one of the most vital factors to consider for long-
term conservation of vegetatively propagated plants. In
the present study, out of the 39 ISSR markers tested, 25
primers (64.10%) amplified in both the accessions (BC4
and BCJ), generating a total of 113 bands per accession,
with an average of 4.52 bands per primer. Among the
tested primers a maximum of eight bands were generated
by primers IS-6, UBC-825, and UBC-841; while, single
bands were generated by primers IS-65, UBS-814, and
UBC-820. Out of the total 113 bands amplified, 105 were
found to be monomorphic (92.92%). Size of the amplified
fragments ranged from 150 to 1000 bp. Jaccard’s similar-
ity co-efficient based analysis revealed genetic similarity
ranging from 94 to 99.8% between the tested plants of an
accession. No significant variation was observed in the
banding patterns of the control and cryoconserved plants,
indicating the robustness of the developed protocols.
Representative amplification profiles of ISSR primers are
presented in Fig. 3. Similar molecular analysis of genetic
stability of cryoconserved plants have been carried out
in several plant species and have revealed no instability
(Agrawal et al. 2014; Kaya and Souza 2017; Sharma et al.
2017, 2021). In B monnieri, genetic stability studies have
also been reported in in vitro conserved, encapsulated and
cryoconserved shoot tips using biochemical and molecular
(RAPD and ISSR) markers (Muthiah et al. 2013; Sharma
et al. 2012). In our previous study, in B. monnieri, clonal
fidelity of plants regenerated after cryoconservation by
vitrification technique was proved by RAPD markers
(Sharma et al. 2017). Thus, our results of genetic stability
assessment using ISSR markers are in conformity with
our previous reports in plant species including B monnieri
(Sharma et al. 2012, 2021, 2022).
Figure 4 represents the outline of the protocol stand-
ardized in the current study. In the earlier reported pro-
tocol (Sharma et al. 2017), approximately 26 wk were
needed for obtaining shoot tips for cryoconservation. In
contrast, present protocol requires only 4 to 12 wk, thus
cut-down the total period by 12 to 20 wk with signifi-
cantly enhanced post-thaw regrowth of approximately
80% using DV and 40% using V compared to 0 to 20%
reported in previous study (Sharma et al. 2011, 2017).
In most of the accessions, less difference of post-thaw
regrowth was observed in shoot tips sourced from cul-
tures pretreated on MS medium with 0.3 M sucrose for
4 to 16 wk. This ensures larger time window and ready
availability of more shoots for cryobanking. The results
comply with the previous report on sweet potato (Wilms
et al. 2020), wherein generic droplet-vitrification protocol
gave > 40% regeneration rate in > 70% of the cultivars.

Table 5.  Predicted numbers of shoot tips required to be cryocon- (0.5 X 0.5 mm) excised from 4 to 16 wk donor plants pretreated on
served to ensure a 95 or 99% reliability of recovering at least one Murashige and Skoog medium supplemented with 0.3 M sucrose
viable plant using V and DV in six accessions of Bacopa mon- (SM medium) and precultured on SM medium for 2 d at 25 °C and
nieri (L.) Wettst. (BC4, BCJ, BCM, BC1, BC5, BC7). Shoot tips dehydrated with PVS2 for 35 min at 0 °C prior to cryopreservation

Accn 4 wk 8 wk 12 wk 14 wk
V DV V DV V DV V DV
95% 99% 95% 99% 95% 99% 95% 99% 95% 99% 95% 99% 95% 99% 95% 99%

BC4 11 14 5 5 11 14 6 7 11 14 6 7 18 24 6 7
BCJ 11 14 5 5 11 14 5 6 11 14 6 7 18 24 6 7
BCM 11 14 5 6 11 14 6 7 14 18 6 7 18 24 6 7
BC1 11 14 5 6 11 14 5 6 11 14 6 7 18 24 6 7
BC5 11 14 5 6 11 14 5 6 14 18 6 7 18 24 6 7
BC7 11 14 5 6 11 14 5 6 14 18 6 7 18 24 6 7

13
 Gowthami et al.
Figure 3.  Representative gel image of amplification of two acces-
sions with ISSR primer UBC-826; Lane L: 100 bp DNA marker;
1–18 BC-1 (1–3—cryoconservation control at 0 ºC; 4–6—cryocon-
servation control at 25 ºC; 7–9 –liquid nitrogen (LN)—vitrification at
0 ºC; 10 -12 LN-vitrification at 25 ºC; 13–15 LN- droplet-vitrifica-
Fig. 4  Outline of the improved and simple shoot tip cryoconserva-
tion protocol for cryobanking of Bacopa monnieri (L.) Wettst. germ-
plasm standardized in the present study. (A) Pretreatment of donor
plants on Murashige and Skoog medium supplemented with 0.3 M
sucrose (SM medium) for 4 to 16 wk (B) Excision of shoot tips (SS)
(0.5 × 0.5 mm) (C) Preculture of excised shoot tips on SM medium
for 2 d under standard culture conditions (SCC) (D) Plant vitrifica-
tion solution 2 (PVS2) dehydration for 35 min at 0 ºC using labtop
cooler (E) Placement of 7 to 8 shoot tips in each drop (2 min before
the end of the PVS2 incubation time) on a aluminum foil with PVS2
droplets (F) Direct plunging of aluminum foil strips with shoot tips in
13
tion at 0 ºC; 16–18 LN-droplet-vitrification at 25 ºC); 19–36 – BC-2
(19–21—cryoconservation control at 0 ºC; 22–24—cryoconservation
control at ­25o C; 25–27 – LN—vitrification at 0 ºC; 28–30—LN vitri-
fication at 25 ºC; 31–33—LN-droplet-vitrification at 0 ºC; 34–36- LN
droplet-vitrification at 25 ºC).
the PVS2 droplets into LN and immediately placed into sterile cryo-
vials filled with LN in cryostorage box (G) Storage in LN (H-I) Rapid
rewarming of shoot tips by rapidly removing the frozen aluminum
foil strips from the cryovials and immediately transferring into an
10 mL of unloading solution in for 20 min in sterile Petri dishes at
room temperature (J) Placement of shoot tips on a sterile dry filter
paper placed in sterile Petri dishes (K) Plating of shoot tips on 10 mL
of SM medium in Petri dishes for regrowth (L) Transfer of growing
shoot tips onto the multiplication medium (MM) in glass test tubes
(M) Ex vitro transfer of regrown shoot tips.
 SHOOT TIP CRYOCONSERVATION BACOPA MONNIERI
Conclusion
The distinct advantages of protocol developed in this paper
include the high regrowth rate of ≥ 80% in comparison to the
0 to 20% in previous methods with simplicity of the protocol,
including decreased pretreatment duration for cryoconserva-
tion (pretreatment for 4 wk instead of 24 wk), quick regrowth
(2 to 3 wk instead of 8 to 24 wk) and plantlet development (8
wk instead of 12 to 18 wk). The protocol also has wider appli-
cability across genotypes and genetic stability of regenerated
plantlets from cryoconserved shoot tips as assessed by ISSR
markers. Thus, the improved DV protocol standardized here
has the potential for long-term conservation of germplasm of
the precious high value medicinal plant, B. monnieri.
Acknowledgements The authors thank the Indian Council of Agricul-
tural Research (ICAR) and Director, ICAR-NBPGR, New Delhi, India,
for providing the funding and research facilities under an in-house
project, for carrying out this work. The technical assistance provided
by Mr Sanjeev is gratefully acknowledged.
Author Contribution Cryopreservation protocol development: NS
and RG equally contributed and share first authorship, RG, NS and
AA conceived, planned and managed the experiments, analyzed the
results prepared and edited the manuscript, RG, RC and JSK prepared
explants. Genetic stability assessment: EVM performed genetic stabil-
ity experiments. AA managed experiments and critically edited the
manuscript.
Data Availability All data generated or analysed during this study are
included in this published article.
Declarations
Competing Interest The authors declared no conflicts of interest.
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