Journal of Genetic Engineering and Biotechnology (2014) 12, 95–101

H O S T E D BY
Academy of Scientific Research & Technology and
National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology
www.elsevier.com/locate/jgeb

ARTICLE

Indirect organogenesis from various

explants of Hildegardia populifolia (Roxb.)
Schott & Endl. – A threatened tree species
from Eastern Ghats of Tamil Nadu, India
A.R. Lavanya a, S. Muthukrishnan a, M. MuthuKumar a,
J.H. Franklin Benjamin b, T. Senthil Kumar c,*, V. Kumaresan a, M.V. Rao a

a
Department of Plant Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
b
Botanical Survey of India, Southern Regional Centre, Coimbatore, Tamil Nadu, India
c
Department of Industry University Collaboration, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India

Received 23 April 2014; revised 6 August 2014; accepted 20 September 2014

Available online 29 October 2014

KEYWORDS Abstract Hildegardia species are an important resource for fiber industry. This investigation was
Hildegardia populifolia; conducted to develop a plant regeneration protocol for Hildegardia populifolia (Roxb.) Schott &
Glutamine; Endl. via indirect organogenesis Callus was obtained from leaf, internode and petiole explants,
In vitro plant regeneration; among these explants internode explant gave best result on MS medium supplemented with differ-
Threatened endangered tree; ent concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). The highest percentage (100%) of
Indirect organogenesis; regeneration was obtained with benzyladenine (BA) (2.0 mg/l) + indole-3-acetic acid (IAA)
Micropropagation; (0.1 mg/l) + glutamine (25 mg/l) + thidiazuron (TDZ) (0.5 mg/l) from internode explants. Shoot-
Tissue culture; lets were highly rooted on MS medium supplemented with 3.0 mg/l indole-3-butyric acid (IBA).
In vitro In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facil-
itate further development of reliable procedures for this genus.
ª 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research &
Technology.

1. Introduction A narrow endemic [2], H. populifolia was known to be repre-

sented by a sole surviving population comprising about 20
Hildegardia populifolia (Roxb.) Schott & Endl. is a medium trees in Kalrayan Hills of Tamil Nadu [1]. This species is under
sized deciduous tree of the family Sterculiaceae confined to threat due to factors not apparent at present. But it is assumed
tropical forests of Tamil Nadu and Andhra Pradesh in India. that anthropogenic interference, habitat losses and other
intrinsic and extrinsic factors might have been the reason for
* Corresponding author. their poor regeneration ability and low seed viability [21].
E-mail address: [email protected] (T. Senthil Kumar). According to IUCN [14] list of threatened species, H. populifo-
Peer review under responsibility of National Research Center, Egypt. lia is critically endangered. Sarcar and Sarcar [29] located 13
http://dx.doi.org/10.1016/j.jgeb.2014.09.002
1687-157X ª 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
96 A.R. Lavanya et al.

mature trees and 11 young natural regenerations of this species cultures were subcultured on the fresh medium after
in dry mixed deciduous forests of Parigam Reserved Forests in 15–20 days.
Kalrayan Hills of Kallakurichi Forest Division, Tamil Nadu
and they assessed its status as threatened. This endemic species 2.3. Callus initiation
was evaluated in its entire range and placed under IUCN
category, critically endangered [27]. This tree is easily recogniz- Explants including internode, (1 cm in length), petiole (0.5 cm
able by its pale green bark. The fiber extracted from the bark is in length), and leaf (0.5 · 0.5 cm) were excised from 45 day old
used for domestic purposes and leaf extract is known to have in vitro germinated seedlings and placed horizontally on MS
healing properties [28]. medium. In this experiment, the effects of cytokinins and aux-
In vitro regeneration is an efficient means of ex situ ins, both separately and in combination were studied on callus
conservation of plant diversity [9,15,5] because with this tech- initiation and its proliferation. Auxins such as 2,4-dichloro-
nology many endangered species can be quickly propagated phenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) and
and preserved using minimal of plant material. Moreover this indole-3-butyric acid (IBA) (0.1, 0.5, 1.0, 2.0 and 3.0 mg/l),
technique has the unique advantage of propagating the desired and cytokinins benzyladenine (BA) and (kinetin) Kin (0.5,
taxon, independent of season, reproducing barriers and germi- 1.0, 2.0 and 3.0 mg/l) either alone or in combinations of
nation hurdles. H. populifolia is ideal candidate for in vitro IAA + BA, IAA + Kin, and Kin + BA. For further callus
regeneration with its intrinsic poor seed viability and regener- proliferation supplements such as adenine, adenine sulfate,
ation capabilities. Available literature [3,16,8] reveals that an glutamine and sodium citrate (10, 25 and 50 mg/l) were used.
in vitro protocol for this vulnerable tree has been carried out These culture conditions were used in all the experiments men-
through distinct in vitro regeneration approaches i.e. indirect tioned below unless stated. Data of frequency (%) of callus
organogenesis. Protocols for organogenesis and somatic formation and its fresh weight were recorded after 45 days of
embryogenesis of this plant from seedling derived axillary mer- culture.
istem and mature tree derived nodal meristem are standardized
[3]. The present study describes successful plant regeneration
2.4. Shoot organogenesis from callus
via adventitious indirect organogenesis from internode, petiole
and leaf segments of H. populifolia.
Well-established hard and compact callus (0.5 g fresh weight)
2. Materials and methods were grown on MS medium supplemented with BA (2.0 mg/
l) + IAA (0.1 mg/l) + glutamine (25 mg/l) for shoot organo-
2.1. Seed germination and explant preparation genesis. Calli were transferred to shoot organogenesis media,
consisting of MS basal media supplemented with TDZ (0.1,
0.5, 1.0, 2.0 and 3.0 mg/l) and activated charcoal (5, 10 and
Seeds of H. populifolia were collected on November 2004 from 25 mg/l), in combinations of TDZ at 0.5 mg/l. Cultures were
Kalrayan Hills which 300–600 m, Eastern Ghats of Tamil transferred on fresh media after 20th day of inoculation. The
Nadu. Chemical scarification was carried out by soaking the percent of shoot organogenesis from callus and average
seeds in concentrated H2SO4 (98%) for 5, 15, 30, 45, 60 and number of shoots per inoculum were recorded on the 35thday
90 min and subsequently rinsing with tap water for 30 min. after transferring the callus on shoot organogenesis media.
The scarified seeds were then disinfected with 70% ethyl alco-
hol for 5 min and followed by aqueous solution of 0.1% mer-
2.5. Formation of adventitious root
curic chloride for 10 min. Then the seeds were rinsed 4–5 times
with sterile distilled water. Sterilized seeds were aseptically
inoculated in MS medium supplemented with GA3 (1.0 mg/l) Regenerated shoots (2–4 cm long) obtained from micropropa-
and cotton soaked with sterile water. The inoculated seeds gated plantlets were cultured on MS basal medium fortified
were incubated under optimal culture condition. The in vitro with either of IBA, NAA, or IAA (0.1, 0.5, 1.0, 2.0, and
raised seedlings were transferred to earthen pots containing 3.0 mg/l) for adventitious rooting. Data were recorded on
soil and sand (1:1) maintained in the garden. The explants were percentage of rooting, number, and length of the roots after
collected from 45 day old seedlings and cut into 10–15 mm 4 weeks of transfer onto the rooting media.
fragment as explants.
2.6. Hardening of regenerated plants
2.2. Media and culture condition
Rooted plantlets were removed from the medium and washed
The basic culture medium (BCM) consists of Murashige and in sterile distilled water to remove all the traces of agar and
Skoog [20]. MS medium was fortified with 30 g/l sucrose basal callus. The plantlets were then transferring to plastic pots
(Himedia, India) and gelled with 0.8% agar (Himedia, India), (5–6 cm diameter) containing garden soil mixed of vermiculite
and the pH of the medium was adjusted to 5.7 ± 0.2 with and sand (2:1:1). The plastic pots were covered with polyethyl-
0.1 N NaOH or 0.1 N HCl after addition of the growth ene bags to maintain the relative humidity of about 70–80%.
regulators. The medium was dispensed in culture tube and These pots were maintained at 25 ± 2 C with a 16-h photope-
autoclaved at 121 C, for 30 min. All the cultures were riod and a light intensity of 25 lmol m 2 s 1for 2 weeks in the
maintained in a sterilized culture room at 26 ± 2 C, under culture room, and the pots were then transferred to a shade
16 h photoperiods provided by cool white fluorescent light (60 lmol m 2 s 1) in the third week. Plantlets were then trans-
(60 lmol 2 s 1) and with 55–60% relative humidity. The ferred to glass house in the fourth week.
Indirect organogenesis from various explants of Hildegardia populifolia (Roxb.) 97

2.7. Statistical analyses epicotyls cultured on MS medium supplemented with 1.5 mg/l
BAP, whereas the highest number of shoots per responding
All the experiments consisted of 25 explants and each was explant was recorded on medium containing 0.7 mg/l BAP
repeated four times unless otherwise stated. Data were statisti- and 0.1 mg/l NAA. Likewise Gikloo et al. [10] tested the
cally analyzed by using either one or two-way analysis of var- different explants and obtained best result in leaf explants
iance (ANOVA). Mean comparisons were made by least from Silybum marianum. In contrast, in Origanum vulgare,
significant difference at the 5% probability level. The arcsine among the cotyledon, hypocotyls, and root segments tested,
square root transformation was applied to proportional data cotyledons proved to be the best source for the production
before ANOVA [11]. of compact and modulated callus [23]. Similarly Gurel et al.
[13] reported that hypocotyls and cotyledon explants produced
significantly more callus than petiole or leaf explants of Beta
3. Results and discussion
vulgaris.
The concentration of PGR required inducing a maximum
3.1. Effect of explant types and PGR concentration on callus percentage of callus induction varied with the explant type.
formation Internode segments showed the maximum response of 95%
callus production when cultured on MS medium supplemented
Effect of explant types and plant growth regulator (PGR) con- with BA (2.0 mg/l), IAA (0.1 mg/l) and glutamine (25 mg/l),
centration on callus formation and in vitro shoot organogene- whereas in petiole explants, maximum callus formation of
sis of H. populifolia were studied in leaf, petiole, and internode 85% was also observed when cultured on MS medium contain-
explants cultured on MS-basal media as shown in Fig. 1; ing BA (2.0 mg/l), IAA (0.1 mg/l) and glutamine (25 mg/l).
Fig. 2a and b. The percentage of callus formation and the per- Leaf explants required relatively higher concentration of
centage of explant regenerating shoots varied significantly 2 mg BAP in combination with IAA (0.1 mg/l) and glutamine
(P < 0.001), depending on the concentration of plant growth (25 mg/l) for obtaining the maximum response of callus induc-
regulators (PGR) present in the medium. There was also a sig- tion (Table 2). Differential responses by different explant types
nificant interaction between the two factors (P < 0.001), indi- on the same medium may be because of the differences in the
cating that the effects of PGR concentration on in vitro endogenous level of plant growth hormone in different
adventitious shoot regeneration are dependent on explant explants [25]. Certainly, callus morphology varied among the
types. There was no significant difference among the different different explants. Internode-derived callus was green in color,
types of explants in terms of the percentage of callus forma- compact in texture, and fast-growing, whereas the callus
tion. However, the percentage response of callus formation formed from petiole explants was green white in color, friable
was significantly increased when IAA was added to the med- in texture, and slow growing. Callus formed from leaf was
ium in combination with BAP (Table 1). Similarly Rajeswari green in color, friable in texture, and slow-growing. With
and Paliwal [26] in Albizia odoratissima among the epicotyls, regard to growth rates, the size of callus after 45 day of culture
petiole, cotyledon segments tested, the best response in terms was greater from internode explants than that of callus formed
of the percentage of shoot regeneration was obtained from from either petiole or leaf.

100

90

80

70

60

50
Internode
40 Peole

30 Leaf

20

10

0
1
2
3

3
2

1
2
3

1
2
3
0.5

0.5

0.5

0.5
IAA 0.1
2,4-D 0.1

NAA 0.1

IBA 0.1

Figure 1 Callus induction from various explants of 45 days old in vitro seedlings grown on MS medium supplemented with auxins, after
45 days.
98 A.R. Lavanya et al.

Figure 2 Organogenesis of Hildegardia populifolia: Callus induction: (a) internode; (b) petiole. Shoot bud induction: (c) internode; (d)
petiole. Shoot regeneration: (e) internode; (f) petiole. Rooting: (g) internode; (h) petiole. (i) hardening.

3.2. Shoot organogenesis from callus internode explants which showed a significantly better
response for the number of shoots regenerated compared to
In internode explants, shoot bud differentiation were observed petiole explants (Table 3). Gubis et al. [12] also reported that
with less callus proliferation and callus and shoot formation hypocotyl and epicotyl segments of Lycopersicon esculentum
occurred simultaneously (Fig. 2c and d). Conversely, in petiole produced the maximum shoot regeneration rate whereas
explants, callus formation usually preceded shoot organogene- cotyledons showed a poor response on regeneration medium
sis. In this study, data on the percentage response of shoot containing 1 mg/l zeatin and 0.1 mg/l IAA. Molina [19] also
regeneration, shoot number, and percentage of callus forma- reported that stems and petioles segments of Salvia canariensis
tion were recorded after 45 days of culture. Of the different produced the maximum shoot regeneration rate whereas stem
explant types tested, internode segments showed a maximum showed a poor response on regeneration medium contain
of 55% shoot regeneration when cultured on MS medium con- 4.44 lM BAP and NAA lM. Likewise Martin et al. [18]
taining 0.5 mg TDZ. However, no significant difference was reported that leaf and internode explants of Ophiorrhiza pro-
observed in the percentage of shoot regeneration between strata produced the maximum shoot regeneration in high
internode and petiole explants (Fig 2e and f). Likewise Thakur range of BAP in the MS medium. At high concentration, auxin
et al. [31] obtained 75% shoot regeneration frequency in inhibits the development of the primordial or axillary buds and
Populus deltoides using petiole explants. Leaf explants showed induces the formation of callus in the presence of activated
no shoot regeneration under any of the conditions tested in charcoal in media, the shoots elongated better on media
this study. A maximum of 1.8 shoots were regenerated from containing lower concentrations of PGRs [6,17]. To promote
Indirect organogenesis from various explants of Hildegardia populifolia (Roxb.) 99

Table 1 Callus proliferation from 45 days old in vitro seedlings grown on MS medium supplemented with cytokinins and auxins, after
25 days.
Plant growth regulators (mg/l) Internode Petiole Leaf
Percentage of Nature Percentage Nature Percentage Nature
response (%) of callus of response (%) of callus of response (%) of callus
BA
0.5 76.0fg GC 70.0g GC 40.0g GC
1.0 81.3e GC 77.3ef GC 46.6e GC
2.0 90.0c GC 85.0cd GC 50.0d GC
3.0 84.0de GC 80.0e GC 44.0ef GC
KN
0.5 54.6k GFC 40.0l GFC 28.0hi GFC
1.0 60.0j GFC 45.0k GFC 35.0h GFC
2.0 50.0l GFC 37.3lm GFC 30.0h GFC
3.0 45.3m GFC 32.0n GFC 26.6i GFC
BA IAA
2.0 0.01 85.3d GCN 80.0e GCN 50.6cd GCN
2.0 0.05 93.3ab GCN 85.3c GCN 56.0c GCN
2.0 0.1 95.0a GCN 90.0a GCN 65.0a GCN
2.0 0.5 90.0c GCN 89.3ab GCN 60.0b GCN
BA IBA
2.0 0.01 77.3f GCN 60.0ij GCN 56.0c GCN
2.0 0.05 80.0ef GCN 65.0h GCN 60.0b GCN
2.0 0.1 72.0h GCN 61.3i GCN 53.3cd GCN
2.0 0.5 66.6i GCN 58.6j GCN 49.3de GCN
GC – green compact; GFC – green friable callus; GCN – green compact nodular.
Values are mean of 25 replicates per treatment and repeated thrice. Values with the same superscript are not significantly different at 5%
probability level according to DMRT.

Table 2 Callus proliferation from 45 days old in vitro seedlings grown on MS medium supplemented with BA (2.0 mg/l), IAA (0.1 mg/
l) and additives, after 25 days.
Additives (mg/l) Internode Petiole Leaf
Percentage Nature Percentage Nature Percentage Nature
of response (%) of callus of response (%) of callus of response (%) of callus
Glutamine
10 90.0b GCN 80.0b GCN 68.0ab GFC
25 95.0a GCN 85.0a GCN 70.0a GFC
50 89.3bc GCN 78.6bc GCN 65.3b GFC
Sodium citrate
10 78.6de GCN 77.3c GCN 58.6cd GFC
25 80.0d GCN 80.0b GCN 65.3b GFC
50 74.6fg GCN 72.0d GCN 60.0c GFC
Adenine
10 65.0ij GFC 55.0fg GFC 40.0gh GFC
25 61.3k GFC 50.6h GFC 37.3h GFC
50 58.6kl GFC 46.6j GFC 30.0i GFC
Adenine sulphate
10 75.0f GFC 60.0e GFC 55.0e GFC
25 70.0h GFC 56.0f GFC 49.3f GFC
50 66.0i GFC 49.3hi GFC 42.6g GFC
GFC – green friable callus; GCN – green compact nodular.
Values are mean of 25 replicates per treatment and repeated thrice. Values with the same superscript are not significantly different at 5%
probability level according to DMRT.

shoot elongation and reduce the basal callusing to adding acti- with an average shoot number (2.5) and nearly 55% with an
vated charcoal into the medium. The percentage of explants average shoot length (Table 3). Anuradha and Pullaiah [3]
showing axillary bud growth on the culture medium supple- used shoot tip, node and mesocotyl explants for direct shoot
ment with 10 mg/l activated charcoal can reach to over 70% regeneration, whereas present experiment leaf, internode and
100 A.R. Lavanya et al.

Table 3 Shoot regeneration response from 45 days old in vitro seedlings grown on MS medium supplemented with BA (2.0 mg/
l) + IAA (0.1 mg/l) + glutamine (25 mg/l), TDZ and charcoal after 45 days.
TDZ (mg/l) Activated Internode Petiole
charcoal (mg/l)
Percentage of Shoot number Percentage of Shoot number
shoot response (%) shoot response (%)
0.1 52.0bc 1.5c 36.0cd 1.0bc
0.5 55.0b 1.8b 40.0b 1.2b
1.0 49.3d 1.3cd 34.6d 1.0c
2.0 0.0 0.0 0.0 0.0
3.0 0.0 0.0 0.0 0.0
5 50.0c 1.2d 35.0bc 1.2b
10 70.0a 2.5a 55.0a 1.8a
25 55.0b 1.7bc 40.0b 1.0bc
Leaf – no response.
Values are mean of 25 replicates per treatment and repeated thrice. Values with the same superscript are not significantly different at 5%
probability level according to DMRT.

Table 4 Rooting response of in vitro raised shoots from 45 days old in vitro seedlings grown on MS medium supplemented with
auxins, after 45 days.
Plant growth regulators (mg/l) Internode Petiole
Rooting Root Root Rooting Root Root
response (%) number length (cm) response (%) number length (cm)
IBA
0.1 60.0fg 2.9fg 2.2f 49.3e 2.1f 1.3g
0.5 66.6d 3.1f 2.5de 52.0d 2.6d 1.6ef
1.0 72.0c 3.6de 3.0bc 56.0c 2.9bc 1.9cd
2.0 77.3ab 4.0b 3.1b 61.3b 3.0b 2.5b
3.0 80.0a 4.5a 3.5a 65.0a 3.8a 2.7a
NAA
0.1 53.3i 2.5h 2.0g 42.6g 1.5h 1.2gh
0.5 61.3f 3.0fg 2.6d 48.0ef 1.8g 1.5ef
1.0 65.0de 3.9bc 2.8d 50.0de 3.0b 2.1c
2.0 60.0fg 3.7d 2.2f 46.6f 2.5de 2.0cd
3.0 56.0h 2.8fg 1.9gh 40.0gh 2.0fg 1.7e
Values are mean of 25 replicates per treatment and repeated thrice. Values with the same superscript are not significantly different at 5%
probability level according to DMRT.

petiole explants were used for shoot regeneration through cal- roots (4.5 ± 0.1) were observed in the shoots obtained from
lus formation. Moreover glutamine, sodium citrate, adenine internode explants (Table 4; Fig. 2g and h). MS medium with
and adenine sulfate was discussed in this experiment. high concentration of IBA favoured better in vitro rooting for
internode derived calluogenesis. Likewise Pandey and Sushma
3.3. Rooting of regenerated shoots [24] obtained more roots using IBA for rooting of Quercus
leucotrichophora. While IAA showed weak roots with callusing
Leafy shoots were transferred to MS basal media containing at the base (data not shown). A similar response was also
different concentrations of IBA, NAA and IAA for rooting. observed by Shriram et al. [30] in Helicteres isora.
45 day after inoculation, root formation was noticed from
the basal cut portion of the shoot. The percentage of shoot 3.4. Acclimatization
forming roots, number of roots per shoot, and length of root
were recorded. Rooting method was followed for inducing In vitro formed plantlets were transferred to soil, sand and ver-
roots from shoots regenerated from internode and petiole miculite mix (2:1:1) and were maintained in culture room
explants in this study. Roots developed on the shoots after under plastic sheets for 25–30 days. Periodical removal of plas-
20 days in most of the cultures. However, the data for the tic sheets, so as to lower down the high atmospheric humidity
percentage of rooting and the number of roots were scored gradually, was observed as an effective way for the acclimati-
after 45 days of culturing. No significant differences were zation process as more than 85% of the plantlets survived
observed in the rooting responses of microshoots harvested through this. Plantlets were watered with 1/4 – strength MS
from internodes and petiole explants, but the maximum per- basal medium devoid of sucrose and meso-inositol at 3 days
centage of rooting (80 ± 5.0), and the maximum number of intervals for a period of 3 weeks. The acclimatization plantlets
Indirect organogenesis from various explants of Hildegardia populifolia (Roxb.) 101

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