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Regeneration and transformation studies in Terminalia chebula Retz.


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This study presents in vitro regeneration of Terminalia chebula Retz. to obtain complete plantlets from juvenile explants (hypocotyl and cotyledon). Dried seeds were inoculated on MS basal medium after surface sterilization with Bavistin (0.2%) alone and followed by HgCl2 (0.1%), resulted in maximum (75%) germination. Hypocotyl showed 90% and cotyledon 75% callus induction on MS basal medium containing 1.0 mg/l 2, 4-D after 30 days of inoculation. Shoot regeneration was recorded only from cotyledonary callus on shoot induction medium comprising 1.5 mg/l BAP with 0.10 mg/l NAA with maximum 36.67% shoot regeneration. Maximum (43.75%) rooting was reported in 1/2 strength MS medium with 0.5% activated charcoal. Transgenic callus was produced through Agrobacterium tumefaciens mediated genetic transformation carrying gus and npt-II gene from cotyledonary explants. Co-cultivation (72 h) preceded by pre-conditioning (72 h) was found best for callus induction. Successful integration of gus gene was reported.
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Journal of Scientific & Industrial Research
Vol. 72, Sept - Oct 2013, pp. 563-571
*Author for correspondence
Regeneration and transformation studies in Terminalia chebula Retz.
Yashveer Singh Verma*, Kamlesh Kanwar and Satya Vrat Bhardwaj
Department of Biotechnology, Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan 173 230, India
Received 19 March 2013; revised 22 May 2013; accepted 29 May 2013
This study presents in vitro regeneration of Terminalia chebula Retz. to obtain complete plantlets from juvenile explants
(hypocotyl and cotyledon). Dried seeds were inoculated on MS basal medium after surface sterilization with Bavistin (0.2%)
alone and followed by HgCl2 (0.1%), resulted in maximum (75%) germination. Hypocotyl showed 90% and cotyledon 75% callus
induction on MS basal medium containing 1.0 mg/l 2, 4-D after 30 days of inoculation. Shoot regeneration was recorded only from
cotyledonary callus on shoot induction medium comprising 1.5 mg/l BAP with 0.10 mg/l NAA with maximum 36.67% shoot
regeneration. Maximum (43.75%) rooting was reported in ½ strength MS medium with 0.5% activated charcoal. Transgenic callus
was produced through Agrobacterium tumefaciens mediated genetic transformation carrying gus and npt-II gene from cotyledonary
explants. Co-cultivation (72 h) preceded by pre-conditioning (72 h) was found best for callus induction. Successful integration of
gus gene was reported.
Keywords: Agrobacterium tumefaciens, Hypocotyl, Cotyledon, Regeneration, Terminalia chebula
Terminalia chebula Retz. (Family, Combretaceae),
commonly known as Harad, is an indigenous,
multipurpose and deciduous tree of great economical
. It occurs in Northern tropical wet evergreen
forests, tropical seasonal swamp forests, Southern and
Northern tropical deciduous forests
. Fruit and dried flesh
surrounding seeds are the most important product. Seeds
contain tannin (30-32%), which varies with season of
collection and locality
. T. chebula is always listed first
in the Ayurvedic metiria medica
. Fruits have antiamoebic
and are used in fevers, cough, asthma, urinary
diseases, piles, worms and rheumatism, and scorpion-
. Triphala (Bel leri c myrobal ans, Embeli c
myrobalans and Chebulic myrobalans) is an important
Ayurvedic formulation used in the treatment of liver and
kidney dysfunctions
. Natural regeneration of T. chebula
from seeds in situ and ex situ is extremely low
and is a
slow growing tree compared to other species of
Termi nal ia
. In vitro propagation has also been
. Direct sowing of seeds results in queer,
inadequate germination and low survival of seedlings, all
of which contribute to high production cost of seedling
stock. Micropropagation of T. chebula has already been
from shoot buds of mature tree. Reports on
induction of callus are also there.
This study presents indirect regeneration in T.
chebula Retz. using juvenile explants [hypocotyl (Hy)
and cotyledon (Co)].
Experimental Section
Seed kernels obtained after excising hard seed coat
were surface sterilized with sodium bavistin (0.2%)
solution for different time durations (0-15 min)
followed by washings with autoclaved distilled water,
by 0.1% HgCl
and then again 5-6 washings with
autoclaved distilled water under aseptic conditions.
Surface sterilized seed kernels were inoculated on MS
basal medium (full strength) for germination. In vitro
grown seedlings (15-20 days old) were used as a source
of explant (Co & Hy) to carry out regeneration studies.
Callus induction (CI) was achieved on Murashige and
Skoogs (MS)
basal medium
containing agar (0.8%),
sucrose (3%) and supplemented with different
concentrations of 2,4-D (2,4- dichlorophenoxy acetic
acid). Type, growth and colour of callus were observed.
All constituents of media were procured from SISCO
Research Laboratories Pvt Ltd, Mumbai, while 2, 4-D
was procured from Merck Chemicals Pvt Ltd.
Subculturing of callus was done on same medium for
two times at an interval of 4 weeks for proliferation. All
cultures were kept in culture room at 26 ± 2°C under 16
h photoperiod at 20 µmol m
. Regenerative callus
was transferred to shoot induction medium [MS basal
medium + different concentrations of BAP
(6-benzyloaminopurine) alone and in combination with
NAA (α- naphthalene acetic acid)]. Shoot regeneration%,
number and length of shoots were noted. Micro shoots
(1.5-2.0 cm long) were taken after 6 weeks from shoot
induction medium and transferred on a different media
for root induction. Shoot induction%, average number of
shoots and average shoot length were recorded for each
subcultured shoot. Similar observations were recorded
in case of rooting
In vitro rooting of micro shoots is done by dipping
them in pre-autoclaved IBA solution for 2 h under dark
aseptic conditions and thereafter subculturing in half-
strength MS medium supplemented with different
concentrations of activated charcoal. In present study,
different concentrations of activated charcoal (0.1-1.0%)
in ½ strength MS medium were tested for in vitro rooting
(Fig. 1). Agrobacterium tumefaciens strain LBA 4404
was used for genetic transformation experiment. In co-
cultivation experiment, explants were inoculated on MS
medium supplemented with 2,4-D for different time
durations and pre conditioning was done by culturing
explants on same medium devoid of Agrobacterium.
All experiments were laid out in a completely randomized
design (CRD)
. Significance level for F-test was 5%.
Results and Discussion
In vitro Regeneration in T. chebula Retz.
To achieve surface sterilization of seed kernels, 0.2%
bavistin alone for 10 min resulted in 73.33%
uncontaminated surviving cultures. However, 0.1% HgCl
dipping for 5 min resulted in higher number (75%) of
uncontaminated surviving cultures after 15 days of
incubation (Table 1). After treatment with each surface
sterilant, seed kernels were washed 5-6 times with
autoclaved distilled water. Surface sterilized seed kernels
were established on solid MS basal medium under 16 h
photoperiod. Germination of Anoge issus pendula
Edgew. viable seeds on MS medium has also been
. High rate of germination frequency in T.
chebula embryos has been obtained on MS medium
supplemented with 0.5 mg dm
gibberellic acid (GA). In
Morus alba, zygotic embryos inoculated on MS basal
medium supplemented with sucrose (30 g/l) showed
97.0% germination and formed seedling
Indirect Shoot Regeneration from Cotyledon (Co) and
Hypocotyl (Hy)
Auxin is the primary hormone used to produce callus.
Often 2,4-D is used to produce callus. In some species,
high concentration of auxin and low concentration of
cytokinin in medium promotes abundant cell proliferation
with callus formation
. Callus is produced on explants
in vitro as a result of wounding and in response of
hormones endogenous or exogenously
. In present study,
Co and Hy explants, which were excised from 3 weeks
old in vitro raised seedling, were subjected to different
concentrations of 2,4-D supplemented in MS medium
for CI. Among Co and Hy explants (Table 2), Hy that
showed 90% CI in treatment C
is the best explant;
Table 1Effect of different duration of 0.2% bavistin (alone) and
0.2% bavistin in combination with 0.1% HgCl2 (5 min) on surface
sterilization of seed kernels (Figures in parentheses are arc sine
Treatment Duration 0.2% Bavistin 0.2% Bavistin
min and 0.1% HgCl2
5 min
B16 46.67 (43.09) 60.00 (50.79)*
B28 63.33 (52.78) 68.33 (55.77)
B310 73.33 (58.93) 75.00 (60.08)
B412 68.33 (55.77) 70.00 (56.84)
B515 53.33 (46.91) 63.33 (52.74)
CD0.05 6.64 4.79
S.E. 2.98 2.15
Fig. 1In vitro germination in Terminalia chebu la (Different
stages of germination after 3rd and 4th week on half strength MS
highest CI recorded in Co was 75% for the same
treatment C
. Similarly, Hy explants were found better
for callusing in Morus alba and M. indica
. Better
CI and proliferation from Co explants than leaf explants
of Punica granatum L. cv. Ganesh is also reported
However, in present study, response of CI reduced for
both explants with further increase in concentration of
2,4-D (Table 2). CI% was more in Hy explants as
compared to Co part. Best recognized treatment for CI
was 1.0 mg/l 2,4-D in MS medium (Table 2, Fig. 2).
Therefore subculturing for proliferation of callus was
done on the medium containing 1.0 mg/l 2,4-D for two
times at an interval of 4 weeks for proliferation. In present
study, although CI% was higher in Hy explant as
compared to Co explant but rate of proliferation and
differentiation of callus was higher in Co explant as
compared to Hy explant. All cultures were incubated at
25±2°C under 16 h photoperiod.
Shoot Bud Induction and Development
Small callus pieces (0.5-0.8 cm
) from Co and Hy
explants were cultured on MS medium with different
concentrations of BAP alone (1.0-2.0 mg/l) and in
combination with NAA (0.05-0.15 mg/l) for in vitro shoot
induction. Co derived callus exhibited a high potential
for shoot differentiation while Hy derived callus did not
show any differentiation. Maximum shoot bud
differentiation in Co derived callus in pomegranate on
MS medium supplemented with 1.0 mg/l BAP+ 0.5 mg/
l NAA is also reported
. BAP alone had no effect on
shoot regeneration (Table 3) till 6 weeks. Highest shoot
regeneration (36.67%) in case of Co was found in
treatment D
containing 1.50 mg/l BAP + 0.10 mg/l
NAA, which was superior to all other combinations. It
showed maximum number (3) of shoots per callus clump
with maximum average shoot length (1.56 cm). Lowest
shoot regeneration (1.67%) was observed in D
containing 1.00 mg/l BAP + 0.05 mg/l NAA with least
number of shoots (0.33) and shoot length (0.53 cm).
Unlike Co derived calli, the calli obtained from Hy explants
did not respond at all to in vitro shoot bud induction and
turned brown on prolonged culture (Fig. 3). In another
, best shoot multiplication response was obtained
from nodal explants of T. arjuna on modified MS medium
containing 4.44 μM BA (benzyladenine) and 0.5 μM
NAA. Shoot induction from calluses is reported
(98%) in Co derived callus cultured on MS medium
supplemented with 0.5 μM NAA + 5.0 μM BA. In vitro
shoot proliferation from nodal segments in T. chebula
Table 2Effect of different concentrations of 2, 4-D on callus induction from cotyledon (Co) and hypocotyl (Hy) explants after 4 week
of incubation (Figures in parentheses are arc sine transformed)
Treatment 2,4-D Callus induction Type of callus Growth of callus Colour of callus
conc %
mg/l Co Hy Co Hy Co Hy Co H y
C1 0.0 0.00 0.00 — — — — —
C2 0.5 63.33 (52.78) 55.00 (47.88)*C F + + G YG
C3 1.0 75.00 (60.08) 90.00 (71.95) C F +++ ++ G YG
C4 1.5 68.33 (55.77) 83.33 (65.96) C F ++ ++ G YG
C5 2.0 56.67 (48.84) 78.33 (62.41) C F + + G YG
C6 2.5 50.00 (45.00) 68.33 (55.77) F F + + G YG
C7 3.0 45.00 (42.12) 51.67 (45.96) F F + + G YG
CD0.05 4.47 5.15
S.E. 2.08 2.40
C, compact; F, friable; G, greenish; YG, yellowish green; -, no growth; +, slow; ++ moderate; +++, fast
Fig. 2Callus induction from cotyledon explant (Well
proliferated callus after 6 weeks of culturing on MS medium
supplemented with 1.0 mg/l 2, 4-D)
on WPM supplemented with 1.50 mg/l BAP + 0.05 mg/
l NAA was also observed
. However, in present study,
best medium for shoot bud induction and proliferation is
MS medium supplemented with 1.5 mg/l BAP +0.10 mg/
l NAA (Fig. 4). D
treatment containing 1.50 mg/l BAP
+ 0.10 mg/l NAA was found to be best for in vitro shoot
Table 3Effect of different concentration of BAP alone and in combination with NAA for shoot induction from callus explant after 4
weeks of incubation (Figures in parentheses are arc sine transformed)
Treatment BAP mg/l NAA mg/l Shoot regeneration% Number of Shoots Shoot length cm
D1 1.0 0.00 0.00 ( 0.00)*0.00 0.00
D2 1.5 0.00 0.00 ( 0.00) 0.00 0.00
D3 2.0 0.00 0.00 (0.00) 0.00 0.00
D4 1.0 0.05 1.67 (4.30) 0.33 0.53
D5 1.0 0.10 3.33 ( 8.61) 0.67 0.87
D6 1.0 0.15 8.33 ( 13.74) 1.00 1.00
D7 1.5 0.05 23.33 (28.86) 2.00 1.17
D8 1.5 0.10 36.67 ( 37.26) 3.00 1.56
D9 1.5 0.15 21.67 ( 27.71) 2.00 1.33
D10 2.0 0.05 15.00 ( 22.60) 1.57 1.16
D11 2.0 0.10 13.33 ( 21.34) 1.33 1.13
D12 2.0 0.15 10.00 (18.44) 1.00 0.93
CD0.05 2.72 4.02 0.16
SE 5.61 8.30 0.35
Fig. 3Callus induction from hypocotyl explant (Browning of callus on shoot regeneration medium after 6 weeks of incubation; on MS
medium supplemented with1.0 mg/l 2,4-D )
Fig. 4Shoot induction and proliferation
a) Shoot bud development after subculturing on shoot induction medium (1.5 mg/l BAP and 0.10 mg/l NAA)
b) Shoot bud elongation after 6 weeks of incubation on shoot induction medium
Figure. 4 b
Figure. 4 a
regeneration from Co derived calli and statistically
In vitro Root Induction
Microshoots (length, 1.5-2.0 cm) were isolated,
excised and transferred to root regeneration medium for
root induction to get complete plantlets. Root induction
initiated within 15-20 days in culture, and within 6 weeks
well developed root system was obtained (Fig. 5). Rooting
in M. alba on MS medium supplemented with 0.05%
activated charcoal is also reported
. However, in present
study, microshoots of T. chebula obtained by indirect
regeneration system were rooted on ½ strength MS
medium + 0.5% activated charcoal after IBA (2 mg/ml)
treatment. Out of 4 treatments, maximum (43.75%)
rooting was obtained with 2.2 number of roots per shoot
and root length 2.15 cm in treatment E
containing ½
strength MS medium + 0.5% activated charcoal
(Fig. 5). But on further increasing concentration of
activated charcoal in ½ strength MS medium, rooting
decreased (37.50%) with 1.56 number of roots per shoot
and root length of 1.77 cm. In ½ strength MS medium
without activated charcoal, microshoots did not respond
to rooting at all (Table 4).
Genetic Transformation of T. chebula Retz.
Of several recombined strains of A. tumefaciens,
LBA 4404 has proved to be most successful
Escherichia coli β- glucuronidase was developed as a
reporter system for transformation of plants to overcome
difficulties faced in using other reporter genes
. An
efficient technique for introducing cloned genes into plant
cells using Agrobacterium was also standardised
Woody species tend to be difficult and often inefficiently
transformed due to lack of proper regeneration system
A. tumefac i ens strain LBA 4404 carrying β-
glucuronidase and neomycin phosphotransferase-II
marker genes were used for genetic transformation
Table 4Effect of different concentration of activated charcoal on root induction in microshoots after 4 weeks of incubation (Figures in
parentheses are arc sine transformed)
Treatment Composition Rooting% Number of roots Root length cm
E1½ strength MS medium 0.00 0.00 0.00
E20.1% activated charcoal + ½ MS 31.25 ( 33.75) 1.30 1.25
E30.5% activated charcoal +½ MS 43.75 (41.25) 2.20 2.15
E41.0% activated charcoal +½ MS 37.50 (37.50) 1.56 1.77
S.E. 4.84
C.D. 0.05 2.17
Fig. 5In vitro root induction in microshoot on ½ strength MS medium + 0.5% activated charcoal after 3 weeks of incubation
studies to develop putative transgenic callus in T. chebula
Retz. using Co (Fig. 2). In present study, genetic
transformation in T. chebula was carried out up to callus phase.
Effect of Pre-conditioning and Co-cultivation Duration on
Transformation Frequency
A pre-conditioning time of 72 h followed by co-
cultivation for 72 h resulted in highest transformation
frequency for CI through Co explants. During pre-
conditioning, explants undergo a physiological and
developmental shift to enter morphogenic competency.
After T-DNA insertion, recipient cells have already
entered regeneration pathway
. Co-cultivation
experiments included inoculation of Co explants on MS
medium supplemented with 1.0 mg/l 2,4-D for 24, 48
and 72 h.. Maximum transformation rate (7.77) was
recorded in treatment F9 with 2.33 explants showing CI
when 72 h of pre-conditioning was followed by 72 h of
co-cultivation (Table 5). However, in F6 treatment, pre-
conditioning for 48 h followed by 72 h of co-cultivation
resulted in decreased transformation rate (4.44) with 1.33
explants showing CI. Low response was recorded when
48 h of co-cultivation was preceded by different durations
(24,48 & 72 h) of pre-conditioning, while no response
was recorded when 24 h of co-cultivation was preceded
by different durations (24,48 & 72 h) of pre-conditioning.
Co-cultivation for 2-3 days is standard for most of the
transformation protocols, as longer periods have
Table 5Effect of pre-conditioning and co-cultivation of explants on transformation frequency in MS medium (Figures in parentheses
are square root +1 transformed)
Treatment Pre-conditioning Co-cultivation Number of Explants Transformation
h h explants producing frequency
F1 24 24 30 0.00 0.00 (1.00)
F2 24 48 30 0.00 0.00 (1.00)
F3 24 72 30 1.00 3.33 (195)
F4 48 24 30 0.00 0.00 (1.00)
F5 48 48 30 0.33 1.11 (1.36)
F6 48 72 30 1.33 4.44 (2.31)
F7 72 24 30 0.00 0.00 (1.00)
F8 72 48 30 0.66 2.22 (1.72)
F9 72 72 30 2.33 7.77 (2.95)
C.D. 0.05 2.1 2.1
S.E. 0.41 0.36
Fig. 6- a Fig. 6 -b
Fig. 6Genetic transformation and callus induction from cotyledon explant
a) Co-cultivation of cotyledon explants on MS medium + 1.0 mg/l 2, 4-D (for 72 hrs)
b) Putative transformed callus on selective medium after 4 weeks of culturing
frequently resulted in Agrobacterium overgrowth
Maximum transformation frequency with 72 h of co-
cultivation in almond has also been reported
Selection of Putative Transgenic Callus
To observe growth and regeneration on selective
medium, co-cultivated explants were transferred to
selective medium containing antibiotics (30 mg/l
kanamycin + 500 mg/l cefotaxime) alongwith 2,4-D for
CI. A selective advantage was given to transformed cells
through introduction of npt-II gene conferring resistance
against kanamycin, which allows transformed cells to
grow where non-transformed cells were unable to grow.
The npt- II gene encodes enzyme neomycin
phosphotransferase-II, which inactivates sugar containing
antibiotics by phosphorylation
. Co-cultivated as well
as control Co explants were transferred to selective CI
medium. Out of 30 explants, maximum (2.33%) explants
showed CI on selective medium after 6 weeks (Table 5,
Fig. 6). Phosphorylation of kanamycin by npt-II enzyme
prevents its action and therefore, only transformed
bacteria and plant tissue can grow effectively on
kanamycin containing media. To eliminate A.
tumefaciens from plant cultures, another antibiotic
cefotaxime is used because it is a broad spectrum
antibiotic for bacterial inhibition
. Control and some of
co-cultivated explants became completely necrotic within
a week of culture on selective medium. After 3 weeks
on CI medium comprising of 1.0 mg/l 2,4-D supplemented
with 500 mg/l cefotaxime and 30 mg/l kanamycin, callus
was formed on cut surface of Co explant (Fig. 6b). Data
was recorded after 6 weeks on selective medium and a
transformation frequency of 7.77 was observed.
However, inoculated control explants did not survive at
all on selective medium. Control showed higher CI% on
non-selective medium as compared to the growth of
Agrobacterium inoculated explants on selective medium.
This may be consequence of antibiotic stress.
Confirmation of Gene Integration
Polymerase chain reaction was carried out to confirm
transfer of gus gene from A. tumefaciens into genome
Table 6Specific (designed) primer used in present study for amplification of gus gene in transgenic
callus of T. chebula Retz.
Sl No. Primer Sequence (5' 3')
Fig. 7PCR analysis of putative transformed callus Lane 1 represents DNA size marker of 100 bp, lane 2 containing the sterile water
and lane 3 containing reaction mixture were taken as negative controls, lane 4 containing plasmid pBl121 was taken as positive control,
whereas lane 5 contain the DNA of none transformed control C and lanes 6-8 contain the DNA of transformed samples T1, T2 and T3
of cells (callus) of T. chebula Retz. Total genomic DNA
was isolated from randomly selected 3 callus samples
by already standardised method
. Gene specific primers
(Table 6) were used to amplify a 0.7 kb fragment of gus
gene by PCR. Stable integration of transgene in citrus
plants was confirmed by PCR analysis in similar studies
Integration of transgene (gus gene) into citrus genome
was confirmed by polymerase chain reaction
product was visualized after electrophoresis in 1.5%
agarose gel (Fig. 7). Lane 1 representing DNA size
marker of 100 bp, lane 2 containing sterile water and
lane 3 containing reaction mixture were taken as negative
controls, lane 4 containing plasmid pBl121 was taken as
positive control, whereas lane 5 contains DNA of none
transformed control C and lanes 6-8 contain DNA of
transformed samples T
, T
and T
(Fig. 7). Lane 4
containing plasmid pBl121 showed the band of amplified
gus gene, while lane 2, 3 and 5 did not show any such
band. Out of 3 putative transgenic callus samples in lanes
6-8 ,only lane 6 and 8 showed amplification of band of
integrated gene that is at par with band of positive control,
thus showing confirmation of gus (Fig. 7). Similarly,
transgenic nature of transformants was demonstrated
by PCR analysis in Jatropha curcas
This study reports for the first time A. tumefaciens
mediated genetic transformation of T. chebula up to
callus phase using gus gene from Co explant on selective
medium containing 30 mg/l kanamycin and 500 mg/l
cefotaxime. Major gap still exists for providing superior
planting material with stably inherited traits associated
with many important industries. Also, because of deviation
from allopathic towards traditional Ayurvedic treatment,
demand for Terminalia will always rise. Therefore,
present research provides a platform for mass
propagation of T. chebula and further carrying out
genetic transformation studies using agronomically
important traits in this species.
1 Chadha Y R, The Wealth of India: Dictionary of Indian Raw
Materials and Ind ustrial Products (CSIR, New Delhi) 1989,
2 Luna R K, Chamoli N & Nautiyal D P, Effect of growth
stimulatory substances on seed germination and subsequent
growth of seedlings of Terminalia bellirica Roxb., Indian J For,
29 (2006) 361-366.
3 Luna R K, Plantation Trees (International Book Distributers,
Dehradun) 1996, 794-797.
4 Warrier P K, Nambiar V P K & Ramankutty C, Indian Med
Plan ts, vol 5 (Orient Longman Ltd, Madras, India) 1997,
5 Sohni Y R & Bhatt R M, Activity of a crude extract formulation
in experimental hepatic amoebiasis and in immunomodulation
studies, J Ethnopharmacol, 54 (1996) 119-124.
6 Singh M P & Panda H, Medicinal Herbs with Their Formulations,
vol 2 (Daya Publishing House, Delhi) 2005, 824-827.
7 Chatterjee A & Pakrashi S C, The Treatise on Indian Med icinal
Plants, vol 3 (National institute of Science Communication, CSIR,
New Delhi, India) 1997, 203-204.
8 Shankar U, A case of high tree diversity in a sal (Shorea robusta)
dominated lowland forest of Eastern Himalaya: floristic
composition, regeneration and conservation, Curr Sci, 81 (2001)
9 Bhardwaj S D & Chakraborty A K, Studies on time of seed
collection, sowing and pre-sowing seed treatments of Terminalia
bellirica Roxb. and Terminalia chebula Retz., Indian For, 120
(1994) 430-439.
10 Shyamkumar B, Anjaneyulu C & Giri C C, Multiple shoot
induction from cotyledonary node explants of Terminalia chebula,
Biol Plant, 47 (2004) 585-588.
11 Anjaneyulu C, Shyamkumar B & Giri C C, Somatic embryogenesis
from callus cultures of Terminalia chebula Retz. ; an important
medicinal tree, Tree Struct Funct, 18 (2004) 547-552.
12 Kanwar K, Deshmukh A J, Raj Deepika & Kaushal B, Efficient
in vitro rapid axillary bud proliferation from mature Terminalia
chebula Retz., a medicinal tree, Tree For Sci Biotechnol, 1 (2007)
13 Murashige T & Skoog F, A revised medium for rapid growth and
bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962)
14 Shyamkumar B, Anjaneyulu C & Giri C C, Genetic transformation
of Terminalia chebula Retz. and detection of tannin in transformed
tissue, Curr Sci, 92 (2007) 361-367.
15 Cochran W G and Cox G M, Experimental design 2nd ed.
(Wiley,New York) 1992.
16 Joshi R, Shekhawat N S & Rathore T S, Micropropagation of
Anogeissus pendula Edgew. - an arid forest tree, Indian J Exp
Biol, 29 (1991) 615-618.
17 Sarvesh A, Reddy T P & Kavikishor P B, Plant regeneration
from cotyledons of niger (Gu izotia abyssin ica Cass cv.
Ootacamund), Plant Cell Tissue Organ Cult, 32 (1993) 131-135.
18 Nishi K, Jaiswal U & Jaiswal V S, Induction of somatic
embryogenesis and plant regeneration from leaf callus of
Terminalia arjuna Bedd., Curr Sci, 75 (1998) 1052-1085.
19 Kathiravan K, Shajahan A & Ganapathi A, Regeneration of
plantlets from hypocotyl derived callus of Morus alba, Israel J
Plant Sci, 43 (1995) 259-262.
20 Sahoo Y, Pattnaik S K & Chand P K, Plant regeneration from
callus cultures of Morus indica L. derived from seedlings and
mature plants, Sci Hort, 69 (1997) 85-90.
21 Murkute A A, Patil S, Patil B N & Mayakum ari V,
Micropropagation in pomegranate, callus induction and
differentiation, South Indian Hort, 50 (2002) 49-55.
22 Pandey S, Singh M, Jaiswal U & Jaiswal V S, Shoot initiation
and multiplication from a mature tree of Terminalia arjuna Roxb.
In vitro Cell Dev Biol Plant, 42 (2006) 389-393.
23 Cid L P B, Machado A C M G, Carvalheira S B R C & Brasileiro
A C M, Plant regeneration from seedling explants of Eucalyptus
grandis x E. urophylla, Plant Cell Tissue Organ Cult, 56 (1999)
24 Kanwar K, Kaushal B, Abrol S & Raj Deepika, In vitro
regeneration from cell suspension culture in Rob ini a
pseudoacacia L., Biol Plant, 52 (2007) 187-190.
25 Agarwal S & Kanwar K, Comparison of genetic transformation
in M orus alba via different regeneration systems, Plant Cell
Rep, 26 (2007) 177-185.
26 Choudhary M L & Chin C K, Decapitated seedling: a novel way
of transgenesis in Petunia hybrida L., Ind ian J Exp Biol, 32
(1994) 922-924.
27 Bosela M J, Sch nurr J P, Cheng Z M & Sargent W A,
Agrobacterium mediated transformation of three elite hybrid
aspens, Hort Sci, 32 (1997) 535.
28 Thakur A K, Sharma S & Srivastava D K, Plant regeneration and
genetic transformation studies in petiole tissue of Himalayan
poplar (Populus ciliata Wall.) Curr Sci, 89 (2005) 664-668.
29 Jefferson R A, Kavanagh T A & Bevan M W, GUS fusions: b-
glucuronidase as a sensitive and versatile gene marker in higher
plants, EMBO J, 6 (1987) 3901-3907.
30 Fraley R T, Rogers S G, Horsch R B, Sanders P S, Flick J S et al,
Expression of bacterial genes in plant cells, Proc Natl Acad Sci
USA, 80 (1983) 4803-4807.
31 Herrera E L, Kyozuka J, Floyd R B, Bateman K S, Tanaka H et
al, Insect and herbicide resistant transgenic Eucalyptus, Mol
Breed, 6 (2000) 307-315.
32 Zambryski P, Joos H, Genetello C, Leemans, J, Van Montagu M
et al, Ti plasmid vector for the introduction of DNA into plant
cells without alteration of their normal regeneration capacity,
EMBO J, 2 (1983) 2143-2150.
33 Hassig B E, Nelson N D & Kidd G H, Trends in the use of
culture in forest improvement, Biotechnol, 5 (1987) 52-59.
34 Bhatnagar S & Khurana P, Agrobacterium tumefaciens-mediated
transformation of Indian mulberry, Morus indica cv. K2: a time-
phased screening strategy, Plant Cell Rep, 21 (2003) 669-675.
35 Zaldivar J M, Ballina G H, Guerrero R C, Aviles B E & Godoy
H G C, Agrobacterium mediated transient transformation of
annatto (Bixa orellana) hypocotyls with the gus reporter gene.
Plant Cell Tissue Organ Cult, 73 (2003) 281-284.
36 Kanwar K, Bhardwaj A & Sharma D R, Genetic transformation
of Robinia pseudoacacia by Agrobacterium tumefaciens, Indian
J Exp Biol, 41 (2003) 149-153.
37 Agarwal S, Kanwar K, Saini N S & Jain R K, Agrobacterium
tumefaciens mediated genetic transformation and regeneration of
Morus alba L., Sci Hort, 100 (2004) 183-191.
38 Cerevera M, Pina J A, Juarez J, Navarro L & Pena L,
Agrobacterium -mediated transformation of citrange: factors
affecting transformation and regeneration, Plant Cell Rep, 18
(1998) 271-278.
39 Costa M, Miguel C & Oliveira M M, Improved conditions for
Agrobacterium mediated transformation of almond, Acta Hort,
738 (2007) 575-281.
40 Cui Mi, Ezura H, Nishimura S, Kamada H & Handa T, A rapid
Agrobacterium-mediated transformation of Antirrhinum majus
L. by using direct shoot regeneration from hypocotyl explants,
Plant Sci, 166 (2004) 873-279.
41 Doyle J J & Doyle J L, A rapid DNA isolation procedure from
small quantities of fresh leaf tissues, Phytochem Bull, 19 (1987)
42 Xiuping Z, Demou Li, Xiaoying L, Keming L & Yan P, An
improved procedure for Agrobacterium-mediated transformation
of Trifoliate Orange (Poncirus trifoliata L. Raf. ) via indirect
organogenesis, In vitro Cell Dev Biol Plant, 44 (2008) 169-177.
43 Duan Y X, Guo W, Meng H J, Tao N G & Deng X, High efficient
transgenic plant regeneration from embryogenic calluses of Citrus
sinensis, Biol Plant, 51 (2007) 212-216.
44 Meiru Li, Hongqing Li, Huawu J, Xiaoping P & Guojiang Wu,
Establishment of an Agrobacterium mediated cotyledon disc
transformation method for Jatropha curcas, Plant Cell Tissue
Organ Cult, 92 (2008) 173-181.
... The transformed callus was analyzed for the presence of tannins using thin layer chromatography, which indicated the presence of tannic acid in the transformed callus. Verma et al. (2013) carried out in vitro regeneration of T. chebula to obtain complete plantlets from juvenile explants (hypocotyls and cotyledon). Dried seed were inoculated on MS medium after surface sterilization with Bavistin (0.2%) alone and followed by HgCl 2 (0.1%), resulted in maximum (75%) germination. ...
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as 'Harar' belongs to the family Combretaceae. It is found in deciduous forests throughout the greater part of India, China, Myanmar, Sri Lanka, Vietnam, Bangladesh, etc. In India, it is distributed throughout the greater part except in arid zones. The fruits are common constituent of 'Triphala', capable of imparting youthful vitality and receptivity of mind and sense and are extensively used for clinical research, tanning and furniture purposes and also contain 1.73% nitrogen and 2.75% Calcium. The demand for its fruit has increased tremendously, because of its medicinal value; its fruits are sold at a price of ` 10-60 kg-1. However, the poor germination capacity, lack of natural regeneration and knowledge regarding its propagation are the limiting factors for its adoption in agroforestry systems. There is need of more productive planting stock with lower juvenile period and comparatively large fruit size.
... There are many reports available on micropropagation of medicinally important plant of Combretaceae members such as Terminalia bellerica (Chittora et al. 2008; Rathore et al. 2008; Suthar et al. 2009; Mehta et al. 2012; Dangi et al. 2012; Dangi et al. 2014),T. cattapa(Phulwaria et al. 2012b), T. chebulaAnjaneyulu et al. 2004;Anjaneyulu and Giri 2008;Verma et al. 2013), T. arjuna(Pandey et al. 2006;Arumugam and Gopinath 2011;Gupta et al. 2014;Choudhary et al. 2015), ...
... Our lab is associated with the area of research on biotechnological interventions in T. chebula for rapid multiplication of this important medicinal tree for forestry (Shyamkumar et al., 2003;Anjaneyulu et al., 2008;Shyamkumar and Giri 2011;Anjaneyulu and Giri 2011). There has been a constant need to develop protocols for genetic transformation in tree species in general and T. chebula in particular for genetic improvement (Giri et al., 2004;Shyamkumar et al., 2007;Dangi et al., 2012;Verma et al., 2013;Zuo et al., 2018). In the present communication, we report biochemical analysis and genetic transformation studies using somatic embryogenesis culture system of T. chebula. ...
... Well-developed root systems were observed after 4 weeks, with 27.3% rooting with an average root length and number of roots per micro-shoot of 1.72 cm and 2.53, respectively (Table 4; Figure 1G). Low salt concentrations in the medium have been shown to enhance the rooting of shoots in several plant species (Thimmappaiah, Shirly, & Iyer, 2007;Rai, Jaiswal, & Jaiswal, 2009;Kanwar et al., 2010Kanwar et al., , 2015Verma, Kanwar, & Bhardwaj, 2013). With successive subculturing of in vitro-raised Values in parentheses are arc sine transformed values. ...
Sequential subculturing leads to a gradual physiological change in cells that may be termed 'rejuvenation'. The effect of repetitive subculturing on callus induction and shoot regeneration from leaf explants of Punica granatum L. 'Kandhari Kabuli' were investigated. Surface sterilised leaves were cultured on 1.0x Murashige and Skoog (MS) medium supplemented with 4.0 mg l(-1) a-naphthaleneacetic acid (NAA) and 2.0 mg l(-1) 6-benzyladenine (BA) for callus induction. Shoots were regenerated from callus on 1.0x MS medium supplemented with 1.5 mg l(-1) BA, 0.5 mg l(-1) kinetin, and 0.25 mg l(-1) NAA. Subculturing of callus onto fresh medium maintained the rate of shoot formation and substantially increased the production of shoot buds up to the second subculture. Following further subculture passages, a lower shoot regeneration potential from callus was observed. A maximum shoot bud induction from callus of 63.9% was observed at the second subculture passage. The rate of multiplication of in vitro shoots increased until the fourth subculture, then became constant. Similarly, in vitro rooting of micro-shoots increased up to the third subculture, followed by a decline during further subculturing.
Seeds of Terminalia chebula, collected from Punjab and adjoining states of Haryana, Himachal Pradesh and Jammu and Kashmir were subjected to different pre-sowing seed treatments to overcome dormancy. The fruits from different locations had different shapes and sizes but the taste and odour was same. The fruits and seeds from Haryana were big and heavier than other locations. The seeds of Punjab and Haryana exhibited higher germination. The pre-sowing treatment with 50% concentrated sulphuric acid for 2 min was most effective to enhance germination. Seed vigour and seedling recovery per cent declined with storage and enhanced with treatment of concentrated sulphuric acid followed by boiling water, cold water and cowdung slurry treatments. The results for micro-propagation of T. chebula revealed that MS + BAP (6-benzylaminopurine 1 mg/l) + kinetin (0.5 mg/l) + GA3 (0.5 mg/l) + charcoal (0.4 g/l) media promoted maximum bud break (71.63%), mean shoot length (25.00 mm) and number of shoots per explant (1.60).
Conference Paper
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An improved system for Agrobacterium-mediated almond transformation was developed. A 100 fold increase in efficiency (12.3%) in comparison to the available transformation method (0.1%) was observed in spite of the lower regeneration ability of the almond explants (20-30%, instead of 70-90% in the existing method). Key modifications included use of 150 μΜ acetosyringone (AS) during the induction period (21 days), and a different selection scheme. Bacteria were directly applied onto wounds. After three days of co-cultivation, bacteria were removed by washing with a 200 μg ml-1 cefotaxime solution and explants were then transferred to fresh induction medium with 300 μg ml-1 cefotaxime, 150 μM AS and 15 μg ml-1 kanamycin. Kanamycin was maintained at 15 μg ml-1 during sub-cultures on shoot elongation medium and gradually raised after shoot transfer to micropropagation medium (one subculture at 15 μg ml-1, one sub-culture at 30 μg ml-1, and the following at 50 μg ml-1). Surviving shoots were propagated on 50 μg ml-1 kanamycin for 2.5 years. Transformed shoots tested positive by PCR, GUS assay and Southern blotting. The improved strategy is being used with cultivar material and is available for gene function analyses in almond.
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Three elite hybrid aspen, Populus grandidentata × P. canescens , P. tremuloides × P. tremula , and P. tremuloides × P. davidiana , have been transformed with Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying kanamycin resistance and GUS genes. The leaves of micropropagated shoots were co-cultivated with Agrobacterium for 65 to 72 hr and then transferred to callus-induction medium with 80–120 mg/L kanamycin in the dark. After 2 weeks, the leaves were transferred to shoot-induction medium under 18-hr photoperiod. Regenerated shoots were verified for transformation by histochemical staining and PCR. Transformed shoots rooted and were transplanted to soil. The three hybrid clones differed widely in their medium requirements for regeneration and in their competence for transformation. The leaves of P. grandidentata × P. canescens callused vigorously on a wide variety of media. In a typical transformation experiment, 30% to 60% of infected leaves produced putatively transformed calli (up to 10 calli per leaf). The origin of these calli and the frequency of shoot formation depended on the Agrobacterium strains. The calli from EHA105-infected leaves produced shoots within six weeks of co-cultivation and at high frequencies (70% to 90%). However, the calli from LBA4404-infected leaves produced shoots more slowly and at much lower frequencies (5% to 10%). Delaying selection for 2 weeks was found to lower the transformation frequency. Putatively transformed calli were obtained from P. tremuloides × P. tremula , and P. tremuloides × P. davidiana hybrids at frequencies of only 2% to 3%. The calli regenerated from P. tremuloides × P. davidiana leaves were very small, but they continued to grow upon being transferred to shoot-induction media and have started to produce shoots. The calli from leaves of P. tremuloides × P. tremula were much larger and they produced shoots more quickly. This transformation protocol is currently being used to introduce rooting genes into these hybrids to improve their rooting from hardwood cuttings.
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POPLARS occupy a unique and important position in the rural economy of India. Among the indigenous poplars, Himalayan poplar, i.e. Populus ciliata is a large, deciduous, dioecious and fast-growing tree of temperate and subtemperate regions of the Himalayas. Its wood is used for making general purpose plywood, packing cases, crates, support doors, matches, artificial limbs, fine paper and newsprint. Ho w- ever, Himalayan poplar is severely affected by a large number of insect pests, which lead to a considerable yield loss. Secondly, high lignin content in this species makes the operational costs of de -lignification process quite expe n- sive in pulp and paper industries. Being an economically important crop, application of plant tissue culture and plant genetic engineering in Himalayan poplar cultivation is of special value, to obtain improved or desired traits like disease and insect resistance and development of reduced lignin content. Efforts devoted to the use of explants of mature trees of proven worth for propagation through tissue culture technique have been limited to the work of only a few investigators 1-6
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Genetic transformation of Terminalia chebula Retz. was carried out using Agrobacterium tumefaciens strain C-58. Explants such as cotyledon, hypocotyl, excised mature zygotic embryo, cotyledonary node, in vitro leaf and shoot were used for genetic transformation. Different experimental methods were followed for infecting the explants. Cotyledon and hypocotyl explants showed swelling response on MS basal medium subsequent to genetic transformation. About 32.5 ± 2.5% cultures showed swelling response when 8-12 d in vitro pre-cultured cotyledon explants were used. Swelling response was not observed in non-transformed control cotyledon explants. Callus induction was observed in one of the swollen cotyledon explants infected with the bacterial suspension grown on media containing 0.1 mM acetosyringone. Callus initiation was not observed in cotyledon explants without co-cultivation, which were kept as control. The transformed callus was subjected to nopaline assay using paper electrophoresis. The analysis indicated the transformed nature of the callus with the presence of nopaline and its absence in non-transformed control callus. Transformed callus grown on fresh MS basal medium showed more than two-fold increase in the growth after four weeks of culture compared to normal control callus. Normally no growth was observed in untransformed control callus. The transformed callus was analysed for the presence of tannins using thin layer chromatography, which indicated the presence of tannic acid in the transformed callus. Genetic transformation of T. chebula and detection of tannin in transformed callus are reported here. This can be used to study the tannin biosynthetic pathway using biochemical and molecular approach.
A protocol for effective plant regeneration via somatic embryogenesis has been developed for Terminalia arjuna Bedd. Calluses were initiated from leaves of mature trees on Murashige and Skoog's medium (MS) supplemented with 5 mg l-1 2,4-dichlorophenoxyacetic acid, 0.01 mg l-1 kinetin, 3% sucrose and 0.8% agar. The calli showed differentiation of globular structures when transferred to the MS basal medium. Globular structures enlarged and produced secondary globular structures and/or somatic embryos. Continued production of globular structures, their differentiation into embryos and germination of embryos occurred on the MS medium with 3% sucrose and 0.8% agar. The plantlets were hardened and transferred to the soil. Such in vitro raised plants showed luxuriant growth in field condition.
Plantlets were regenerated from hypocotyl callus of Morus alba cv. MR2. Calli were established from hypocotyl segments on Murashige and Skoog (MS) medium supplemented with indoleacetic acid (0.5 mg/1) and benzyladenine (BA) (0.5 mg/1). They were transferred to MS medium with different concentrations of naphthaleneacetic acid NAA and BA for four weeks. Adventitious shoot buds were observed by transferring callus onto fresh Linsmaier and Skoog (LS) medium containing NAA (0.5 mg/1) and BA (0.75 mg/1). Shoots produced in vitro were rooted on MS medium with indolebutyric acid (0.75 mg/1).
An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector pBI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.