morphological change due to effects of acute gamma ray on wishbone flower (torenia fourmieri) in...

*Corresponding author (Anchalee Jala). Tel/Fax: +66-2-5644440-59 Ext. 2450. E-mail: [email protected]. 2011. International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. Volume 2 No.4. ISSN 2228-9860. eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

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

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Morphological Change Due to Effects of Acute Gamma Ray on Wishbone Flower (Torenia fourmieri) In Vitro Anchalee Jalaa* a Department of Biotechnology, Faculty of Science and Technology, Thammasat University, THAILAND A R T I C L E I N F O

A B S T RA C T

Article history: Received 17 June 2011 Received in revised form 01 August 2011 Accepted 03 August 2011 Available online 03 August 2011 Keywords: Wishbone Flower, Gamma Rays, Acute Irradiation, Morphology, Tissue Culture

Young shoot tips 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 and new shoots were grown on MS medium supplemented with combination of 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). Gamma irradiation had various effects on growth of Torenia fournieri. Higher dosage of gamma irradiation reduced plant height, number of roots, number of leaves, leaf length, leaf width, petiole length and number of 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

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

376 Anchalee Jala

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


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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 Number

of callus a Visual Observation

of callus

Shoot formation NAA (mg/l)

BA (mg/l)

Shoot height(cm)

No. new shoot(shoot)

No.nodes /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

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

A B C

378 Anchalee Jala

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 of gamma ray

Number of leaves**

Plant height (cm)**

Number of root NS

Root length (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 of gamma ray

Leaf length (cm) *

leaf width (cm) *

petiole length (cm)*

leaf arrangement 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


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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.

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 gamma Ray

Abaxial epidermis** Adaxial epidermis **

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

2A 2B 2C

380 Anchalee Jala

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

3B3A 3C


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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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Peer Review: This article has been internationally peer-reviewed and accepted for publication

according to the guidelines given at the journal’s website.

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.

morphological change due to effects of acute gamma ray on wishbone flower (torenia fourmieri) in...

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