Plant Cell, Tissue and Organ Culture 67: 55–62, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

55

Effect of vitamins and inorganic micronutrients on callus growth and

somatic embryogenesis from leaves of chilli pepper

S. Kintzios∗ , J. B. Drossopoulos & Ch. Lymperopoulos

Department of Plant Physiology, Faculty of Agricultural Biotechnology, Agricultural University of Athens, Iera
Odos 75, 11855 Athens, Greece (∗ requests for offprints; Fax: 003-01-529 4286; E-mail: skin@aua.gr)

Received 28 July 2000; accepted in revised form 24 April 2001

Key words: callus culture, Capsicum annuum, globular somatic embryos, mineral nutrition, Solanaceae

Abstract
The effect of different vitamins and inorganic micronutrients on callus growth and the induction and proliferation
of somatic embryos from young mature, fully expanded leaves of chilli pepper (Capsicum annuum L.) was in-
vestigated. Explants were cultured on a solid Murashige and Skoog (MS) medium supplemented with 8% (w/v)
sucrose, 12.9 µM 6-benzyladenine, 9 µM 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 thiamin.HCl in various
combinations of eleven different vitamins. Alternatively, explants were cultured onto a solid medium containing
MS macro- and micronutrients except for the salts of Mn, Zn, I, Cu and Co which were added at either the standard
MS concentration or at a tenfold increased (Cu, Co) or decreased (Mn, Zn, I) concentration. The results indicated
that somatic embryogenesis from pepper leaves is favoured by the addition of nicotinic acid to the culture medium
and the increase of copper concentration (an average induction of 70.2 globular embryos/mm2 of explant surface,
9.2% higher than control), without reducing embryo maturation and germination.

Abbreviations: BA – 6-benzyladenine; 2,4-D – 2,4-dichlorophenoxyacetic acid; MS – Murashige and Skoog basal

medium (1962); PGR – plant growth regulators; PPFD – photosynthetic photon flux density

Introduction vitro grown seedlings (Kintzios et al., 1998) or field-

Somatic embryogenesis, the development of adventi-
tious embryos from somatic (sporophytic) tissue, can
be valuable for the micropropagation of plant species.
In chilli pepper (Capsicum annuum), investigations
on somatic embryogenesis have been focused on the
effect of the donor plant genotype, plant growth reg-
ulators and explant source (Harini and Lakshmi Sita,
1993; Kintzios et al., 1998). Direct induction of so-
matic embryogenesis in pepper has been demonstrated
on immature zygotic embryos (Harini and Lakshmi
Sita, 1993; Binzel et al., 1996) on MS medium, sup-
plemented with 2,4-D, coconut water and at least at
8% w/v sucrose. Embryo germination ensued on me-
dium supplemented with 1 mg l−1 gibberellic acid.
In addition, the effect of light on induction of callus
and somatic embryogenesis from pepper mature leaf
explants (cv. ‘Colombo’) has been studied on either in
grown plants (Kintzios et al., unpublished data, 2001).
These studies indicate that somatic embryos can be
optimally induced from leaf segments (derived from
the first leaf of plants) after incubation in darkness
for 3 weeks and then transfer to light (250 µmol m−2
s−1 ) and grown on a solid MS medium supplemented
with 9 µM 2,4-D + 12.9 µM 6-BA. Under these condi-
tions, about 18% of induced globular stage embryos
develop to the mature cotyledonary stage (embryos
capable of germination). There have been few re-
ports on the effect of vitamins and inorganic elements
on the development of plant tissue cultures. For ex-
ample, Asamo et al. (1996) found that thiamin and
riboflavin favoured induction of embryogenic callus
from seeds of Zoysia japonica. Montoro et al. (1995)
reported on the effect of calcium on callus friability
and somatic embryogenesis in Hevea brasiliensis. We
have recently demonstrated that certain vitamins and
56
inorganic micronutrients significantly promote callus
growth and somatic embryogenesis from rose (Rosa
hybrida) leaves (Kintzios et al., 2000).
Therefore, we decided to investigate the effect of
different vitamins and inorganic micronutrients on cal-
lus growth and on the induction and proliferation of
somatic embryos from young mature, fully expanded
leaves of pepper.
Materials and methods
Explant source and preparation
The commercial pepper cv. ‘Colombo’ was used in
the study. Donor pepper seedlings (3–5 days old) were
potted into a 1:1 peat:perlite mixture and grown in the
glasshouse (26±2 ◦ C, 200 µmol m−2 s−1 16-h pho-
toperiod, cool white fluorescent lamps) until explant
removal. Explants were derived from the median re-
gion (without the midvein) of the first fully expanded,
green leaves (young mature leaves) on non-flowering
donor plants. Leaves were dissected from each plant,
surface sterilized for 10 min in 0.2% sodium hypo-
chlorite solution, containing 2% Tween-80, and rinsed
four times in sterile distilled water.
Vitamin treatments
Explants were cultured on solid MS (Murashige and
Skoog, 1962) basal medium (0.8% w/w agar) supple-
mented with 8% (w/v) sucrose, 12.9 µM BA, 9 µM
2,4-D and 0.5 mg l−1 thiamin.HCl in combination with
one or all of the following vitamins:
1. Pyridoxine.HCl (0.5 mg l−1 )
2. Nicotinic acid (0.1 mg l−1 )
3. Biotin (0.005 mg l−1 )
4. Pantothenic acid (0.5 mg l−1 )
5. Adenine sulfate (5 mg l−1 )
6. Cystein (10 mg l−1 )
7. Glycine (0.1 mg l−1 )
8. Casein hydrolysate (5 mg l−1 )
9. Cyanocobalamin (B12) (0.3 mg l−1 )
10. Myo-inositol (100 mg l−1 )
11. Combinations of all above vitamins 1–10.
There was no control treatment without vitam-
ins, since thiamin is considered an essential factor for
plant tissue culture (Gamborg, 1984) (as we have also
verified in preliminary experiments).
Micronutrient treatments
Explants were cultured on solid medium containing
MS macros, 8% (w/v) sucrose, 0.8% (w/v) agar,
0.5 mg l−1 thiamin.HCl, 0.5 mg l−1 pyridoxin.HCl,
0.05 mg l−1 nicotinic acid, 100 mg l−1 myo-inositol,
12.9 µM BA and 9 µM 2,4-D, and micronutrients ex-
cept for the salts of manganese, zinc, iodine, copper
and cobalt, which were added at either the standard
MS concentration or at a tenfold increased (copper,
cobalt) or decreased (manganese, zinc, iodine) con-
centration, according to the formulations in Table 1.
The selected variation of micronutrient concentration
was arbitrary, since no information was available on
the actual requirements of the cultures. Control media
(solid) consisted of MS inorganic salts and vitamins
(pyridoxine.HCl, nicotinic acid, thiamin.HCl, glycine,
and myo-inositol), 8% (w/v) sucrose, 12.9 µM BA and
9 µM 2,4-D. All reagents were purchased from Sigma
(St. Louis, Mo., USA). Media were adjusted to pH 5.8
using 1 N NaOH or 1 N HCl and autoclaved at 121 ◦ C
for 20 min. Vitamin solutions were filter-sterilized and
added to autoclaved media when cooled. Media were
poured into polystyrene 100 × 20 mm Petri dishes (30
ml dish−1 ). Five explants were inoculated on each me-
dium and each experiment was replicated three times.
Inoculated dishes were sealed with ParafilmT M and
incubated at 25 ◦ C, initially in darkness (for 3 weeks),
and then under illumination at a photosynthetic photon
flux density (PPFD) of 250 µmol m−2 s−1 with a 16-h
photoperiod from cool white fluorescent lamps.
Somatic embryo development, maturation and
germination
After 4 weeks in culture, callus tissues with somatic
embryos at the globular stage were aseptically re-
moved from culture. The fresh weight of the tissues
was measured and the number of globular embryos
per mm2 of explant surface was recorded. The tissues
were then transferred onto fresh control medium con-
taining 3% sucrose at the same temperature and light
conditions for further embryo development. Embryos
at the torpedo-shaped stage germinated on the same
medium.
Data analysis
Results were assessed by a standard analysis of vari-
ance for a completely randomized design, using MS-
STATISTICA software. Means were separated by
Student-Newman-Keul’s (SNK) Multiple Range test.
 57
Table 1. Micronutrient formulation of 12 culture induction media, designed to examine the effect of micronutri-
ents on callus growth and somatic embryogenesis of pepper

Medium Manganese sulfate Zinc sulfate Potassium iodide Copper sulfate Cobalt chloride
MnSO4 .4H2 O ZnSO4 .7H2 O KI CuSO4 .5H2 O CoCl2 .6H2 O
(mg l−1 ) (mg l−1 ) (mg l−1 ) (mg l−1 ) (mg l−1 )

1 15.6 8.6 0.8 0.025 0.025

(Control)
2 15.6 8.6 0.08 0.025 0.025
3 15.6 8.6 0.8 0.25 0.025
4 15.6 8.6 0.8 0.025 0.25
5 1.56 8.6 0.8 0.025 0.025
6 1.56 8.6 0.08 0.025 0.025
7 1.56 8.6 0.8 0.25 0.025
8 1.56 8.6 0.8 0.025 0.25
9 15.6 0.86 0.8 0.025 0.025
10 15.6 0.86 0.08 0.025 0.025
11 15.6 0.86 0.8 0.25 0.025
12 15.6 0.86 0.8 0.025 0.25

Results and discussion
Callus induction and somatic embryo formation
Formation of a yellowish friable embryogenic callus
tissue was observed on leaf explants after 2 weeks
of culture on a solid medium. The rate of callus
formation was unaffected by the different vitamin and
micronutrient treatments. Somatic embryo formation
was observed 3 weeks after callus induction.
Effect of vitamins on callus growth and somatic
embryogenesis
Maximum callus growth was obtained on medium
containing either pyridoxine (treatment 2) or myo-
inositol (treatment 11) (Table 2, Figures 1 and 2). Both
treatments yielded more callus than the control (treat-
ment 1) which contained only thiamin. Significantly
more somatic globular embryos were induced when
cultures were initially incubated in a medium contain-
ing nicotinic acid (treatment 3) than in any other treat-
ment. Glycine (treatment 8) was the least favourable
vitamin for both callus growth and somatic embryo
proliferation. Explants cultivated on medium supple-
mented with all vitamins (treatment 12) or on control
medium gave a satisfactory embryogenic response.
In our previous study with young mature leaves of
rose (Rosa hybrida) (Kintzios et al., 2000), we also
found that maximum callus growth was obtained with
either pyridoxine or myo-inositol, while the addition
of nicotinic acid (or cysteine) to the culture medium
favored somatic embryo induction. Interestingly, gly-
cine had the least positive effect on callus culture of
both species.
Potential explanations of the positive effects of
these vitamins include the following:
Nicotinic acid is the core component of the elec-
tron carrier coenzyme nicotinamide adenine dinuc-
leotide (NAD+) and its phosphate derivative (NADP+)
which participate in numerous oxidation-reduction re-
actions, particularly during glycolysis and oxidative
phosphorylation (Metzler, 1977).
Pyridoxin (Vitamin B6 ) is involved in the trans-
fer of amino groups and amino acid metabolism. It
indirectly promotes nicotinic acid synthesis through
biosynthesis of 3-hydroxyanthranilic acid from 3-
hydroxykynurenin (Jakubke and Jeschkeit, 1975). The
hexahydroxycyclohexane myo-inositol is a component
of galactinol, a specific precursor of cell wall polysac-
charides. Thus, myo-inositol could be essential during
cell division and/or enlargement. Additionally, myo-
inositol phosphate esters, such as the hexaphosphate
phytic acid, are important sources of mineral nutrients
(magnesium, calcium) (Metzler, 1977).
Effect of inorganic micronutrients on callus growth
and somatic embryogenesis
Increasing the concentration of Cu in the culture me-
dium by tenfold (relative to standard MS medium con-
centrations) increased callus growth (especially when
58

Figure 1. Growth (expressed as callus fresh weight) of callus cultures derived from pepper leaf explants on MS + 12.9 µM BA + 9 µM 2,4-D
in response to different vitamin treatments (∗ ). Bars indicate standard errors.

Figure 2. Relative somatic globular embryo production (globular embryos/mm2 explant surface) from pepper leaf explants on MS + 12.9
µM BA + 9 µM 2,4- D in response to different vitamin treatments(∗ ). Bars indicate standard errors. ∗ (1) Control medium (MS medium)
(2)Pyridoxine.HCl (0.5 mg l−1 ) (3) Nicotinic acid (0.1 mg l−1 ) (4) Biotin (0.005 mg l−1 ) (5) Pantothenic acid (0.5 mg l−1 ) (6) Adenine sulfate
(5 mg l−1 ) (7) Cysteine (10 mg l−1 ) (8) Glycine (0.1 mg l−1 ) (9) Casein hydrolysate (5 mg l−1 ) (10) Cyanocobalamin (B12) (0.3 mg l−1 )
(11) Myo-inositol (100 mg l−1 ) (12) All vitamins 2–11.
 59
Table 2. Analysis of variance of callus growth and somatic globular embryo
production from pepper leaf explants on MS + 12.9 µM BA + 9 µM 2,4-D in
response to treatment with different vitamins and mineral elements

Source of variation df Callus growth Embryo production

(fresh weight) (embryos/mm2 )
Mean square Mean square

a. Vitamins:
Vitamin treatment 11 9.201552∗∗ 1543.6182∗∗
Error 168 0.065773 7.6883
Total 179

Mineral elements:
Mineral element treatment 11 3.2877∗∗ 627.3∗∗
Error 168 0.2428 4.6751
Total 179

df = degrees of freedom, ∗∗ = p<0.01.

Figure 3. Growth (expressed as callus fresh weight) of callus cultures derived from pepper leaf explants on MS + 12.9 µM BA + 9 µM 2,4-D
in response to different micronutrient treatments (∗ ). Bars indicate standard errors.

standard MS levels of Mn were retained) and somatic
embryo production (treatment 3) (Table 2, Figures 3
and 4). Increasing the Co concentration had varying
effects on somatic embryogenesis depending on the
manganese concentration: the number of globular em-
bryos was increased when the Mn concentration was
decreased by tenfold (treatment 8), otherwise a reduc-
tion in somatic embryogenesis was observed, relative
to the control (treatment 4). On the other hand, de-
creasing the Mn, Zn or I concentration (treatments 2,
5 and 9, respectively) did not affect negatively cal-
lus growth and somatic embryogenesis in comparison
with the control medium.
Callus growth and somatic embryogenesis from
rose leaves is maximized at increased Cu concentra-
tions (Kintzios et al., 2000).
60

Figure 4. Relative somatic globular embryo production (globular embryos/mm2 explant surface) from pepper leaf explants on MS + 12.9 µM
BA + 9 µM 2,4-D in response to different micronutrient treatments (∗ ). Bars indicate standard errors. ∗ (1) Control medium (2) 1/10 × iodine
(3) 10 × copper (4) 10 × cobalt (5) 1/10 × manganese (6) 1/10 × manganese, 1/10 × iodine (7) 1/10 × manganese, 10 × copper (8) 1/10 ×
manganese, 10 × cobalt (9) 1/10 × zinc (10) 1/10 × zinc, 1/10 × iodine (11) 1/10 × zinc, 10 × copper (12) 1/10 × zinc, 10 × cobalt.

Copper ions lie at the active centers of a ver-
satile group of enzymes, as a site for reaction with
O2 and participating in a variety of redox reactions
(Marschner, 1995). According to Sandmann and Bo-
erger (1983), three forms of protein exist in which
Cu is the metal component (Cu-proteins); i.e. blue
proteins, non-blue proteins and multicopper proteins.
Generally, more than 50% of the Cu localized in
chloroplasts is bound in plastocyanin, a component of
the electron transport chain of photosystem I. Under
Cu deficiency, there is a close relationship between
the Cu content of leaves and the plastocyanin content.
Copper is also a component of other chloroplast en-
zymes and is required for the synthesis of quinones,
thus affecting the activity of PSII via electron transport
inhibition (Droppa et al., 1984). Since Cu is also a
component of the Cu–Zn superoxide dismutase, it is
directly involved in the mechanism of detoxification
of O2 generated in photosynthesis (Elstner, 1982). A
significant promotive effect of light intensity on cal-
lus induction and somatic embryogenesis has been
reported for a number of vegetable and ornamental
species, such as cucumber (Cucumis sativus) (Cade
et al., 1988; Colijn-Hooymans et al., 1988), melon
(Cucumis melo) (Gray et al., 1993; Kintzios and Tara-
vira, 1997) pepper (Capsicum annuum) and rose (Rosa
hybrida) (Kintzios et al., 1998). Experiments with
common mallow (Malva sylvestris) tissue cultures
have also demonstrated that higher light intensities are
favourable for somatic embryo proliferation (Kintzios,
2001).
Addition of Cu could have promoted callus induc-
tion and somatic embryogenesis by direct promotion
of polyamine biosynthesis. Diamine oxidase is a Cu-
containing flavoprotein catalyzing the degradation of
polyamines, such as spermidine to form putrescine
(Federico et al., 1990). Increased putrescine and sper-
midine levels have been observed during somatic em-
bryogenesis of various plant species (Montague et al.,
1978; Fienberg et al., 1984; Gaspar et al., 1996).
Dahleen (1995) reported that increased Cu levels were
associated with improved callus induction and plant
regeneration from barley somatic embryos.
Contrary to the Cu functions mentioned above, in-
creasing copper content has been inversely correlated
with shoot induction from tissue cultured plants, pos-

sibly due to higher polyphenol oxidase and peroxidase
activities (Schum et al., 1988).
Embryo development, maturation and germination
Globular somatic embryos that were subcultured on
fresh ‘standard induction medium’ were able to de-
velop to heart- and torpedo-shaped forms (1–2 mm
and 3–5 mm long, respectively). Embryo germination
(at a rate of 1.1 regenerated plants per 100 embryos
subcultured), indicated by the elongation of shoot
structures from each embryo and subsequent root ini-
tiation, took place on the same medium. Further devel-
opment of globular somatic embryos to cotyledonary
and torpedo-shaped stages and their germination was
not affected by any previous vitamin or micronutrient
treatment.
About half of the reported embryo induction me-
dia used across all plant species are MS-based media
(Brown et al., 1995), while an absolute requirement
for macronutrients such as potassium and iron EDTA
has been frequently indicated. Although often hav-
ing an unidentified function, vitamins and organic
compounds have been shown empirically to stimulate
callus growth and/or embryogenesis. Thiamine is con-
sidered essential for plant cells in culture (Gamborg,
1984) and nicotinic acid, pyridoxine and myo-inositol
are standard components of MS and B5 (Gamborg et
al., 1968) media. Amino acids like glutamine and alan-
ine can act as replacements of ammonium chloride as
a nitrogen source, supporting growth and embryogen-
esis (Kamada and Harada, 1979, 1984; Nomura and
Komamine, 1985, 1995). In carrot, alanine acceler-
ated cell division during the earlier stages of somatic
embryogenesis.
When considering nutrient supply in the medium,
we must remember that the plants in vitro do not have
direct access to the entire volume of the medium. The
availability of nutrients is further limited by the water
potential of the medium (–0.7 Mpa for MS), which is
much lower than in a soil under normal conditions.
Suspension cultures are consequently more suitable
for studying nutrient uptake, as the only factor is the
uptake across the cell membrane (Leifert et al., 1995).
Therefore, it is impossible to discuss the nutrient sup-
ply of tissue culture systems without considering the
chemical, physical and biological factors involved. In
addition, one of the difficulties in experimentation
with micronutrients is that they occur as contaminants
in other medium ingredients so that it is difficult to de-
termine precise cause and effects. However, provided
61
that the concentration of each of those ingredients was
the same in all treatments, in vitro qualitative effects
could be ascribed to individual micronutrients, even
though the actual concentration of them in the medium
remains unknown.
In conclusion, the results of the present study in-
dicate that somatic embryogenesis from leaves of the
pepper cv. ‘Colombo’ can be optimized by addition of
thiamin and nicotinic acid to the culture medium while
other vitamins can be reduced or even omitted and
either an increase of copper concentration or a con-
comitant increase of cobalt and decrease of manganese
concentrations by tenfold. Under these conditions,
approximately 72 globular embryos/mm2 of explant
surface can be obtained, a value that is higher than
for the control (64 globular embryos mm−2 ), while
the embryo germination rate remains the same (1.1%).
Future experiments will focus on the analysis of endo-
genous vitamin and micronutrient concentrations, in
order to define more precisely the media requirements
for optimal expression of somatic embryogenesis in
this species.
Acknowledgement
The authors wish to thank Prof. P. Kaltsikes (Labor-
atory of Plant Breeding and Biometry) for his kind
advice on the statistical analysis of the results of the
present study.
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