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International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
©2016 RS Publication, rspublicationhouse@gmail.com Page 411
Micropropagation for Ehretia laevis Roxb. A rare Indian medicinal plant
Admuthe N. B.1
and Nalwade A. R.2
1, 2 Department of Botany, Annasaheb Awate Arts, Commerce and Hutatma Babu Genu
Science College, Manchar, Dist.-Pune Affiliated to Savitribai Phule Pune University
Maharashtra, INDIA
ABSTRACT
An efficient protocol was developed for shoot proliferation and plant regeneration of
Ehretia laevis Roxb. a rare medicinal plant, through in vitro culture using nodal explants.
Best shoot induction was observed on MS with 2.5 mg/l BAP, in which nodal explants
produce maximum number of shoots per culture were investigated. The highest number of
shoots and maximum average shoot length are recorded on MS medium supplemented with
2.5 BAP. Leaves segment were used as an explants MS with 2,4‐D were used for callus
induction. MS with 1.5 mg/l 2, 4-D had strong effect on callus formation. In vitro raised
shoots rooted on MS with IBA. MS with 1.4 mg/l IBA was the most appropriate condition for
rooting. The cultures were incubated at 3000 ‐5000 lux for 16 / 8 hrs light/dark at 25 ± 2ºC.
The well developed in vitro rooted plantlets were hardened successfully in the potting
mixture containing mixture of soil rite and vermiculite in the ratio of 1: 1. Pots were
regulararly watered and fed with liquid fertilizer. The plantlets were transferred to large pots
containing a mixture of soil and compost (2:1) and maintained in the polyhouse. The
hardening of plants occurred within 60-65 days. After hardening plantlets were maintained in
the shadenet. The survival rate of regenerated plantlets was 90%.
Key words: Ehretia laevis, explants, Shoot, Callus, rooting, plantlets.
INTRODUCTION
Ehretia laevis Roxb. is a rare Indian medicinal plant and member of Boraginaceae
family is widely used medicinal plant. The Ehretia laevis Roxb. is high valued medicinal
plant and becoming rare in the state of Maharashtra. It has a religious importance among
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
©2016 RS Publication, rspublicationhouse@gmail.com Page 412
Hindus. It is growing luxuriantly growing at Alandi near the Dnyaneshwar temple. It is a
highly valued plant cited in the literature of Nath Sampradaya. Dan and Dan (1982) have
estimated the triterpenoids in the bark of Ehretia laevis Roxb. Literature survey revealed
wide biological activity of family Boraginaceae. The inner bark of Ehretia laevis Roxb. is
used as food. Leaves are applied to ulcers and in headache. Fruit is astringent, anthelmintic,
diuretic, demulcent, expectorant and used in affections of urinary passages, diseases of lungs
and spleen. Powdered kernel mixed with oil is a remedy in ringworm. Seeds are anthelmintic.
The use of medicinal plants is increasing worldwide. Thapliyal and Yadav ( 2003) isolated
new napthoquinone derivative named lewisone was isolated from the aerial parts of Ehretia
laevis Roxb., collected from Northern region, along with the known compounds, n-
octatricontane, baurenol acetate , baurenol and ursolic acid. Torane et al. (2010) carried out
preliminary phytochemical screening and the nutritive factors like fats, proteins,
carbohydrates, vitamin-C, E, A, riboflavin and thiamine and reported that it has been proved
a good source of nutritional supplement. They carried out quantitative determination of trace
elements such as calcium, iron, potassium, phosphorus present in the leaves. The vital role of
trace elements in the human body has been established.
Torane et al. (2011) studied in vitro antioxidative activity of phenolic and flavonoid
contents of leaves of medicinal plant- Ehretia laevis. They estimated the total phenolic and
flavonoid content and evaluated in vitro antioxidant activity of methanolic extract of Ehretia
laevis Roxb. Torane et al. (2011) estimated antioxidant capacity of leaves and stem, total
phenolics, and flavonoid contents. They also evaluated in vitro antioxidant activity of
alcoholic extract of Ehretia laevis Roxb. Torane et al. (2011) quantified dioctyl phthalate
(DOP), a bioactive molecule from Ehretia laevis Roxb. by High Performance Thin Layer
Chromatographic (HPTLC) method. According to the World Health Organization (WHO),
approximately 80% of the world’s population currently use herbal medicines. In vitro cell and
tissue culture methodologies are envisaged as a mean for germplasm conservation to plant
species, rapid mass propagation for large scale regeneration, and for genetic manipulation
studies. In vitro propagation techniques may help in conservation of biodiversity of locally
used medicinal plants. In‐vitro propagation of plants holds tremendous potential for the
production of high quality plant based medicines (Murch et al. 2000).
There are no studies on the micro propagation of Ehretia laevis Roxb. or any member
of this genus. This paper describes for the first time a successful protocol on in vitro
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
©2016 RS Publication, rspublicationhouse@gmail.com Page 413
regeneration and field survival of Ehretia laevis Roxb. from nodal cuttings. The present
investigation was carried out to develop a simple, fast and efficient protocol for rapid
micropropagation of Ehretia laevis Roxb. by using nodal sector explant.
MATERIALS AND METHODS
The investigation was carried out in the Plant Tissue Culture Laboratory in Annasaheb
Awate Arts, Commerce and Hutatma Babu Genu Science College, Manchar, Dist.-Pune
Maharashtra, during the year 2011 to 2015.
Collection of Plant Material
Herbarium preparation
The flowering twig of Ehretia laevis Roxb. was collected from Pune, Maharashtra,
India. The taxonomic identification is accomplished with the help of the Flora of Presidency
of Bombay (Cook, T 1958) and Flora of Maharashtra State (Singh, N. P. et. al 2000) for
identification. The herbarium is prepared (Jain S. K. Rao R. R 1976) and it was identified and
authenticated at Botanical Survey of India, Pune, Maharashtra, India. Its voucher number is
BSI / WRC / TECH / 2013 / 156.
Explant preparation
Nodal cuttings of Ehretia laevis Roxb. were collected from healthy stocks of wild
plants.
Surface Sterilization of Explant
The shoot segments were washed under running tap water for 30 min, after that
immersed in liquid detergent Teepol (1% v/v ) for 2-3 min and washed thoroughly with
distilled water. Subsequently they were sterilized on the surface with 0.1 % HgCl2 solution
for 10 minutes and again washed well in distilled water for 3-4 times to remove the traces of
HgCl2.The explants were dipped in 70% ethanol for 30 sec. before application of the
sterilization treatments. After sterilizing the explants, they were rinsed in sterilized double
distilled water 3-4 times to remove all traces of the sterilizing substances. All steps of the
sterilization procedure were done under aseptic conditions inside the laminar airflow culture
cabinet.
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
©2016 RS Publication, rspublicationhouse@gmail.com Page 414
Sterilization of non- living articles
The sterilization procedure of non living articles such as nutrient medium, glass goods,
distilled water, instruments ( wrapped with brown paper ) is by autoclaving under stream at a
pressure of 15 lb/in2 and a temperature of 120
0 C for 15 minutes.
Culture Medium Preparation
The all explants were implanted on sterile medium consisting of salts and vitamins of
(Murashige and Skoog, 1962) supplemented with 0.8% (w/v) agar and 3% (w/v) sucrose. An
efficient media was developed and used alongside the conventional MS medium to regenerate
nodal cutting explants. Explants will only grow in vitro on a suitable artificially prepared
nutrient medium which is known as culture medium.
The nodal explants were aseptically placed in test tube (25 mm × 150 mm) containing MS
supplemented with different concentrations of growth regulators and sucrose (3%). The pH of
the medium was adjusted to 5.8 using 0.1N HCl or 0.1N NaOH before autoclaving and
solidified with 0.8% agar. The medium 20 ml was dispensed in each culture tube. The
medium was sterilized at 15 lb/in2 for 15 min.
Culture condition
After the treatments, the explants were cultured MS medium. Each culture tube with one
explant. All cultures were incubated in 16 hrs light/ 8 hrs dark photoperiod under light
intensity of 3000 ‐5000 lux at 25 ± 2°C with 80% relative humidity.
Shoot initiation:
Explants of Ehretia laevis Roxb. were cultured in vitro on MS medium supplemented
with BAP each at concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg/l, in
addition to control explants grown on a hormone-free MS medium. Optimum concentration
of BAP was recorded. After 4 weeks the data were recorded number of shoot/ explants, shoot
length (cm), and number of leaves /explants.
Callus induction:
Leaves segment explants of Ehretia laevis Roxb. were cultured in vitro on MS medium
supplemented with 2,4-D each at concentrations of 0.5,1.0,1.5, 2.0, 2.5, 3.0 mg/l, in addition
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
©2016 RS Publication, rspublicationhouse@gmail.com Page 415
to control explants grown on a hormone-free MS medium. Optimum concentration of 2,4-D
was recorded. After 4 weeks the data were recorded.
Sub-culturing:
Shoot and callus multiplication was continued by repeated sub-culturing on optimum
concentrations of PGRS till sufficient number of shoot and sufficient amount of callus were
obtained. The results were recorded and photographed.
Root initiation:
This experiment was carried out to study the effect of MS with different Indole 3-butyric
acid (IBA) concentrations 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4 mg/l, in
addition to control explants grown on a hormone free MS medium for root formation. After 4
weeks from culturing the explants, the data were recorded the number of roots/shootlets, root
length (cm), plant height (cm), and number of leaves/shoot. Optimum concentration of IBA
was recorded. Shoots were then cultured at optimum concentration of IBA in MS medium for
rooting.
Hardening of plantlets:
Plantlets were thoroughly washed with tap water to remove all traces of the medium.
These were planted in small pots filled with a mixture of soilrite and vermiculite (1:1). These
were covered with polythene bags and kept in the polyhouse to maintain the humidity and
temperature for first 30 days. Pots were regulararly watered and fed with liquid fertilizer. The
polythene bags were removed and plantlets were transferred to large pots containing a
mixture of soil and compost (2:1) and maintained in the polyhouse for next 30 days. The
hardening of plants occurred within 60-65 days. After hardening plantlets were maintained in
the shadenet. After this the plants were subjected to field trials for completing their
acclimation. Hardened plantlets were cultivated in the college campus.
Statistical analysis
The data were summarized as the means of three replicates with standard deviation as
the measures of variability. One-way ANOVA test was performed to determine significant
differences due to various treatments. Fisher’s LSD (Least significant difference) was used as
post hoc test to as certain significant differences among treatments at p= 0.05. Statistical
analysis and graphical data presentations were carried out by using Sigma stat (ver.3.5).
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
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RESULTS AND DISCUSSION
Effect of different concentration of Benzyl Aminopurine (BAP) with MS media on Shoot
Induction and multiplication:
For high frequency regeneration Ehretia laevis Roxb. nodal segments were used. Nodal
explants were used in MS supplemented with BAP 0.5 to 5.0 mg/l. Cultures were maintained
at 25 ± 2ºC at 16 hrs light and 8 hrs dark per day of fluorescent light 3000 - 5000 lux. The
nodal explants were cultured on MS supplemented with BAP (2.5 mg/l) produced high range
of multiple shoot after 4 weeks. MS with 2.5mg/l BAP was suitable for best shoot
regeneration. Best shoot multiplication was obtained from nodal explants in MS
supplemented with 2.5 mg/l BAP. Subcultures were made on the same medium after 4 weeks.
Similar results were reported by Faisal M. et al (2007) in Tylophora indica (Burm f.) Merrill
plants.
Table No. 1: Effect of Cytokinin BAP on shoot proliferation from nodal shoot explants
of Ehretia laevis Roxb
MS + BAP
(mg/l)
No of shoot/explant Shoot length (cm) No of leaves/explant
0.0 1.01±0.04 1.54±0.12 3.0±0.12
0.5 1.7±0.09 2.01±0.10 6.8±0.34
1.0 2.7±0.19 2.43±0.17 8.8±0.62
1.5 3.4±0.10 2.73±0.08 9.4±0.28
2.0 3.7±0.22 2.95±0.18 10.5±0.63
2.5 5.1±0.31 2.34±0.14 10.6±0.64
3.0 3.9±0.16 2.49±0.10 10.5±0.42
3.5 3.3±0.23 2.49±0.17 10.3±0.72
4.0 2.9±0.09 2.33±0.07 10.1±0.30
4.5 2.7±0.14 2.03±0.10 10.5±0.53
5.0 2.3±0.07 1.87±0.06 10.4±0.31
SEM± 0.14 0.10 0.39
F-value 132.39 27.97 70.98
P-value 0.01 0.01 0.01
LSD0.05 0.28 0.20 0.78
Data are means of three replicates ± standard deviation. Significant difference due to
treatments was assessed by Fisher’s LSD as a post-hoc test.
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
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Effect of different concentration 2,4-Dichlorophenoxyactic acid (2,4-D) with MS media
on Callus Induction:
Callus initiation was observed on MS media supplemented with 2,4-D 0.5 to 3.5
mg/l. The overall callus induction frequency on MS medium the maximum callus yield was
obtained after 4 weeks on MS with 1.5 mg/l 2,4-D. The similar result got in the range of 2,4-
D(1.5 to 3.0 mg/l) by Arumugam A. et al (2011) in Withania somnifera (L) plants.
Table No. 2: Effect of Auxin 2, 4-D on callus induction from leaves explants of
Ehretia laevis Roxb.
MS+2,4-D (mg/l) Fresh weight (in gm) Dry weight (in gm)
0.0 0.633±0.06 0.060±0.01
0.5 2.425±0.24 0.237±0.02
1.0 2.850±0.34 0.280±0.03
1.5 3.400±0.27 0.332±0.03
2.0 3.066±0.34 0.300±0.03
2.5 2.250±0.25 0.217±0.02
3.0 1.950±0.18 0.187±0.02
SEM± 0.21 0.02
F-value 38.04 39.09
P-value 0.01 0.01
LSD0.05 0.42 0.04
Data are means of three replicates ± standard deviation. Significant difference due to
treatments was assessed by Fisher’s LSD as a post-hoc test.
Effect of different concentration Indole 3-butyric acid (IBA) with MS media on Roots
Induction:
The elongated shootlets derived from Ehretia laevis Roxb. were transferred on to MS
supplemented with different increasing concentrations of Indole 3-butyric acid (IBA) from
0.2 to 2.4 mg/l for rooting. The best root induction was found on 1.4 mg/l IBA. The success
of IBA in promoting efficient root induction has been reported in Bacopa monnieri (L.)
Pennel plants by Mehta J. et al (2012).
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Table No. 3: Effect of Auxin IBA on root induction from shoot of Ehretia laevis Roxb.
MS+ IBA
(mg/l)
No. of roots/
shootlets
Root length (in
cm)
Plant height (in
cm)
No. of leaves
/shoot
0.0 0.5±0.02 0.80±0.03 1.10±0.04 2.4±0.10
0.2 1.9±0.09 1.05±0.05 2.55±0.13 4.6±0.23
0.4 2.6±0.18 0.93±0.07 2.43±0.17 4.6±0.32
0.6 3.4±0.10 1.50±0.04 2.85±0.09 7.4±0.22
0.8 4.4±0.26 2.10±0.13 2.82±0.17 8.0±0.48
1.0 5.5±0.33 2.72±0.16 2.87±0.17 7.4±0.44
1.2 6.0±0.24 2.30±0.09 3.12±0.12 9.0±0.36
1.4 8.0±0.56 3.37±0.24 3.70±0.26 10.1±0.71
1.6 5.3±0.16 2.92±0.09 2.60±0.08 7.8±0.23
1.8 4.4±0.22 2.20±0.11 2.10±0.11 6.2±0.31
2.0 3.6±0.11 2.22±0.07 2.02±0.06 6.0±0.18
22 2.9±0.17 1.80±0.11 2.26±0.14 6.2±0.37
2.4 2.1±0.15 1.73±0.12 1.96±0.14 3.4±0.24
SEM± 0.19 0.09 0.11 0.29
F-value 211.12 141.32 62.88 115.58
P-value 0.01 0.01 0.01 0.01
LSD0.05 0.38 0.18 0.22 0.58
Data are means of three replicates ± standard deviation. Significant difference due to
treatments was assessed by Fisher’s LSD as a post-hoc test.
Acclimatization:
The rooted plantlets were removed from the culture tubes. The plantlets were thoroughly
washed with tap water to remove all traces of the medium. These were planted in small pots
filled with a mixture of soil rite and vermiculite (1:1). These were covered with polythene
bags and kept in the polyhouse to maintain the humidity and temperature for first 30 days.
Pots were regulararly watered and fed with liquid fertilizer. The polythene bags were
removed and plantlets were transferred to large pots containing a mixture of soil and compost
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
©2016 RS Publication, rspublicationhouse@gmail.com Page 419
(2:1) and maintained in the polyhouse for next 30 days. The hardening of plants occurred
within 60-65 days. After hardening plantlets were maintained in the shade net. The survival
rates of the hardened plants were found to be 90%. The present study describes a well
documented and reliable protocol of Ehretia laevis Roxb. from nodal explants with much
higher rate of multiplication.
CONCLUSION
The present study contributes to the first report for effective successful regeneration of
Ehretia laevis Roxb. There are no studies on the micro propagation of Ehretia laevis Roxb. or
any member of this genus. This paper describes for the first time a successful protocol on in
vitro regeneration and field survival of Ehretia laevis Roxb. from nodal cuttings. The present
investigation was carried out to develop a simple, fast and efficient protocol for rapid micro
propagation of Ehretia laevis Roxb. by using nodal sector explants. The efficient in vitro
propagation method reported here can be commercially utilized for basic tool for commercial
cultivation, immediate conservation, improved multiplication and reforestation of Ehretia
laevis Roxb.
ACKNOWLEDGEMENTS
Authors are thankful to the Principal, Annasaheb Awate Arts, Commerce and Hutatma
Babu Genu Science College, Manchar, Dist.- Pune and Rayat Shikshan Sanstha, Satara for
providing laboratory facilities.
PLATE
A flowering Photo Ehretia laevis Roxb. MS+2.5mg/l BAP Root induction MS+1.4mg/l IBA
Callus induction MS+1.5mg/l 2,4-D Hardening I A Hardening I B
International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
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Plantlets in Polythene bag Plantlets in Pots
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International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
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International Journal of Advanced Scientific and Technical Research Issue 6 volume 3, May –June 2016 Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
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