<i>In Vitro</i> Organogenesis of <i>Quisqualis indica </i>Linn.

doi:10.4236/ajps.2012.39154

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American Journal of Plant Sciences, 2012, 3, 1272-1282

http://dx.doi.org/10.4236/ajps.2012.39154 Published Online September 2012 (http://www.SciRP.org/journal/ajps)

In Vitro Organogenesis of Quisqualis indica Linn.

—An Ornamental Creeper

Jaydip Mandal1*, Undurthy Laxminarayana2

1Department of Education in Science and Mathematics, Regional Institute of Education, National Council of Educational Research

and Training, Bhopal, India; 2Department of Education, Regional Institute of Education, National Council of Educational Research

and Training, Mysore, India.

Email: *[email protected]

Received July 7th, 2012; revised August 3rd, 2012; accepted August 13th, 2012

ABSTRACT

Shoot organogenesis and plant regen eration were achiev ed on callu s derived from leaf section and stem base explants of

Quisqualis indica (Combretaceae). In vitro cultures were established using nodal segments obtained from mature

field-grown shrubby plants. For the development of optimized protocol, different types and concentrations of plant

growth regulators were used to induce adventitious shoot regeneration via callus from leaf section and one-node stem

base explants obtained from in vitro regenerated micro shoots and direct field-grown newly flush-off shoots. The TDZ

was considered to be the best among the cytokinins (6-benzyladenine (BA), 6-(



, dimethylallyamino purine) (2-iP)

and thidiazuron (TDZ) added to the Murashige and Skoog’s medium (MS) for adventitious shoot productions. A com-

bination of 1.0 mg/L TDZ and 0.5 mg/L GA3 was most effective in stimulating callus induction and ad ventitious shoot

regeneration from the leaf section derived calli with an average of 6 shoots per callus exp lant and an av erag e of 8 shoots

per callus explant originated from one-node stem base explants. In vitro raised shoots were sub- cu ltu red on MS medium

supplemented with 1.0 mg/L BA and 0.5 mg/L GA3 for further shoot growth. Maximum rooting of in vitro regenerated

shoots was obtained on MS medium supplemented with either 0.5 mg/L indole-3-acetic acid (IAA) or indole-3-butyric

acid (IBA) individually or a combination of 0.5 mg/L IAA and 0.5 mg/L IBA. Plantlets raised in vitro were acclima-

tized and subsequently transferred to experimental field.

Keywords: Organogenesis; Quisqualis indica; Shoot Regeneration; Tissue Culture

1. Introduction

Rangoon Creeper scientifically known as Quisqualis in-

dica originated from South East Asia and occurs all over

Africa, Philippines, Vietnam, Malaysia, India, Bangla-

desh and Thailand. It has bright colored fragrant flowers

and is one of the most stunning ornamental of the family

Combretaceae. Quisqualis indica is grown as an orna-

mental garden plant for it’s horizontally orientation to

pendulous white, pink and red flowers that give out dis-

tinct perfume. The flowers con tain high quantity of poly-

phenol that are believed to be strong antioxidants benefi-

cial for human health [1-3]. This species is known to

have free radical scavenging activity and alleviating

flatulent distension of abdomen like that of the medicinal

properties of Terminalia chebula, T. belerica and Em-

blica officinalis [4-6]. The plant parts such as roots,

flowers and seeds of the plant are used for curing diar-

rhea, fever, rickets, rheumatism and nephritis [2,7]. The

leaves, fruits and seeds of the plant have been used as

anthelmintic for expelling round worms and thread worms

[8-10].

This ornamental shrub is conventionally propagated

through seeds and cuttings. However, according to Lam-

bardi and Rugini [11] propagation through seeds renders

undesirable variation whereas shoot cuttings of many

genotypes do not respond to root inducing medium.

These difficulties may be overcome using in vitro

tissue culture techniques. Plant tissue culture techniques

are considered as easy and reliable for rapid up-scaling of

shoot regeneration of elite g enotypes independent of sea-

sonal and environmental influences. In addition, produc-

tion of transgenic plants relies on successful establish-

ment of in vitro regeneration methods based on organo-

genesis and thus can complement conventional breeding

technique.

Regeneration protocols have been reported for micro-

propagation of many species such as Cinnamomum cam-

phora ([12], Terminalia chebula [13], Saussurea obva-

llata [14], Terminalia bellirica [15], Terminalia arjuna

[16], Aloe polyphyla [17], Vaccinium species [18], Pha-

*Corresponding author.

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper 1273

seolus vulgaris [19], Punica granatum [20], Trifolium

alexandrinum [21] from axillary meristems and shoot

tips as well as shoot organogenesis fr om excised leaf ex-

plants, cotyledon and embryo axis with different plant

growth regul at ors.

In vitro systems may offer the tools for rapid multipli-

cation and conserv ation of genetic stocks and availability

of wild Quisqualis germplasm for its medicinal and or-

namental use besides its potential use in genetic engi-

neering and plant breeding. However, there has been no

report on the shoot organogenesis of Quisqualis indica.

The aim of the present study was to establish an efficient

regeneration protocol through callus mediated organo-

genesis of leaf and stem base explants of Quisqualis in-

dica.

2. Materials and Methods

2.1. Culture Initiation

Nodal segments from newly flush off shoots of adult

climber plants of Quisqualis indica were collected in

December 2009, February 2010 and March 2011 and

thoroughly washed under running tap water for 10 min

and surface sterilized with 0.1% HgCl2 for 5 min under

aseptic condition in Laminar air flow. After five wash-

ings in sterile double distilled water for 10 min, the cut

ends of the nodal segments were trimmed and cultured

on MS [22] Murashige and Skoog, 1962) medium sup-

plemented with the 1.0 mg/L benzyladenine (BA), 0.5

mg/L gibberellic acid (GA3), 3% sucrose and gelled with

0.8% agar in 20  150 mm glass tubes and 100 ml coni-

cal flasks. The pH was adjusted to 5.8 with 1 M HCl or 1

M NaOH and autoclaved at 121˚C for 20 min. Cultures

were incubated in a culture room maintained at 29˚C 

2˚C under a 16/8 h photosynthetic photon density of 40

µmol·m–2·s–1 provided by cool white fluorescent tubes

(40 W; Philips, India). Shoots of in vitro plants were cut

into nodal segments and subcultured on MS medium

supplemented with 1.0 mg/L 6-benzyladenine (BA) and

0.5 mg/L gibberellic acid (GA3). Every three weeks,

newly formed shoots were subjected to the same proce-

dure of cutting nodal segments and subculture on the

fresh medium.

2.2. Callus Induction

Field-grown matured plants as well as three-week old in

vitro regenerated shoots were the sources of leaf section

and one-node stem base explants. Explants from field

grown plants were surface sterilized following the pro-

cedures used above for cu lture initiation. The petiole was

removed and two cuts were made on each leaf. Stem base

with one-node was used as another explant. The surface

sterilized leaf section explants were inoculated with ad-

axial or abaxial side in contact with the medium. The

stem base with one-node was inoculated with lower base

inserted into the medium. The MS medium was gelled

with 0.8% agar and differ ent types and concentrations of

plant growth regulators such as cytokinins, 6-benzylade-

nine (BA) or 6-(



, dimethylallylamino purine) (2-iP) or

thidiazuron (TDZ) at the concentrations of 0.5 mg/L or

1.0 mg/L either individually or in combination with ei-

ther 0.25 mg/L naphthaleneacetic acid (NAA) or 0.5

mg/L gibberellic acid (GA3). Preliminary trials suggested

that there were no marked differences in the calli induc-

tion potentials between the explants of field-grown and

in vitro shoots, subsequent experiments based on ex-

plants of field-grown plants were considered. Every three

weeks the developed calli were subdivided and subcul-

tured on the same medium.

2.3. Shoot Regeneration and Proliferation

Isolated calli (0.5 - 1.0 cm2) from leaf section and basal

portion of one-node stem base explants were placed on

MS medium supplemented with 0.5 mg/L or 1.0 mg/L

BA or TDZ either individually or in combination with

0.25 mg/L α-naphthaleneacetic acid (NAA) or 0.5 mg/L

gibberellic acid (GA3) for regeneration of shoots for six

weeks. Shoots of 5 - 8 mm in length were separated from

the calli explants and subcultured on MS supplemented

with 1.0 mg/L BA and 0.5 mg/L gibberellic acid (GA3)

for elongation and multiple shoot regeneration for four

weeks. A total of five explants per conical flasks were

used and each treatment was replicated four times. The

experiment was repeated three times. The explants were

incubated for six weeks to the same photoperiod, light

intensity and temperature used for culture initiation. Data

on percentage callus induction, percentage of organo-

genic calli, and number of shoots per callus explant and

shoot length was recorded after four weeks of incubation

and presented in Figures 1-4.

2.4. In Vitro Rooting of Shoots and Hardening

Six-week old in vitro shoots (2 - 3 cm) developed from

calli were cultured on MS medium supplemented with

0.5 mg/L or 1.0 mg/L IAA or IBA either individually or

in combination for rooting for a period of four weeks

following a procedure for rooting of micropropagated

shoots of Quisqualis indica (unpublished, data not

shown). After this incubation period, in vitro raised

plantlets with healthy root systems and without roots

were thoroughly washed under running tap water and

planted in plastic cups containing a 1:1 (v/v) mixture of

sterilized vermiculite and garden soil moistened with 1/2

MS strength. These cups were wrapped with polyethy-

lene bags ensuring high humidity and kept under the

same growth conditions as for culture initiation for fur-

ther development. Thereafter, the plants were nurtured

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper

1274

Figure 1. Mean % of callus induction of leaf section explants of Quisqualis indica on MS medium supplemented with different

combination and concentrations of plant growth regulators. Different letter(s) indicate a significant difference between

treatments at P ≤ 0.05 according to Tukey test.

Figure 2. Mean % of callus induction of stem base explants of Quisqualis indica on MS medium supplemented with different

combination and concentrations of plant growth regulators. Different letter(s) indicate a significant difference between

treatments at P ≤ 0.05 according to Tukey test.

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper 1275

(a)

(b)

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper

1276

(c)

Figure 3. (a)-(c) Organogenic responses of stem base explants of Quisqualis indica on MS medium supplemented with differ-

ent combination and concentrations of plant growth regulators after 30 d. (a) % Organogenic calli regenerating shoots; (b)

Mean number of shoots per callus; (c) Mean shoot length (cm). Different letter(s) indicate a significant difference between

treatments at P ≤ 0.05 according to Tukey test.

(a)

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper 1277

(b)

(c)

Figure 4. (a)-(c) Organogenic responses of leaf section explants of Quisqualis indica on MS medium supplemented with dif-

ferent combination and concentrations of plant growth regulators. After 30 d. (a) % Organogenic calli regenerating shoots; (b)

Mean number of shoots per callus; (c) Mean shoot length (cm). Different letter(s) indicate a significant difference between

treatments at P ≤ 0.05 according to Tukey test.

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper

1278

under a shade (50% light cut off) for a period of two

weeks before transfer to the experimental field

2.5. Data Analysis

All the experiments were carried out in a completely

randomized design and repeated thrice. Means and stan-

dard error of means for all the dependent variables such

as, callus induction shoot regener ation shoot number and

shoot length under different plant growth regulator con-

centrations were computed and found out the significant

differences between means using Tukey test.

3. Results

3.1. Culture Initiation

Shoot cultures of Quisqualis indica were initiated suc-

cessfully from nodal segments with 100% bud break.

Preliminary experiments suggested the inclusion of BA

and GA3 in the MS medium for bud break an d shoot pro-

liferation. The high est bud brea k (100%) w ith an av erage

of 20 shoots per node explant was observed on the MS

medium supplemented with 1.0 mg/L BA and 0.5 mg/L

GA3 within two weeks of inocul at i on. I n contrast , 75% o f

the explants developed multiple shoots with an average

of 2 - 3 shoots per node explant on MS medium supple-

mented with 1.0 mg/L TDZ or 2-iP in combination with

0.5 mg/L GA3 (data not shown). The MS medium sup-

plemented with 1.0 mg/L BA and 0.5 mg/L GA3 was

chosen for further up-scaling of in vitro shoot regenera-

tion because of its increased shoot formation potential.

Figure 5. (a)-(h) In vitro plant regeneration of Quisqualis

indica. (a) Callus formation from leaf section explants after

30 d of culture on MS medium supplemented with 1.0

mg·l–1 TDZ 2 iP and 0.5 mg·l–1 GA3. (b) Shoot regeneration

from leaf section callus after 21 d culture on MS medium

supplemented with 1.0 mg·l–1 TDZ and 0.5 mg·l–1 GA3; (c)

Shoot elongation after 30 d following subculture on the

same medium; (d) Induction and proliferation of multiple

shoots from one-node stem base derived callus culture after

30 d culture on MS medium supplemented with 1.0 mg·l–1

TDZ and 0.5 mg·l–1 GA3. (e) Elongation of regenerated

shoots after 30 d of culture on MS medium supplemented

with 1.0 mg·l–1 BA and 0.5 mg·l–1 GA3; (f) In vitro shoot

produced healthy root after 30 d of culture on MS medium

supplemented with 0.5 mg·l–1 IAA and 0.5 mg·l–1 IBA; (g)

Rooting of regenerated shoot after 45 d of culture on MS

medium supplemented with 0.5 mg·l–1 IAA; (h) An estab-

lished plant of Quisqualis indica after 30 d of transfer to

garden soil. Bars = 0.7 cm (a); 2 cm (b); 2 cm (c); 1 cm (d, e,

f, g), and 1 cm (h).

3.2. Callus Induction

Leaf section and one-node stem base explants were in-

cubated on different media to promote the induction of

calli (Figures 1 and 2). Callus was induced from both the

explants: the leaf section explants produced the higher

frequency of callusing than the one-node stem base ex-

plants. Initiation of callus was observed within seven

days from cut ends of leaf section and basal cut end re-

gion of one-node stem base explants on MS medium

supplemented with plant growth regulators. Leaf section

explants showed the highest percentage of callus induc-

tion (100  0) on MS medium supplemented with 0.5

mg/L 2-iP and 0.5 mg/L GA3 or 1.0 mg/L TDZ in com-

bination with 0.25 mg/L NAA or 0.5 mg/L GA3 after

four weeks (Figure 1). In contrast, the highest callus

induction frequency (86.6  2.3) (significantly different,

P < 0.05) from basal cut end of one-node stem base ex-

plants was observed on MS medium supplemented with

1.0 mg/L TDZ and 0.5 mg/L GA3 after four weeks (Fig-

ure 2). The calli were compact and green in color (Fig-

ures 5(a), (b) and (d)). Thus the results of the investiga-

tion showed higher fr equency of callus inductio n through

leaf section explants than the one-node stem base ex-

plants (Figures 1 and 2).

3.3. Shoot Regeneration and Proliferation

In the present investigation, the highest frequency of or-

ganogenic calli (91.1  2.5) (significant differences, P <

0.05) was obtained from leaf section explants on MS

medium supplemented with 1.0 mg/L TDZ and 0.5 mg/L

GA3 inducing an average of 5.4 shoots per callus explant

with an average shoot length of 3.19 cm (Figures 4(a),

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper 1279

(b), (c), 5(b) and (c)), whereas the organogenic calli

(91.8  2.3) (significant differences, P < 0.05) derived

from one-node stem base explant induced an average

number of 7.3 shoots per callus explant with an average

shoot length of 2.18 cm on this medium (Figures 3(a),

(b), (c) and 5(d)).

The total number of regenerants increased as the con-

centrations of TDZ increased in combination with 0.5

mg/L GA3 or 0.25 mg/L NAA (Figures 3(b) and 4(b)).

In the present investigation, 71.6% of the organogenic

calli derived through leaf section explants on MS me-

dium supplemented with 1.0 mg/L TDZ and 0.25 mg/L

NAA produced an average of 2.1 shoots per callus ex-

plant averaging a length of 11.7 cm (Figures 4(b) and

(c)). The regenerated shoots were excised as node ex-

plants and subcultured on the MS medium supplemented

with 1.0 mg/L BA and 0.5 mg/L GA3 for further shoot

proliferation and elongation growth (Figure 5(e)).

3.4. In Vitro Rooting of Shoots and Hardening

In vitro shoots of Quisqualis indica regenerated via shoot

organogenesis rooted spontaneously on MS basal me-

dium. Besides, the MS medium supplemented with dif-

ferent concentrations of IAA or IBA either individually

or in combination induced root initiation (Figures 5(f)

and (g)). IAA at a concentration of 1.0 mg/L showed

decrease in root production while IBA at this level was

inhibitory to root induction. The inhibitory action was

possibly due to the less quick metabolism of IBA than

that of IAA [23]. The action of IAA and IBA at a lower

concentration of 0.5 mg/L was found to increase the

number of roots per explant. In vitro raised plantlet was

acclimatized and maintained in experimental field where

90.0 % of plantlets survived (Figure 5(h)).

4. Discussion

In the present investigation, the MS medium supple-

mented with 1.0 mg/L BA and 0.5 mg/L GA3 was chosen

for further up-scaling of in vitro shoot regeneration be-

cause of its increased (20 shoots per node) shoot forma-

tion potential. This result was similar to the report of

Tzitzikas et al. [24] who achieved regeneration of 22

shoots per node explant of Pisum sativum on MS3 me-

dium supplemented with 1.0 mg/L GA3 and 1.0 mg/L

BAP. In contrast, in the present investigation, the less

percentage and poor shoot regeneration (2 - 3 shoots) on

MS medium containing 1.0 mg/L 2-iP and 0.5 mg/L GA3

was possibly due to phytotoxicity of cytokinin (2-iP).

The similar results of less percentage of shoot regenera-

tion and stunted shoot growth through nodal segments of

Vaccinium species [18] were obtained on woody and

plant medium [25] supplemented with 25 µM 2-iP.

The source of explants is an important factor in deter-

mining the ability to induce callus. This suggests th at the

endogenous hormone levels as well as hormone respon-

sivity vary among the different organs. Thus the results

of the investigation showed higher frequency of callus

induction through leaf sectio n explants than the one-node

stem base explants. Similar to these results, the addition

of TDZ to MS medium resulted to successful calli induc-

tion from leaf explants of Echinaceae purpurea [26].

Stimulated callus growth was observed from epicotyl

explants of Phaseolus lunatus [27] on MS containing 0.5

mg/L TDZ and 0.05 mg/L IAA. The best calli induction

response from root segments of Dorem ammoniacum was

reported by Irvani et al. [28] on MS medium supple-

mented with 2.0 mg/L BA and 1.0 mg/L NAA whereas

cotyledon explants of Punica granatum [20] showed the

highest frequency of callus induction on MS medium

supplemented with 21 µM NAA and 9 µM BA.

On the other hand, similar to present investigation,

calli formation from cut ends of slender stem of Old-

enlandia umbellata has been reported by Siva et al. [29].

The successful induction (85%) of callus like tissue from

base of node stem explants of Pisum sativum was re-

ported by Tzitzikas et al. [23] on MS3 medium supple-

mented with 2.2 mg/L TDZ. Physiological gradient in

explants hold s key to th e freque ncy of callu s indu ction as

observed in different species including Fagus sylvatica

[30].

The balance between cytokinins and auxins holds key

to the differentiation of organogenic calli into shoot bud

induction and plantlet development during shoot or-

ganogenesis [31,32]. In the present investigation, it was

observed that the total number of regenerants increased

as the concentrations of TDZ increased in combination

with 0.5 mg/L GA3 or 0.25 mg/L NAA. This was in line

with the observations on differentiation of organogenic

callus into plantlets in Hovenia dulcis [33] on MS me-

dium supplemented with 0.23 µM gibberellic acid and

0.46µM kinetin. Similarly maximum number of shoots

was achieved from leaf section callus of Hyptis suave-

olens [34] on MS medium containing 0.5 mg/L BA and

0.5 mg/L GA3. In the present investigation, 71.6% of th e

organogenic calli derived through leaf section explants

on MS medium supplemented with 1.0 mg/L TDZ and

0.25 mg/L NAA produced an average of 2.1 shoots per

callus explant which was consistent with the previous

reports such as, Saussurea obvallata [14] inducing 12

shoots per callus explant on MS medium supplemented

with 5.0 µM BA and 1.0 µM NAA, Astragalus cariensis

[35] producing 23 shoots per callus explant on MS sup-

plemented with 4.0 mg/L BA and 0.5 mg/L NAA, Kos-

teletzkya pentacarpos [36] showing high shoot induction

on medium containing 1.0 mg/L kinetin and 2.0 mg/L

In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper

1280

IAA. Similarly Tan et al. [37] could induce plantlet re-

generation in Vanilla planifolia from leaf and node de-

rived callus on MS medium supplemented with 1.0 mg/L

BA and 0.5 mg/L NAA. There were reports of the best

shoot regeneration response from leaf explants of Brun-

felsia calycina [38] on MS medium supplemented with

2.85 µM IAA and 4.44 µM BA or 4.54 µM TDZ and

hypocotyl derived organogenic calli of Dorem ammo-

niacum [28] on MS medium with 2.0 mg/L BA and 0.2

mg/L IBA. Likewise, the MS medium supplemented with

cytokinins and auxins (TDZ and NAA) induced adventi-

tious shoot regeneration in many species such as, Prunus

serotina [39], Aechmea fasciata [40] and Echinacea pur-

purea [26]. Ghimire et al. [41] obtained highest mean

number (10.65) of shoots per explant from in vitro leaves

of Drymaria cordata on medium containing 1.0 mg/L

BA and 0.1 mg/L NAA. Similar to the present results of

indirect shoot organogenesis, successful callus initiation

and proliferation and adventitious shoot production

through nodal explants of silver maple and Acer species

were achieved by Preece et al. [42] and Marks and Sim-

pson [43] respectively. This was ascribed to the auxin

accumulation at the excision sites by downward move-

ment which in turn causes cell proliferation especially in

the presence of cytokinins [43].

The Rooting efficiency of IAA or IBA has been docu-

mented for many medicinal and ornamental plants [16,

20]. The inhibitory action of IBA at 1.0 mg/L compared

to IAA was possibly due to the less quick metabolism of

IBA than that of IAA [23]. The action of IAA and IBA at

a lower concentration was found to increase the number

of roots per explant. This result was consistent with the

previous report on ro ot ing of Ailanthus triphysa [44].

5. Conclusion

In the present investigation, the TDZ treatment was su-

perior to that of 2-iP and BA in combination with either

GA3 or NAA in terms of inducing the formation of mul-

tiple shoots from organogenic callus derived from leaf

section and one-node stem base explants of this orna-

mental shrubby climber Quisqualis indica. This was at-

tributed to the special property of TDZ and NAA to ful-

fill the capability of the ratios of cytokinin and auxin

responses in plant species for achieving adventitious

shoot production from callus tissues. The development of

an efficient plant tissue culture system for the micro-

propagation of Quisqualis indica through organogenic

callus derived from leaf section and one-node stem base

explants holds promise to facilitate conservation and the

commercial production of this plant for its medicinal and

ornamental use, besides its potential use in plant breeding

and the production of transgenic plants through genetic

engineering.

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