b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 4 2 e4 6

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Short communication

Improved in vitro rooting and acclimatization of Jatropha

curcas plantlets

Dibya D. Toppo, Gaurav Singh, D.K. Purshottam, Pratibha Misra*

Plant Transformation Laboratory, National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR),
Lucknow 226001, India

article info abstract

Article history: An improved protocol has been developed for micropropagation of Jatropha curcas to
Received 4 March 2010 obtain quality planting material on large scale. To obtain improved shoot growth,
Received in revised form rooting and acclimatization of in vitro plants, multiplication medium was modified.
13 April 2012 Shoots multiplied in Murashige and Skoog’s (MS) medium containing 15 mg dm
3 each of
Accepted 15 April 2012 L-glutamine and L-arginine along with 50 mg dm
3 Augmentin
and 15 mg dm
3 coconut
Available online 15 May 2012 water (CW), besides 0.5 mg dm
3 benzyladenine (BA), 0.1 mg dm
3 indole-3-butyric acid
(IBA) and 10 mg dm
3 adenine sulphate (AdS), rooted well under in vitro conditions. The
Keywords: addition of 15 mg dm
3 coconut water from green coconut in the multiplication medium,
Acclimatization produced healthy shoots with broad leaves, resulting in improved rooting of 85% as against
Augmentin
52% reported earlier. The use of Augmentin
in the medium not only controlled bacterial
Coconut water contamination, but also resulted in better survival and growth of plants during hardening.
Jatropha curcas These rooted shoots e plantlets e were acclimatized in the soilrite with 100% transplant
Rooting success where they grew vigorously under nethouse conditions.
ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction known characters. The present study overcomes hitherto

Millions of hectares of Jatropha plantation are being projected
to be undertaken in the coming years worldwide. In view of
Jatropha’s plant life being as much as 50 years, the quality of
planting material is bound to have long term tangible ramifi-
cations. Jatropha has a lot of interspecific genetic diversity [1]
as well as diversity among different accessions of Indian
varieties [2]. The inter-variety spread of oil yield between
Jatropha varieties ranges between 30 and 37%. Therefore, use
of elite variety for planting material can assure a 20% more
biodiesel yield. Field-grown material from elite mature trees is
preferred for collecting explants due to its genetic stability and
problem of browning of in vitro cultures of Jatropha [3] and
achieves a robust protocol for large scale propagation of tissue
raised plants of its elite variety. There are many recent
reviews published on Jatropha [4e7], which describes all the
biotechnological interventions for its improvement. Most of
the reports on in vitro clonal propagation were achieved
through organogenesis [8e10], but few are through somatic
embryogenesis [11,12]. Although shoot multiplication is good
in Jatropha curcas, but the growth of regenerating shoots, low
rooting percentage and difficult acclimatization of tissue
raised plantlets, restricts clonal propagation for quality
planting material. In the present communication, improved

Abbreviations: AdS, adenine sulphate; BA, benzyl adenine; CW, coconut water; IBA, indole-3-butyric acid; MS medium, Murashige and
Skoog’s medium; mMS, modified Murashige and Skoog’s medium.
* Corresponding author. Tel.: þ91 (0) 522 2297923; fax: þ91 (0) 522 2205836.
E-mail addresses: pratibhaflora@yahoo.com, pratibhamisra@nbri.res.in (P. Misra).
0961-9534/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2012.04.014
 b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 4 2 e4 6 43

Fig. 1 e (a & b) In vitro cultures of J. curcas; (a) mMS medium without CW and Augmentin
; (b) mMS medium with
50 mg dmL3 Augmentin
and 15% CW.

percentage of rooting and perfect survival of acclimatized
plants in the field has been shown due to the addition of CW,
Augmentin
and AdS in the multiplication medium.
2. Material and methods
The plants of J. curcas were procured from CSMCRI,
Bhavnagar, India, located at 21.7700 N, 72.1500 E and grown
in NBRI campus. Nodal explants were collected from 2nd e 5th
node from the shoot tip and established from a 2 years-old-
high yielding C2 accession of J. curcas [8,13]. Shoots were
multiplied in Murashige and Skoog’s [MS [14],] medium, con-
taining 15 mg dm
3 each of L-glutamine and L-arginine and
50 mg dm
3 Augmentin
(mMS medium). The medium was
supplemented with a combination of 0.5 mg dm
3 BA and
0.1 mg dm
3 IBA along with 10 mg dm
3 AdS. Effect of fresh
coconut water (10e25%) was seen on multiplication and
growth of shoots. Fresh coconut water was collected from
green coconuts and filtered with sterilized double cheese
cloth. The pH of all the tissue culture media was adjusted to
5.8 prior to the addition of agar and autoclaved at 121  C for
20 min. All the media were solidified with 8 g dm
3 agar.
Cultures were incubated at 25  2  C under 16 h light and 8 h
dark period. Light at a photon flux of 60 mmol photon m
2 s
1
was supplied by fluorescent tube lights fitted on culture racks.
For rooting, initially, half MS or full MS basal media were
used supplemented with different concentrations of IBA
(0.25e1.0 mg dm
3). Later, half mMS or full mMS media,
without AdS and CW was used with similar concentrations of
IBA to obtain maximum rooting percentage along with growth
of rooted plantlets. After root induction the plantlets were
subcultured in the hormone-free medium for their further
growth. The rooted shoots e plantlets e were transferred
directly to the pots filled with sterilized soilrite. During initial
10e15 d of hardening, high humidity was maintained by
covering the plants with acclimatization hood of plexiglass
(Basco Pvt. Ltd., India) and irrigated with tap water. For next
15 d, acclimatization hoods were raised slightly decreasing the
humidity slowly. After 4 wk, plants were kept in nethouse at

Table 1 e Effects of CW on growth of regenerating shoots.

Treatments *Avg. height of *Avg. number ***Avg. length of ***Avg. width Callus General health
(Conc. mg dm
3) regenerated of leaves  SE leaves (cm)  SE of leaves formation of the shoots
shoots (cm)  SE after 30 d
(cm)  SE

Control 2.5  0.21 7  1.0 2.5  0.18 2.0  0.12 þ Leaf necrosis
CW 10 4.0  0.38 10  2.0 3.8  0.35 2.5  0.18 þ Leaves green and
healthy
CW 15 4.5  0.42 12  2.4 4.0  0.36 3.0  0.21 þ Leaves green and
healthy
CW 20 3.8  0.34 9  1.8 3.5  0.28 2.5  0.16 þþþ Leaves yellowish
green

*Average of longest shoots in each of 5 replicate cultures.

**Average of total number of fully expanded leaves in each of 5 replicate cultures.
***Average of middle whorl of leaves in 5 replicate cultures.
44 b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 4 2 e4 6

Table 2 e Effects of composition and dilution of basal media on in vitro rooting.

Basal medium IBA concentration Rooting (%) Associated General health of
(mg dm
3) callus the plantlets

Full MS 0.25 58 þþ Shoots were green

0.5 64 þþþ and healthy
1.0 68 þþþ
Half MS 0.25 75 þ Shoots were weak
0.5 80 þ with yellowing of leaves
1.0 82 þ
Full mMSa 0.25 64 þ Shoots were fresh,
0.5 68 þ green and healthy
1.0 72 þ
Half mMS 0.25 79 e Shoots were fresh,
0.5 85 e green and healthy
1.0 83 e

a MS medium containing 15 mg dm
3 each of L-glutamine and L-arginine and 50 mg dm
3 Augmentin
.

28  2  C. Initially the plantlets were covered with polythene
for 1 wk and slowly the humidity was decreased by removing
the polythene under nethouse conditions.
3. Results and discussion
Shoots did not grow well and started necrosis in mMS medium
beyond 20 d of culture incubation (Fig. 1a), but proliferated
well in mMS medium having 15 mg dm
3 L-glutamine and L-
arginine supplemented with 0.5 mg dm
3 BA and 0.1 mg dm
3
IBA, 10 mg dm
3 AdS and 50 mg dm
3 Augmentin
(Fig. 1b).
In J. curcas AdS has already been reported beneficial for
somatic embryo development [12] as well as on increasing
shoot number and shoot length [9] similar to our observations
of better leaf and shoot growth. In other plants, like, Leucaena
leucocephala [15] and Hemidesmus indicus [16] addition of AdS
was found beneficial for leaf growth. Increase in leaf size
appears to be an important factor for plant’s overall growth
which develops strength in the plantlets to withstand the
septic conditions of outside environment. Addition of fresh
coconut water improved the general health and growth of the
regenerated shoots (Table 1). Shoots were more vigorous and
sturdier with increased number and size of the leaves, in the
presence of CW to the medium.
Among different concentrations of CW used, 15% concen-
tration was selected as the optimum concentration, beyond
which a large callus was developed at the base of the shoots.

Fig. 2 e (a) Rooting in excised shoots, with Augmentin
(L) and without Augmentin
(R); (b) 4-Wk-old potted plants with
Augmentin
(L) and without Augmentin
(R); (c & d) 8-Wk-old potted plants in the field without Augmentin
(c); and with
Augmentin
(d).
 b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 4 2 e4 6 45

Table 3 e Comparison of in vitro-shoots grown in the presence and absence of Augmentin
.

Parameters studied for regenerated shoots Shoots grown in the Shoots grown in the
presence of Augmentin
absence of Augmentin

In vitro multiplication Number of shoots 5 3

Height of shoots 5 3
Number of leaves 10 7
In vitro rooting Percentage of rooting (%) 100 70
Number of roots 9 4
Acclimatization Percentage of 100 70
of shoots acclimatized shoots (%)
Growth of shoots after Excellent growth Not much growth with
4 wk with larger leaves comparatively smaller leaves
Field performance Height of shoots More than 30 cm Less than 15 cm
after 8 wk Overall growth of shoots Shoots grew fresh, Shoots grew green and
green and healthy healthy with an average
with an average of 8 cm leaf length
of 12 cm leaf length

Shoots grew stronger in the presence of 50 mg dm
3

Augmentin
, which not only protected the shoots from
any bacterial contamination but helped in growing healthy
shoots also.
In J. curcas rooting of in vitro regenerated shoots was
reported as low as 52% [9]. Sometimes ex vitro rooting has been
performed but there also the percentage was very low [17].
The results of in vitro rooting are shown in Table 2. Between
two concentrations of media used, 85% shoots rooted in half
MS medium as compared to 60% in full MS medium. However,
shoot growth was much better in full MS medium as
compared to half MS medium, which may be due to low salt
concentration.
In the second set of experiment, half mMS medium was
used instead of half MS having high nitrogen contents in the
form of glutamine and arginine. Shoots not only grew healthy
in half mMS medium but also rooted 85% in the presence of
0.5 mg dm
3 IBA within 3 wk of incubation (Fig. 2a). When
plantlets from an Augmentin
containing medium were
transferred in the pots, the survival rate was 100% and the
growth of the plants was also vigorous (Table 3).
Augmentin
was used in the medium to suppress the
bacterial growth, but at the same time it supported growth of
plant system with regard to cell division (plant growth) and
cell differentiation (shoot organogenesis) as reported in many
plant systems [18]. This may be due to the antibiotic coverage
to the plants during their early stage of acclimatization when
they are prone to any biotic or abiotic stress present in their
rhizosphere.
Once the plant is fully acclimatized, i.e. approximately 4
months of transplantation, it could withstand any kind of
biotic or abiotic stress and survived (Figs. 2be1d).
4. Conclusion
It may be concluded that the shoots which were multiplied in
the presence of CW and Augmentin
, acclimatized 100% in
soilrite. Whereas, shoots grown in their absence, were having
smaller leaves and became susceptible to different soil
microbes resulting in higher mortality of tissue raised plants.
Acknowledgements
The authors are thankful to the Director, National Botanical
Research Institute (Council of Scientific and Industrial Research),
Lucknow, for providing necessary infrastructure facilities.
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