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American Transactions on
Engineering & Applied Sciences
http://TuEngr.com/ATEAS
Role of 2,4-D on Callus Induction and Shoo...
rose bushes, but they come in a variety of types and colors and are a very carefree grower with great
disease resistance. ...
4. Results and Discussion
After two weeks, all clean cultured from explants were cultured on MS medium supplemented
with d...
and 1.0 mg/l BA was the best medium for regenerating new shoot (100%) from callus as showed in
Table 2.
Table 2: Average o...
When compared the average height of plant, root induction in statistics, it was found that
plant height and percentage of ...
transferring the explants to MS medium free from any PGRs was successful for root formation in
Damask rose. 2,4-D is one o...
damascena Mill. from mature bushes using thidaizuron. Journal of Horticultural Science
and Biotechnology. 76:30–34.
Mello,...
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Role of 2,4-D on Callus Induction and Shoot Formation to Increase Number of Shoot in Miniature Rose In Vitro

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Stem node of Miniature Rose with axillary bud were used as explants. These explants cultured on MS medium supplemented with different concentrations of 2,4-D. It was found that MS medium supplemented with 0.5 mg/l 2,4-D gave the highest number of green callus. The callus cultured on MS medium supplemented with different combinations of NAA and BA to form new shoot and root. From the result, we are able to find the highest number of young shoots that were induced from callus when cultured callus on MS medium supplemented with NAA and BA. When subcultured all new shoots with the same size to MS medium supplemented with different concentrations of NAA and BA, and 2,4- D for six weeks. The result was significant difference (P≤0.5) when compared the average height of plant and percentage of root formation, but their duration time for flowering were not significant different.

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Role of 2,4-D on Callus Induction and Shoot Formation to Increase Number of Shoot in Miniature Rose In Vitro

  1. 1. American Transactions on Engineering & Applied Sciences http://TuEngr.com/ATEAS Role of 2,4-D on Callus Induction and Shoot Formation to Increase Number of Shoot in Miniature Rose In Vitro Anchalee Jala a* a Department of Biotechnology, Faculty of Science and Technology, Thammasat University Pathumtani, 12121 A R T I C L E I N F O A B S T R A C T Article history: Received February 28, 2014 Received in revised form June 11, 2014 Accepted June, 20 2014 Available online June 24, 2014 Keywords: Mini rose; Duncan’s New Multiple Range Test; NAA; BA. Stem node with axillary bud were used as explants. These explants cultured on MS medium supplemented with different concentrations of (0.1, 0.2, 0.5, 2, 5, 10 mg/l) 2,4-D. It was found that MS medium supplemented with 0.5 mg/l 2,4-D gave the highest number of green callus. The callus cultured on MS medium supplemented with different combination of NAA (0.1, 0.5, 1.0, 2.0 mg/l) and BA (0.1, 0.5, 1.0, 2.0 mg/l) to form new shoot and root. The result showed that the highest number of young shoots was induced from callus when cultured callus on MS medium supplemented with 1.0 mg/l NAA and 1.0 mg/l BA. When subcultured all new shoots with the same size to MS medium supplemented with different concentration of (0.5 and 1.0 mg/l) NAA and (0.5 and 1.0 mg/l) BA, and (0.1, 0.5 and 1.0 mg/l) 2,4- D for 6 weeks. The result was significant difference (P≤0.5) when compared the average height of plant and percentage of root formation, but their duration time for flowering were not significant different. 2014 Am. Trans. Eng. Appl. Sci. 1. Introduction A miniature roses are true roses with perfectly placed in a pink pail. The metal pail is embossed with a delicate floral pattern. Most mini roses also have smaller flowers than standard 2014 American Transactions on Engineering & Applied Sciences. *Corresponding author (Anchalee Jala). Tel/Fax: +66-2-5644440-59 Ext. 2450. E-mail address: anchaleejala@yahoo.com. 2014. American Transactions on Engineering & Applied Sciences. Volume 3 No.3 ISSN 2229-1652 eISSN 2229-1660 Online Available at http://TuEngr.com/ATEAS/V03/0207.pdf. 207
  2. 2. rose bushes, but they come in a variety of types and colors and are a very carefree grower with great disease resistance. Overall height of plant is approximately 10 inches. Clonal propagation through tissue culture offers great potential for the establishment plantlets from inducing callus to form new shoots (Jala, 2012; 2013b). In this paper was present for mass production and for a rapid in vitro clonal propagation system for this rose, which would be ideal for flowering in bottles. 2. Materials and methods 2.1 Plant materials Stem nodes with axillary buds of miniature rose were used as explants. These explants having height about 1–1.5cm, were washed with tap water and followed by15% and 10% clorox solution for 10 and 15 minutes, respectively, then followed by washing materials three times with sterile distilled water. Finally these stem nodes with axillary bud were inoculated on MS medium supplemented with combination of 0.1 mg/l NAA and 0.1 mg/l BA, 2% sucrose, 0.25% gelrite at pH 5.7 and autoclaving at 121o C for 20 min. The cultures were maintained at 25 ± 2° C under 16-hour photoperiod with illumination provided by cool fluorescent lamps at an intensity of 60 µmolm-2 sec-1 (TLD 36 w/853350 lm Phillips Thailand). Callus induction: Cleaned explants were cultured on MS medium supplemented with different concentration of (0.1, 0.2, 0.5, 2, 5, 10 mg/l) 2,4-D, to enhance callus induction. Shoot formation from callus: All callus were cultured on MS medium supplemented with combination of (0.1, 0.5, 1.0 and 2.0 mg/l) NAA and (0.10.5, 1.0 and 2.0 mg/l) BA for inducing shoot formation. Duration time on flowering: Young shoots with the same size (about 3cm) were subcultured on MS medium supplemented with combination of (0.5,1.0 mg/l) NAA and (0.5,1.0 mg/l) BA and vary concentrations of( 0.1,0.5,1.0 mg/l) 2,4-D to form flowers. 3. Statistical Analysis Experiments were set up in Completely Randomized Design (CRD) with 6 treatments; each treatment was consisted of 20 and 25 replicates for the first and the second experiment, respectively. The test of statistical significance was done by applying Dancan1955 at 5% confidence level using SAS statistical software. 208 Anchalee Jala
  3. 3. 4. Results and Discussion After two weeks, all clean cultured from explants were cultured on MS medium supplemented with different concentration of 2,4-D. It was showed that cluster of callus were formed and proliferated around the base of explants as showed on Table 1 and Figure 1. Table 1: Callus induction from explants which cultured on MS medium supplemented with different concentration of 2,4 -D for 6 weeks. MS medium supplemented with Proliferation*(score) Color of callus Characteristic of callus 2,4-D 0.1mg/l 4.24a Pale green Big cluster on explants 2,4-D 0.5mg/ 4.98a green Biggest cluster on explants 2,4-D 1.0 mg/ 2.36b Pale green few callus around explants 2,4-D 2.0 mg/ 2.22b yellowish few callus around explants 2,4-D 5.0 mg/ 1.24c creamy A little callus had occur 2,4-D 10.0mg/ 1.02c white A little callus had occur Score of callus formation: 1 – less (diameter about 0.1-0.3cm) 3 – medium(diameter about 0.4-1.0 cm) 5 – high diameter about 1.1-1.5 cm) *Significant different (P≤0.5) abc - compared their by Duncan’s New Multiple Range Test (P≤0.5) This experiment showed that low concentration of 2,4-D induced callus at the base of explants but high concentration of 2,4-D(5.0 and 10.0 mg/l) gave less callus. It showed that 0.1- 0.5 mg/l 2,4-D suitable for inducing callus and giving the biggest cluster of callus(Figure1). Callus proliferated around the explants and their color was green. This callus was used as inoculum for shoot induction. Figure 1: Cluster of callus occurred on MS medium supplemented with: a – 0.5 mg/l 2,4 – D b – 0.1 mg/ 2,4 - D After transferred cluster of callus to MS medium supplemented with different combination of (0.1, 0.5, 1.0, 2.0 mg/l) NAA and (0.1, 0.5, 1.0, 2.0 mg/l) BA for 6 weeks. It showed that callus was induced and formed young shoots (Table 2). MS medium supplemented with 1.0mg/l NAA a b 1cm 1cm *Corresponding author (Anchalee Jala). Tel/Fax: +66-2-5644440-59 Ext. 2450. E-mail address: anchaleejala@yahoo.com. 2014. American Transactions on Engineering & Applied Sciences. Volume 3 No.3 ISSN 2229-1652 eISSN 2229-1660 Online Available at http://TuEngr.com/ATEAS/V03/0207.pdf. 209
  4. 4. and 1.0 mg/l BA was the best medium for regenerating new shoot (100%) from callus as showed in Table 2. Table 2: Average of new shoots formed from callus in each treatment after cultured for 6 weeks. MS medium % new shoot from callus NAA (mg/l) BA(mg/l) 0.1 0.1 18.6 b 0.1 0.5 26.2ab 0.5 0.5 42.9ab 1.0 0.5 42.9ab 1.0 1.0 100a 2.0 1.0 28.2ab 1.0 2.0 14.3b 2. 0 2.0 11.6b * *Different letters indicate significant difference at p ≤ 0.05 ( Duncan, 1955) These new shoots are regenerated from callus. According to Jala (2013a), histological analysis revealed that callus was formed from hypertrophied cortical parenchyma cells of the explants. Some of these cell unbent was divided, while the surrounding cell accumulated starch. Callus was capable of shoot bud regenerated after 70 days. New shoots (with the same size) were subcultured to MS medium supplemented with different combination of (0.5 and 1.0 mg/l) NAA and (0.5 and 1.0 mg/l ) BA and (0.1.0.5 and 1.0 mg/l) 2,4- D for 6 weeks. It was found that all new shoots were grew up, we measured the average of plant height, root formation, flowering induction, and duration time for flowering after subcultured as showed in Table 3. Table 3: Average of plant height, percentage of root induction, flower induction and duration time for flowering after subcultured within 6 weeks. MS medium with Plant height(cm) * % Root induction * % Flower induction ns Duration time for flowering after subcultured nsNAA(mg/l) BA(mg/l) 0.5 0.5 3.44a 50a 30 27.0 1.0 0.5 3.45a 40a 30 27.65 1.0 0.1 3.42a 50a 40 25.5 2,4-D 0.1 mg/l 0 2.69b 30ab 0 0 2,4-D 0.5 mg/l 0 2.54b 20ab 0 0 2,4-D 1.0 mg/l 0 0.97c 10b 0 0 * Different letters indicate significant difference at p ≤ 0.05 ( Duncan, 1955) ns = non-significant difference 210 Anchalee Jala
  5. 5. When compared the average height of plant, root induction in statistics, it was found that plant height and percentage of root formation was significant difference (p≤0.5) but percentage of flower induction and duration time for flowering were not significant difference. Plantlets are cultured on MS medium supplemented with different concentrations of NAA and BA, their percentages of flower induction were about 30–40% and the same as duration time for flowering about 25.5 – 27.5 days. It was found that flower induction was occurred only in MS medium supplemented with different combination of (0.5 and 1.0 mg/l) NAA and (0.5 and 1.0 mg/l) BA (Figure 2). Figure 2: Plant with flower (arrow) on MS medium supplemented with 0.1 mg/l BA and 1.0 mg/l 2,4-D. 5. Discussion Based on the results obtained in this study, low concentration of 2,4-D can induced callus. Plantlets can be regenerated from these calli by indirect organogenesis (Mello et al., 2001). BA at concentrations of 0.1-0.5 mg/l in combination with 0.5 mg/l NAA was the most suitable treatment for in vitro multiplication of Miniature rose. Results were similar to those obtained by other investigators for other rose species (Campos and Pais, 1990, Khosh-Khui and Sink, 1982, Kumar et al., 2001, Salehi and Khosh-Khui, 1996 and Skirvin et al., 1990). The shoot proliferation and multiplication were decreased when increased concentration of NAA and BA followed the same pattern as Kim et al (2003) and Davies (1980), but different from Carelli and Echeverrigaray (2002). According to the results, application of MS medium supplemented with different concentrations of NAA and BA 0.1, 0.5, and 1.0 mg/l 2,4-D for six weeks followed by *Corresponding author (Anchalee Jala). Tel/Fax: +66-2-5644440-59 Ext. 2450. E-mail address: anchaleejala@yahoo.com. 2014. American Transactions on Engineering & Applied Sciences. Volume 3 No.3 ISSN 2229-1652 eISSN 2229-1660 Online Available at http://TuEngr.com/ATEAS/V03/0207.pdf. 211
  6. 6. transferring the explants to MS medium free from any PGRs was successful for root formation in Damask rose. 2,4-D is one of the important phenolic compounds with auxinic effect, at low concentrations and acts as a rooting cofactor and prevents breaking of endogenous auxin by oxidase enzyme results in rooting, similar to Hudson et al. (2002). 6. Conclusion Stem node with axillary bud cultured on MS medium supplemented with 0.5 mg/l 2,4-D enhanced callus induction and the callus color was green. When cultured callus on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, it was found that callus regenerated to form the highest number of young shoots on MS medium supplement with 1.0 mg/l NAA and 1.0mg/l BA. It was found that all new shoots grew up. MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA gave the average of plant height and percentage of root formation. But the duration time for flowering in each treatments was not significant different. 7. References Campos P.S. and M.S.S. Pais. (1990). Mass propagation of the dwarf rose cultivar Rosamini. Sci Hortic. 43:321–30. Carelli BP, S. Echeverrigaray. (2002). An improved system for the in vitro propagation of rose cultivars. Sci Hortic. 92:69–74. Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11(1), 1-42. Davies,D.R.(1980) Rapid propagation of roses. In vitro Sci. Hort., 13(4):169-172. Jala, A. and W. Patchpoonporna. (2012). Effect of BA NAA and 2,4-D on Micropropagation of Jiaogulan (Gynostemma pentaphyllum Makino). International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. V3(4):363-370. Jala, A.(2013a). Callogenesis and Organogenesis from inflorescence segments of Curcuma alismatifolia and Curcuma hybrid ‘Laddawan’. American Transactions Engineering & Applied Sciences. 2(3):213 -222. Jala, A.(2013b). Potential of Benzyl Adenine, Naphthalene Acetic Acid and Sucrose Concentration on Growth, Development, and Regeneration of New Shoot and cormel on Gladiolus. American Transactions on Engineering & Applied Sciences.2(4):277- 285. Kim, C.K., J.Y. Oh, S.O. Jee and J.D. Chung. (2003). In vitro micropropagation of Rosa hybrida L. J. Plant Biotechnology. 5(2):115-119. Khosh-Khui M, Sink KC.(1982). Callus induction and culture of Rosa. Sci. Hortic.17:361-370. Kumar, A., A. Sood, U.T. Plani, A.K. Gupta, and L.M.S. Plani. (2001).Micropropagation of Rosa 212 Anchalee Jala
  7. 7. damascena Mill. from mature bushes using thidaizuron. Journal of Horticultural Science and Biotechnology. 76:30–34. Mello,M.O., M. Melo, B.Appezzato-Da-Gloria. (2004). Histological analysis of the callogenesis and organogenesis from root segments of Curcuma zedoaria Roscoe. Scientia Agricola. 57:703-713. Murashige and Skoog. (1962). A revised medium for rapid growth and bioassays with tissue culture. Physilogia Plantarum. 15:473-497. Salehi H. and M. Khosh-Khui. (1997). Effects of explant length and diameter on in vitro shoot growth and proliferation rate of miniature roses. J Hortic Sci. 72:673–6. Skirvin R.M, M.C. Chu and H.J. Young.(1990). Rose. In: Ammirato P.V., Evans D.R.,Sharp W.R., Bajaj Y.P.S., editors. Handbook of Plant Cell Culture Vol. 5. New York: McGraw Hill, New York: Springer; pp. 716–743. 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 address: anchaleejala@yahoo.com. 2014. American Transactions on Engineering & Applied Sciences. Volume 3 No.3 ISSN 2229-1652 eISSN 2229-1660 Online Available at http://TuEngr.com/ATEAS/V03/0207.pdf. 213

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