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

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

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

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
209

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

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
211

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
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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.
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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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