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

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 Jala
a

a*

Department of Biotechnology, Faculty of Science and Technology, Thammasat University, THAILAND

ARTICLEINFO 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 A B S T RA C T

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 0.50.8 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
*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. eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

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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. MaterialsandMethods
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 25 1C 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
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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 NAA BA (mg/l) (mg/l) 0 0 0.1 0.1 0.5 0.1 0.5 0.5 1.0 0.1 1.0 0.5 Number of callus a 1 1.6 2.1 4.1 2.6 1.7 Visual Observation of callus swollen Small and creamy Medium and creamy Big and light green Medium and light yellow Medium and light brown Shoot height(cm) 1.2 1.17 1.12 1.10 1.16 Shoot formation No. new No.nodes shoot(shoot) /plant(node) 0.9 1.2 2.2 1.4 3.4 2.4 2.1 2.2 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
*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. eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

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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) 190.26 a 6.30.12 a 11 2.5 0.21c 5 grays 8 0.19c 2.30.24 c 8 2.10.18 c 10grays 10 0.21c 2.5 0.18c 8 2.0 0.17 c 15grays 110.18 bc 2.8 0.16c 8 3.40.19 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 Duncans 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 of gamma ray (cm) * (cm) * 0 gray 2.190.16b 1.300.45a 5 grays 1.460.17a 1.180.15b 10grays 1.560.15a 1.120.12 b 15grays 1.720.16a 1.040.12b 20grays 1.840.19a 0.780.11c * significant difference (p 0.05) petiole length (cm)* 1.200.10a 1.240.1a 1.020.12 b 0.970.1b 0.840.12c leaf arrangement on node (leaves)* 2.000.00a 2.170.77b 2.400.56b 2.440.64b 2.620.46b

a b c- Average compared mean within column by Duncans 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 ( p0.05). 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
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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.

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 ( p0.01) (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 Control (0 gray) 5 grays 10grays 15grays 20grays Abaxial epidermis** 24.60.60a 18.40.74b 16.40.64b 15.81.28c 13.80.80c Adaxial epidermis ** 38.23.41a 35.82.51 34.92.46b 34.62.31b 33.62.39b

** highly significant difference (p 0.01) a b c- Average compared with the mean within column by Duncans multiple range test at P0.05
*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. eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

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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). 1990; and Kobayashi et al, 1995). Stem segment of T. fourmieri could tissue of regenerated adventitious bud (Ishioka and Tanimoto, 1992; Tanimoto and Harada, 1986 and However, acute gamma ray affected the 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
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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 0.50.8 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.

7. Reference
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*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. eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

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Tanimoto, S., Harada, H. (1986). Involvement of calcium in adventitious bud initiation in Torenia stem segments. Plant and Cell Physiology 27, 1-10. Tanimoto, S., Harada, H. (1990). Wishbone flower. Chapter 31 In: Handbook of Plant Cell Culture, Vol.5. McGraw-Hill, New York, pp 763-782. Thohirah Lee Abullah, Johari Endan and Mohd Nazir. (2009). Change in Flower Development, Chlorophyll Mutation and Alteration in Plant Morphology of Curcuma alismatifolia by Gamma Irradiation. Amer. J. Applied Sci. 6(7):1436-1439. Trewavas, A.J. and Cleland, R.E. (1983). Is plant development regulated by changes in concentration of substances or by changes in the sensitivity? Trends in Biochemical Science 8, 354-357. Vanegas, P.E., Cruz-Hernandez, A., Valverde, M. E. and Paredes-Lopez, O. (2002). Plant regeneration via organogenesis in marigold. Plant Cell, Tissue and Organ Culture. 69, 279-283. Yamazaki, T. (1985). A Revision of the Genera Limnophila and Torenia from Indochina. Journal of Faculty of Science University of Tokyo III 13, 575-624

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 journals website.

*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. eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf

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