This article was downloaded by: [Bahagian Terbitan Bersiri]

On: 17 February 2010

Access details: Access Details: [subscription number 919216201]
Publisher Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-
41 Mortimer Street, London W1T 3JH, UK

Acta Agriculturae Scandinavica, Section B - Plant Soil Science

Publication details, including instructions for authors and subscription information:
http://www.informaworld.com/smpp/title~content=t713394126

Effects of NAA and vitamin B2 on in vitro rooting of Citrus

Christos Chatzissavvidis a; Chrysovalantou Antonopoulou b; Ioannis Papadakis b; Ioannis Therios b;
Kortessa Dimassi b
a
Department of Agricultural Development, Democritus University of Thrace, Orestiada, Greece b
Laboratory of Pomology, School of Agriculture, Aristotle University, Thessaloniki, Greece

First published on: 02 July 2009

To cite this Article Chatzissavvidis, Christos, Antonopoulou, Chrysovalantou, Papadakis, Ioannis, Therios, Ioannis and
Dimassi, Kortessa(2010) 'Effects of NAA and vitamin B2 on in vitro rooting of Citrus', Acta Agriculturae Scandinavica,
Section B - Plant Soil Science, 60: 2, 189 — 192, First published on: 02 July 2009 (iFirst)
To link to this Article: DOI: 10.1080/09064710902785300
URL: http://dx.doi.org/10.1080/09064710902785300

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial or
systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or
distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses
should be independently verified with primary sources. The publisher shall not be liable for any loss,
actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly
or indirectly in connection with or arising out of the use of this material.
 Acta Agriculturae Scandinavica Section B
Soil and Plant Science, 2010; 60: 189
192

SHORT COMMUNICATION

Effects of NAA and vitamin B2 on in vitro rooting of Citrus

CHRISTOS CHATZISSAVVIDIS1, CHRYSOVALANTOU ANTONOPOULOU2,

IOANNIS PAPADAKIS2, IOANNIS THERIOS2 & KORTESSA DIMASSI2
1
Department of Agricultural Development, Democritus University of Thrace, Orestiada 68200, Greece, 2Laboratory of
Pomology, School of Agriculture, Aristotle University, Thessaloniki 54124, Greece
Downloaded By: [Bahagian Terbitan Bersiri] At: 15:03 17 February 2010

Abstract
The effects of riboflavin combined with a-naphthaleneacetic acid on the in vitro rooting of sour and trifoliate orange
explants were studied. After four weeks in culture, there was a reduction in both the rooting percentage and root length of
sour orange explants. When riboflavin was added to the nutrient medium containing 0.1 mg dm 3 a-naphthaleneacetic
acid, the rooting percentage of sour orange explants was reduced. However, in the treatments containing 1.0 mg dm 3
a-naphthaleneacetic acid the root length increased. Rooting in trifoliate orange explants was not distinctly affected by
riboflavin. Increasing riboflavin concentration in the nutrient medium leads to a) the rooting percentage was affected
(reduced) only in sour orange explants treated with 0.1 mg dm 3 NAA; b) the root length either decreased (0.1 mg dm 3
NAA) or increased (1.0 mg dm 3 NAA); and c) the root number was reduced in most treatments. Finally, it is strongly
recommended that riboflavin in vitro should be extensively studied, as it has immense economic significance for commercial
laboratories when it is added to the nutrient medium with the aim to reduce exogenous auxin levels, rather than transferring
cultures to a growth-regulator-free medium.

Keywords: a-naphtaleneacetic acid, auxin, micropropagation, riboflavin, sour orange, trifoliate orange.

Introduction (Bachelard & Stone, 1962; Drew & Miller, 1989;

Sour orange (Citrus aurantium L.) is one of the most
commonly used Citrus rootstocks in the Mediterra-
nean region (Georgiou & Gregoriou, 1999). Another
is trifoliate orange (Poncirus trifoliata L.), which is a
relative of the genus Citrus. Both are used extensively
in citriculture and are propagated through different
procedures.
Micropropagation protocols have been described
for a number of Citrus species. There have been many
attempts to improve rhizogenesis by adding growth
regulators to the culture medium in various concen-
trations and combinations. Furthermore, there are a
great number of possible growth-regulating sub-
stances which have not been extensively tested
(George, 1996). Little research has been conducted
concerning the effectiveness of vitamins on in vitro
rooting of Citrus. Riboflavin, occasionally used in
tissue culture media, has been found to stimulate
rooting of Eucalyptus, papaya, and walnut explants
Gruselle & Boxus, 1990) or to inhibit root formation
of papaya explants (Drew & Smith, 1986). Further-
more, the inclusion of a-naphthaleneacetic acid
(NAA) in an in vitro medium was found to be
beneficial for the rooting not only of Citrus (Al-Wasel,
2000; Bordon et al., 2000) but for other species as
well (Moshen, 2004; Tian et al., 2007).
As the results for other species appear to be
variable and contradictory, the present experiment
was conducted in order to investigate the effects of
riboflavin in combination with two NAA concentra-
tions on the in vitro rooting and growth of sour and
trifoliate orange explants.
Materials and methods
Apical shoot tips (10
15 mm) of sour and trifoliate
orange were obtained from previous subcultures on
a full-strength MS (Murashige & Skoog, 1962)

Correspondence: Dr C. Chatzissavvidis, Department of Agricultural Development, Democritus University of Thrace, Pantazidou 193 str., Orestiada 68200,
Greece. Fax: 302552041191. Tel: 302552041113. E-mail: cchatz@agro.duth.gr.

(Received 6 December 2008; accepted 28 January 2009)

ISSN 0906-4710 print/ISSN 1651-1913 online # 2010 Taylor & Francis
DOI: 10.1080/09064710902785300
 190 C. Chatzissavvidis et al.

medium and were transferred to 10 cm3 of the same
rooting medium. Riboflavin was added in five con-
centrations: 0, 0.5, 1.0, 1.5, and 2.0 mg dm 3. All
media contained 0.1 and 1.0 mg dm 3 NAA, sucrose
30 g dm 3, and agar Oxoid No. 3, 6 g dm 3. The pH
of the media was adjusted to 5.8 prior to autoclaving
at 121 oC for 20 min. Cultures were maintained at
2691 oC under cool white fluorescent light (Phillips,
90 mmol m 2 s1) with a 16 h photoperiod. After
four weeks in culture, the microcuttings were sepa-
rated into shoots and roots, counted, and weighed.
Each treatment included fifteen replications (culture
vessels) with one microcutting in each 25 100 mm
test tube.
The ex vitro acclimatization procedure of the
plantlets was similar to that applied by Shiyab et al.
(2003). At the end of three weeks, the survival
Downloaded By: [Bahagian Terbitan Bersiri] At: 15:03 17 February 2010
(reduced) only by the presence of 1.0 mg dm 3
riboflavin, independently of NAA levels (Figure 2A).
The root number per explant was reduced with the
addition of riboflavin in the sour orange for both
NAA treatments (Figure 1B), and in the trifoliate
orange treated with 1.0 mg dm 3 NAA (Figure 2B).
Root fresh weight was negatively affected with the
addition of riboflavin in sour orange explants for both
NAA levels (Figure 1C), whereas in the trifoliate
orange it was reduced only in treatment containing
1.0 mg dm 3 riboflavin and 0.1 mg dm 3 NAA
(Figure 2C). From ANOVA, riboflavin significantly
affected root number, as well as the root fresh and dry
weight of the trifoliate orange explants (data not
shown). It was found that the addition of riboflavin
reduced the root length of sour orange explants
treated with 0.1 mg dm 3 NAA and increased it in
the treatments containing 1.0 mg dm 3 NAA

percentage was recorded and plants were transferred

to a greenhouse (Tmin 18 oC, Tmax 38 oC, Tmean 30 oC)
for further growth and development.
The experimental layout was completely rando-
mized and the experiment was repeated twice. The
reported data are the means of the two experiments.
The means were subjected to analysis of variance
(ANOVA) and compared by using the Duncan
multiple-range test (P 50.05).
Results and discussion
The addition of riboflavin to the nutrient medium
containing 0.1 mg dm 3 NAA led to a decrease in the
rooting percentage of sour orange explants, whereas
there was no effect when NAA concentration was 1.0
mg dm 3 (Figure 1A). However, the rooting percen-
tage in trifoliate orange explants was affected
(Figure 1D). The same trend was observed in half
of the riboflavin-treated trifoliate orange explants
(Figure 2D). The greater root length observed in
explants treated with 1.0 mg dm 3 in combination
with the higher riboflavin concentrations could be
attributed to the negative correlation between root
number and root length. In all treatments, no calli
developed, the microshoots of both species were
green and vigorous, and new shoots appeared after
their buds burst. Rooted plantlets (75
85%) were
successfully acclimatized and there were no statistical
differences in survival percentages among treatments
(data not shown). The acclimatized plantlets were
healthy and were successfully grown in the green-
house.
Riboflavin, like other chemical compounds, is also
used in tissue culture media (George, 1996) but is

A B
5
Root number
Rooting (%)

100 b A A A A A 4 B
a a 3
50 a a 2 A A
b a A aA
1 a a
0 0
0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0
Riboflavin (mg dm-3) Riboflavin (mg dm-3)
C D
60
Root length (cm)

8
Root f.w. (mg)

B AB b
6 B
40 b A B B B
A A 4 a
a a
20 a a a a a
2 A
0 0
0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0
Riboflavin (mg dm-3) Riboflavin (mg dm-3)
Figure 1. Effect of riboflavin and NAA (0.1 mg dm 3 I, 1.0 mg dm3 j) on the rooting% (A), root number per explant (B), root fresh
weight (C), and root length (D) of sour orange at the end of the experiment. Within the same NAA treatment, means followed by the same
letter are not significantly different (Duncan multiple-range test, P50.05).
 In vitro rooting of Citrus microcuttings 191
A B
5

Root number
Rooting (%)
100 B B cB 4
cd B
bc 3
A bc
50 2 B
a b ab A bA
1 a A ab A
0 0
0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0
Riboflavin (mg dm-3) Riboflavin (mg dm-3)
C D
40 8

Root length (cm)

Root f.w. (mg)

30 A
b A 6
B
b A b A b b B
20 A 4 AB AB
ab ab
10 a 2 A a a
0 0
0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0
Riboflavin (mg dm-3) Riboflavin (mg dm-3)
Figure 2. Effect of riboflavin and NAA (0.1 mg dm 3 I, 1.0 mg dm3 j) on the rooting% (A), root number per explant (B), root fresh
Downloaded By: [Bahagian Terbitan Bersiri] At: 15:03 17 February 2010

weight (C), and root length (D) of trifoliate orange at the end of the experiment. Within the same NAA treatment, means followed by the
same letter are not significantly different (Duncan multiple-range test, P 50.05).

not a basic component of the MS media. In most of
the treatments reported above, riboflavin either
reduced or had no effect on root initiation and
growth of sour orange and/or trifoliate orange
explants. Our data are consistent with those reported
by Antonopoulou et al. (2005), who found that the
effects of riboflavin were deleterious to root initiation
for GF677 (Prunus persica Prunus amygdalus)
rootstocks. In contrast, 1.0 mg dm 3 of riboflavin
added to the Driver Kuniyuli Walnut (DKW) media
promoted rooting of Juglans regia and Paradox
microcuttings (Gruselle & Boxus, 1990). In addi-
tion, riboflavin promoted rooting of Eucalyptus
explants (Bachelard & Stone, 1962; Trinidade,
1997). Unlike the findings of an experiment with
GF677 explants (Antonopoulou et al., 2005), in the
present study the addition of riboflavin did not have
a negative effect on shoot growth (data not shown).
The fact that during rooting the shoots’ basal
region was maintained under light could explain
the negative effects of riboflavin. Riboflavin was
found to catalyse auxin photooxidation (Drew
et al., 1991). In treatments with 0.1 mg dm 3
NAA, riboflavin probably catalysed auxin photoox-
idation, explaining the low percentage of rooting.
Alternatively, in the liquid MS medium with 0.1 mg
dm 3 NAA, trifoliate orange explants presented a
high number of roots as well as an increase in root
length (Al-Wasel, 2000). The findings of Gorst et al.
(1983) are in line with ours, namely that the
presence of riboflavin in the nutrient medium under
conditions of exposure to light sensitizes the oxida-
tion of the synthetic indole-3-butyric acid (IBA),
reducing the amount of auxin in the medium. Even
though NAA is more photooxidative tolerant than
other auxins (Stasinopoulos & Hangarter, 1990), as
regards the sour orange explants treated with 1.0 mg
dm 3 NAA (part of which could be photooxidized),
in our study a significant part remained stable and
was high enough to stimulate rhizogenesis. Similarly,
this same NAA concentration in the nutrient med-
ium led to a high percentage of rooting in the in vitro
cultures of sour orange (Bordon et al., 2000). A
proposed technique to avoid any possible negative
effects that riboflavin may have on rooting is to inject
riboflavin onto the agar surface one day following the
establishment of the culture on the rooting medium
(Drew et al., 1993).
References
Al-Wasel, A. (2000). Micropropagation of trifoliate orange root-
stock (Poncirus trifoliata Raf.). HortScience, 41, 1052.
Antonopoulou, C., Dimassi, K., Therios, I., & Chatzissavvidis, C.
(2005). Inhibitory effects of riboflavin (Vitamin B2) on the in
vitro rooting and nutrient concentration of explants of peach
rootstock GF 677 (Prunus amygdalus x P. persica). Scientia
Horticulturae, 106, 268
272.
Bachelard, E.P., & Stone, B.S. (1962). A possible link between
root initiation and anthocyanin formation. Nature, 194,
209
210.
Bordon, Y., Guardiola, J.L., & Garcia-Luis, A. (2000). Genotype
affects the morphogenic response in vitro of epicotyl seg-
ments of Citrus rootstocks. Annals of Botany, 86, 159
166.
Drew, R.A., & Miller, R.M. (1989). Nutritional and cultural
factors affecting rooting of papaya (Carica papaya L.) in vitro.
Journal of Horticultural Science, 64, 767
773.
Drew, R.A., & Smith, N.G. (1986). Growth of apical and lateral
buds of papaya (Carica papaya L.) as affected by nutritional
and hormonal factors. Journal of Horticultural Science, 61,
535
543.
Drew, R.A., Simpson, B.W., & Osborne, W.J. (1991). Degradation
of exogenous indole-3-butyric acid and riboflavin and their
influence on rooting response of papaya in vitro. Plant Cell
Tissue & Organ Culture, 26, 29
34.
 192 C. Chatzissavvidis et al.
Drew, R.A., McComb, J.A., & Considine, J.A. (1993). Rhizogen-
esis and root growth of Carica papaya L. in vitro in relation to
auxin sensitive phases and use of riboflavin. Plant Cell Tissue
& Organ Culture, 33, 1
7.
George, E.F. (1996). Plant propagation by tissue culture in practice-
Part I. Exegetics Limited Press, London, UK.
Georgiou, A., & Gregoriou, C. (1999). Growth, yield and fruit
quality of ‘Shamouti’ orange on fourteen rootstocks in
Cyprus. Scientia Horticulturae, 80, 113
121.
Gorst, J.R., Slaytor, M., & De Fossard, R.A. (1983). The effect of
indole-3-butyric acid and riboflavin on the morphogenesis of
adventitious roots of Eucalyptus ficifolia F. Meull. grown in
vitro. Journal of Experimental Botany, 34, 1503
1515.
Gruselle, R., & Boxus, P. (1990). Walnut propagation. Acta
Horticulturae, 284, 45
52.
Moshen, Khe, (2004). Comparison, determination and optimiz-
ing the conditions required for rhizome and shoot formation,
and flowering of in vitro cultured calla explants. Scientia
Horticulturae, 101, 305
313.
Downloaded By: [Bahagian Terbitan Bersiri] At: 15:03 17 February 2010
Murashige, T., & Skoog, F. (1962). A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiologia
Plantarum, 15, 473
497.
Shiyab, S.M., Shibli, R.A., & Mohammad, M.M. (2003).
Influence of sodium chloride salt stress on growth and
nutrient acquisition of sour orange in vitro. Journal of Plant
Nutrition, 26, 985
996.
Stasinopoulos, T.C., & Hangarter, R.P. (1990). Preventing
photochemistry in culture media by long-pass light filters
alters growth of cultured tissues. Plant Physiology, 93,
1365
1369.
Tian, L., Sibbald, S., Subramanian, J., & Svircev, A. (2007).
Characterization of Prunus domestica L. in vitro regeneration
via hypocotyls. Scientia Horticulturae, 112, 462
466.
Trinidade, H. (1997). In vitro studies on Eucalyptus globulus
rooting ability. In Vitro Cellular Developmental Biology-Plant,
33, 1
5.