RESEARCH ARTICLE

In Vitro Plant Regeneration of Cymbopogon jwarancusa (Jones)Schult from Meristematic Base of Spikelet

Pooja Mehandru • H. S. Gehlot • N. S. Shekhawat •

J. B. Vibha • Rachana Dinesh

Received: 2 April 2013 / Revised: 22 August 2013 / Accepted: 22 October 2013 / Published online: 11 March 2014

� The National Academy of Sciences, India 2014

Abstract Cymbopogon jwarancusa is a multiple stress

(es)-tolerant, C4 and aromatic grass of extremely arid parts

of the Thar Desert. Genetic amelioration of this cross

pollinated species necessitates development of an effective

in vitro plant regeneration protocol. Therefore base of the

spikelet comprising a zone of actively dividing meriste-

matic cells was used as source(s) of totipotent/pluripotent

cells for callogenesis. These explants produced competent

and highly regenerative cultures on agar gelled MS med-

ium containing 22.62 lM of 2, 4-D. The cultures multi-

plied optimally on MS medium with 18.1 lM of 2, 4-D,

4 % sucrose and 0.5 % glucose. The callus organized to

globular embryos on plant growth regulator-free MS

medium followed by emergence of coleoptile and rhizo-

genesis. Incorporation of 4.65 lM of Kinetin in the

regeneration medium enhanced the rate of plant regenera-

tion. About 40–45 plantlets were obtained from a single

explant derived cultures. Of the in vitro raised plantlets,

98 % survived under the greenhouse conditions. The plants

matured and set viable seeds.

Keywords Acclimatization � Aromatic grass �Somatic embryogenesis � Plant regeneration

Introduction

Advances in plant and cell culture of cereals and grasses

are moderate due to limitations like genotype, explant type

and having short regeneration ability [1]. Such approaches

in aromatic grasses particularly Cymbopogon are limited.

Choice of explant plays a critical role in in vitro culture

particularly in case of monocots. Immature tissues and cells

derived from a source close to the embryonic state are the

choice of tissue type competent for regenerating whole

plants. Therefore immature embryo, mature seed and

immature inflorescence [2, 3] have been used as explants

for most studies. These are a heterogeneous mass of tissue

which yields both competent and non-competent cultures

requiring the difficult task of selection of competent cul-

tures. For establishment of morphogenic cultures there is a

need for explant(s) that are meristematic and source of

homogenous mass of cell/tissue. Various type of explants

such as inflorescence, rhizome, leaf node, nodal shoot

segments and seedling explants have been used for in vitro

propagation of Cymbopogon including the induction of

organogenesis, somatic embryogenesis, plant regeneration

and soma clonal variations in callus cultures [4–6]. How-

ever, these require longer duration for induction of mor-

phogenic callus, as explants respond slowly [5, 7, 8]. The

percentage survival of in vitro raised plants has been

reported to be low [5]. Based on earlier reports, whereby

the base of arrow (peduncle) which is a repertoire of rap-

idly dividing and uniform population of meristematic cells

has been used to establish highly regenerative cultures in

Lasiurus sindicus [9] and Pennisetum glaucum

[10].Therefore the base of spikelet comprising actively

dividing totipotent cells was used as explant in this study

for callogenesis in Cymbopogon jwarancusa (Jones)

Schult, locally known as Khavi grass. This is a native

P. Mehandru (&) � N. S. Shekhawat � J. B. Vibha � R. Dinesh

Biotechnology Centre, Department of Botany, Jai Narain Vyas

University, Jodhpur 342033, Rajasthan, India

e-mail: lalitpooja@hotmail.com

H. S. Gehlot

BNF & Stress Biology Laboratory, Department of Botany, Jai

Narain Vyas University, Jodhpur 342001, Rajasthan, India

123

Natl. Acad. Sci. Lett. (March–April 2014) 37(2):131–135

DOI 10.1007/s40009-013-0210-7

aromatic grass of the Thar Desert, particularly the extreme

arid environment of Bikaner, Churu, Jaisalmer and Jodh-

pur. It yields Khavi grass oil. It is also useful in diseases of

blood, skin, vomiting, abdominal tumors, unconsciousness

and fever [11].This grass species also acts as a good soil

binder and has high fodder value. Till date there is no

report of in vitro culture in this grass species.

Materials and Methods

The meristematic base of the spikelet was harvested from

the plants maintained and managed in NBPGR, Regional

Station, Jodhpur. The explants (10–15 mm long) compris-

ing meristematic region enclosed within the sheathing leaf

base were pretreated with 0.01 % (w/v) of antibiotic solu-

tion (streptomycin ? tetracycline) (Hi Media Laboratories

Private Limited, Mumbai, India) for 10 min and 0.1 % (w/v)

solution of Bavistin (BASF India Limited, Mumbai, India)

for 15 min with intermittent washing after each step under

aseptic condition. These were surface sterilized with 0.1 %

(w/v) solution of HgCl2 (Hi-Media, India) for 3 min fol-

lowed by thorough washing with sterile water. The surface

sterilized explants were finally treated with chilled 0. 2 %

(w/v) solution of ascorbic acid for 15 min. The sheathing

leaf base was removed before inoculation on Murashige and

Skoog [12] medium containing 8.0 g l-1 agar (Bacterio-

logical grade, Qualigens Fine Chemicals, Mumbai, India),

MS basal salts, vitamins with myoinositol (100 mg l-1),

sucrose (4 %), glucose (0.5 %) and supplemented with

various concentrations (13.57–45.24 lM) of 2, 4-D for

initiation of competent culture. The pH of the medium was

adjusted to 5.8 before gelling with agar and autoclaving at

1.1 kg cm-2 for 15 min. The culture tubes were incubated

in growth chamber at temperature 26 ± 2 �C under diffused

light (15–20 lmol m-2 s-1 PFD). Approx. 100 mg of callus

was then transferred to MS medium containing

9.05–22.62 lM of 2, 4-D with 4 % sucrose and 0.5 % glu-

cose for callus proliferation. The proliferated callus was

further transferred to full strength, half strength or quarter

strength plant growth regulator free MS medium and to MS

medium with various concentrations of Kinetin

(2.32–13.92 lM) for shoot regeneration. The cultures were

incubated under full light conditions (40–50 lmol m-2 s-1

PFD) in growth chamber at 26 ± 2 �C, 60 % RH. In vitro

raised plantlets were transferred to autoclaved screw cap

bottles (250 ml) containing 1/3 volume of soil rite. The

plantlets were nurtured with one-fourth strength MS med-

ium and were kept in green house at 28 ± 2 �C, 70 % RH.

Acclimatized plants were then transferred to polybags

containing garden soil before consequent field transfer.

The experiments were set up in completely randomized

block design and repeated thrice with a minimum of 20

replicates per treatment. The data was analyzed statistically

using SPSS v.17 (SPSS, Chicago, USA). The significance

of differences among means was carried out using [13]

Duncan’s multiple range test at P \ 0.05.

Results and Discussion

An efficient in vitro regeneration of plants from single cells

is a pre requisite for cellular and molecular manipulations

[14]. In the present study, intercalary meristem of the base

of the spikelet was selected as the explant for induction of

callus (Fig. 1a). Treatment of explants with antibiotic

solution was required to prevent bacterial contamination as

the explants were harvested during rainy season while

treatment of the explant with an antioxidant such as

ascorbic acid was essential since explants turned brown in

culture and deteriorated. Ascorbic acid was also incorpo-

rated in the medium in order to check the browning of the

cultures.

Effect of 2, 4-D on Callus Initiation

Meristematic base of the spikelet responded within

8–10 days, exhibiting signs of enlargement from the

intercalary meristematic regions of the explant (Fig. 1b)

on MS medium with various concentrations

(13.57–45.24 lM) of 2, 4-D. High concentration of 2, 4-D

is required for initiation of cell division in the meristematic

tissues that are under development pressure of the mother

tissues. Early molecular events during callus initiation

involve differential expression of genes mainly related to

hormone homeostasis and signaling, transcriptional and

post transcriptional regulations, protein phosphorelay cas-

cade and DNA or chromatin modification [15]. Numerous

studies have shown that 2, 4-D is the most widely used

growth regulator for morphogenic calli induction irre-

spective of the explant in conformity to our results [8].

Maximum explant response was observed on MS medium

supplemented with 22.62 lM of 2, 4-D (Table 1; Fig. 1b).

The callus induced from the meristematic base of the

spikelet was nodular, greenish yellow, friable, slow grow-

ing and highly morphogenetic.

Optimization of Medium for Callus Proliferation

For amplification, compact, nodular and yellowish callus

induced from the explant was separated and a substantial

stock of proliferative calli was built up by sub culturing it

for 4–5 cycles on MS medium containing additives, 4 %

sucrose, 0.5 % glucose and 18.10 lM of 2, 4-D (Fig. 1c).

The concentration of 2, 4-D required for the maintenance

of competent cultures was lower as compared to that

132 P. Mehandru et al.

123

needed for the induction of embryogenic cultures in C.

jwarancusa. After establishment of competent cultures the

requirement of 2, 4-D declines. Carbohydrates are not only

the source of energy but they even influence and maintain

the osmolarity of the culture medium. Increased osmotic

value helps in maintenance of morphogenic capacity of

callus [1] by preventing onset of embryogenesis associated

with significant decline in osmolarity of the medium [16].

Plantlet Regeneration

The transition from dedifferentiated status to embryogenic

and/or organogenic status is a complex process comprising

several phases, including dedifferentiation, cell reactiva-

tion, cell division and various metabolic and develop-

mental re-programming steps. These maturation processes

Fig. 1 Induction and proliferation of morphogenic calli from mer-

istematic base of spikelet in C. jwarancusa. a Inflorescence of C.

jwarancusa including the meristematic base used as explant for

culture initiation (scale bar 1.5 cm). b Morphogenic calli induced

from the base of the spikelet after 8–10 days of culture on MS

medium with 22.62 lM of 2, 4-D supplemented with 4 % sucrose and

0.5 % glucose (scale bar 0.8 cm). c Compact, nodular and yellowish

green proliferative callus induced from the explant on MS medium

with 18.1 lM of 2, 4-D supplemented with 4 %sucrose and 0.5 %

glucose (scale bar 1.4 cm). d Globular embryo like structures on

phytohormone-free MS medium (scale bar 2 mm). e Regeneration of

embryogenic tissues into green shoots on phytohormone-free MS

medium. (scale bar 2 mm). f Plantlet regeneration from competent

cultures on MS medium with 4.65 lM Kinetin. (scale bar 1.4 cm).

g In vitro regenerated plantlets of C. jwarancusa (scale bar 1.8 cm).

h Acclimatized plantlets of C. jwarancusa in poly bags containing

garden soil under greenhouse conditions (scale bar 7 cm). i Normal

growth and flowering of in vitro raised plants of C. jwarancusa (scale

bar 12 cm)

Table 1 Effect of different concentrations of 2, 4-D on callus

induction from meristematic base of spikelet of C. jwarancusa on MS

medium

Concentration of 2,4-D (lM) Explant response (%)

0.0 0.0

13.57 66.3 ± 0.32c

22.62 77 ± 1.65a

31.6 73.9 ± 1.96b

36.1 52.6 ± 1.28d

45.24 39.2 ± 0.69e

Data was recorded after 2 weeks of culture. Each treatment was

repeated three times. Basal medium: MS ? 4 % Sucrose ? 0.8 %

agar ? 0.5 % glucose

Values represent mean ± SE. Mean followed by same letter are not

significantly different at the 0.05 level of confidence as per DMRT

[13] test (P \ 0.05)

In Vitro Plant Regeneration of C. jwarancusa 133

123

are under the control of a number of signaling pathways,

which integrate genetic, metabolic and hormonal signals

[16]. Synthetic auxins like 2, 4-D which is used for

induction and proliferation of embryogenic cultures, are

usually less metabolized by the cells than other auxins.

Therefore, it is necessary to transfer embryogenic cultures

to a medium lacking auxin to stimulate further growth [17].

The cultures initially showed optimum callus growth fol-

lowed by organization into globular embryo like structures

(Fig. 1d) on PGR free MS medium of various strengths

(Table 2) within 20–25 days. On further subculture these

continued to grow up to 10–15 days along with the emer-

gence of coleoptile (Fig. 1e). About 4–5 plantlets were

regenerated from approximately 100 mg of embryogenic

culture on full strength MS medium without PGRs. These

attained a height of 7–8 cm within 30 days. Incorporation

of Kinetin in the MS medium resulted in improvement of

somatic seedling regeneration in C. jwarancusa and

reduced the duration of its full growth in concurrence with

earlier report [18]. Maximum regeneration was observed

on MS basal medium containing 4.65 lM of Kinetin

(Table 3). Approximately 11–13 plantlets regenerated per

100 mg of embryogenic callus (Fig. 1f) inoculated. The

regenerated plantlets attained a height of 8–11 cm within

30 days (Fig. 1g). The plantlets hardened successfully

under greenhouse conditions with 98 % ±0.38 survival.

After full growth these were transferred to poly bags

containing garden soil (Fig. 1h). Seedlings showed profuse

and vigorous growth on transfer to pots in the nursery. The

plants developed inflorescence after sufficient vegetative

growth, followed by seed setting (Fig. 1i). Our results

suggest that the base of spikelet is a potential source of

competent/totipotent cells for establishment of embryo-

genic cultures. It can also be used as a suitable target for

direct genetic transformation in graminaceous plants and as

a source of cells for protoplast isolation.

Acknowledgments Pooja acknowledges WOS-A fellowship from

DST while Vibha acknowledges UGC post-doctoral fellowship for

women. We gratefully acknowledge the financial support provided by

Department of Biotechnology (DBT) Department of Science and

Technology (DST) and University Grant Commission (UGC) for

establishment of regional facility for micropropagation and green-

house for arid region.

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Concentration

(mg l-1)

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plantlets per 100 mg

callus (mean ± SD)

Shoot length (cm)

mean ± SD

MS 4.0 ± 0.5a 7.8 ± 0.42a

MS � 2.5 ± 0.70b 6.8 ± 0.42b

MS � 1.9 ± 0.56c 6.5 ± 0.52b

Data was recorded after 4 weeks of culture

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from regenerative calli of C. jwarancusa on MS medium ? additives

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Kinetin (lM)

Number of plantlets per

100 mg callus (mean ± SD)

Shoot length (cm)

(mean ± SD)

2.32 7.9 ± 0.73c 8.4 ± 0.51e

4.65 12.6 ± 0.96a 11.2 ± 0.46a

6.97 10.4 ± 0.69b 9.9 ± 0.31c

9.28 9.5 ± 0.52c 10.5 ± 0.52b

11.62 8.5 ± 0.70c 9.2 ± 0.44d

13.92 8.1 ± 0.73c 10.08 ± 0.47c

Data was recorded after 4 weeks of culture

Mean followed by same letter are not significantly different at the

0.05 level of confidence as per DMRT [13] test (P \ 0.05)

134 P. Mehandru et al.

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