8 ff3 fs2 fc0 sc0 ls2 ws21">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
Copyright © 2012 SciRes. AJPS
In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper
Copyright © 2012 SciRes. AJPS
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)
Copyright © 2012 SciRes. AJPS
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)
Copyright © 2012 SciRes. AJPS
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.
Copyright © 2012 SciRes. AJPS
In Vitro Organogenesis of Quisqualis indica Linn.—An Ornamental Creeper
Copyright © 2012 SciRes. AJPS
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
Copyright © 2012 SciRes. AJPS
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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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: *jaydipmandal07@yahoo.com
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.
Copyright © 2012 SciRes. AJPS
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.
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