Journal Journal

of Applied
Journal of Applied Horticulture, 22(1): 80-84, 2020
Appl Horticulture
DOI: 10.37855/jah.2020.v22i01.16 ISSN: 0972-1045

Influence of plant growth regulators on plant regeneration from

epicotyl and hypocotyl explants of Caesalpinia bonduc (L.) Roxb
– an ethnomedical plant

A. Thirumurugan, T. Senthil Kumar and B.D. Ranjitha Kumari*

Department of Botany, Bharathidasan University, Tiruchirappalli - 620 024, India. *E-mail: ranjithakumari2004@yahoo.co.in

Abstract
This study demonstrates the morphogenic potential of Caesalpinia bonduc (L.) Roxb, a traditional medicinal plant of the family
Fabaceae/ Caesalpinaceae. The present study was designed to examine the effect of phytohormones on plant regeneration from epicotyl
and hypocotyl explants of C. bonduc. The dormancy of the seeds was overcomed by acid scarification.. Of the two explants tested,
92 percent frequency of shoot regeneration and maximum number of shoots (3.6 ± 0.3), were noticed from the epicotyl explants on
Murashige and Skoog (MS) medium fortified with 6-benzyladenine (BA) 3.0 mg/L, Indole– 3- acetic acid (IAA) 1.0 mg/L and Poly
vinyl pyrrolidone 100 mg/L at pH 5.8. Elongated shoots were individually rooted on half strength MS medium supplemented with
1.5 mg/L Indole -3-butric acid (IBA) and exhibited 93 % frequency of root development. The in vitro raised plantlets were potted
and acclimatized under culture condition for four weeks and transferred to the green house. This efficient protocol will be helpful for
propagation of woody climber plants belongs to the family Caesalpinaceae and could be used for genetic transformation study.
Key words: Hypocotyl, epicotyl, plant regeneration, H2SO4, polyvinyl pyrrolidone.

Introduction alkaloids, glycosides, saponin and isoflavones (Shukla et al.,

2009; Singh and Raghav, 2012; Billah et al., 2013; Shelar et al.,
Medicinal plants are an important source of life saving drugs 2014).
for human kind, especially in developing countries. It is well
known that, synthetic drugs associated with health hazards and Conservation of medicinal plants through in vitro proliferation,
their toxicity has renewed the interest in the use of plant-based in situ and ex situ methods was reported by Govindaraj et al.
drugs. Caesalpinia bonduc (L.) Roxb., (Family-Fabaceae/ (2008); Radhakrishnan et al. (2009). There are number of reports
available on the successful plant regeneration from juvenile
Caesalpiniaceae), a prickly shrub or woody vine reaching a
epicotyl explants of various woody legumes including Tetrapleura
length of 10 m or more is also known as kalarchikai (Tamil). The
tetraptera (Ayisire and Amoo, 2004), Parkia biglobosa (Amoo
name of the species (Bonduc) is derived from the Arabic word
and Ayisire, 2005), Albizia odoratissima (Rajeswari and Paliwal,
“Bonduce” meaning a “little ball” which indicates the globular
2008) and Piliostigma thonningii (Ayisire et al., 2009). There
shape of the seed (Kannur et al., 2012). The plant is threatened
are as yet no successful report on epicotyl explants regeneration
and widely distributed throughout the world especially in tropical
from germinating seeds of C. bonduc. Therefore, in the present
parts of India, Sri Lanka and Myanmar sparsely distributed in the investigation epicotyl and hypocotyl explants of C. bonduc were
deciduous forests of the Western Ghats of India. It is also reported studied for regeneration.
to be critically endangered in Malaysia.
C. bonduc is one of such medicinal plants well-known for its Materials and methods
health benefits in folk medicine. The secondary metabolite The mature dried seeds of C. bonduc (Fig.1a & b) were collected
compound properties might be responsible for spectrum of from Kollidam river bank, Tiruchirappalli District, Tamil Nadu,
phytochemical and pharmacological activities of C. bonduc. It is India. The plant was authenticated with the type specimen
extensively used in Ayurveda, Siddha, Unani and homoeopathy available in the Rapinat Herbarium. St. Joseph’s College
since last two decades. Different parts of the plant are used to (Autonomous), Tiruchirappalli, Tamil Nadu. The dormancy of the
treat asthma, chronic fever, cough, headache and stomach upset seeds was overcomed by acid scarification using conc. H2SO4 for
(Chopra and Nayar, 1956; Nandakarni and Nandakarni, 1976). 45 min. The treated seeds were washing under running tap water
Recently conducted studies reported that, different parts of plant for about 10 min. Then seeds were sterilized with detergent for
possess significant anti-inflammatory, analgesic, anti-bacterial, 5 minutes followed by washed under running tap water for 10
anti-diarrheal and cytotoxic activity. In addition to this, plant is minutes. Immersed in an aqueous solution of 5 % Teepol for 5
known to offer multiple pharmacological benefits such as anti- min, later the seeds were treated with 4 % of Sodium hypochloride
pyretic, anti-diabetic, anti-carcinogenic, anti-fungal, anti-filarial, solution for 5 minutes and washed with distilled water followed
immunomodulatory, insecticidal, anxiolytic and antioxidant by 0.1 % of mercuric chloride and washed with sterile water 3
effects. C. bonduc is known to contain phenolic compounds, times.
Journal of Applied Horticulture (www.horticultureresearch.net)
 Plant regeneration from epicotyl and hypocotyl explants of Caesalpinia bonduc 81

analysis of variance (ANOVA) to assess treatment differences and

interactions using SPSS programme (16.0). The mean values of
treatments were subjected to DMRT (Duncans Multiple Range
Test) (P≤0.05). Data are represented as Mean ± Standard Error
of three replications.

Results and discussion

Shoot multiplication: Establishment of an efficient in vitro
regeneration system is dependent on the type of explant used.
Shoot proliferation from the epicotyl and hypocotyl explant
was visible after 15-20 days of inoculation. In vitro generated
explants of C. bonduc were used for multiple shoot induction
Fig. 1. a: C. bonduc plant. b: C. bonduc seeds
and proliferation. MS medium supplemented with different
Shoot initiation and multiplication: The treated seeds were concentrations of cytokinins such as TDZ, BAP, and KIN (1.0-4.0
inoculated on the MS medium (Murashige and Skoog, 1962) mg/L) were used for shoot induction (Table 1 and 2). Interestingly,
supplemented with 3 % (w/v) sucrose and 0.8 % (w/v) agar. The all cytokinins used in the present study showed similar shoot
medium was adjusted to pH 5.7 with 1 N NaOH or 1 N HCl prior response. Similar studies are also reported by Eapen and George
to the autoclave at 121 ºC for 20 min. For direct multiple shoot (1993) in groundnut, Hossain et al. (1994) in Aegle marmelos,
induction, 10-12 days of the epicotyl, hypocotyl explants were Mendoza and Futsuhara (1990) in mungbean, Pandey and Bansal
excised and inoculated on the medium fortified with 100 mg/L (1992) in soybean and Barna and Wakhlu (1994) in chickpea,
polyvinyl pyrrolidone (PVP) to arrest phenolic contamination, Venkatachalam et al. (1998) in groundnut and in Celastrus
BAP, Kin, TDZ and IAA for the shoot induction. The explants paniculatus Willd. by Moola and Ranjitha Kumari (2019).
were supplemented with the different concentrations of BAP, Kin,
TDZ and IAA compared with control (without any hormone). All Effect of cytokinins on shoot multiplication of C. bonduc: In
the cultures were incubated at 25± 2 oC under cool –white light this study, different concentrations of cytokinins with auxin were
with a 16 hrs photoperiod and 8 hrs dark period and subcultured used for shoot multiplication. Among the different concentrations
on fresh media at 15-20 days of interval. tested, the highest shoot multiplication was achieved at BAP+IAA
3.0+1.0 mg/L concentration with number of shoots (3.6±0.3), and
Rooting, hardening and acclimation: Data were collected at the high frequency of response (92±0.5 %) obtained from epicotyl
end of 45 days of culture on percentage shoot regeneration and explants (Table 3) whereas in hypocotyl explants (Table 4), the
number of shoots per responding explant. For the root initiation highest shoot multiplication was achieved at BAP+IAA 3.0+1.0
the shoots were transferred to the half strength medium 3 % (w/v) mg/L concentration with maximum number of shoots (2.6 ±
sucrose and 0.8 % agar (w/v) supplemented with Indole -3-butyric 0.08) and highest frequency percentage (82±1.1). In comparison
acid (IBA) (0.5-2.0 mg/L) or NAA (0.5-2.0 mg/L) and control with hypocotyl explants, epicotyl explants showed highest
(without any hormone). The in vitro raised plantlets were potted regeneration efficiency in all aspects.
and acclimatized under culture condition for four weeks before
Explants tested in the present study failed to elicit response in a
their transfer to green house.
hormone-free medium. MS supplemented with 17.75 µMol BAP
Statistical analysis: Data were analyzed by using one-way and 2.46 µMol IBA induced a mean of 3.40±1.07 frequency root
Table 1. Effect of various concentrations of BAP, KN, TDZ on survival rate and shoot multiplication from epicotyl explants of C. bonduc
Concentration BAP Kin TDZ
Response (%) No. of shoots Response (%) No. of shoots Response (%) No. of shoots
1.0 26 ± 0.6f 1.4 ±0.03d 20 ± 0.5 g 1.0 ± 0.0c 20 ± 0.5 g 1.0 ± 0.0c
2.0 36 ± 0.3 f
1.5 ±0.05 cdā
33 ± 0.3ef
1.6 ±0.3 abc
33 ± 0.3ef
1.6 ± 0.3 abc
3.0 56 ± 0.3e 2.1± 0.0a 56 ± 0.3bc 2.3 ± 0.3a 56 ± 0.3bc 2.3 ± 0.3a
4.0 40 ± 0.5e 1.6 ±0.06bc 46 ± 0.3cd 1.3 ± 0.3bc 46 ± 0.3cd 1.3 ± 0.3bc
Means in each column followed by the same superscript letters are not significantly different according to DMRT at P < 0.05
Table 2. Effect of various concentrations of BAP, KN, TDZ on survival rate and shoot multiplication from hypocotyl explants of C. bonduc
Concentration BAP Kin TDZ
Response (%) No. of shoots Response (%) No. of shoots Response (%) No. of shoots
1.0 24 ± 0.5 i
0.8 ±0.1 g
26 ± 0.5 i
1.06 ± 0.03 f
26 ± 0.5 i
1.06 ± 0.03f
2.0 25.3 ± 0.3i 1.0 ± 0.03f 27.3 ± 0.3ki 1.46 ±0.03cd 27.3 ± 0.3ki 1.46 ±0.03cd
3.0 34 ± 1.1i 1.6 ± 0.0bc 31.3 ± 1.7ij 1.8 ± 0.1b 31.3 ± 1.7ij 1.8 ± 0.1b
4.0 30 ± 1.1jk 1.2 ± 0.0ef 30.3 ± 0.3jk 1.3 ± 0.08de 30.3 ± 0.3jk 1.3 ± 0.08de
Means in each column followed by the same superscript letters are not significantly different according to DMRT at P < 0.05

Journal of Applied Horticulture (www.horticultureresearch.net)

82 Plant regeneration from epicotyl and hypocotyl explants of Caesalpinia bonduc

Table 3. Effect of BAP, Kin, TDZ with IAA concentrations on survival rate and shoot multiplication from epicotyl explants of C. bonduc
Concentration BAP + IAA Kin + IAA TDZ + IAA
Response (%) No. of shoots Response (%) No. of shoots Response (%) No. of shoots
1.0 +0.5 36 ± 0.3 f
1.4 ± 0.03 egf
26 ± 0.3g
1.3±0.03 fgh
40 ± 0.5gh
1.2± 0.1h
2.0 +0.5 66 ± 0.6d 1.6 ±0.05cd 40 ± 0.5de 1.5 ± 0.0def 46 ± 0.8fg 1.38±0.1fgh
3.0+0.5 73 ± 0.3c 2.6 ± 0.08ae 76 ± 0.3a 2.4 ± 0.03b 70 ± 0.5bc 2.3± 0.0b
4.0+0.5 63 ± 0.8c 1.7 ± 0.03c 66 ± 0.3ab 1.6 ±0.03cd 50 ± 0.5ef 1.2± 0.03gh
1.0+1.0 60 ± 0.5d 2.0 ± 0.0 cd 55 ± 0.5e 1.5± 0.1c 63 ± 0.3cd 2.0 ± 0.0abc
2.0+1.0 83 ± 0.3a 2.3 ± 0.3bc 65.3 ± 1.4d 1.2±0.3d 83 ± 0.3ab 2.3 ± 0.3ab
3.0+1.0 92 ± 0.5 a
3.6 ± 0.3 a
83 ± 1.1 a
2.2±0.3 a
90 ± 0.3 a
3.0 ± 0.5a
4.0+1.0 76 ± 0.3a 3.0 ± 0.5ab 72 ± 1.1c 1.9±0.3b 80 ± 0.5ab 1.6 ± 0.3bc
Means in each column followed by the same superscript letters are not significantly different according to DMRT at P < 0.05
Table 4. Effect of different concentrations of BAP, Kin, TDZ with IAA on survival rate and shoot multiplication from hypocotyl explants of C.
bonduc (L.) Robx.
Concentration BAP + IAA Kin + IAA TDZ + IAA
Response (%) No. of shoots Response (%) No. of shoots Response (%) No.of shoots
1.0 +0.5 37.6 ±0.8 h
1.3±0.3 cd
41± 1.1 gh
1.3 ± 0.3 bc
45±3.0 ef
1.0 ± 0.0c
2.0 +0.5 40 ± 1.1gh 1.3 ± 0.3cd 43.3 ±2.8fg 1.6 ± 0.3abc 48.3 ±1.8e 1.3 ± 0.3bc
3.0+0.5 46 ± 1.1f 1.6 ± 0.3cd 46.6 ± 1.2f 2.3 ± 0.3a 54 ± 2.0d 2.3 ± 0.3ab
4.0+0.5 39 ± 0.5h 1.0 ± 0.0cd 40.6±0.6gh 1.0 ± 0.0c 46.3 ±1.7ef 1.3 ± 0.3bc
1.0+1.0 53 ± 0.5e 1.9±0.05e 30 ± 0.5fg 1.3 ± 0.3bc 64± 1.7c 1.8±0.03e
2.0+1.0 64 ± 1.5d 2.2±0.0c 43 ± 0.8cd 1.0 ± 0.0c 84.6 ± 1.4a 1.9±0.03de
3.0+1.0 82 ± 1.1 a
2.6± 0.08 a
56 ± 0.3 bc
2.0 ± 0.0 ab
86.6 ± 1.4 a
2.3±0.06bc
4.0+1.0 76 ± 1.1b 2.4±0.0b 50 ± 0.5 cd 1.3 ± 0.3bc 74 ±1.5b 2.06±0.03d
Means in each column followed by the same superscript letters are not significantly different according to DMRT at P < 0.05
explant (Kumar et al., 2012). Owing to phenolics, most of woody strength MS medium supplemented with different concentrations
species explants do not induce morphogenic response in culture ranging from 0.5-2.0 mg/L of auxins such as NAA and IBA. The
condition (Furze and Cress well, 1985; Bonga, 1987). Many percentage of root and number of roots per shoot were calculated
woody legume plants supplemented with BAP alone in different (Table 5 and Fig. 2). Out of two auxins tested, IBA was found to
concentrations or in combination with auxin induced shoot bud be most effective for root induction. The highest number of roots
organogenesis (Subotic et al., 2009). BAP and IAA induced per shoot (5.3±0.3) and high frequency (93 %) were obtained in
callus regeneration was reported in Acacia sinuate (Vengadesan IBA at 1.5 mg/L. Whereas, the lowest number of roots per shoot
et al., 2000), Citrullus lanatus (Chaturvedi and Bhatnagar, (1.3±0.3) and low frequency (50 %) were obtained in NAA (0.5
2001), Argyrolobium roseum (Khanna et al., 2006) and Vigna mg/L).
subterranea (L.) Verdc. (Hilaire et al., 2009).
Ozean et al. (1992) reported that Indole-3-butyric acid is
Rooting considered as the most effective growth regulator for induction
Root induction from in vitro elongated shoots of C. bonduc: of roots in legumes. Similar findings about IBA-induced rooting
For root induction, in vitro elongated shoots of C. bonduc were
were observed in other leguminous species such as Acacia
excised from regenerating cultures and transferred to half-
sinuata (Vengadesan et al., 2000), Swainsona salsula (Yang
Table 5. Influence of IBA and NAA on rooting of in vitro formed shoots et al., 2001), Cassia angustifolia (Agarwal and Sardar, 2007)
on half MS medium.
and Vigna subterranea (L.) Verdc. (Hilaire et al., 2009). The
PGR (mg/L) Percentage Number of roots acclimatization of plantlets resulted in a survival rate of 95 %.
IBA NAA response explants
During the period of 6 months in the greenhouse, there was no
0.5 0.0 63 ± 0.3 cd
1.6 ± 0.3cd morphological abnormalities or detectable phenotypic variation
1.0 0.0 73 ± 0.3 bc
2.3 ± 0.3cd
in regenerated plants when compared with mother plant.
1.5 0.0 93 ± 0.3a 5.3 ± 0.3a
2.0 0.0 56 ± 0.3de 1.3 ± 0.3d The present study developed a time-saving protocol for rapid
0.0 0.5 50 ± 0.5e 1.3 ± 0.3d and high-frequency regeneration method for C. bonduc by using
0.0 1.0 53 ± 0.3de 1.6 ± 0.3cd
0.0 1.5 76 ± 0.3b 4.3 ± 0.3b
epicotyl and hypocotyl (non-meristematic) explants. Out of the
0.0 2.0 50 ± 0.5 e
2.6 ± 0.3c two explants tested, epicotyl explants were found to be best for
Means in each column followed by the same superscript letters are shoot multiplication using combination of BAP alongwith IAA
not significantly different according to DMRT at P < 0.05 (3.0+1.0 mg/L). For root induction, IBA at 1.5 mg/L concentration
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 Plant regeneration from epicotyl and hypocotyl explants of Caesalpinia bonduc 83

Fig. 2. Direct plant regeneration of in vitro cultured Epicotyl explants of C. bonducella (L.) Roxb. (a) Endosperm, (b) Germination of
Endosperm, (c) Shoot initiation from epicotyl explants, (d) Development of shoots, (e, f and g) Shoot multiplication and elongation, (h)
Rooting, (i) Hardening of plant

showed best response. Total regeneration process from the Barna, K.S. and A.K. Wakhlu, 1994. Whole plant regeneration of Cicer
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A.A. Mia, 2013. Antibacterial, antidiarrhoeal and cytotoxic activities
used for initiation would contribute to enhance the number of of methanol extract and its fractions of Caesalpinia bonducella (L.)
shoots per explant. Hence, the present study concludes that, Roxb leaves. BMC Complementary Alternative Medicine,13(1): 101.
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