Morphological Change Due to Effects of Acute Gamma Ray on Wishbone Flower (Torenia fourmieri) In Vitro

2011 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies.

International Transaction Journal of Engineering,
Management, & Applied Sciences & Technologies

http://www.TuEngr.com, http://go.to/Research

Morphological Change Due to Effects of Acute Gamma Ray on
Wishbone Flower (Torenia fourmieri) In Vitro
a*
Anchalee Jala
a
Department of Biotechnology, Faculty of Science and Technology, Thammasat University,
THAILAND

ARTICLEINFO A B S T RA C T
Article history: Young shoot tips were used as explants and cultured on
Received 17 June 2011 MS medium supplemented with varying concentrations of (0.1,
Received in revised form
01 August 2011
0.5, 1.0 mg/l) BA and (0.1, 0.5, 1.0 mg/l) NAA. Calli and new
Accepted 03 August 2011 shoots were grown on MS medium supplemented with
Available online combination of 0.5 mg/l BA and 0.5 mg/l NAA. New shoots
03 August 2011 averaging 0.5–0.8 cm were irradiated with varying doses of
Keywords: gamma rays (5, 10, 15, 20 grays). Gamma irradiation had
Wishbone Flower, various effects on growth of Torenia fournieri. Higher dosage of
Gamma Rays,
Acute Irradiation, gamma irradiation reduced plant height, number of roots, number
Morphology, of leaves, leaf length, leaf width, petiole length and number of
Tissue Culture guard cells at abaxial and adaxial epidermis surface. Plant
morphology and flower development was also modified.

2011 International Transaction Journal of Engineering, Management, &
Applied Sciences & Technologies. Some Rights Reserved.

1. Introduction
Wishbone flower or Torenia fournieri is a member of the Scrophulariaceae family. It is
generally a perennial plant which is normally grown as an annual shrub. With an approximate
height of 12 inches, it is preferably planted along with many other similar species to ensure a
widespread flowering bed. The plant looks like a nice little green shrub. The mature plant is
densely branched and decorated with shiny green leaves and delicate cup-shaped flowers. It is
grown as a pot plant or used for decorating and landscaping. Demand for the plant has
*Corresponding author (Anchalee Jala). Tel/Fax: +66-2-5644440-59 Ext. 2450. E-mail:
anchaleejala@yahoo.com. 2011. International Transaction Journal of Engineering,
Management, & Applied Sciences & Technologies. Volume 2 No.4. ISSN 2228-9860.
375
eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

continued to increase. Induced mutation has been reported to be an efficient technique to
achieve the desirable characters in flowers and ornamental plants (Maluszynski, 1995).

Gamma rays generally influence plant growth and development by inducing genetic,
biochemical, physiological, morphological and anatomical change in cells and tissues (
Gunckeland Sparrow, 1961). Various effects of gamma rays on ornamental plants are
observed in the different generations after mutation induction. M1 generation is
heterogeneous with different mutations for different plants. It also exhibits non-heritable
direct effects on mutagens such as sterility. Chimeric heterozygous at mutations are change of
genetic material that may be transferred from M1 to the following generations (Gaul. H.,
1977).

The objective of this study was to use an In Vitro mutation technique to improve
wishbone flower in order to select suitable colors for growing and to study morphological
change after transplanting to soil.

2. Materials and Methods
Young shoot tips of wishbone flower were used as explants materials. These explants
were sterilised with 5.25% calcium hypochlorite and cultured on MS medium (Murashige and
Skoog, 1962) containing 30 g/l sucrose, 2.5 gm/l gelrite and supplemented with varying
concentrations (0, 0.1, 0.5 mg/l) BA (Benzyl adenine) and (0, 0.1, 0.5, 1.0 mg/l) NAA
(Naphthaline acetic acid). After calli were formed and multiplying shoots developed, the
culture was irradiated with varying doses of gamma ray (0, 5, 10, 15, 20 grays). Following
irradiation, M1V1 shoots were immediately cut into small pieces, each piece had 2 nodes with
average length of 0.5-0.8 cm and subcultured into fresh medium with the same formula
(MS+0.5 mg/l BA and 0.5mg/l NAA) at 4 weeks interval from M1V1 to M1V4 . All these
were maintained at 25º ± 1ºC under 16 hr cool white, fluorescent light (1600 luxs)
(Dooley,1991). The plants were transferred to soil to observe their growth.

Data collection was undertaken of plant height, number of roots, root length, number of
leaves, leaf length, leaf width, petiole length, leaves arrangement on node. Guard cells from
abaxial and adaxial epidermis surface were examined by light microscope. Each slide was
randomly sampled to determine guard cell frequency by using images viewed under

376 Anchalee Jala

magnification of 10x40.

3. Results
After young shoot tip explants had been cultured on MS medium with six different
concentrations of BA and NAA for 8 weeks, the effective results were obtained as shown in
Table 1. After one week some explants swelled, turned green, and Calli were formed and
proliferated new shoots within 8 weeks. Samples which cultured in 0.5 mg/l BA and 0.5 mg/l
NAA (Figure 1a) were the best. This was followed by a combination of 0.1mg/l BA and 1.0
mg/l NAA. MS medium supplemented with 0.5 mg/l BA and 0.5 mg/l NAA was used for
multiplication of new shoots (Figure 1b).

Table 1: Callus induction from shoot tip explants of Torenia fournieri cultured on MS
medium with combinations of NAA and BA for 8 weeks.
MS medium Shoot formation
Number Visual Observation
NAA BA Shoot No. new No.nodes
of callus a of callus
(mg/l) (mg/l) height(cm) shoot(shoot) /plant(node)
0 0 1 swollen - - -
0.1 0.1 1.6 Small and creamy 1.2 0.9 1.2
0.5 0.1 2.1 Medium and creamy 1.17 2.2 1.4
0.5 0.5 4.1 Big and light green 1.12 3.4 2.4
1.0 0.1 2.6 Medium and light yellow 1.10 2.1 2.2
1.0 0.5 1.7 Medium and light brown 1.16 1.2 1.6
a- Callus growth was graded by an index of 1 – 5: 1 - indicating no callus formation
3- indicating medium sized, and 5 - indicating the biggest sized of callus formation

A B C

Figure 1: Effects of BA and NAA in MS medium on calli induced and shoot regenerated on
wishbone flower explants:
(A): calli induction, (B): shoot regeneration, (C) : young shoots before irradiated gamma rays

New young shoots (0.5-0.8cm) (Figure 1C) (M1V1) irradiated with gamma rays were
subcultured fourfold (M1V4) in the same medium every 4 weeks. The elongated shoots (about
377

6-8 cm) with roots were transferred for hardening. The well developed healthy plants were
transplanted to soil in greenhouse. When plantlets developed, their morphological change
was shown in each concentration of gamma rays. The effect was shown in dwarf, small or
curved leaves. There was highly significant difference (p ≤0.01) in each parameter. When
concentration of gamma rays was increased, number of leaves decreased and 5 grays was the
lowest averaging 8 leaves per plantlet (Table 2). The height of plants decreased when
concentration of gamma ray was increased. At 5 -15 grays was the lowest averaging 2.3- 2.8
cm per plant). However, the length of roots increased with increased concentration of gamma
ray. At 20 grays gave the longest root length averaging 4 cm.

Table 2: Average number of roots, leaves, plant height ,root length from M1V4 irradiated
with acute gamma ray in each concentration after being cultured for 10 weeks.
Concentration Number Plant height Number of Root length
of gamma ray of leaves** (cm)** root NS (cm)**
Control (0 gray) 19±0.26 a 6.3±0.12 a 11 2.5 ±0.21c
5 grays 8 ±0.19c 2.3±0.24 c 8 2.1±0.18 c
10grays 10 ±0.21c 2.5 ±0.18c 8 2.0 ±0.17 c
15grays 11±0.18 bc 2.8 ±0.16c 8 3.4±0.19 b
20grays 14 ±0.19b 5.0 ±0.20b 9 4.0 ±0.20a
** highly significant difference (p≤ 0.01), NS: non-significant difference
a b c- Average compared mean within column by Duncan’s multiple range test at (p≤ 0.01)

Table 3: Average number of leaf length, leaf width, petiole length, leaves arrangement
on node, from plantlet after transplanted to In Vivo condition for 8 weeks
Concentration Leaf length leaf width petiole length leaf arrangement
of gamma ray (cm) * (cm) * (cm)* on node (leaves)*
0 gray 2.19±0.16b 1.30±0.45a 1.20±0.10a 2.00±0.00a
5 grays 1.46±0.17a 1.18±0.15b 1.24±0.1a 2.17±0.77b
10grays 1.56±0.15a 1.12±0.12 b 1.02±0.12 b 2.40±0.56b
15grays 1.72±0.16a 1.04±0.12b 0.97±0.1b 2.44±0.64b
20grays 1.84±0.19a 0.78±0.11c 0.84±0.12c 2.62±0.46b
* significant difference (p≤ 0.05)
a b c- Average compared mean within column by Duncan’s multiple range test at (p≤ 0.05)

After 8 weeks, the sixth leaf from the base was measured and all parameters (leaf length ,
leaf width, petiole length, and leaves arrangement) showed significant difference ( p≤0.05).
Leaf length in plants irradiated with gamma ray was shorter than control, and the same
applied to leaf width, and petiole length (Table 3). The arrangement of leaves on node was
abnormal. In control it was opposite (2 leaves per node) but some plants irradiated with

378 Anchalee Jala

gamma ray exhibited whorl (3-4 leaves per node). High dosage of gamma ray also gave
abnormal leaf shape. Leaves in Figure 2B shown whorl arrangement, while some leaves in
Figure 2C appeared to be small and long, in heart or lanceolate shape.

2A 2B 2C

Figure 2: Effects of gamma irradiation on stem (Flat - 2A),
leaves with whorl arrangement (2B), leaves having small, long and heart-shaped (2C).

Under In Vivo condition, after 10 weeks, they were in bloom. The number of pollen sacs
in each treatment did not show any significant difference. Some plantlets had a few more
pollen sacs than control. The shape of filaments was abnormal. Some were curved, shorter or
longer than control (Figure 3). The flowers were dark purple and darker (Figure 3C) than
control. High dosages of gamma rays made wishbone flower stem flat. The number of guard
cells at abaxial and adaxial epidermis showed highly significant difference ( p≤0.01) (Table
4). Abaxial epidermis (24.6cells) and adaxial epidermis (38.2 cells) from control (not
irradiated) had the highest number of guard cells. The results showed that when gamma ray
dosage increased, the number of guard cells at abaxial and adaxial epidermis decreased. The
guard cells from 20 grays were smaller than control.

Table 4: Average number of guard cells on abaxial and adaxial epidermis surface of
wishbone flower irradiated with acute gamma ray after transplanting 8 weeks.
Concentration of Abaxial epidermis** Adaxial epidermis
gamma Ray **
Control (0 gray) 24.6±0.60a 38.2±3.41a
5 grays 18.4±0.74b 35.8±2.51
10grays 16.4±0.64b 34.9±2.46b
15grays 15.8±1.28c 34.6±2.31b
20grays 13.8±0.80c 33.6±2.39b
** highly significant difference (p≤ 0.01)
a b c- Average compared with the mean within column by Duncan’s multiple range test at P≤0.05
379

3A 3B 3C

Figure 3: Effect of gamma irradiation on flower: color was darker than control and position
of pollen sac were abnormal
(A): filament in control was erect,
(B): filaments with high dosage of gamma rays was curve, or
(C): shorter than control

4. Discussion
Callus induction from young shoot tip explants and regeneration for shoot multiplication
in T. fournieri preferred BA and NAA at 0.5 mg/l. This may suggest that bud formation
required cytokinin and auxin. A conjunction of BA and NAA evoked a better response in
shoot multiplication than NAA alone and this is probably due to the difference in endogenous
levels of growth regulators in this plant or to a difference in sensitivity (Trewavas and
Cleland, 1983). Such a synergistic effect of NAA and BA is in concurrence with the results
in other ornamental plants such as Tagetes (Belarmino, 1992), Lilium (Liu, 1986) and
Dianthus (Jethwani, 1993). Shoot tip could induce new shoots which is in concurrence with
the report of Dianthus chinensis (Kantia and Kothari, 2002). NAA and BA in several
combinations resulting in callusing and shoot multiplication on callus suggest that the normal
endogenous growth substance levels are conductive to bud formation. A similar result was
observed in plant regeneration of Dianthus barbatus through organogenesis in callus induced
from leaf explants (Pareek and Pareek, 2005). Stem segment of T. fourmieri could
regenerated adventitious bud (Ishioka and Tanimoto, 1992; Tanimoto and Harada, 1986 and
1990; and Kobayashi et al, 1995). However, acute gamma ray affected the tissue of
wishbone plantlets . When plants grew up, sizes of leaves, stem, and root were recorded. The
number of leaves increased, similar to those observed by Chutinthorn (1979) which reported
this in the study of many ornamental plants. When concentration of gamma ray increased,
plant growth decreased and abnormal characters occurred. Some died as a result of high

380 Anchalee Jala

dosage of gamma ray. This result were the same as Jala (2005) in the study of petunia found
that growth rate and rate of survival decreased when the plant was exposed to high dosage of
gamma ray. Plant height increased in response to an increase of dosage of gamma rays. This
was the same as for Curcuma alismatifolia (Thohirah et al., 2009).

5. Conclusion
Young shoot tips of Wishbone flower were used as explants and cultured on MS medium
supplemented with varying concentrations of (0.1, 0.5, 1.0 mg/l) BA and (0.1, 0.5, 1.0 mg/l)
NAA. Calli were formed and proliferated new shoots with in 8 weeks in 0.5 mg/l BA and 0.5
mg/l NAA. New shoots averaging 0.5–0.8 cm were irradiated with varying doses of gamma
rays (5, 10, 15, 20 grays) and subcultured fourfold (M1V4) in the same medium every 4
weeks. The elongated shoots (about 6-8 cm) with roots were transferred for hardening. The
well developed healthy plants were transplanted to soil in greenhouse. Gamma irradiation
exerted various effects on growth of Torenia fournieri. When gamma ray dosage increased,
plant height, number of roots, number of leaves, leaf length, leaf width, petiole length and
number of guard cells at abaxial and adaxial epidermis decreased. Sizes of guard cells from
20 grays were smaller than control.

6. Acknowledgement
A very special thank you is due to Professor Dr. Thana Na-Nakara for insightful
comments, helping clarify and improve the manuscript.

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Dr.Anchalee Jala is an Associate Professor in Department of Biotechnology, Faculty of Science and
Technology, Thammasat University, Rangsit Campus, Pathumtani , Thailand. Her teaching is in the areas of
botany and plant tissue culture. She is also very active in plant tissue culture research.

Peer Review: This article has been internationally peer-reviewed and accepted for publication
according to the guidelines given at the journal’s website.

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Morphological Change Due to Effects of Acute Gamma Ray on Wishbone Flower (Torenia fourmieri) In Vitro

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