AsPac J. Mol.

2014
Mol. Biol.
Biol.Biotechnol.
Biotechnol.
Vol. 22 (2), 2014
Vol. 22 (2) : 191- 198

191

Micropropagation of Tecomella undulata (Sm.) Seem. and genetic fidelity testing

of in vitro raised plants
Suman Kumari and Narender Singh*
Department of Botany, Kurukshetra University, Kurukshetra-136119, India
Received 4th April 2014 / Accepted 12th June 2014

Abstract.
An efficient protocol has been developed for plant regeneration from shoot apex explants of Tecomella
undulata. Explants were isolated from 2-3 year old plants, cultured on a shoot induction media, and fortified with different
concentrations (0.25, 0.5, 1.0 or 2.0 mg l-1) of auxins (NAA, IAA or 2, 4-D) and cytokinins (BAP or Kinetin). The greatest
number of shoots (3.0) was obtained on the medium fortified with BAP (1.0 mg l-1). The greatest root induction response
(63.8%) was observed on MS half strength semisolid medium supplemented with 5.0 mg l-1 NAA. The regenerated plantlets were
acclimatized and transferred to soil for normal growth under field conditions and 60% survived. Random amplified polymorphic DNA
markers were used to analyze the genetic fidelity of these in vitro raised plants. Out of the forty three RAPD decamers
screened; only ten primers resulted in two to twelve scorable bands. These ten RAPD primers generated 51 amplicons in total,
ranging from 200-2,500 bp in size with an average of 5.1 bands per primer. The amplification products were monomorphic in
micropropagated plants and similar to the mother plants, confirming the genetic fidelity of in vitro raised plantlets and
corroborating the fact that in vitro multiplication through direct organogenesis is the safest method for multiplying true to type
plants.
Keywords: Genetic fidelity, In vitro propagation, Molecular markers, RAPD, Tecomella undulata.

INTRODUCTION
Tecomella
undulata
(Sm.)
Seem.
family
Bignoniaceae, commonly known as Rohida or Desert
Teak, is an important deciduous, ornamental and
medicinal tree. It is naturally distributed in the drier parts
of Arabia, Southern Pakistan and North-West India. In India, it occurs mainly in Maharashtra, Gujarat, Rajasthan,
Punjab and Haryana (Kirtikar and Basu, 1984). Rohida has
been identified as an important agro forestry tree species
in the desert of Western Rajasthan and Southern Haryana
mainly due to its high survival rate even in extreme drought
conditions and its fire resistant properties (Shankaranarayan
and Nanda, 1963). It is also known as desert teak due to
its high quality wood, which is excellent as firewood and
for producing charcoal. T. undulata plays an important role
in environmental conservation, soil binding and providing
shelter for wildlife. Its leaves and bark are traditionally used
as a remedies for typhoid fever, urinary disorders,
gonorrhea, leucoderma, diabetes, syphilis and eczema (Ullah
et al., 2010).
This tree is on the verge of extinction due to
indiscriminate felling for fuel and timber coupled with
poor regeneration and slow growth (Arya et al., 1992).
Conventional methods of propagation from seed are
unreliable because of disease, pest species, poor
germination rates and also slow growth. Additionally, this

plant is highly cross-pollinated and it shows wide genetic

variability in seed-raised plants, so. Therefore, alternative
methods such as micropropagation are required for mass
multiplication and conservation of this valuable medicinal
tree. In vitro mass multiplication of T. undulata has been
successfully attempted by Rathore et al. (1991), Nandwani
et al. (1995) and Robinson et al. (2005). In the present
study we undertook in vitro regeneration of T. undulata
using shoot apex explants and subsequently tested the genetic
fidelity of in vitro-raised plants of T. undulata using
molecular markers.In vitro culture has long been recognized
as an efficient tool for rapid clonal multiplication. Besides
allowing multiplication in limited time and space, the
technique of tissue culture circumvents the limitations posed
by
the
somaclonal
variation
arising
during
micropropagation due to the long duration of in vitro
culture. Alterations in auxin-cytokinin concentrations,
explants source, genotypes used and the stress created by
the in vitro environment may, together or independently,
be responsible for inducing somaclonal variation (Modgil
* Author for correspondence: : Narender Singh, Department of Botany,
Kurukshetra University, Kurukshetra-136119, India.
Email - nsheorankukbot11@gmail.com.

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AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

et al., 2005; Yadav et al., 2012; Yadav et al., 2013a). It is

therefore imperative to establish genetic uniformity in such
plants by ensuring strict quality checks at various stages of
in vitro culture especially in the case of trees which have a
long life cycle.
Traditionally morphological description,
physiological supervision, karyological analysis and
biochemical estimations have been used to detect
any type of genetic variation, but modern molecular
markers have complemented traditional methods in
attempts to detect and monitor the genetic fidelity of tissue
culture-derived plantlets (Tyagi et al., 2007). Unfortunately,
morphological characterization is a time-consuming
process, based on characters which can be affected by the
environment, and so is very difficult to predict genetic
identity with a high reliability. In comparison to these
methods, molecular markers are stable, heritable, highly
reproducible, detectible in all tissues, reliable, easy to
access, and independent of the developmental environment
(Agarwal et al., 2008). RAPD is becoming a widely
employed method for the detection of genetic stability since
it has the advantage of being technically simple, quick to
perform and requires only small amounts of DNA, without the
need for any sequence information to design the primers and
radioactive probes as required in RFLP (Lakshmanan
et al., 2007). Thus, they are suitable for the assessment of the
genetic fidelity of in vitro-raised clones. The present study
was undertaken to assess the probability of induction of
somaclonal variation in the in vitro-raised plants
of T. undulata using RAPD. This is the first
report on the use of genetic markers to establish
genetic fidelity of in vitro raised plants of T. undulata

MATERIALS AND MATHODS

Explant collection and disinfection. Shoot apex explants
were collected from 2-3 year old plants growing in the
Botanical Garden of Kurukshetra University, Kurukshetra,
India, and washed under running tap water with liquid
detergent. The explants were then surface-sterilized inside
a Laminar Air Flow chamber with 0.1% mercuric chloride
for 3-4 minutes followed by washing with sterilized double
distilled water 4-5 times.
Culture media and conditions. Murashige and Skoog
(1962) medium supplemented with 3% (w/v) sucrose and
0.8% (w/v) agar was used for the present study. The pH of
the medium was adjusted to 5.8 using 0.1 N NaOH and
0.1 N HCl before autoclaving at 120 C for 20 minutes.
After the inoculation, the culture tubes were incubated at
252 C with a 16 hour light and 8 hour dark cycle. All the
experiments were repeated 3 times with 24 replicates.
Shoot induction and multiplication.
The surface-

sterilized explants were inoculated on MS media

supplemented with various concentrations (0.25, 0.5, 1.0,
or 2.0 mg l-l) of different auxins (IAA, NAA or 2, 4-D) and
cytokinins (BAP and Kn) for culture initiation. The explants
producing shoots were subcultured onto the fresh media
every 4 weeks. A treatment comprising MS medium without
growth regulators served as a control.
Root formation and acclimatization. Regenerated shoots
(2-3 cm) were cultured for root formation on MS medium
supplemented with various concentrations (1.0-5.0 mg
l-1) of auxins (NAA, IAA, and NAA). Observations on the
proportion rooting, number of roots per shoot and root
length were recorded after 10 weeks. Regenerated plantlets
were removed from the culture tubes, washed thoroughly
to remove the nutrient medium and subsequently
transplanted to plastic cups containing sterile soil and
sand in a 3:1 ratio. Potted plantlets were covered with
transparent plastic bags to ensure high humidity and watered
every 2 days for 2 weeks with half strength MS salt solution.
Thereafter, bags were removed in order to acclimatize the
plantlets to field conditions. After 4 weeks, acclimatized
plantlets were transferred to pots containing normal garden
soil and maintained in the greenhouse under normal day
length conditions.

DNA extraction and PCR amplification conditions.

Clonal fidelity of in vitro-raised clones was tested using
RAPD markers. Total genomic DNA was extracted from
the leaves of the mother plant, in vitro raised shoots at
3rdand 6th subculture stage, and seven randomlyselected micropropagated plantlets using the modified CTAB
method (Doyle and Doyle, 1990). Quantity and quality
of purified total DNA was assessed by spectrophotometer
(NanoDrop-1000, V-3.1.1). DNA concentration was also
rechecked by visual assessment of band intensity in
comparison with Lambda DNA of known concentration
using an 0.8% agrose gel. The DNA samples were diluted to
25 ng l-1 in TE (Tris-EDTA) buffer and stored at 4 C for
further analysis.
For RAPD analysis, 43 primers obtained from the
University of British Columbia, Vancouver, Canada were
used for screening. The PCR reactions were performed
in a 25 l reaction mixture containing 10 assay buffer
(2.5l), Taq DNA polymerase (0.5 units), dNTPs (2.0 l)
(Bangalore Genei), primers 0.2 M (4.0 l), and 50 ng
of template DNA in 14.5l double distilled water. The
PCR reactions were carried out in a DNA thermal cycler
(Model-CGI-96, Corbett Research, Australia) using a
single primer in each reaction. The PCR amplification
conditions for RAPD consisted of an initial extended
denaturation step at 94C for 4 minutes followed by
44 cycles of denaturation at 94 C for 1 minute, primer
annealing at 37 C for 1 minute and elongation at 72 C for

AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

2 minutes, followed by a final step of extension at 72 C for

4 minutes. The PCR reaction products were mixed with 4l
of 6 DNA loading buffer and fractionated on 1.2 % agarose
for RAPD which was prepared in 1 TBE buffer containing
0.5 g l-1 Ethidium bromide. The amplified products were
electrophoresed and after separation gels were documented
using the Avigene Gel Doc system (Avigene, Korea).
Scoring and data analysis.
Observations based on
percentage of cultured samples showing axillary bud
induction, the number of shoots per explant, and shoot
lengths were recorded after 10 weeks. The results were
analyzed statistically using SPSS ver. 12 (SPSS Inc., Chicago,
IL, USA). The significance of differences between means was
carried out using Duncans multiple range test at P=0.05.
Only clear and reproducible bands were scored for the data
analysis, but a major band corresponding to a faint band
in other replicates was included in the analysis. RAPD data
were scored for the presence (1) or absence (0) of a band, each
corresponding to a l DNA EcoRI/ product. Bands with the
same molecular weight and mobility were considered to be
a single locus. The Hind III double digest marker was used
as a standard for the estimation of molecular weight of the
total number of RAPD alleles and polymorphic alleles, and
the average number of alleles per primer were calculated.
RESULTS AND DISCUSSION
Propagation through tissue culture offers a viable
alternative to normal propagation because it can also be
used as a complimentary strategy for conservation and
utilization of genetic resources. Further, in vitro plant
regeneration from vegetative parts is an easy and
economical way to obtain a large number of consistently
uniform and true-to- type plants within a short span of time.
Shoot tips were excised from healthy young soft shoots of
the mother plant and used as explant material for a study to
establish the best conditions for their in vitro propagation.
When shoot apex explants were cultured on MS medium
supplemented
with
various
growth
regulators,
the emergence of adventitious shoot buds was observed
5-9 days after inoculation (Table 1). The medium without
growth regulator (negative control) induced no regeneration
response. Therefore it was concluded that the use of
phytohormone is essential for shoot-root proliferation
and multiplication. MS medium supplemented with IAA,
2, 4-D and NAA (0.25-2.0 mg l-1) was unable to induce
multiple shoot formation from shoot apex explants
(Table 1). However, the medium containing BAP and Kn
(0.25-2.0 mg l-1) induced multiple shoots (Table 1). Of the two
cytokinins tested, BAP-treated explants achieved higher
regeneration than those treated with Kn. The fortification
of 1.0 mg l-1 BAP yielded the greatest number of shoots

193

(Table 1). Similar reports about the effectiveness of BAP in

inducing shoot multiplication from shoot apex segments of
Sapindus emarginatus has been reported by Srinivas et al.
(2014).
After development of shoot apices into plantlets, plants
were subcultured for induction of multiple shoots on
MS medium fortified with the same concentration of
BAP (1.0 mg l-1) (Table 1, Figure 1B). In many plant
species micropropagation requires two media shoot
multiplication
medium
and
shoot
elongation
medium, making the micropropagation procedures
cumbersome and uneconomical (Debergh and Maene, 1981).
In our present work, shoot multiplication and subsequent
elongation were achieved on the same medium. Shoot
generation potential was maintained up to the sixth
subculture on shoot induction medium by regular
subculturing.
In order to induce rooting, shoots with excellent
growth were transferred to MS half-strength medium
supplemented with different concentrations (1.0 - 5.0 mg
l-1) of NAA, IAA or NAA (Table 2). Out of the three auxins
tested, MS half strength medium supplemented with NAA
(5.0 mg l-1) was found to be the most effective for root induction
(Table 2, Figure 1C). Root induction was associated with callus
induction from the basal region of the shoot;
moreover this callus formation increased in proportion to the
concentration of auxins. Roots were thus produced by a
two step method in this plant. However, Burger (1989) in
Paulonia tomentosa (a member of Bignoniaceae family)
observed root induction from a single step method.
Initially shoots were transferred to MS half strength medium
supplemented with auxins for 48 hours, then these shoots were
transferred to auxin-free half-strength medium. An initial
dark period of one week favoured root induction. Low light
treatment could have caused partial photochemical
degradation of the medium resulting in decreased
availability of auxins and nutrients (Stasinopoulos and
Hangarter, 1990). Plantlets with well developed roots
were successfully transferred to soil and sand (3:1) and
hardened off in a growth chamber for 4 weeks (Figure 1E).
Similar potting mixture was used in the propagation of Acacia
sinuta by Vengadesan et al., (2002). 60% of plantlets
survived after transfer to garden soil under field conditions
(Figure 1F). Three month old plants were transferred to the
field and all of them survived and did not show any detectable
variation in morphological characters such as plant height,
width of main stem, number of branches, or leaf width when
compared with their respective donor plants.
The present investigation was also conducted to
ascertain the clonal fidelity of the in vitro regenerated plants
and mother plant using RAPD markers. Total genomic
DNA was extracted from the leaves of the mother plant,
in vitro raised shoots at 3rd and 6th subculture stages and
seven randomly selected micropropagated plantlets using
the modified CTAB method (Doyle and Doyle, 1990) and
selected primers were used to confirm the genetic stability of
regenerated plantlets.

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AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

Table 1. Effect of auxins and cytokinins on shoot apex explants of Tecomella undulata.
Medium and
conc. of growth
regulator
(mg l-1)

Days required
for response
(mean value)

Per cent
Response*

No. of shoots
per explants*

Shoot
length*
(cm)

Control

0.00

0.00

0.000.00g

0.000.00h

MS+IAA(0.25)
(0.5)
(1.0)
(2.0)

7.0
6.4
6.0
5.0

72.21.93
70.80.00
68.71.97
77.71.97

de

1.50.08
1.70.12cd
1.70.10cd
1.40.08f

fg

0.430.03
0.560.13f
0.650.04f
0.790.05f

+
+
+
+

MS+NAA(0.25)
(0.5)
(1.0)
(2.0)

9.2
9.0
8.6
7.8

75.61.97
77.70.00
72.21.97
80.51.93
-

1.80.12cd
1.50.11de
1.60.12cde
1.60.15cde

0.470.04fg
0.630.05f
1.20.10e
0.530.04f

+
+
+
+

MS+2,4D(0.25)
(0.5)
(1.0)
(2.0)

0.00
0.00
0.00
0.00

0.000.00
0.000.00
0.000.00
0.000.00

0.000.00g
0.000.00g
0.000.00g
0.000.00g

0.000.00h
0.000.00h
0.000.00h
0.000.00h

MS+BAP(0.25)
(0.5)
(1.0)
(2.0)

7.5
7.0
6.3
6.0

80.51.97
83.30.00
84.71.97
88.81.93

1.90.15c
2.80.13ab
3.00.04a
2.50.12b

2.40.09ab
2.30.19ab
2.50.04a
1.90.10c

++
++
++
++

MS+KN (0.25)
(0.5)
(1.0)
(2.0)

7.3
7.0
6.5
6.5

65.21.93
70.80.00
72.21.93
66.60.00

1.40.09f
1.60.17cde
1.80.49cd
1.90.06c

2.20.12b
2.40.02ab
2.50.11a
1.80.08cd

+
+
+

0.29

0.23

F20,42=20.37

F20,42=122.6

LSD (P<0.05)
ANOVA

Callus
growth

* Values are Means S.E of three independent experiments. Data were recorded after 10
week of culture.
- No Response, + Poor growth, + + Moderate growth, + + + Good growth.
Mean values within a column sharing the same subscript letter are not significantly
different at P<0.05 according to the Duncans Multiple Range Test.

Out of the forty three RAPD decamers screened, only

ten primers resulted in two to twelve scorable bands.
These ten RAPD primers generated 51 amplicons in total,
ranging from 200-2500 bp in size. The greatest number
of bands (12) was obtained with the marker -GCC 117
(Figure 2A). The number of bands amplified from the
selected primers varied, with 4 amplified from primer -GCC
113 (Figure 2C), 7 from primer -GCC139 (Figure 2B), and
5 bands produced with the primer -GCC123 (Figure 2D),
with an average of 5.1 bands per RAPD primer (Table 3,
Figure 2). The amplification products were monomorphic in
regenerated plants and similar to those of the mother plant.
No polymorphism was detected, revealing the genetic

uniformity of these micropropagated plants of

T.undulate, confirming that micropropagation of T.undulata
using shoot apex explants maintains genetic fidelity even
after prolonged periods under in vitro conditions. The
absence of genetic variation demonstrated using RAPD markers has been reported in several previous investigations, such as
micropropagated plants of Populus deltoids (Rani et al.,
1995), Paulownia tomentosa (Rout et al., 2001), Tectona grandis
(Gangopadhyay et al., 2003), Capparis decidua (Tyagi et
al., 2010), Simmondsiachinensis (Kumar et al., 2011),
Eucalyptus tereticornis (Aggrawal et al., 2012), Gloriosa
superba (Yadav et al., 2013b) and Spilanthes acmella
(Yadav et al., 2014).

AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

195

Table 2. Effect of auxins on root induction of regenerated shoots of Tecomella undulata.

Medium and conc.
of growth regulator
( mg l-1)

MS medium

MS Medium
(Half Strength

Days required
for root
induction
(mean value)

Per cent
Response*

No. of
roots per
shoot*

Root length*
(cm)

IAA+ 1.0
2.0
3.0
4.0
5.0

NAA+ 1.0
2.0
3.0
4.0
5.0

0.00
0.00
13.4
12.2
11.5

0.000.00
0.000.00
45.80.00
55.51.97
63.83.90

0.000.00
0.000.00
0.860.18
1.20.29
2.20.12

0.000.00
0.000.00
0.960.34
1.40.43
4.30.61

IBA + 1.0
2.0
3.0
4.0
5.0

*Values are Means S.E of three independent experiments. Data were recorded after 10 weeks of
culture.

Figure 1. Various stages of plant regeneration of Tecomella undulata using shoot apex explants. (A) 30 days old
culture showing shoots bud initiation on MS medium containing 1.0 mg l-1 BAP. (B) Proliferated shoots cultured on
MS medium supplemented with 1.0 mg l-1 BAP for elongation and multiplication. (C) Root induction on gelled MS
medium supplemented with 5.0 mg l-1 NAA. (D) Rooted plantlet. (E) In vitro-raised plants after transfer to plastic
cups just before transfer to earthen pots. (F) Plantlet after transferred to the field conditions.

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AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

Table 3. List of primers, their sequences, and number and size of amplified fragments generated by random amplified
polymorphic DNA (RAPD).
Primer
name

Primer sequence
(5-3)

Annealing
temp.

GCC121

ATACAGGGAG

37 oC

GCC113

ATCCCAAGAG

Number Approximate
of total
size range
amplicons
(bp)

Approximate size of each

amplicon (bp)

831-1375

831, 1090, 1375

890-1300

890, 960, 1170, 1300

200-2500

200, 300, 350, 400, 701, 900, 1100, 1202, 1580,

1904, 2047, 2500

37 C

GCC117 TTAGCGGTCT

37 C

12

GCC129

GCGGTATAGT

37 oC

600-905

600, 730, 905

GCC135 AAGCTGCGAG

37 oC

710-1864

710, 790, 998, 1257, 1390, 1864

650-2227

650, 800, 960, 1260, 1410, 2000, 2227

600-1650

600, 900, 1003, 1200, 1650

500-947

500, 730, 947

350-1850

350, 500, 810, 1100, 1250, 1850

GCC139

CCCAATCTTC

GCC123 GTCTTTCAGG
GCC103 GTGACGCCGC
GCC104

GGGCAATGAT

37 C
37 C
37 C
37 C

CONCLUSION
The results presented in this paper demonstrate that
RAPD technique proved to be effective in generating
reproducible results useful in assessment of genetic
fidelity of micro-propagated plants of T.undulata.
These results further support the feasibility of this
protocol which could be successfully used for the mass
multiplication and germplasm conservation of this valuable
tree.
ACKNOWLEDGEMENTS
The authors wish to express their sincere thanks to
Kurukshetra University Kurukshetra, Haryana (India) for
providing the laboratory facilities, financial assistance and
other institutional support.

Figure 2. RAPD profiles of mother plant, in vitro

regenerated shoots at 3rd and 6th subculture stage
and of micropropagated plants of Tecomella
undulate. Amplification was with forward primers:
(A)
A-RAPD-GCC117
(5-TTAGCGGTCT-3),
(B)
B-RAPD-GCC139
(5-CCCAATCTTC-3),
(C) C-RAPD-GCC 113 (5-ATCCCAAGAG-3), and
(D)
D-RAPD-GCC123
(5-GTCTTTCAGG-3).
First lane: DNA EcoRI/ HindIII double digest
marker; lane 1, mother plant; lanes 2 and 3, in vitro
raised shoots at 3rd and 6th subculture stage; lane from
4-10, 7 randomly selected micropropagated plants.

REFERENCES
Agarwal, M., Shrivastava, N. and Padh, H. 2008. Advances
in molecular marker techniques and their applications
in plant sciences. Plant Cell Reports 27: 617-631.
Aggarwal, A., Kumar, A., Sharma, J. and Reddy, M.S. 2012.
Factors affecting micropropagation and acclimatization
of an elite clone of Eucalyptus tereticornis Sm. In Vitro
Cellular & Developmental Biology 48(5): 521-529.

AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

Arya, S., Toky, O.P., Harris, S.M. and Harris, P.J.C. 1992.
Tecomella undulata (Rohida): a valuable tree of the Thar
Desert. International Tree Crops Journal 7: 141-147.
Burger, D.W. 1989. Empress tree (Paulonia tomentosa
Steud) In: Bajaj Y. P. S. (Ed.) Trees 2, Biotechnology in

Agriculture and Forestry 5: 359-369.
Debergh, P.C. and Maene, L.J. 1981. A scheme for

commercial propagation of ornamental plants by tissue
culture. Scientia Horticulturae 14: 335-345.
Doyle, J.J. and Doyle, J.L. 1990. Isolation of plant DNA
from fresh tissue. Focus 12: 13-15.
Gangopadhyay,
G.,
Gangopadhyay,
S.B.,
Poddar, R., Gupta, S. and Mukherjee, K,K. 2003.

Micropropagation of Tectona grandis: Assessment of
Genetic Fidelity. Biologia Plantarium 46(3): 459-461.
Kirtikar, K. R. and Basu, B.D. 1984. Indian medicinal
plants, Second Edition, pp. 1841, International Book
Distributors, Dehradun.
Kumar, S., Mangal, M., Dhawan, A.K. and Singh, N. 2011.
Assessment of genetic fidelity of micropropagated
plants of Simmondsia chinensis (Link) Schneider using
RAPD and ISSR markers. Acta Physiologiae Plantarum
33: 2541-2545.
Lakshmanan, V., Venkataramareddy, S.R. and Neelwarne,
B. 2007. Molecular analysis of genetic stability in

long-term micropropagated shoots of Banana

using RAPD and ISSR markers. Electronic Journal of

Biotechnology 10: 1-8.
Modgil, M.K., Mahajan, S.K., Chakrabarti, D.R., Sharma,
A. and Sobti, R.C. 2005. Molecular analysis of genetic
stability in micropropagated apple rootstock MM106.
Scientia Horticulturae 104: 151-160.
Nandwani, D., Mathur, N. and Ramawat, K.G. 1995. In
vitro shoot multiplication from cotyledonary node

explants of Tecomella undulata. Gartenbauwisseschaft
60: 65-68.
Rani, V., Parida, A. and Raina, S.N. 1995. Random

amplified polymorphic DNA (RAPD) markers for

genetic analysis in micropropagated plants of Populus
deltoids Marsh. Plant Cell Reports 14(7): 459-462.
Rathore, T.S., Singh, R.P. and Shekhawat, N.S. 1991.
Clonal propagation of desert teak (Tecomella undulata)
through tissue culture. Plant Science 79: 217-222.

197

Robinson, R., Kumari, B. and Beniwal, V.S. 2005. In

vitro shoot multiplication of Tecomella undulata (Sm.)
Seem.- an endangered tree species. Indian Journal of
Plant Physiology 10(4): 372-376.
Rout, G.R., Reddy, G.M. and Das, P. 2001. Studies on in
vitro clonal propagation of Paulownia tomentosa Steud.
and evalution of genetic fidelity through RAPD marker.
Silvae Genetica 50: 208-212.
Shankaranarayan, K.A. and Nanda, P.C. 1963.

Cytotaxonomy of Tecomella undulata Seem. Annals of
Arid Zone 1: 174-175.
Srinivas, D., Venkateshwarlu, M., Thirupathi, M.,

Sreenivas, A., Rajender, K. and Jaganmohan, K.
R. 2014. Micropragation of axillary shoot buds in

Sapindus emarginatus Vahl. International Journal of
Multidisciplinary and Current Research 2: 313-316.
Stasinopoulos, T.C. and Hangarter, R.P. 1990. Preventing
photochemistry in culture media by long-pass light

filters alters growth of cultured tissues. Plant Physiology
93: 1365-1369.
Tyagi, P., Khanduja, S. and Kothari, S.L. 2010. In vitro

culture of Capparis decidua and assessment of clonal
fidelity of the regenerated plants. Biologia Plantarum
54(1): 126-130.
Tyagi, R.K., Agrawal, A., Mahalakshmi, C. and Hussain,
Z. 2007. Low-cost media for in vitro conservation of

turmeric (Curcuma longa L.) and genetic stability

assessment using RAPD markers. In Vitro Cellular and
Developmental Biology of Plants 43: 51-58.
Ullah, M.O., Hamid, K., Rahman, K.A. and

Choudhuri, M.S.K. 2010. Effect of Rohitakarista
(RHT), an ayurvedic formulation, on the lipid profile
of rat plasma after chronic administration. Biology and

Medicine 2(2): 26-31.
Yadav, K. Kumar, S. and Singh, N. 2014. Genetic fidelity
assessment of micropropagated Spilanthes acmella (L.)
Murr. by RAPD and ISSR markers assay. Indian Journal
of Biotechnology 13(2): 274-277.
Yadav, K. Aggarwal, A. and Singh, N. 2013a.

Arbuscular mycorrhizal fungi (AMF) induced

acclimatization,
growth
enhancement
and

colchicine content of micropropagated Gloriosa

superba L. plantlets. Industrial Crops and Products

45: 88-93.

198

AsPac J. Mol. Biol. Biotechnol. Vol. 22 (2), 2014

Yadav, K. Aggarwal, A. and Singh, N. 2013b. Evaluation

of genetic fidelity among micropropagated plants of

Gloriosa superba L. using DNA-based markers - a

potential medicinal plant. Fitoterapia 89: 265-270.
Yadav, K. Aggarwal, A. and Singh, N. 2012. Actions
for ex situ conservation of Gloriosa superba L. - an

endangered ornamental cum medicinal plant. Journal
of Crop Science and Biotechnology 15: 297-303.

198