Plant Cell Tiss Organ Cult (2017) 129:313324
DOI 10.1007/s11240-017-1179-6
ORIGINAL ARTICLE
In vitro shoots fromroot explant, their encapsulation, storage,
plant recovery andgenetic fidelity assessment ofLimonium hybrid
Misty Blue: aflorist plant
ShankhamalaBose1 JoydeepKarmakar1 DevanandP.Fulzele2 UtpalBasu1
TapasKumarBandyopadhyay1
Received: 6 September 2016 / Accepted: 23 January 2017 / Published online: 7 February 2017
Springer Science+Business Media Dordrecht 2017
Abstract Limonium Misty Blue is an interspecific
hybrid of Limonium latifolium and L. bellidifolium and has
a huge demand in floriculture business as both fresh and
dry flowers with stunning purple-blue blooms. The propa-
gation only through vegetative means restrict the popu-
larization of this plant to the flower growers. We therefore
optimized an efficient micropropagation protocol for direct
organogenesis from root explants, as leaf is not conduc-
ible to respond in culture. 61.43% of root explants directly
formed shoot buds on their surface after 4-weeks of culture
in media containing MS, 43.82 mM sucrose 2.22 M
BA and 1.07M NAA. The shoot buds failed to differen-
tiate into healthy shoots unless the previous medium was
replaced by full strength MS, and 87.64mM sucrose along
with 0.44 M BA and 1.07 M NAA. Encapsulations of
juvenile shoots were carried out by 3% sodium alginate
and 100 mM C aCl2 which were again successfully stored
at 4C for 30 days along with 56.79% of plant recovery
in MS+0.44 M BA+4.5 M IBA+87.64 mM sucrose
containing medium. 150 synthetic seed derived full grown
plants were successfully acclimatized in green house,
where a total of 101 plants survived after secondary hard-
ening. The ISSR analysis revealed genetic homogeneity of
synthetic seed derived hardened plants.
* Tapas Kumar Bandyopadhyay
tapas@klyuniv.ac.in; bantapas@hotmail.com
1
Department ofMolecular Biology andBiotechnology,
Faculty ofScience, University ofKalyani, Kalyani,
WestBengal741235, India
2 with stunning purple-blue blooms has a huge demand in
Plant Biotechnology & Secondary Metabolites Section,
NA&BTD, Bhaba Atomic Research Centre, Mumbai400085,
India
Keywords Limonium Misty Blue Root Direct
organogenesis Histology Synthetic seed ISSR
Abbreviations
MS Murashige and Skoog (1962)
MS Half-strength Murashige and Skoog
NAA -Napthalene acetic acid
BA 6-Benzyladenine
Kn Kinetin
2-iP 6-(,-Dimethylallyalamino) purine
IBA Indole-3-butyric acid
ISSR Inter simple sequence repeat
PCR Polymerase chain reaction
CTAB Cetyl trimethylammonium bromide
DMSO Dimethyl sulfoxide
Introduction
The genus Limonium encompasses a wide range of exotic
flowering species in Plumbaginaceae family. Commonly
known by names as sea-lavender, statice or marsh-rosemary
the members of this genus are distributed in various parts
of Europe, Asia, Africa, Australia and North-America.
Since most species of Limonium are reported to be salt and
drought tolerant (Rathinasbapathi 2000), they can be easily
grown on coastal areas, salt marshes, saline soil and water
deficient areas where other flowering plants are not condu-
cive to grow. With increasing demand of Limonium culti-
vars as cut flower, the interspecfic hybrid of Limonium lati-
folium and L. bellidifolium, Misty Blue was first released
in 1984 (Kunitake and Mii 1994). This particular hybrid
floriculture business as both fresh and dry flowers. Com-
mercially one bunch containing ten stems of Misty Blue
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costs around US $18 (http://www.wholesaleflowersover-
night.com, accessed 1st September 2016). Conventional
propagation through root cuttings took 68 months time
and usually gave 2030% success (Topoonyanont et al.
2000); therefore, invitro culture techniques have been used
by many companies, that produce Limonium flowers, for
multiplying their stocks and as well as for breeding pur-
poses (Harazy etal. 1985; Kunitake and Mii 1990).
Although many protocols for in vitro regeneration of
different species of Limonium have been reported, most
of them are based on axillary bud proliferation from node
(Martn and Prez 1992, 1995) and inflorescence axis
(Topoonyanont et al. 2000; Xiao and Kozai 2006; Dam
etal. 2013).
But the global cut flower industry flourishes based on
novelty. Recent advances in biotechnology including the
direct insertion of genes into plants have offered the scope
to develop improved germplasm by overcoming the dif-
ficulties to cross distant taxa (Kon et al. 2013). Genetic
engineering is providing a valuable tool for expansion
of flowering gene pool to promote the generation of new
commercial varieties (Tanaka et al. 2005). However, very
few reports (Mercuri et al. 2001; Igawa et al. 2002; Yuan
et al. 2014) are available for the application of genetic
transformation of Limonium for the development of new
cultivars. The development of such technologies largely
depends on efficient regeneration of plantlets from suitable
explant (Kon et al. 2013). The lack of efficient in vitro
regeneration systems is the principal bottleneck that ham-
pers genetic transformation of Limonium because only few
reports are available for direct (Seelye et al. 1994; Huang
etal. 2000; Jeong etal. 2001; Dam etal. 2010) or indirect
organogenesis and somatic embryogenesis (Aly etal. 2002;
Igawa et al. 2002). However, only two reports on in vitro
propagation (Lian etal. 2002; Topoonyanont etal. 2000) of
this important interspecific hybrid, Limonium Misty Blue
and lack of information on repetitive somatic embryogen-
esis or organogenesis of the same restrict the large scale
propagation in bioreactor and further research in the field of
genetic transformation. On the above background this inter-
specific Limonium hybrid was chosen to develop a protocol
for organogenesis from suitable explant. In most of the pre-
vious works noted above on Limonium tissue culture, leaf
was found to be most suitable explant for establishment of
culture and further regeneration of plantlets either through
organogenic or embryogenic pathway in different species,
as leaves are available in abundance throughout the year
and easy to sterilize. In our preliminary study with Limo-
nium hybrid Misty Blue, leaf explant did not show any
kind of morphogenetic response, as an alternative, roots
obtained from mature plant were tried for direct organo-
genesis. Recently the use of roots as a source of explants
for induction of organ formation have been reported in a
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Plant Cell Tiss Organ Cult (2017) 129:313324
number of species (Atta et al. 2009; da Cruz et al. 2014;
Jani etal. 2015) and regeneration of shoots from roots have
become a valuable system to study the molecular regula-
tion of cell potency and competency (Shemer etal. 2015).
However, it has some advantages over other explants as
in vitro transgenic hairy roots, obtained after inoculation
with Agrobacterium rhizogenes, may be used for recur-
rent organogenesis for long term multiplication (Gutierrez-
Pesce etal. 1998) and generation of plants with improved
phenotype (Handa 1992).
The artificial seed technology is becoming popular due
to its comprehensive application in germplasm conserva-
tion and for exchanges between countries in the floricul-
ture trade (Siew et al. 2014). The artificially encapsulated
somatic embryos, shoot buds, axillary shoot or any other
meristematic tissues can be used as functional mimic seed
for sowing because it possesses the ability to convert into
a plant under in vitro or ex vitro conditions (Sharma and
Shahzad 2012), and can be cryopreserved using encapsula-
tion-dehydration technique as in mint (Martn etal. 2015),
chrysanthemum (Teixeira da Silva etal. 2015), apple (Feng
et al. 2013) etc. Moreover, feasible commercialization of
encapsulation technology requires the direct sowing of
synthetic seeds in non-aseptic conditions. Very recently,
a successful protocol for both production and sowing of
chrysanthemum synthetic seeds in non-aseptic conditions
(Hung and Dung 2015) opened up a new vista in com-
mercialization of synthetic seed technology in ornamental
horticulture. Since Limonium hybrid Misty Blue is sterile
and does not form any viable seeds therefore, encapsulated
shoots with efficient endosperm can substantiate the need
of zygotic seeds.
Assessment of clonal fidelity is an important step among
the invitro regenerated plants for their large scale propaga-
tion. Irrespective of the advantages of invitro propagation,
genetic instability has been documented in tissue culture-
generated plants (Larkin and Scowcroft 1981). Of several
molecular markers used for evaluation of genetic fidelity of
in vitro regenerated plants, inter-simple sequence repeats
(ISSR) marker analysis are the simple, quick and cost-
effective method (Lakshmanan et al. 2007). Recently this
marker system have been successfully used to study genetic
homogeneity among invitro regenerated plantlets of many
species (Rai et al. 2012; Ramakrishnan et al. 2014; Agar-
wal et al. 2015; Ramrez-Mosqueda and Iglesias-Andreu
2015; Saha etal. 2015; Viehmannova etal. 2016).
In this background, our main objective of this study was
to optimize an efficient protocol for induction of shoots
from in vivo root of Limonium hybrid Misty Blue, their
encapsulation to form synthetic seeds, storage and conver-
sion in artificial culture medium. The protocol was further
improved by ISSR marker analysis to evaluate genetic
homogeneity of regenerated plantlets. To our knowledge in
Plant Cell Tiss Organ Cult (2017) 129:313324 315
this particular hybrid, it is the first report on direct organo-
genesis from root and production of synthetic seeds.
Materials andmethods
Plant material andsurface sterilization
Young plantlets of Limonium Misty Blue hybrid plants,
obtained from Spa Flora Pvt. Ltd, (Bengaluru-560095,
India), were grown under greenhouse condition in
earthen pots filled with sterile cocopeat and soilrite mix-
ture in 3:1 ratio and optimum care was taken to avoid any
fungal and bacterial contamination. After proper cleaning
with running tap water and treating with Tween 20 [1%
(v/v)] and sodium hypochloride solution [3% (v/v)], a
three months old green-house grown plant designated as
Fig.1Direct initiation and subsequent development of shoot
buds from root explant of Limonium Misty Blue a A 4-month old
plant from hydroponic culture used for collection of root explant,
b clump of shoot buds c single shoot buds formed directly on the
wounded surfaces of root in 1/2 MS +
2.22 M BA + 1.07 M
NAA+43.82mM sucrose containing medium, d formation of shoot
buds on the tip (black arrow) and base (white arrow) of lateral roots,
mother plant, was cultured in hydroponics supplemented
with modified Hoagland Solution (Hoagland and Arnon
1950) and 4.92 M IBA. The hydroponic culture was
incubated in plant tissue culture growth room at 2325C
and 5070% relative humidity under a 16-h photoperiod
with a light intensity of 55 molm2s1 (Philips, India)
maintaining sterile condition as far as possible. The
freshly developed stout roots, obtained from a single one-
month old hydroponics culture, were used as explants for
subsequent experimentation on organogenesis (Fig. 1).
The juvenile root tips, having length of 45 cm, were
thoroughly washed under running tap water for 15 min,
and then dipped in 1% (v/v) teepol solution for 5 min.
The roots were subsequently treated with 0.1% (w/v)
mercuric chloridesolution for 10min (Himedia, India) in
the laminar air flow cabinet and rinsed with sterile dou-
ble distilled water for at least five times to ensure proper
e a shoot bud in maturation medium (MS+0.44 M BA+1.07 M
NAA +87.64 mM sucrose) with contrasting pigment deposition
(arrow) after 2-weeks marks the differential growth pattern, f leaf
primordium with chlorophyll deposition at the top (arrow) of highly
differentiated shoot bud after 3-weeks, g 4 weeks old matured micro-
shoot with distinct leaf, ready for encapsulation
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316
removal of any traces of mercuric chloride and cleansing
agents. Surface sterilized roots were then excised in small
pieces (approx. 2 cm each) and pricked several times
using a sterile scalpel to facilitate rapid organogenesis.
Induction ofshoot bud formation andmaturation
The root explants were cultured on basal MS
(Murashige and Skoog 1962) solid media containing
43.82 mM sucrose (Merck, India), and 0.8% (w/v) agar
powder (Bacteriological grade, Himedia, India) with dif-
ferent plant growth regulators (PGRs), i.e., 6-benzylad-
enine (BA), kinetin (Kn), 6-(, -dimethylallyalamino)
purine (2-iP) and naphthalene acetic acid (NAA) either
singly or in combinations. However, in the preliminary
experiments (data not shown) it was observed that the
media supplemented only with NAA, BA, 2-iP or Kn
failed to initiate organogenesis after long day of culture,
therefore the combined treatment of NAA (1.07 M)
along with BA (0.442.22M), 2-iP (0.492.46M) and 2028C with 55 mol m2 s1 irradiance and 7080%
Kn (0.462.32M) were studied further.
After 4 weeks of culture, the explants with shoot buds
were transferred to another set of media for transition into
mature shoots which consisted of full strength MS basal
salt formulation (Murashige and Skoog 1962), 87.64mM
sucrose, 0.8% (w/v) agar along with different combina-
tions of BA (0.0442.22M) and NAA (1.07M). house by a process called secondary hardening. The plants
Encapsulation ofjuvenile shoots
The juvenile shoots approximately 510 mm were dis-
sected from root explant and used for synthetic seed pro-
duction. For encapsulation, the gelling matrix was pre-
pared with different percentages (14%, w/v) of sodium
alginate (Sigma, USA) in liquid MS with 87.64 mM
sucrose, and 50125 mM CaCl2 (Merck, India) as com-
plexing agent. All the chemicals and glass goods used
in this experiment were sterilized in autoclave and the
whole encapsulation procedure was done under laminar
air flow hood to avoid contaminations. At first the juve-
nile shoot-tips were suspended in sodium alginate solu-
tion and then carefully dropped into C aCl2 solution using
a pipette. For completion of the complexation process
the beads in CaCl2 solution was stirred at 120 rpm for
15min using a rotary shaker (Sonar, Delhi, India), which
resultedin the formation of firm spherical artificial beads
with encapsulated shoot-tips. Thereafter, CaCl2 solution
was decanted off and beads were washed thrice with ster-
ile distilled water to remove any traces of excess calcium
chloride.
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Plant Cell Tiss Organ Cult (2017) 129:313324
Recovery ofplantlets fromtheencapsulated shoots
After storing at various temperatures (0, 4 and 25C) for
certain time interval (15, 30, 45 and 60days) the artificial
seeds were inoculated in solidified MS basal medium sup-
plemented with 0.0442.22 M BA and 4.5 M IBA and
87.64 mM of sucrose for plant recovery from synthetic
seeds. The recovery percentage was scored at second week
from the date of inoculation.
Acclimatization ofrecovered plantlets
Healthy plantlets germinated from artificial seeds were
randomly selected and gently washed with sterile distilled
water for removal of the adherent media. For primary hard-
ening, these plantlets were grown in protrays containing
three types of sterilized potting mixture of cocopeat and
coarse sand in 1:1, 2:1 and 3:1 ratios respectively. Plants
in pro-trays were incubated in a plant growth chamber at
relative humidity; over-head watering was done using
hand sprayer as and when required. After 2 weeks, they
were transferred to a polythene growth chamber with a
photon flux density of 200 mol m2 s1, relative humid-
ity of 70100% and were maintained at 2528C. The
primary hardened plants were then established in green-
were removed from the plastic pots without hampering
the root ball and planted on 15cm earthen pots containing
soil:sand:organic manure (1:1:1). The potted plants were
kept within greenhouse under 50% shade provided by Agro
shade net (B & V Agro Irrigation Co., India). A 12-h pho-
toperiod with light intensity of 200molm2s1, relative
humidity 70% and temperature 282C were maintained.
Histology
Explants with different organogenesis stages were
first fixed in FAA (95% ethanol:glacial acetic
acid:formaldehyde:water, 10:1:2:7) followed by dehydra-
tion through a graded ethanol series (20, 30, 50, 70, 90,
and 100%) for 20 min at each step with three changes.
The tissues were embedded in paraffin wax and thin sec-
tions (10m) were cut using a rotary microtome (HM 340E
Electronic Rotary Microtome, Thermo Fisher, USA). For
staining purpose, the achieved section strips were dipped
into hematoxylene and eosine (HE) stains, then gently
washed with distilled water and dried at 42C. The strips
were further washed with xylol, mounted with DPX (BDH)
and visualised under compound microscope (Leica, DME,
Germany).
Plant Cell Tiss Organ Cult (2017) 129:313324 317
DNA extraction andPCR amplification conditions
Total genomic DNA was extracted using CTAB extraction
method (Doyle and Doyle 1990) from the synthetic-seed
generated secondary hardened plants and the mother plant
from where explants were collected. Following RNase
treatment the quality of extracted DNA was assessed on
0.8% agarose gel and finally the quantification of DNA
was done using spectrophotometer (Nanodrop 2000,
Thermo Scientific, USA). The DNA samples were diluted
to 125ngl1 with TE (TrisEDTA) buffer before use and
stored at 4C for ISSR experimentation.
For ISSR analysis, 30 micro-satellite based prim-
ers (Integrated DNA Technologies, USA) as reported by
Ding etal. (2013) in Limonium sinense, were screened and
among them 20 primers were used for the genetic diversity
analysis of 9 synthetic seed derived hardened plants (F1-
F9) in respect with the mother plant (Mo) from where ini-
tial explants were collected. The PCR reactions were per-
formed in a 25 l volume of reaction mixture containing
1 Taq buffer [NEB, USA], 1U of Taq DNA polymerase
[NEB,USA], 200M of each dNTPs (NEB, USA), 0.2M
primers, 75ng of template DNA and 4% DMSO (Himedia,
India). The PCR reactions were carried out in 2720 thermal
cycler (Applied Biosystems, USA) using a single primer in
each reaction. The differential base composition of primer
affects the annealing temperature therefore annealing tem-
perature for each of the primers was optimised.
The PCR amplification conditions were consisted of ini-
tial denaturation at 94C for 4min followed by 40 cycles of
PCR reaction consisting of denaturation at 94C for 1min,
primer annealing at specified temperature (as mentioned in
the table) for each primer for 1min and elongation at 72C
for 2min. A final extension at 72C for 7min was carried
out. The PCR reaction products were mixed with 4 l of
6 DNA loading buffer and resolved in presence of 100bp
DNAladder (GCC Biotech Pvt. Ltd., India) on 1.8% aga-
rose gel matrix containing 0.5 g l1 ethidium bromide.
Gels were visualized and documented using Gel-documen-
tation system (Bio-Rad XRS+, Bio-Rad, USA).
Experimental design andstatistical analysis
Forty surface sterilized roots (1520 cm) were randomly
taken for explant preparation to induce shoot buds. The
explants were cultured in jam bottle having diameter of
6cm; the shoot bud differentiation were noticed and aver-
age number of shoot buds were counted under a stereo
zoom microscope (SZH, Olympus, Japan). All the experi-
ments were set up in a completely randomized block design
with ten explants per experiment, each with three replicates.
The pH of all the above mentioned media was adjusted to
5.7 with 0.1N NaOH and autoclaved at 121C for 15min.
The cultures were maintained at 252C, under cool
white fluorescent light (16h photoperiod; 55 molm2s1,
Philips, India) and 70% humidity. For encapsulation pur-
pose, ten shoots regenerated from root were chosen for each
experiment having three replicates. The recovery percent-
age of synthetic seeds were calculated after storage at 0, 4
and 25C for 15, 30, 45, 60days. A simultaneous control
experiment for plant recovery was carried out on 0th day
at 25C. Primary hardening was done with random 150
synthetic seed generated plantlets and percentage survival
was scored after 30days (Table3). Thereafter, 132 primary
hardened plantlets were transferred to green house for sec-
ondary hardening. All percentage data were arcsine trans-
formed. Duncans multiple range test (DMRT) was done
using the SPSS software (Statistical Package for the Social
Sciences, IBM, USA) version 20 to distinguish differences
between treatment means at =0.05 level.
For ISSR analysis, the PCR reactions were repeated
thrice for each primer to ensure the reproducibility of
results and only clear and reproducible bands were scored
for the data interpretation using XLSTAT 2016 (Addin-
soft, USA). During analysis a major band along with a faint
band in repetition was also included. The estimation of
amplicon size of the ISSR product in this experiment was
accomplished with the help of 100 bp DNA ladder (GCC
Biotech Pvt. Ltd., India).
Result anddiscussion
Induction ofshoot formation
A detailed study on organogenesis from mature roots of
Limonium hybrid Misty Blue and its successive encap-
sulation into synthetic seeds was successfully revealed
through this study. This is the first report on organogenesis
from root explants of any species in Plumbaginaceae fam-
ily. The root explants collected from a 4-month old hydro-
ponic culture (Fig. 1a) induced shoot bud differentiation
either in clump (Fig.1b) or singly after 2 weeks of culture
(Fig. 1c). The competency of shoot bud differentiation
is so high that they are even formed on lateral root tip or
their base (Fig.1d). The response of organogenic differen-
tiation of this explant mainly depended on the concentra-
tions of PGRs in culture medium (Table1). In the present
findings, the medium ( MS basal+43.82 mM sucrose)
supplemented with NAA-2-iP induced the lowest percent-
age (0.009.97%) of response in comparison with Kine-
tin-NAA (18.4331.31%) or BA-NAA (40.9861.34%)
supplemented medium. However, the highest percentage
(61.34%) of responding explants was recorded after 4 week
of incubation in BA (2.22M) and NAA (1.07M) supple-
mented medium (Table1). In this respect, it is noteworthy
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318 Plant Cell Tiss Organ Cult (2017) 129:313324

Table1The Role of PGRs on the induction of shoot buds from root

explant of Limonium hybrid Misty Blue
PGR concentration (M) Percentage of
explant forming
NAA BAP 2-iP KIN shoot buds

0 0 0 0 0
1.07 0.44 0 0 40.98
0.88 0 0 43.28
2.22 0 0 61.34
1.07 0 0.49 0 9.97
0 0.98 0 0
0 2.46 0 0
1.07 0 0 0.46 31.31
0 0 0.93 24.35
0 0 2.32 18.43

All the percentage values were arcsine transformed. Data were taken
from 10 different explants per treatment having three replicated
means. All the cultures were grown in basal MS medium supple-
mented with 43.82mM sucrose and data were taken after 4 weeks of
culture

to mention that strength MS basal medium was more
responsive than full strength MS basal medium (data is not
included) regarding organogenic response of root explants.
The above mentioned observations suggest that root may be
a good choice as an explant. One of the important condi-
tions for explant selection is their easy and throughout year
availability. Generally, Limonium Misty Blue grew as a
rosette with many leaves arranged spirally with very short
internodes (Topoonyanont et al. 2000); therefore the leaf,
root and inflorescence can be used as explants for organo-
genesis. Although Topoonyanont et al. (2000) reported
shoot regeneration from inflorescence by reversion and
produced only 2.5 to 4 newly developed vegetative shoots
per explant by applying 2mg/L Kn and 2mg/L (3-indolyl)
acetic acid (IAA) in basal MS medium but the availabil-
ity of inflorescence depends on season which restricts the
year round production of this important cut flower generat-
ing plant. Between root and leaf explant, in our preliminary
experiment, leaf of this particular hybrid has been failed to
regenerate plants either through organogenic or embryo-
genic pathway, though the direct somatic embryogenesis
from leaf of Limonium sinensis (Dam etal. 2010) had been
reported earlier from our laboratory.
Maturation ofshoot buds
The juvenile shoot buds did not differentiate into shoots
when they were continued to grow in same culture
medium at second subculture; furthermore the explants
were dedifferentiated into calli. To avoid callus formation
the explants with attached shoot buds were transferred
Fig.2The role of plant growth regulators on transition of shoot buds
into mature shoots of Limonium Misty Blue plants. Note below
the figure. In all the experiment the basal medium was MS medium
supplemented with 87.64 mM sucrose and 0.8% (w/v) agar. Data
were scored after 4-weeks of culture. Values are expressed as the
meanSE of average number of matured shoots per explant. Three
replicated experiments were performed with ten explants per treat-
ment. Means with same letter within a column indicate statistical
insignificance at P0.05 level according to Duncan multiple range
test (DMRT)
into maturation media. In this particular culture medium
the top portion of shoot buds were extended much faster
than the basal portion, the contrasting pigment deposi-
tion suggest the differential growth pattern of the marked
shoot bud (Fig.1e). In the next 2-weeks, the shoot buds
further transformed into shoots with distinct leaf primor-
dia (Fig. 1f) and fully differentiated leaf (Fig. 1g). Dur-
ing maturation the BA and NAA containing medium
was found more suitable than the medium devoid of
any PGRs, in this context statistically higher response
(10.330.88 shoots/explant) was observed in culture
medium containing BA (0.44 M) and NAA (1.07 M)
after 4-weeks of culture (Fig.2).
Various reports are available on organogenesis from root
explants by application of different cytokinins (Atta et al.
2009; da Cruz etal. 2014; Jani etal. 2015). But here in our
study with Limonium hybrid Misty Blue, combination of
BA along with NAA was found to be suitable for induction
of organogenetic response of root explants, which is con-
sistent with the findings, where these two hormonal com-
binations induced shoot organogenesis (Phulwaria et al.

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Plant Cell Tiss Organ Cult (2017) 129:313324 319

2013; Sukhumpinij etal. 2010; Patel etal. 2014) from dif-

ferent explants in other plants.

Encapsulation ofjuvenile shoots

The encapsulation of regenerated shoots via sodium algi-

nate has been proved to be the most efficient technique for
mass propagation of ornamental plants as well as short
term conservation of important germplasms (Kulus and
Zalewska 2014). In this context, Limonium Misty Blue
which is a hybrid plant, does not form any viable seeds
therefore; encapsulated shoots with efficient endosperm can
substantiate the need of zygotic seeds. A literature review
indicates no report on artificial seed formation in Limo-
nium Misty Blue as well as in other Limonium species.
In this respect, the newly formed shoots from the matura-
tion media were excised and encapsulated in sodium algi-
nate beads (Fig. 3a). The concentration of alginate during
encapsulation of shoots is an important parameter because
it performs as a protective guard from physical damage dur-
ing storage; simultaneously it must allow splitting the artifi-
cial seed coat during germination. In the present investiga-
tion, 3% sodium alginate complexed with 100 mM CaCl2
formed firm, round and uniform beads; the concentration
lower than 3% failed to produce a turgid covering, whereas,
higher concentration (4%) resulted in very hard covering,
which was difficult to rupture. The concentration of sodium
alginate was properly judged when beads were allowed to
convert into plantlets in MS medium containing 0.44 M
BA and 4.5 M IBA. 3% sodium alginate encapsulated
shoots (60.67%) sprouted within 5-days and produced full-
fledged rooted plant after 10 days (Fig. 3b). This particu-
lar combination of 3% sodium alginate and 100mM CaCl2
(Table2) has successfully been used in several plants like
Phyllanthus amarus (Singh et al. 2006), Cucumis sativus
(Adhikari et al. 2014), Ocimum kilimandscharicum (Saha
etal. 2015) Ledebouria revoluta (Haque and Ghosh 2016)
for encapsulation purpose.

Recovery ofplantlets fromtheencapsulated shoots

In another experiment on storage of encapsulated shoots,

highest recovery percentage was obtained after 15 (61.34%)
and 30 days (56.79%) respectively at 4C, which was very
close to the recovery percentage (65.65%) of plantlets in
control condition (25C at 0 day). In contrast, the recov-
ery percentage was significantly lowered after storage
at 0C and 25C for 15, 30, 45 and 60 days respectively
(Table3). It is assumed that partial desiccation during stor-
age for longer period of time result in lower conversion
ratios (Faisal and Anis 2007). Similar higher conversion
frequency after storage at 4C was noted in other plants
Fig.3Encapsulation of juvenile shoots, plant recovery and harden-
ing. a The firm, round and uniform beads after encapsulation with 3%
sodium alginate with 100mM CaCl2, enlarged view of bead at inset;
b conversion of encapsulated shoots in MS+0.44 M BA+4.5 M
IBA medium after storage at 4C for 30 days, note the emergence
of shoot (sh) and root (rt), c the rooted plants derived from synthetic
seeds are ready for transplantation in green house, d one month old
primary hardened plantlets in a portray, e two month old secondary
hardened plantlets in earthen pots

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320 Plant Cell Tiss Organ Cult (2017) 129:313324

Table2The role of different combinations of sodium alginate and BA produced significantly longest shoot (31.960.47mm)
calcium chloride for encapsulation of juvenile shoots and plant recov- along with highest number of roots (9.120.10) after stor-
ery of Limonium hybrid Misty Blue
age at 4C (Table4). These well- developed plants (Fig.3c)
Concentration of Concentration of cal- Germination of were then transferred in the green house for hardening.
sodium alginate (%) cium chloride (mM) artificial seeds During hardening, the statistical analysis of 150 plants
(%)
revealed 69.73% of survivality after 4 weeks in potting
1 50 28.66 media containing cocopeat:coarse sand (2:1), which was
75 37.46 significantly higher in comparison to other two treatments
100 31.31 (Table 5). Primary hardening depends on two important
125 26.57 parameters one, porosity and two, water holding capacity
2 50 43.28 of the substrate. In this respect, 2:1 ratio of cocopeat and
75 46.72 coarse sand was most efficient because it promoted growth
100 49.02 of tender roots of tissue cultured Limonium plants. Moreo-
125 31.31 ver, the use of low cost and easily available coco peat and
3 50 31.31 coarse sand may be an important aspect for large scale pro-
75 52.54 duction. Several workers reported the suitability of sand
100 60.67 and cocopeat mixture during hardening of Stereospermum
125 43.28 personatum (Shukla et al. 2009) Anthurium andraeanum
4 50 46.72 (Bhattacharya et al. 2016), Santalum album (Singh et al.
75 49.02 2016). In the second phase of hardening, 132 healthy plant-
100 40.98 lets with sufficient root system (Fig.3d) were transplanted
125 35.06 in the earthen pot for secondary hardening. Out of which
101 plants survived and showed luxurious growth (Fig.3e).
All the shoots were encapsulated in presence of liquid MS with
87.64 mM sucrose. All the percentage values were arcsine trans-
formed of three independent experiments Histology

A number of histological studies reported the origin of

Table3The relation between storage and plantlets recovery from the shoot bud from the pericycle of root explants (Arora etal.
encapsulated shoots of Limonium hybrid Misty Blue 2011; Rocha et al. 2012), which is inconsistent with the
Storage (days) Percentage of germination of artificial current finding where shoot bud differentiation occur from
seeds cortical cells of root tissue (Fig.4a) as reported by Rao and
0C 4C 25C Mohanram (1981) in Limnophila indica and Suboti and
Grubii (2007) in Centaurium erythreae. Within explant
0 65.65 some of the cells of that region started frequent division
15 45.0 61.34 40.98 and turned into shoot bud primordium; the cells in this
30 39.23 56.79 35.06 primordium are characterized by their small size, compact
45 21.13 43.28 24.35 nature (due to absence of inter cellular spaces) and darkly
60 0 35.06 0

All the percentage values were arcsine transformed of three inde-

pendent experiments. All the culture media were supplemented with Table4The role of plant growth regulators on the length of shoot
basal MS along with 0.44M BA and 1.07M NAA and 87.64mM and number of roots of recovered plantlets of Limonium hybrid
of sucrose and data was scored after 15days Misty Blue
Plant growth regulators (M) Length of shoots (mm) No. of roots
(Rai etal. 2012; Faisal and Anis 2007; Adhikari etal. 2014; No PGR 16.630.61d 5.130.09c,d
Saha etal. 2015; Viehmannova etal. 2016). 1.07 NAA+0.044 BA 23.760.27c 7.040.09b,c
1.07 NAA+0.44 BA 31.960.46a 9.120.09a
Rooting andacclimatization ofrecovered plantlets 1.07 NAA+0.88 BA 26.040.52b,c 7.940.11b
1.07 NAA+2.22 BA 18.340.33c,d 5.560.17c
The ultimate success of any tissue culture work is the
acclimatization of plants in the natural condition. During The basal medium was supplemented with MS along with 87.64mM
pre-hardening the concentration of PGRs in conversion of sucrose. All values are the meanSE of three independent experi-
ments. Means followed by same letter indicate statistical insig-
medium affected the shoot and root growth. The germina- nificance at P0.05 level according to Duncan multiple range test
tion medium supplemented with 4.5M IBA and 0.44M (DMRT). Data scored after 30 days

13
Plant Cell Tiss Organ Cult (2017) 129:313324 321

Table5Primary hardening of artificial seed derived plantlets of section of the root also confirmed multiple shoot devel-
Limonium hybrid Misty Blue opment at different stages when the explant containing
Protray potting medium Primary hardening shoot buds cultured in maturation medium containing
MS+0.44 M BA+1.07 M NAA+87.64 mM sucrose
Total number of Survival per-
plants transferred centage after (Fig.4c). The shoot buds further developed into complete
30days shoot having distinct leaf primordia and apical shoot mer-
istem. In this developmental phase shoots are emerged out
Cocopeat:coarse sand (1:1) 50 61.34
from the explant but they are well connected with mother
Cocopeat:coarse sand (2:1) 50 69.73
tissue (Fig. 4d), which confirmed their organogenic ori-
Cocopeat:coarse sand (3:1) 50 63.43
gin. Considering all aspects such as genetic potentiality,
All percentage values are arcsine transformed of three independent induction, competence and commitment the roots of hybrid
experiments Misty Blue may be consider as good explant for organo-
genic shoot development.

stained nucleus. These triangular cell mass (Fig.4b), which Screening oforganogenic shoots throughISSR marker
is designated as shoot primordium, can clearly be demar-
cated from their surrounding tissue by their protruded The somaclonal variation events can be more frequent
growth on the surface of the explant. The longitudinal in respect to protocol based on adventitious shoot

Fig.4Histological study of shoot bud development from root c the longitudinal section of the root formed multiple shoot buds at
explant of Limonium Misty Blue. a The longitudinal section of different developmental stages when cultured in maturation medium
root showing shoot bud primordium developed at the sub-epidermal comprising of MS+0.44 M BA+1.07 M NAA+87.64 mM
layer of root tissue, these regions are characterized by their small sucrose, d a cross section of a distinct shoot bud, consisting of leaf
size, compact nature and darkly stained nucleus (arrow), b a triangu- primordia (LP) and apical shoot meristem (SP), are clearly attached
lar shoot primordial protrusion (arrow) on the surface of root tissue, with the mother tissue

13

322 Plant Cell Tiss Organ Cult (2017) 129:313324

regeneration than the protocol based on regeneration

from axillay bud; in this respect, the heterogeneous off-
spring may be produced during shoot organogenesis
from root explant of Limonium Misty Blue. Therefore;
the screening of regenerated plants with the molecular
marker is an essential step to validate the micropropa-
gation protocol. In the present investigation the ISSR
markers were used to screen the somaclones. Out of
30 ISSR primers 20 selected primers (Table 6) resulted
in 28 scorable bands, which were selected for further
study. These 20 ISSR primers generated scorable bands
Fig.5A representative ISSR band pattern of artificial seed derived
ranging from 100 to 900 bp in size. The number of
plants of Limonium Misty blue using primer [(AC)8TC]. Mo -
bands for each primer varied from 2 (IS11 and IS15) to mother plant, W- negative control; F1F9 artificial seed derived
8 (IS1 and IS20), with an average of 4.1 bands per ISSR plants, M- 100bp DNA marker
primer. Among all the primers tested, monomorphic
banding pattern (Fig. 5) was observed in all the nine
synthetic seed derived and mother plants. The applica- Conclusion
tion of molecular marker technique especially by ISSR
for clonal fidelity of invitro propagated plants have been In conclusion, the present report describes, a detailed effi-
recommended by several authors in different plant sys- cient, true-to-type in vitro plant propagation method from
tems (Ramrez-Mosqueda and Iglesias-Andreu 2015; the invivo roots of mature plant of a commercially impor-
Saha et al. 2015; Viehmannova et al. 2016). Our result tant florist plant Limonium Misty Blue and also represent
suggests the genetic integrity and true-to-type nature of an efficient germplasm preservation method for this non-
the invitro generated plants in germplasm conservation seed producing variety of Limonium which can be used as
programme. an effective solution for exchange and transportation to dif-
ferent parts of the world.

Table6List of primers used in Serial no. ISSR primer Molecular Am (C) No. of bands Band
the study with their molecular sequence weight appeared appeared In
weights, annealing temperature range [bp]
used for each primers, no. of
band generated with their range 1 (AC)8AT 150 46 8 100900
of appearance with respect to
2 (AC)8TT 170 46 6 100400
100bp DNA marker
3 (AC)8AG 140 45.9 4 200500
4 (AC)8TG 150 45.9 5 100500
5 (AC)8AA 120 45 3 100500
6 (AC)8TA 140 44.8 4 200400
7 (AC)8TC 190 45.9 7 100500
8 (AC)8CA 130 47.5 5 100400
9 (AC)8CC 150 48.6 6 200500
10 (AG)8AA 140 44.3 4 200500
11 (AG)8TA 150 40 2 200300
12 (AG)8TC 160 42.3 4 100300
13 (AC)8GC 150 48.6 2 100200
14 (AC)8CT 150 47.5 4 200500
15 (AC)8GA 140 47.5 2 100300
16 (AC)8GC 180 48.8 5 100300
17 (AC)8GG 130 50 3 100300
18 (AC)8GT 140 47.5 5 100300
19 (TG)8AA 190 45 3 200300
20 (TG)8AC 160 45.9 8 100400

13
Plant Cell Tiss Organ Cult (2017) 129:313324 323
Acknowledgements The financial support from the Department
of Atomic Energy, Board of Research in Nuclear Sciences, Mum-
bai (Project No: 2013/35/42/BRNS/1385 dated 12/08/2013), DST-
PURSE, WBDST FIST, and PRGs for teachers, University of Kaly-
ani, Kalyani, West Bengal, India, are gratefully acknowledged.
Author contributions Conceived and designed the experiments:
TKB, DPF. Performed the experiments: SB, JK (equal contribution)
Analyzed the data: SB, JK, UB, TKB, DPF. Wrote the paper: SB, JK,
and TKB.
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