In Vitro Cell.Dev.Biol.
—Plant (2007) 43:51–58
DOI 10.1007/s11627-006-9000-y
MICROPROPAGATION
Low-cost media for in vitro conservation of turmeric
(Curcuma longa L.) and genetic stability assessment
using RAPD markers
Rishi K. Tyagi & Anuradha Agrawal & C. Mahalakshmi &
Zakir Hussain & Husnara Tyagi
Received: 7 July 2006 / 27 November 2006 / Published online: 9 February 2007 / Editor: A. Vieitez
# The Society for In Vitro Biology 2007
Abstract Up to 73% decrease in cost of media for plant
regeneration and in vitro conservation was achieved in
Curcuma longa cv Prathibha by using inexpensive carbon
source and gelling agent. Laboratory reagent-grade sucrose
was replaced by locally available commercial sugar (market
sugar or sugar cubes) as carbon source and bacteriological
grade agar by isabgol (also named isubgol) as gelling agent.
No adverse effects on shoot regeneration and conservation
on isabgol-gelled low-cost media were observed as com-
pared to that on agar-gelled control medium (CM). Some
33–56% cultures of C. longa survived up to 12 mo. on
isabgol-gelled medium in comparison to only 16% on CM.
Genetic stability of 12-month-old in vitro-conserved plants
was assessed using 25 random amplified polymorphic DNA
(RAPD) primers; no significant variation was observed in
RAPD profiles of mother plants and in vitro-conserved
plantlets on CM and low-cost media.
Keywords Curcuma . Isabgol . Isubgol . Low-cost tissue
culture media . Matric potential . Osmotic potential .
Psyllium husk
Introduction
In vitro techniques have been successfully applied as a
relatively safe method for conservation and international
R. K. Tyagi (*) : A. Agrawal : C. Mahalakshmi :
Z. Hussain : H. Tyagi
Tissue Culture and Cryopreservation Unit,
National Bureau of Plant Genetic Resources,
New Delhi 110012, India
e-mail: rktyagi@nbpgr.ernet.in
e-mail: rishiktyagi@email.com
exchange with limited quarantine of various vegetatively
propagated crop germplasm (Ashmore, 1997) including in
our laboratory also (Mandal et al., 2000). An often-cited
disadvantage of in vitro genebanks is the relatively higher
costs involved as compared to other methods (Jarret and
Florkowski, 1990; Koo et al., 2003). The need for low-cost
plant tissue culture systems, applicable for micropropaga-
tion and in vitro conservation of plant genetic resources, has
been emphasized to allow the large-scale application of
such technology in developing countries (IAEA, 2004).
Recurring costs of micropropagation and in vitro
conservation include those for chemicals that are used in
culture media, i.e., carbon sources, gelling agents, inorganic
and organic supplements, and growth regulators. Sucrose is
usually used as a source of carbon and agar as the gelling
agent, and together they constitute the most expensive
components of the culture media.
The genus Curcuma (family Zingiberaceae) comprises
more than 80 species of rhizomatous perennial herbs and
has a widespread occurrence in the tropics of Asia and
extends to Africa and Australia (Purseglove et al., 1981).
Curcuma longa L. (syn. Curcuma domestica Val.), com-
monly known as turmeric, is an important commercial spice
crop of India, Sri Lanka, Pakistan, Bangladesh, and China,
which has traditionally been used in the Ayurvedic and
Chinese systems of medicine since the ancient times. A
large number of accessions (2,368) of turmeric and related
species (all vegetatively propagated) are maintained in field
genebanks in India; the conservation of these is of high
priority for their use in future crop improvement programs
(Ravindran et al., 2005). Currently, 137 genetically diverse
accessions of Curcuma are conserved as in vitro cultures in
the In Vitro Genebank of National Bureau of Plant Genetic
Resources following periodic subculturing. As the number
of accessions continues to increase in the In Vitro
52 TYAGI ET AL.
Genebank, the cost of conservation on long-term basis
becomes a major concern.
In our study, to reduce the cost of plant regeneration and
in vitro conservation, laboratory reagent (LR)-grade sucrose
was replaced by locally available commercial sugar (market
sugar or sugar cubes with a standard trade name) as carbon
source and bacteriological-grade agar by isabgol (also
named isubgol) as gelling agent. Isabgol or psyllium, the
husk derived from the seeds of the medicinal plant
Plantago ovata Forsk (family Plantaginaceae), has been
successfully used as gelling agent in tissue culture media
(Babbar and Jain, 1998; Jain and Babbar, 2005). The
efficacy of isabgol is due to the presence of mucilaginous
husk in seeds. Mucilage yield amounts to approximately
25% (by weight) of the total seed yield and is obtained by
mechanical milling/grinding of the outer layer of the seed.
The milled seed mucilage is a white fibrous material that is
hydrophilic.
In the present study, we aimed to determine a low-cost
media using readily available and inexpensive alternatives of
carbon sources and gelling agents in culture medium for in
vitro plantlet regeneration and conservation of C. longa cv
Prathibha. The effect of the low-cost substitutes on the genetic
stability of in vitro-conserved plants was also assessed using
random amplified polymorphic DNA (RAPD) markers.
Materials and Methods
Initiation of cultures. In vitro shoot cultures of C. longa cv
Prathibha were established using the rhizome buds collected
from sprouted rhizomes that were harvested from plants
growing in a net house of the National Bureau of Plant
Genetic Resources (NBPGR), New Delhi, India. The buds
(2–3 cm) were excised and thoroughly washed under
running tap water, surface-sterilized using 0.1% (w/v)
mercuric chloride and two or three drops of Tween-20 for
10–15 min, and subsequently thoroughly washed seven or
eight times with sterilized distilled water to remove traces of
mercuric chloride. The sterilized buds were cultured onto
Murashige and Skoog (1962) medium (MS) supplemented
with 2.5 mg l−1 N6 benzyladenine (BA) to obtain the in vitro
shoots, which were further used to study in vitro plant
regeneration and conservation (Balachandran et al., 1990,
Tyagi et al., 2004). In fact, such shoot cultures were
maintained on the above medium for approximately 4 yr
through periodic subculture at 7–8 mo. intervals before using
the explants for the present study.
Plantlet regeneration and conservation. For all experi-
ments, shoots were excised from 4-week-old cultures main-
tained through periodic subculture on MS+2.5 mg l−1 BA.
The basal swollen portion of shoot containing the shoot tip
Table 1. Details of media tested for in vitro plantlet regeneration and
conservation of C. longa
Medium code MS+2.5 mg l−1 BA +
Carbon sourcea Gelling agentb
SAD (CM) Sucrose Agar
MAD Market sugar Agar
CAD Sugar cubes Agar
SID Sucrose Isabgol
MID Market sugar Isabgol
CID Sugar cubes Isabgol
a
Concentration of carbon sources=3% (w/v)
b
Concentration agar=0.7% (w/v), isabgol=3.5% (w/v)
CM=Control medium; MS=Murashige and Skoog’s medium [20]
encircled by whorls of leaves was cut to 1–2 cm above the
base (referred to as shoot tip explant hereafter) and cultured
on various test media. Table 1 presents media codes and
details of various combinations of carbon sources and
gelling agents, which were tested in the present study. The
treatment SAD served as the control medium (CM).
Sucrose [Qualigens, Mumbai, India, (LR grade)] or
inexpensive market crystalline sugar (Mawana Crystal
Sugar™, India) or sugar cubes (Daurala Sugars™, India)
were used as carbon sources. Sucrose (LR) is made from cane
sugar and contains 99.98% sucrose and 0.01% reducing
sugars. Market crystalline sugar (referred to as market sugar
hereafter) is made from cane syrup treated with sulfur dioxide
(sulfitation) or carbon dioxide (carbonation) and contains 96–
97% sucrose and 0.75–1% reducing sugars. A sugar cube is
made from fine grains of refined crystalline sugar by shaking
it mechanically into cubes, and it contains 99.5% sucrose and
0.03% reducing sugars. It is considered to be of higher quality
than crystalline sugar and is hard enough not to break but
dissolves instantly in water (WOI, 1981). The media used for
the present study were either gelled with 0.7% (w/v) agar
(bacteriological grade; Qualigens, Mumbai, India) or 3.5%
(w/v) isabgol (Sat-Isabgol™ Sidhpur, Gujarat, India).
After 12 mo. of conservation, some of the cultures were
drawn for testing their viability and genetic stability; the
remaining cultures were allowed to grow beyond 12 mo. on
their respective media without subculture.
Viability of in vitro-conserved plantlets. After 12–13 mo. of
conservation, plantlets conserved on CM (SAD) and low-cost
media (SID, MID, and CID) were subcultured to test their
viability through shoot regeneration. Some 4–12 shoot tip
explants from each media were subcultured on their respective
parent media (isabgol-gelled) and 4–11 shoot tip explants on
CM (SAD). No attempt was made to record the detailed data;
only the occurrence of shoot and root regeneration was
observed after 8 wk of subculturing the explants.
 LOW-COST MEDIA FOR TURMERIC CONSERVATION
Determination of pH, electrical conductivity, osmotic
potential, and relative matric potential of medium. The
pH of the medium was determined before (original) and after
autoclaving by a digital pH meter (Chemtron, India, model no.
9140). Immediately after autoclaving, 20 ml of each medium
was homogenized with 30 ml of distilled water (DW) using a
magnetic stirrer. Electrical conductivity (EC) (mS cm−1) of
each medium was measured at 25°C by a digital conductiv-
ity meter (Control Dynamics, India). Using the EC values,
osmotic potential (OP) of the same was calculated according
to the formula derived by Janardhan et al. Janardhan et al.
(1975) and adopted by Bhattacharya et al. (1994):
ðEC  0:36  dilution factorÞ
OP ¼ 
0:987
Relative matric potential of each medium was measured
following the method using filter-paper discs (Whatmann
No. 3) as described by Owens and Wozniak (1991). A
precisely moistened filter-paper disc was placed on the
surface of the medium, allowed to equilibrate, removed,
and weighed. The relative gain or loss of water from the
paper discs was a measure of the matric potential of the
medium.
Culture conditions. Unless otherwise stated for a particular
treatment, all other chemicals used for preparing the media
were of LR grade (Qualigens, Mumbai, India) and BA from
Sigma, St. Louis, MO, USA. The pH of all media used for
raising the cultures (Table 1) was adjusted to 5.8 with 0.1 N
NaOH or HCl before adding the gelling agent and auto-
claving. Agar was dissolved in hot (80–90°C) DW, whereas
isabgol was suspended in DW at room temperature (30–32°
C) before autoclaving. Approximately 20 ml of the medium
was dispensed into culture tubes (25×150 mm, Borosil,
India). The medium was autoclaved at 121°C and 106 kPa for
20 min. After autoclaving, culture tubes containing isabgol-
gelled media were stirred manually to suspend the isabgol
uniformly before its gelling. Each culture tube received one
explant and was closed with polypropylene caps and covered
with cling film. Cultures were incubated at 25±2°C and a
light intensity of 40 μmol m−2 s−1, provided by cool white
fluorescent lamps (Philips, India) at a 16-h photoperiod.
Comparison of costs of media. Costs of carbon sources
(sucrose, market sugar, sugar cubes) and gelling agents (agar,
isabgol) were calculated on the basis of prevailing costs and
the actual amount or volume used to prepare 10 l of final
medium and were compared. Initially, the costs were
calculated in Indian rupee (INR) which was converted into
US dollar (US$) by dividing with a factor of 45 (US$ 1=INR
45). Because equal amounts of other chemicals [MS salts,
inorganic and organic supplements, and BA (2.5 mg l−1)]
were used in each treatment, the cost of these chemicals was
53
considered constant for all the media tested and not included
in the total costs for comparison.
Genetic stability assessment. To ascertain whether low-cost
carbon sources and isabgol used in media have any effect
on the genetic stability of plantlets conserved in vitro, a
comparison of RAPD profiles of mother plants (source of
explants used for in vitro plantlet regeneration and
conservation) and the plantlets conserved in vitro for
12 mo. on SAD (CM), MAD, CAD, SID, MID, and CID
(low-cost media) was carried out.
DNA extraction. Total genomic DNA was extracted from
1 g of leaves of three to five plantlets for each treatment
using a modified Saghai-Maroof et al. (1984) method. After
RNAse treatment, the DNA concentrations were deter-
mined following the method of Brunk et al. (1979) using a
fluorometer (Amersham Biosciences, USA) and bisbenzi-
mide (Hoechst dye 33258) as the fluorescent dye. The
DNA samples were stored at −20°C until further use.
Working solutions of genomic DNA (5 ng/μl) were
prepared after dilution with sterile DW and stored at 4°C
for subsequent use in RAPD analyses.
RAPD analysis. PCR reaction was carried out in a DNA
Thermal Cycler (GeneAmp 9600 PCR system, Perkin-
Elmer Cetus, Norwalk, CT, USA). Each 25 μl reaction mix
contained 1× PCR reaction buffer (10 mM Tris–HCl,
50 mM KCl, and pH 8.3), 3 mM MgCl2, 0.5 U of Taq
DNA polymerase, 200 μM each of dATP, dTTP, dCTP and
dGTP (all reagents from Bangalore Genei, India), 0.6 μM
of primer (Operon Technologies, USA), and approximately
50 ng of template DNA.
A total of 200 primers (Operon Technologies, USA) were
screened for RAPD analysis. Out of these, some 25 primers—
OPA 03, OPA 10, OPC 01, OPC 03, OPC 04, OPC 05, OPC
08, OPC 09, OPC 1, OPC 11, OPC 14, OPC 18, OPC 20,
OPD 05, OPD 07, OPD 11, OPD 18, OPK 17, OPK 19, OPL
03, OPM 14, OPO 06, OPO 13, OPO 16, and OPW 12, were
found to be polymorphic and used for further analyses. The
PCR conditions were as follows: initial extended step of
denaturation at 94°C for 5 min followed by 35 cycles of
denaturation at 94°C for 1 min, primer annealing at 36°C for
1 min, primer elongation at 72°C for 2 min, followed by an
extended elongation step at 72°C for 10 min. Reaction
products were mixed with 2.5 μl of 10× loading dye (0.25%
bromophenol blue, 0.25% xylene cyanol, and 40% sucrose,
w/v) using a microfuge. The mixture was electrophoresed on
1.4% agarose gel at 90 V (Bio–Rad subcell GT) followed by
staining with ethidium bromide and photographed under
ultraviolet light using a Digital Imaging System (UltraCam).
The amplification products of the in vitro-conserved
plantlets derived from MID medium were scored across the
54 TYAGI ET AL.

lanes, and they were compared with that of mother plants
and plants conserved on CM. Each band was treated as one
RAPD marker and scored as present or absent from the
photographs. Amplifications were repeated twice.
Data recording and statistical analyses. Each treatment
comprised a set of 12 cultures and was replicated three
times. The experiments were conducted in completely
randomized block design. Observations were recorded at
periodic intervals. The data for plant regeneration were
computed as mean values of the total 36 cultures for the
number of shoots/culture, shoot length (centimeter), num-
ber of roots, and root length (centimeter) up to 8 wk, and
mean values were subjected to Tukey’s test (Table 2).
Shoots were allowed to grow on their respective media
without transfer or subculture and studied for conservation.
Cultures that contained at least two green shoots were
considered as surviving. To determine conservation period,
survival of cultures (per cent) was recorded at 1-month
interval (Fig. 2). ANOVA was carried out for the effect of
carbon sources, gelling agents, and their interaction on in
vitro regeneration and conservation. Data for pH, EC, OP,
and relative matric potential (Table 3) were recorded for
five determinations each, and mean values were subjected
to Duncan’s multiple range test (DMRT) using standard
software SPSS 10.0 to test the significance.
week and gave rise two or three shoot buds per explant. In
3–4 wk, 1- to 2-cm long shoots were obtained; these shoots
were subcultured on the same medium repeatedly to obtain
a sufficient stock of explants for in vitro plantlet regener-
ation and conservation experiments using low-cost media.
Plantlet regeneration. Shoot buds were induced from the
swollen basal portion of the explants in all the six media
tested within 2 wk. After 8 wk of culture initiation, 83.3 to
97% cultures exhibited shoot regeneration ranging from 2.6
to 4.6 shoots per culture. In general, longer shoots were
recorded on isabgol-gelled media (3.2–4.1 cm) in comparison
to agar-gelled media (2.3–3.0 cm) (Table 2). Figure 1a, b
represents 10-week-old cultures showing plantlet regenera-
tion on all the media tested. Rooting was observed in all
shoot-forming cultures on their respective parent media,
ranging from 2.1 roots per shoot on SAD medium to 3.4
roots per shoot on SID medium (Table 2). Generally, the
quality of sugars and type of gelling agent did not influence
the rooting (Fig. 1a, b). ANOVA shows that the differences
in the number of shoots (F=8.8, df=1, P≤0.05), shoot
length (F=24.5, df=1, P≤0.01), root length (F=29.5, df=1,
P ≤ 0.01) due to isabgol used as gelling agent were
significant. In contrast, differences for the above parameters
due to various carbon sources used were nonsignificant at
P≤0.05. Interaction between the carbon source and gelling
agent was nonsignificant for all the above parameters except
shoot length (F=5.8, df=5, P≤0.05).

Results Conservation. All the tested media supported growth

of almost 100% shoot-forming cultures up to 6 mo. After
The isabgol-gelled (3.5%, w/v) media solidified faster than 8 mo., the survival pattern of the cultures maintained on
agar-gelled (0.7%, w/v) media. However, both types of various media tested is shown in Fig. 2a, b. With the
media were firm enough to hold the explant vertically. increase in age of the cultures beyond 8 mo., survival rates
generally declined. In 12-month-old cultures, higher sur-
Initiation of cultures. Rhizome buds, cultured on MS vival (50.0–56.4%) was observed on media gelled with
(3% sucrose) + 2.5 mg l−1 BA, started sprouting within a isabgol (SID, MID, and CID) as compared to 11.2–16.6 %
survival on agar-gelled media (SAD, MAD, and CAD),
Table 2. Plantlet regeneration in C. Longa after 8 wk of culture on with the highest value (56.4%) being recorded on MID
control (SAD) and various low-cost media medium (Fig. 2). ANOVA shows that the differences in
Medium No. of Shoot No. of Root survival of cultures (F=115.3, df=1, P=0.01) due to the
code* shoots/ length (cm) roots/shoot length two gelling agents were highly significant. However, no
culture (cm) significant differences were observed due to either source
of carbon or interactions of carbon source and gelling
SAD 4.6±0.7a 2.3±0.1c 2.1±0.4a 1.1±0.1c
agent. At the end of 14 mo., approximately 22% cultures on
MAD 4.5±0.4ab 2.5±0.1bc 2.7±0.3a 1.2±0.1c
CAD 4.5±0.6ab 3.0±0.3bc 2.6±0.3a 1.4±0.1bc
MID and 12–13% cultures each on SID and CID survived,
SID 2.6±0.5b 4.1±0.4a 3.4±0.1a 1.6±0.2ab whereas no cultures or <5% cultures survived on all the
MID 4.1±0.3ab 3.3±0.2ab 2.7±0.3a 1.9±0.2a agar-gelled media.
CID 3.2±0.6ab 3.2±0.1b 2.9±0.5a 1.9±0.2a
*
Viability of in vitro-conserved plantlets. Upon subculturing
See Table 1 for medium codes.
Values are mean±SE of 36 replicate cultures.
the shoot tip explants excised from 12-month-old in vitro-
Values superscripted with the same letter in each column are not conserved plantlets on low-cost media, shoot regeneration
significantly different on the basis of Tukey’s test analysis (P≤0.05). (two to four shoots per culture) was obtained in almost all
 LOW-COST MEDIA FOR TURMERIC CONSERVATION 55

Table 3. Values of pH, electrical conductivity (EC), osmotic potential (OP), and relative matric potential of different media (at 25°C) used for in
vitro plant regeneration and conservation of C. longa

Medium code pH EC (mS cm−1) OP (MPa) Relative matric potential

Original* Post-autoclave

SAD 4.35 5.75 3.802±0.051a −0.347±0.005a 1.272±0.051a

MAD 4.37 5.74 3.920±0.028a −0.357±0.003a 1.247±0.018a
CAD 4.30 5.72 3.768±0.046a −0.344±0.004a 1.218±0.016a
SID 5.02 5.17 2.530±0.030b −0.231±0.003b 0.780±0.006b
MID 5.04 5.11 2.758±0.129b −0.251±0.012bc 0.782±0.005b
CID 5.10 5.12 2.690±0.028b −0.245±0.003bc 0.798±0.009b

*Original pH of all media was adjusted to 5.8 before autoclaving using 0.1 N NaOH or HCl.
Values are mean±SE of five replicates of each medium.
Values superscripted with the same letter in each column are not significantly different on the basis of DMRT analysis (P≤0.05).

the cultures on parent media as well as on CM (SAD). This pH, electrical conductivity, osmotic potential, and relative
phenomenon was observed after both the first subculture matric potential The changes in pH, EC, OP, and relative
and the second subculture. Irrespective of the media, matric potential of post-autoclaved media and their effects
profuse rooting also occurred within 8 wk (Fig. 1d). on in vitro plantlet regeneration and conservation were

Figure 1. (a–b) Ten-week-old cultures of C. longa cv Prathibha on:
(a) SAD, MAD, CAD agar-gelled media; (b) MID, CID, SID isabgol-
gelled media, all of which showing normal plantlet regeneration; (c)
12-month-old in vitro-conserved cultures on control (SAD) and
various low-cost media; (d) an 8-week-old plantlet regenerated after
subculturing of a shoot tip explant from 12-month-old in vitro-
conserved plantlets on isabgol-gelled medium; (e) RAPD profiles
generated from mother plants (M) and 12-month-old in vitro-
conserved plants on SAD (CM) and MID. Amplification pattern in
lanes 1–3 obtained by primer OPC 05, lanes 4–6 by primer OPC 08,
and lanes 7–9 by primer OPC 10; ( f ) as in (e) except the profiles of
plants on SID. Amplification pattern obtained by using primer OPW
12. Each bar on a, b, c, and d=2.5 cm. See Table 1 for media codes.
56 TYAGI ET AL.

Figure 2. (a–b) Survival (%) of cultures of C. longa cv Prathibha after different conservation periods on various media. See Table 1 for media
codes. Vertical line on each bar indicates SE.

determined (Table 3). The pH of all media was adjusted to
5.8 before autoclaving, and this value decreased (from 5.8
to 5.11–5.75) in all post-autoclaved media; isabgol-gelled
media had lower pH values than those of agar-gelled media
after autoclaving. Values of the EC of agar-gelled media
(3.768 to 3.920 mS cm−1) were higher than those of
isabgol-gelled ones (2.530–2.758 mS cm−1). Consequently,
agar-gelled media elicited lower values of OP (−0.357 to
−0.344 MPa) than those gelled with isabgol (−0.251 to
−0.231 MPa). The relative matric potential values ranged
from 1.218 to 1.272 for agar-gelled media and 0.780 to
0.798 for isabgol-gelled media. Overall, the isabgol-gelled
media exhibited significantly lower relative matric potential
than the agar-gelled media (Table 3).
Comparison of costs of media. Table 4 presents the
comparative costs of the six media tested in the present
study. Agar is the costliest constituent of the media (US
Table 4. Comparative costs of media with various carbon sources and
gelling agents
Medium Costb of 10 l of Decrease (%) in cost in
codea medium (US$) comparison to CM
$64.44/kg) followed by sucrose (US$ 4.67/kg). All media
gelled with isabgol and supplemented with market sugar or
sugar cubes were significantly cheaper in cost as compared
to agar-gelled media supplemented with sucrose. Up to 73%
reduction in cost can be achieved by using MID medium for
in vitro conservation of C. longa cultures.
Genetic stability assessment. A total of 177 scorable bands,
ranging from 0.4 to 2.4 kb with an average of 7.08 bands
per primer, were obtained using 25 primers. The total
number of bands produced by an individual primer ranged
from five in OPA 10 to ten in OPM 14. Using OPA, OPC,
OPD, OPK, OPL, OPM, OPO, and OPW series of primers
(details in the “Materials and Methods” section), amplified
products exhibited monomorphic pattern across all the
plants of C. longa conserved on all the tested media.
Examples of RAPD profiles of mother plants and plants
conserved on SAD, SID, and MID media are shown as
Fig. 1e, f. On the basis of available data, no major genetic
variation was observed due to market sugar and isabgol, the
low-cost alternatives of sucrose and agar, respectively.

SAD 6.612 – Discussion

(CM)
MAD 5.343 19.19 The main objective of the present study was to develop a
CAD 5.565 15.83
low-cost medium for in vitro conservation of turmeric.
SID 3.032 54.14
MID 1.763 73.34
Generally, no adverse effects of quality of sugar and type of
CID 1.985 69.98 gelling agents were observed on the growth of plantlets and
survival of cultures of C. longa. Our observations are in
a
Concentrations used : carbon source 3% (w/v); agar 0.7% (w/v); agreement with the plantlet regeneration in banana on
isabgol 3.5% (w/v)
b
Cost (US$) were calculated as follows: sucrose=4.67/kg; market
sucrose (3%) or commercial-grade-sugar-supplemented
sugar=0.44/kg; sugar cubes=1.18/kg; agar=64.44/kg; media (Ganapathi et al., 1995). More importantly, isabgol-
isabgol=2.66/kg gelled media supported better survival of cultures in
 LOW-COST MEDIA FOR TURMERIC CONSERVATION
comparison to agar-gelled media. The superiority of isabgol-
gelled medium as compared to agar-gelled media in terms of
fresh weight in cultures of chrysanthemum cv “Birbal Sahni”
was also established by Bhattacharya et al. (1994), and in
terms of shoot regeneration in Dendrobium chrysotoxum by
Jain and Babbar (2005). To the best of our knowledge, the
positive role of isabgol in increasing the survival rate as well
as longevity of in vitro-conserved cultures of C. longa, found
in the present study, has not been reported previously.
To examine the question of whether the pH, EC, OP, and
relative matric potential of a particular medium play any role
in the growth of plant, the above parameters of post-
autoclaved media were determined. In our study, the
resulting OP values of the media tested ranged between
−0.357 and −0.231 MPa. Data on EC show that isabgol-
gelled media had significantly lower ionic concentrations
and higher OP than those of agar-gelled media. Our
observations are consistent with those reported by Kimball
et al. (1975) where the cultured tissues of soybean can grow
in a medium with OP values as low as −1.290 MPa. The OP
of the media tested was never less than −0.357 MPa and was
favorable for growth and survival of cultures. Isabgol-gelled
media showed lower relative matric potentials than agar-
gelled media, indicating the less availability of free water in
isabgol matrix. This may be responsible for the slow rate of
transport of water along with nutrients in isabgol-gelled
media, resulting in the slow growth of cultures that survived
for longer period. Owens and Wozniak (1991) also demon-
strated that even the difference in narrow range of matric
potentials causes marked effects on growth of tissues. The
rate of transport of water through gel is controlled
predominantly by the matric potential, and it is not OP but
the matric potential associated with gel structure and
capillarity that determines water and nutrient availability to
the tissues (Cameron, 2006). Additionally, agar is known to
contain high amounts of Na+ (6,900–10,700 mg/kg) as an
impurity (Debergh, 1983), which could be inhibitory to the
growth of cultures for longer periods. The chemical analysis
of isabgol has shown that the husk contains around 22.6%
arabinose and 74.6% xylose (Fischer et al., 2004). The role of
arabinose and xylose has been established in induction of
shoots and flowers in tobacco (Eberhard et al., 1989),
whereas embryogenesis in microspore cultures of wheat
has been promoted by gum arabic extracts (Letarte et al.,
2006). It may be likely that, upon hydrolysis during
autoclaving of media, such compounds may also be playing
a metabolic role in promoting the survival of cultures of
C. longa for longer periods. However, the morphogenetic
and metabolic role of arabinose and xylose from isabgol in
the growth of plants needs to be investigated.
Cost becomes a major concern for the maintenance of
large numbers of cultures and continuously increasing
numbers of accessions in any in vitro genebank. Our results
57
showed that by replacing sucrose with sugar cubes or
market sugar, and agar with isabgol, the cost of the medium
could be decreased considerably. Therefore, we recommend
MS media (with 2.5 mg l−1 BA) supplemented with market
sugar or sugar cubes and gelled with isabgol, i.e., MID and
CID, which not only supports the survival (56 and 50%,
respectively) of the cultures up to 12 mo. but also
considerably decreases the cost of media for in vitro
conservation. Isabgol posed no problem in tissue culture
of C. longa, and it contributes significantly in reducing the
cost of the medium.
RAPD technique, being simple and cost effective, has
been used to assess the genetic variations of tissue culture
plants in numerous studies (Martins et al., 2004) and
references therein), including C. longa (Salvi et al., 2001).
RAPD polymorphism was reported in callus-derived plants
of C. longa (Salvi et al., 2001). However, in our study,
RAPD analysis showed no evidence of polymorphism
between mother plants and the plants of C. longa conserved
for 12 mo. on low-cost media, which may be due to the fact
that plantlets were regenerated directly from organized
shoot tip explants.
The results presented in this paper provide an efficient
and cost-effective in vitro conservation protocol. The
above-recommended media (MID and CID) are being
tested for in vitro conservation of a large number of
genotypes of wild and cultivated species of Curcuma.
Acknowledgments The authors are grateful to the director, NBPGR,
New Delhi, for providing the facilities. Special thanks are accorded to
Dr. Ana M. Vieitez, the associate editor of this journal, whose critique
has greatly helped in the improvement of the manuscript.
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