Regeneration and transformation studies in Terminalia chebula Retz.

Abstract

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.

Full text

563
VERMA et al: IN VITRO REGENERATION OF TERMINALIA CHEBULA Retz.
Journal of Scientific & Industrial Research
Vol. 72, Sept - Oct 2013, pp. 563-571
*Author for correspondence
E-mail: yashveer_verma@hotmail.com
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
Introduction
Terminalia chebula Retz. (Family, Combretaceae),
commonly known as Harad, is an indigenous,
multipurpose and deciduous tree of great economical
importance
1
. It occurs in Northern tropical wet evergreen
forests, tropical seasonal swamp forests, Southern and
Northern tropical deciduous forests
2
. Fruit and dried flesh
surrounding seeds are the most important product. Seeds
contain tannin (30-32%), which varies with season of
collection and locality
3
. T. chebula is always listed first
in the Ayurvedic metiria medica
4
. Fruits have antiamoebic
properties
5
and are used in fevers, cough, asthma, urinary
diseases, piles, worms and rheumatism, and scorpion-
sting
6
. 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
7
. Natural regeneration of T. chebula
from seeds in situ and ex situ is extremely low
8
and is a
slow growing tree compared to other species of
Termi nal ia
9
. In vitro propagation has also been
developed
10,11
. 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
reported
12
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
2
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
Skoog’s (MS)
13
basal medium
14
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

J SCI IND RES VOL 72 SEPT - OCT 2013
564
cultures were kept in culture room at 26 ± 2°C under 16
h photoperiod at 20 µmol m
-2
s
-1
. 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)
15
. 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
2
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
reported
16
. High rate of germination frequency in T.
chebula embryos has been obtained on MS medium
supplemented with 0.5 mg dm
-3
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
17
.
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
18
. Callus is produced on explants
in vitro as a result of wounding and in response of
hormones endogenous or exogenously
19
. 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
3
is the best explant;
Table 1—Effect 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
transformed)
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. 1—In vitro germination in Terminalia chebu la (Different
stages of germination after 3rd and 4th week on half strength MS
medium)

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VERMA et al: IN VITRO REGENERATION OF TERMINALIA CHEBULA Retz.
highest CI recorded in Co was 75% for the same
treatment C
3
. Similarly, Hy explants were found better
for callusing in Morus alba and M. indica
19,20
. Better
CI and proliferation from Co explants than leaf explants
of Punica granatum L. cv. Ganesh is also reported
21
.
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
2
) 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
21
. 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
8
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
4
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
study
22
, 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
23
highest
(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 2—Effect 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. 2—Callus induction from cotyledon explant (Well
proliferated callus after 6 weeks of culturing on MS medium
supplemented with 1.0 mg/l 2, 4-D)

J SCI IND RES VOL 72 SEPT - OCT 2013
566
on WPM supplemented with 1.50 mg/l BAP + 0.05 mg/
l NAA was also observed
24
. 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
8
treatment containing 1.50 mg/l BAP
+ 0.10 mg/l NAA was found to be best for in vitro shoot
Table 3—Effect 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. 3—Callus 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. 4—Shoot 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

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VERMA et al: IN VITRO REGENERATION OF TERMINALIA CHEBULA Retz.
regeneration from Co derived calli and statistically
significant.
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
25
. 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
3
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
26-28
.
Escherichia coli β- glucuronidase was developed as a
reporter system for transformation of plants to overcome
difficulties faced in using other reporter genes
29
. An
efficient technique for introducing cloned genes into plant
cells using Agrobacterium was also standardised
30-32
.
Woody species tend to be difficult and often inefficiently
transformed due to lack of proper regeneration system
33
.
A. tumefac i ens strain LBA 4404 carrying β-
glucuronidase and neomycin phosphotransferase-II
marker genes were used for genetic transformation
Table 4—Effect 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. 5—In vitro root induction in microshoot on ½ strength MS medium + 0.5% activated charcoal after 3 weeks of incubation

J SCI IND RES VOL 72 SEPT - OCT 2013
568
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
34-37
. 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 5—Effect 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
callus
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. 6—Genetic 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

569
VERMA et al: IN VITRO REGENERATION OF TERMINALIA CHEBULA Retz.
frequently resulted in Agrobacterium overgrowth
38
.
Maximum transformation frequency with 72 h of co-
cultivation in almond has also been reported
39
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
40
. 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
40
. 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 6—Specific (designed) primer used in present study for amplification of gus gene in transgenic
callus of T. chebula Retz.
Sl No. Primer Sequence (5' – 3')
1 Forward primer CTA GGA TAA ATT ATC GCG CGG GTG
2 Reverse primer GTT CAA GAT CCT CTG CCG ACT GTC
Fig. 7—PCR 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

J SCI IND RES VOL 72 SEPT - OCT 2013
570
of cells (callus) of T. chebula Retz. Total genomic DNA
was isolated from randomly selected 3 callus samples
by already standardised method
41
. 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
42
.
Integration of transgene (gus gene) into citrus genome
was confirmed by polymerase chain reaction
43
. PCR
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
1
, T
2
and T
3
(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
44
.
Conclusions
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.
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