the effect of carrot extract and activated charcoal

XI th EUCARPIA Meeting on Genetics and Breeding of Capsicum & Eggplant, 2001, Antalya-Turkey
THE EFFECT OF CARROT EXTRACT AND ACTIVATED CHARCOAL ON THE
ANDROGENESIS OF PEPPER
1 Ellialtıoğlu Ş, 2 Kaplan F., 3 Abak K.
1 Department of Horticulture, Faculty of Agriculture, University of Ankara, Turkey
2 Department of Horticulture, Faculty of Agriculture, University of Adnan Menderes, Aydın-Turkey
3 Department of Horticulture, Faculty of Agriculture, University of Çukurova, Adana-Turkey
Abstract
In anther culture, one of the ways to improve embryo formation is to apply thermal shocks at
the beginning of cultures. In pepper, it is induced the embryo formation to keep anthers in the
dark conditions and incubate them at 35 o C in the first 8 days. As an alternative, 29 o C and
continuous light incubation regime is stated. It is known, that activated charcoal has positive
effect on the anther culture of several plant species included peppers. In this study, anther
culture was carried out in Kahramanmaraş local pepper genotype and the effects of two
different incubation conditions and two different basal medium formulations supplemented
with charcoal and carrot extract were investigated. The highest embryo formation and plant
growing were obtained from the anthers which incubated at 29 o C In the continuous light
conditions and cultured on the MS medium without 1% charcoal and carrot extract.
Introduction
Pepper is an important vegetable crop the economic point of view and it is used all over the
world. Turkey is the biggest pepper producer country after China and Mexico on the world.
There are some traditional methods for pepper breeding. But the anther culture has advantages
to short breeding time. On the other hand, complex genotypes are fixed by completely
homozygous plants obtained through dihaploidization of haploids (Bajaj, 1983, Heberle-Bors,
1985, Vagera, 1990). Haploids and diploids can be successfully applied to study pepper
resistance to viruses (Vaulx et al., 1982, Pochard et al., 1983) and transmission of resistance to
Phytophthora capsici (Abak et al., 1982). The experiments following the work of Wang et al.
(1973) did not succeed in obtaining androgenic plants (Vagera, 1990). Sibi et al. (1979)
described androgenesis morphologically and cytologically. They found that uninuclear
microspores were most suitable for inducing androgenesis. By the study of Vaulx et. al.
(1981), the yield of androgenesis in pepper was increased by heat treatment (+35 o C) at the first
stage of anther culture. Munyon et al. (1989) reported that androgenesis occurred in C. annuum
anthers in a continuous warm environment (29 o C) with continuous light. In the study of Vagera
and Havranek (1985) application of the media supplemented with charcoal and carrot extract
has been successfully used to stimulate androgenesis in Severka and Morava pepper cultivars.
The positive effect of charcoal on the anther culture of pepper was described by Chunling
(1992) too.
In this study we aimed to improve the yield of haploid embryos in pepper anther culture by
using different incubation conditions and by application of the two different basal media
formulation supplemented with charcoal and carrot extract.
Materials and methods
As the plant material in the experiments a local genotype of pepper (Capsicum annuum L.)
grown in South Eastern region of Turkey (in Kahramanmaraş) was used. Dried dark red and
hot fruits of these peppers are used as spices. The fruits of this pepper are shown in Figure 1.

Figure 1. The fruits of Kahramanmaraş pepper genotype at the harvesting time.
Anthers were excised from flower buds of greenhouse grown-plant when the corolla was
lightly longer than the calyx, at this stage the majority of microspores were at the late
mononucleate or early binucleate phase.
Two different media compositions were used as the basal medium. One of them was medium C
described by Dumas de Vaulx et al. (1981) with additions 5.0 mg/l 2,4-D and 5.0 mg/l kinetin.
The second medium was MS basal medium with additions 4.0 mg/l NAA and 1.0 mg/l BA.
Except control group 1% activated charcoal was added into the media alone or with 200 ml/l
carrot extract described by Vagera and Havranek (1985). The half of the number of petri dishes
were incubated at 35 o C for 8 days in the dark, than anthers were placed at 25 o C, 30 μE m -2 min
–1 for 4 days and finally the anthers were transferred to medium R containing 0.1 mg/l kinetin.
The other parts of cultures were incubated at 29 o C in the continuous light. Embryos appeared
after 4-6 weeks of culture on the both of the medium. Well developed embryos were
transferred to MS hormone-free medium for further development. Plantlets with normal root
development were transplanted in soil under high humidity.
Results and discussions
A low frequency of androgenesis occurred in vitro in Kahramanmaraş pepper genotype in all
tested media and incubation conditions. The frequency of responding anthers ranged from 0%
to 2.26%. The highest embryo formation was obtained from the anthers which cultured on the
MS medium without charcoal and carrot extract incubated at 29 o C with continuous light
(2.82%) (Table 1).
In the present study, at 29 o C incubation was occurred more effective than the other incubation
regime to induction of embryo formation. The pepper embryos developed from anther cultures
are shown in Fig. 2. Total 37 plants were obtained from the MS medium without AC or CE.
One of them is shown in Fig. 3. The numbers of responding anthers and the frequency of
embryo formation were relatively higher in the DDV medium supplemented, in addition with
charcoal an carrot extract than control. But the embryos in the containing charcoal did not grow
to complete plants. Globular embryoids gradually differentiated to apical and root portions.
The apical portions of the some embryos which obtained from DDV medium containing
charcoal and carrot extract became hypertrophied and a long root grew on the basal part of

plantlets. Some other embryos had no shoots, they had just hairy roots (Fig. 4). They couldn’t
grow to complete plants.
Table 1. Number of cultured anthers, number and frequency of responding anthers and
regenerated embryos and number of mature plants obtained on different media and under
different incubation conditions.
Basal
medium
Adding of
AC and CE
Incubation
regime
N o of cultured
anther
Responding anther
Embryos
Number Frequency(%) Number Frequency(%)
N o of
plants
Control
29 o C 1097 5 0.45 5 0.45 0
35 o C/25 o C 1056 0 0 0 0 0
DDV AC
29 o C 1047 11 1.05 11 1.05 0
35 o C/25 o C 1133 0 0 0 0 0
AC + CE
29 o C 1195 10 0.83 14 1.17 6
35 o C/25 o C 1147 0 0 0 0 0
Control
29 o C 1062 24 2.26 30 2.82 30
35 o C/25 o C 1215 8 0.66 10 0.82 7
MS AC
29 o C 986 0 0 0 0 0
35 o C/25 o C 864 0 0 0 0 0
AC + CE
29 o C 372 0 0 0 0 0
35 o C/25 o C 382 0 0 0 0 0
DDV : Basal medium described by Vaulx et al. (1981); MS: Basal medium described by Murashige and Skoog (1962);
AC: Activated charcoal; CE: Carrot extract
Figure 2. Pollen embryogenesis In Kahramanmaraş pepper genotype on the MS medium
containing 4.0 mg/l NAA and 1.0 mg/l BA at 29 o C in the continuous light condition.

Figure 3. A normal complete plant obtained from the MS medium without AC and CE.
Figure 4. Root formation on anthers in the DDV medium containing AC and CE.
In the experiments of Vagera and Havranek (1985), the low frequency of androgenesis in
Capsicum was increased by using activated charcoal, which not only increased the number of
embryoids, but also prolonged the embryogenic vitality of the culture. In our study, the
concentration of charcoal might be caused unfavorable effect on the anther culture of
Kahramanmaraş pepper genotype. Vagera and Havranek (1985) reported that the embryoids in
anthers in the medium with activated charcoal grew to plants only occasionally. However, they
continued to grow when they were transferred to a medium without activated charcoal.
In conclusion, activated charcoal can improve the yield of androgenesis in some plant species.
But one of the most important factors in anther culture is the genotype of plant material. In our
study, adding of 1% charcoal had positive effect on the frequency of embryo formation from
microspores, but inhibited the growing of embryoids to complete plants. Adding of carrot
extract to the media with charcoal had also no markedly effect on the androgenesis in
Kahramanmaraş Turkish local pepper genotype. The combination of both substances with
DDV basal medium gave some positive results on the embryo formation, but the combination
with MS medium decreased androgenic response.

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