In Vitro Multiple Shoots regeneration in Salvadora oleoides Decne

Surender Kumar1, J.S. Laura*2 and Narender Singh3

1
Department of Botany, K.M. Govt. College Narwana
2
Department of EVS, Maharshi Dayanand University Rohtak
3
Department of Botany, Kurukshetra University Kurukshetra
Corresponding author* - jslmdu@gmail.com
Abstract
A highly efficient micropropagation protocol was developed for Salvadora oleoides using
shoot tip explants obtained from 12-15 days old aseptically grown seedlings. Multiple shoots
were produced by using shoot tip explants on Murashige and Skoogs medium fortified with
different concentrations of cytokinins (Kn and BAP) alone and in combination with auxins (IAA
and NAA). Among individual treatments of cytokinins tested, 3.0 mg l-1 BAP supplemented in
MS medium supported better results. Maximum shoots were induced on MS medium containing
3.0 mg l-1 BAP in combination with 0.5 mg l-1 NAA. Rooting of the plantlets was achieved on MS
medium supplemented with 1.0 mg l-1 NAA. The plantlets showed 80% survival rate during
acclimatization and hardening.

Key words: Shoot tip explants, Salvadora oleoides, Cytokinins, Auxins

Abbreviations: BAP 6-benzylaminopurine, Kn- 6-furfurylaminopurine, IAA- Indole-3-acetic
acids, NAA -Naphthalene acetic acid, IBA Indole-3-butyric acid.

Introduction
Salvadora oleoides is a multipurpose tree found in the arid tracts in the state of Punjab,
Haryana, Gujarat, Rajasthan and south-western parts of Uttar Pradesh in India. It is locally known
as Pilu and Jhal. This species has great ethno-medicinal value. Leaves are used in treatment of
cough and enlarged spleen and are given to horses as purgative. The fruits are sweets and edible.
The fruits contain glucose, fructose, and sucrose and are good source of calcium. The fruits are
also used in the treatment of piles, tumors, bronchitis, low fever, disease of spleen and fed to
cattles to increase their milk yield (Khan, 1996).The fruit are relished by a variety of insects, birds
and rodents. Leaves and fruits are used during child birth and as antidote of snake bites. The seeds
are rich in non edible oil and their fat is used in the treatment of rheumatic pains and as a base of
ointment (Kirtikar and Basu, 2012). Seed oil is widely used in commercial production of
cosmetics, paints, varnish and lubricants. Seed oil of S.oleoides can be used as mosquito
repellants and oil of this plant also shows larvicidal activities in the form of hundred per cent
mortality against mosquitoes like Culex fatigans and Anopheles stephensi (Tare and
Sharma,1991). This species is drought hardy, moderately salt tolerant, sand binder, useful in
restoration of fragile arid tracts, stabilization of sand dunes and afforestation of degraded lands.
Salvadora oleoides provides ecological services such as habitat, food and shelter for varieties of
mammals, reptiles, birds and hence maintain an ecological balance (Khan, 1996).
This species is decreasing very rapidly due to indiscriminate exploitation and low rate of
seed set, poor seed viability and inefficiency to propagate by vegetative means. Therefore, 476
keeping in view the economic and ecological importance of this plant species; efforts were made
to develop an efficient method for rapid in vitro propagation using axenic shoot tip explants via
optimization of basal media, growth regulators and followed by successful outdoor establishment
of regenerated plants.

Materials and Methods

All experiments were conducted in plant tissue culture laboratory, Department of
Environmental Science, Maharshi Dayanand University, Rohtak. Seeds were collected from
mature plants. The seeds were washed with detergent in running tap water to remove all traces of
fruit pulp, followed by selection of healthy seeds. The seeds were then surface sterilized in 90%
ethanol for 3 minutes followed by a treatment of freshly prepared 0.1% HgCl 2 solution for five
minutes. Finally, seeds were washed 4-5 times in sterilized distilled water to remove all traces of
mercuric chloride. MS medium was prepared with 3% sucrose and 0.8% agar-agar. The pH of
medium was adjusted 5.8 prior to adding of agar-agar. The medium was autoclaved for 20
minutes at 15 psi pressure and 1210C temperature. Seeds were inoculated in culture vessels
containing half strength MS medium. Culture conditions were maintained in a culture room at
25 20C temperature under 16/8 hours (light/dark) period with 50 mol m-2 s-1 photon flux
density provided by cool white fluorescent tubes (Philips, India) and 60% relative humidity.
Similar culture conditions and compositions of MS basal medium were used for further
investigation to regenerate plantlets using axenic shoot tip explants.
Shoot tip explants were excised from twelve to fifteen days old aseptically grown
seedlings. Explants were inoculated in culture vessels containing MS medium with and without
growth regulators. MS basal medium without growth regulator served as control. Different
cytokinins (Kn and BAP) in various concentrations were used. Individual concentrations of
cytokinins which gave best results were used in combinations with auxins (IAA and NAA) to
enhance the shoots multiplication and proliferation rate. In vitro regenerated shoots along with
explants were transferred to fresh medium at regular interval for maintaining the healthy growth
of shoots. In vitro regenerated shoots after attaining the height of 2- 4 cm were implanted in
rooting medium augmented with IBA and NAA. In vitro regenerated complete plantlets were
taken out from medium and their roots were washed with the help of fine brush to remove traces
of medium by dipping the roots in tap water. Then individual plantlets were transplanted in a pot
having sterile soil and vermiculite (1:1). Initially the plantlets were kept in laboratory with high
humidity and finally these were shifted to field conditions after acclimatization. Data were
recorded after every 5 weeks. Each experiment was repeated thrice containing ten replicates for
each hormonal treatment. The data were analyzed (mean S.E.) for each treatment.

Results and Discussion

The regeneration of multiple shoots is considered as an important step in standardization
of micropropagation protocol. But optimum requirement for shoot proliferation stage may differ
from species to species. Production of plantlets starting from shoot tip explants taken from
aseptically grown seedlings of Salvadora oleoides has been achieved in present study. Shoot
formation from the shoot tip was seen in all the treatments of Kn and BAP with variable
frequency of regeneration. Generally, cytokinins are required for in vitro axillary shoot induction
and proliferation but type of cytokinins and its optimal concentration varies with the species. 477
Cytokinins are supposed to play a significant role in DNA synthesis as reported by Park (2008).
During present investigation, shoot tip explants responded morphogenetically to a growth free MS
medium with less number of shoots. MS medium augmented with 3.0 mg l-1 BAP individually
was found optimum for shoot induction and proliferation as this concentration gave superior
results individually. BAP showed superior results than Kn in present investigation (Table-1).
Higher concentration of Kn and BAP than 3.0 mg l-1 produced less number of shoots. These
observations further support the well known inhibitory influence of higher concentration of
cytokinins on shoot elongation and regeneration as reported by (Koroch et al. (1997) in Hedeoma
multifolium.
Table 1.- Effects of different concentrations of Kn and BAP on shoot tip explants taken from in vitro raised seedlings.

Concentrations of growth regulators

Media Per cent bud break No. of shoots regenerated per explant (S.E.) Shoot length (cm) (S.E.)
( mg l-1)
Control - 40 10.23 0.80.04
0.5 70 20.43 1.00.08
1.0 80 20.34 1.20.16
1.5 90 20.16 1.30.02
2.0 100 40.24 1.50.41
MS+Kn
2.5 100 50.63 1.70.56

3.0 100 60.28 2.00.54

4.0 70 40.45 1.90.35

0.5 80 20.44 1.20.21
1.0 90 30.18 1.40.60
1.5 100 30.26 1.50.38
MS+BAP 2.0 100 50.14 1.60.32
2.5 100 60.62 1.80.25
3.0 100 80.36 2.10.34

4.0 90 50.08 2.00.04

A stimulatory effect of cytokinins (Kn and BAP) with auxins (IAA and NAA) was also
evaluated. Auxins are known to promote cell division. So combinations of cytokinins with auxins
may proliferate shoots most significantly as observed by Heide (1965), Skoog and
Miller(1957).Optimized concentrations of Kn and BAP (3.0 mg l-1) were used with low
concentrations (0.25-1.0 mg l-1) of auxins. Combined effect of cytokinins with auxins has been
demonstrated by many workers such as Sudha et al.(1998) in Hollostemma annulare, Sreekumar
et al.(2000) in Hemidesmus indicus and Arya et al. (2003) in Leptadenia reticulata. Low levels of
IAA and NAA (0.5 mg l-1) in combination with cytokinins had promotional effects on shoot bud
induction whereas further higher concentrations (1.0 mg l-1 or more) were not found beneficial.
Among two cytokinins optimized, combined effects of BAP with IAA and NAA were better than
Kn with IAA and NAA. Similarly NAA was evaluated better than IAA when used in combination
with cytokinins (Kn and BAP). Since the maximum number of shoots were produced in the
presence of MS + BAP (3.0 mg l-1) and NAA (0.5 mg l-1), this medium was designated as best
shoot induction and multiplication medium (Table-2). Similar results were obtained by Ajith et al.
(1999) in Syzygium travancorium.
According to Gasper and coumans (1987), rooting of the developed shoots is usually
478
achieved in auxins containing medium. Root formation is an energy demanding process and thus
exogenous supply of carbohydrates is required. However, this being the last stage of in vitro
culture, it is important to transform the plant from heterotrophic to autotrophic mode of nutrition.
Thus the supply of exogenous sugars should be reduced at this time. The rooting response
differed according to different concentrations and combinations of auxins used.
The in vitro-regenerated shoots (2.5- 4 cm), having at least 2-4 leaves and 2-3 nodes were
excised from shoot clumps and transferred to rooting medium with and without growth
regulators. Presence of NAA (0.5,1.0, 1.5 and 2.0 mg l-1) in MS full strength medium facilitated
better rhizogenesis as compared to similar concentrations of IBA. In present study maximum
rooting (80%) was achieved on full strength MS medium containing 1.0 mg l-1 NAA with fairly
good length and number of roots (Table-3). Chand and Singh (2004) reported that NAA was very
effective auxins for rhizogenesis in Pterocarpus marsupium . Intermittent callus formation at the
junction of root and shoot was reported. To reduce the callogenesis, the sucrose concentration was
reduced to 20 g l-1 from 30 g l-1.Sucrose at reduced concentrations has also been found optimal
by Das et al.(1993) for rooting in Acacia auriculiformis.
During acclimatization and hardening, shoot elongated and leaves turned green and
expanded. In vitro regenerated complete plants were transferred to small thermocol cups
containing sterilized soil and vermiculite (1:1) and maintained under high humidity in culture
room by covering them with inverted glass beaker and polythene bags. Equal ratio of Soil and
vermiculite has been used by Rao et al.( 2006) in Capsicum annum. Further the plantlets were
transferred to poly house followed by field conditions with eighty per cent survival rate. In
conclusion, BAP individually as well as in combination with NAA can be used for rapid
multiplication of true to type plants of Salvadora oleoides using shoot tip explants.

1 2 3

4 5
Fig.-1-3 Multiple shoots regeneration from shoot tip explants. (1) MS+3.0 mg l-1BAP,
(2)- MS+3.0 mg l-1Kn+0.5 mg l-1NAA, (3) MS + 3.0 mg l-1BAP +0.5 mg l-1 NAA, 479
(4) Root formation on MS+1.0 mg l-1NAA, (5) Acclimatized plant under field conditions
 The present paper describes a prime and easy to use protocol for large scale production of plants
through axenic shoot tip explants of Salvadora oleoides.

Table 2 - Combined effects of cytokinins (Kn and BAP) with different concentrations of auxins (IAA and NAA) on shoot tip explants taken from in vitro raised
seedlings.

Media Concentrations of growth regulators (mg l-1) Per cent bud break No. of shoots regenerated per explant (S.E.) Shoot length (S.E.)

3.0+0.25
100 60.64 2.10.57

3.0+0.5
MS+Kn+IAA 100 70.72 2.30.74

3.0+1.0 100 50.64 2.20.62

3.0+0.25 100 70.67 2.20.48

MS+Kn+NAA 3.0+0.5 100 80.58 2.40.36

3.0+1.0 100 60.37 2.30.94

3.0+0.25 100 80.37 2.20.50

MS+BAP+IAA 3.0+0.5 100 90.46 2.40.56

3.0+1.0 100 60.72 2.20.32

3.0+0.25 100 90.35 2.50.18

MS+BAP+NAA 3.0+0.5 100 100.36 2.60.77

3.0+1.0 100 80.60 2.40.49

Table-3 Effects of different concentrations of auxins (IBA and NAA) on in vitro regenerated shoots for root induction.

Auxins Concentrations of auxins Per cent root formation No. of regenerated roots per shoot (S.E.)

MS full strength
Control - -
(mg l-1)

0.5 - -

1.0 - -

MS+IBA 1.5 40 30.40

2.0 30 30.42

0.5 40 40.54

1.0 80 60.16

MS+NAA
1.5 60 40.45

2.0 60 40.45

480
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