Morphological Change Due to Effects of Acute Gamma Ray on Wishbone Flower (Torenia fourmieri) In Vitro
Upcoming SlideShare
Loading in...5
×
 

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

on

  • 879 views

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 ...

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.

Statistics

Views

Total Views
879
Views on SlideShare
879
Embed Views
0

Actions

Likes
0
Downloads
11
Comments
0

0 Embeds 0

No embeds

Accessibility

Categories

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

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

  • 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/ResearchMorphological Change Due to Effects of Acute Gamma Ray onWishbone Flower (Torenia fourmieri) In Vitro a*Anchalee Jalaa Department of Biotechnology, Faculty of Science and Technology, Thammasat University,THAILANDARTICLEINFO A B S T RA C TArticle history: Young shoot tips were used as explants and cultured onReceived 17 June 2011 MS medium supplemented with varying concentrations of (0.1,Received in revised form01 August 2011 0.5, 1.0 mg/l) BA and (0.1, 0.5, 1.0 mg/l) NAA. Calli and newAccepted 03 August 2011 shoots were grown on MS medium supplemented withAvailable online combination of 0.5 mg/l BA and 0.5 mg/l NAA. New shoots03 August 2011 averaging 0.5–0.8 cm were irradiated with varying doses ofKeywords: gamma rays (5, 10, 15, 20 grays). Gamma irradiation hadWishbone Flower, various effects on growth of Torenia fournieri. Higher dosage ofGamma Rays,Acute Irradiation, gamma irradiation reduced plant height, number of roots, numberMorphology, of leaves, leaf length, leaf width, petiole length and number ofTissue 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 isgenerally a perennial plant which is normally grown as an annual shrub. With an approximateheight of 12 inches, it is preferably planted along with many other similar species to ensure awidespread flowering bed. The plant looks like a nice little green shrub. The mature plant isdensely branched and decorated with shiny green leaves and delicate cup-shaped flowers. It isgrown 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. 375eISSN 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 toachieve 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 areobserved in the different generations after mutation induction. M1 generation isheterogeneous with different mutations for different plants. It also exhibits non-heritabledirect effects on mutagens such as sterility. Chimeric heterozygous at mutations are change ofgenetic 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 improvewishbone flower in order to select suitable colors for growing and to study morphologicalchange after transplanting to soil.2. Materials and Methods  Young shoot tips of wishbone flower were used as explants materials. These explantswere sterilised with 5.25% calcium hypochlorite and cultured on MS medium (Murashige andSkoog, 1962) containing 30 g/l sucrose, 2.5 gm/l gelrite and supplemented with varyingconcentrations (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, theculture was irradiated with varying doses of gamma ray (0, 5, 10, 15, 20 grays). Followingirradiation, M1V1 shoots were immediately cut into small pieces, each piece had 2 nodes withaverage 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 thesewere 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 ofleaves, leaf length, leaf width, petiole length, leaves arrangement on node. Guard cells fromabaxial and adaxial epidermis surface were examined by light microscope. Each slide wasrandomly 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 differentconcentrations of BA and NAA for 8 weeks, the effective results were obtained as shown inTable 1. After one week some explants swelled, turned green, and Calli were formed andproliferated new shoots within 8 weeks. Samples which cultured in 0.5 mg/l BA and 0.5 mg/lNAA (Figure 1a) were the best. This was followed by a combination of 0.1mg/l BA and 1.0mg/l NAA. MS medium supplemented with 0.5 mg/l BA and 0.5 mg/l NAA was used formultiplication 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 weresubcultured 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. 377eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf
  • 6-8 cm) with roots were transferred for hardening. The well developed healthy plants weretransplanted to soil in greenhouse. When plantlets developed, their morphological changewas shown in each concentration of gamma rays. The effect was shown in dwarf, small orcurved leaves. There was highly significant difference (p ≤0.01) in each parameter. Whenconcentration of gamma rays was increased, number of leaves decreased and 5 grays was thelowest averaging 8 leaves per plantlet (Table 2). The height of plants decreased whenconcentration of gamma ray was increased. At 5 -15 grays was the lowest averaging 2.3- 2.8cm per plant). However, the length of roots increased with increased concentration of gammaray. 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 sameapplied to leaf width, and petiole length (Table 3). The arrangement of leaves on node wasabnormal. 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 gaveabnormal leaf shape. Leaves in Figure 2B shown whorl arrangement, while some leaves inFigure 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 sacsin each treatment did not show any significant difference. Some plantlets had a few morepollen sacs than control. The shape of filaments was abnormal. Some were curved, shorter orlonger than control (Figure 3). The flowers were dark purple and darker (Figure 3C) thancontrol. High dosages of gamma rays made wishbone flower stem flat. The number of guardcells at abaxial and adaxial epidermis showed highly significant difference ( p≤0.01) (Table4). Abaxial epidermis (24.6cells) and adaxial epidermis (38.2 cells) from control (notirradiated) had the highest number of guard cells. The results showed that when gamma raydosage increased, the number of guard cells at abaxial and adaxial epidermis decreased. Theguard 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*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. 379eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf
  • 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 control4. Discussion  Callus induction from young shoot tip explants and regeneration for shoot multiplicationin T. fournieri preferred BA and NAA at 0.5 mg/l. This may suggest that bud formationrequired cytokinin and auxin. A conjunction of BA and NAA evoked a better response inshoot multiplication than NAA alone and this is probably due to the difference in endogenouslevels of growth regulators in this plant or to a difference in sensitivity (Trewavas andCleland, 1983). Such a synergistic effect of NAA and BA is in concurrence with the resultsin other ornamental plants such as Tagetes (Belarmino, 1992), Lilium (Liu, 1986) andDianthus (Jethwani, 1993). Shoot tip could induce new shoots which is in concurrence withthe report of Dianthus chinensis (Kantia and Kothari, 2002). NAA and BA in severalcombinations resulting in callusing and shoot multiplication on callus suggest that the normalendogenous growth substance levels are conductive to bud formation. A similar result wasobserved in plant regeneration of Dianthus barbatus through organogenesis in callus inducedfrom leaf explants (Pareek and Pareek, 2005). Stem segment of T. fourmieri couldregenerated adventitious bud (Ishioka and Tanimoto, 1992; Tanimoto and Harada, 1986 and1990; and Kobayashi et al, 1995). However, acute gamma ray affected the tissue ofwishbone plantlets . When plants grew up, sizes of leaves, stem, and root were recorded. Thenumber of leaves increased, similar to those observed by Chutinthorn (1979) which reportedthis 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 foundthat growth rate and rate of survival decreased when the plant was exposed to high dosage ofgamma ray. Plant height increased in response to an increase of dosage of gamma rays. Thiswas 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 mediumsupplemented 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 0.5mg/l NAA. New shoots averaging 0.5–0.8 cm were irradiated with varying doses of gammarays (5, 10, 15, 20 grays) and subcultured fourfold (M1V4) in the same medium every 4weeks. The elongated shoots (about 6-8 cm) with roots were transferred for hardening. Thewell developed healthy plants were transplanted to soil in greenhouse. Gamma irradiationexerted 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 andnumber of guard cells at abaxial and adaxial epidermis decreased. Sizes of guard cells from20 grays were smaller than control.6. Acknowledgement  A very special thank you is due to Professor Dr. Thana Na-Nakara for insightfulcomments, helping clarify and improve the manuscript.7. Reference Ahmed, E.U., Hayashi, T. and Yazawa, S. (2004). Auxins increase the occurrence of leaf- color variants in Caladium regenerated from leaf explants. Scientia Horticulturae 100, 153 - 159.Aida, R., Kishimoto, S., Tanaka, Y. and Shibata, M. (2000). Modification of flower color in Torenia by genetic transformation. Plant Science 153, 33-42.Belarmino, m.M., Abe, T. and Sasahara, T. (1992). Callus induction and plant regeneration in African marigold (Tagetes erecta L.). Japanese Journal of Breeding 42, 835-841.Declerck, V. and Korban, S.( 1995). Shoot regeneration from leaf tissues of Phlox paniculata L. Journal of Plant Physiology. 147, 441-446.*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. 381eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf
  • Gaul, H. (1977). Plant Injury and Lethality. In: Induced Mutations in Vegetatively Propagated Plant, II, IAEA, Vienna, pp:29-36.Ishioka, N. and Tanimoto, S (1992). Adventitious bud induction by protein kinase c activators in Torenia stem segments. Plant Tissue Culture Letters 9, 86-89.Jethwani, V. and Kothari, S.L. (1993). Micropropagation of Dianthus barbatus and D. chinensis through cotyledon node culture. Plant Tissue Culture 3, 91-96.Kantia, A. and Kothari, S.L.(2002). High efficiency adventitious shoot bud formation and plant regeneration from leaf explants of Dianthus chinensis L. Scientia Horticulturae 96, 205-212.Kikuchi, S., Kishii, M., Shimizu, M. and Tsujimoto, H. (2005). Centromere-specific repetitive sequences from Torenia, a model plant for interspecific fertilization and whole-mount FISH of its interspecific hybrid embryos. Cytogenetic and Genome Research 109, 228-235.Kobayashi, S., Amaki, W. and Higuchi, H. (1995). Effects of medium pH on shoot growth and flowering of Torenia internodal stem segments In vitro. Acta Horticulturae. 393, 135-142.Jala, Anchalee. (2005). Effect of gamma ray on morphological of petunia by tissue culture. In TSB Annual meeting at BioThailand 2005: Biotechnology Challenges in the 21st Century at the Queen Sirikit National Convention Center (QSNCC) in Bangkok, Thailand, 2 – 3 November 2005.Ledbetter, D.I. and Preece, J.E.N. (2004). Thidiazuron stimulates adventitious shoot production from Hydrangia quercifolia Bartr. leaf explants. Scientia Horticulturae 101, 121-126.Li, M-L., Wang, X-J. and Li, H-Q. (2006). Establishment of Agrobacterium-mediated transformation system for Torenia. Acta Horticulturae Sinica 33, 105-110.Li, H-Q., Kang, P-J., Li, M-L. and Li M-R. (2007). Genetic transformation of Torenia fournieri using the PMI/ mannose selection system. Plant Cell, Tissue and Organ Culture 90, 103-109.Liu. L. and Burger, D.W. (1986). In vitro propagation of Easter lily from pedicels. HortScience .21, 1437-1438.Martin, K.P., Joseph, D., Madassery, J. and Philip, V.J. (2003). Direct shoot regeneration from lamina explants of two commercial cut flower cultivars of Anthurium andraeanum Hort. In Vitro Cellular Develop Biology Plant. 39, 500-504.Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473-497.Pareek, A. and Pareek, L.K. (2005). De-novo differentiation of shoots of Dianthus barbatus from callus cultures. Journal of Cell and Tissue Research 5, 327-329. 382 Anchalee Jala
  • 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 journal’s 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. 383eISSN 1906-9642. Online Available at http://TuEngr.com/V02/375-383.pdf