the effect of carrot extract and activated charcoal
the effect of carrot extract and activated charcoal
the effect of carrot extract and activated charcoal
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XI th EUCARPIA Meeting on Genetics <strong>and</strong> Breeding <strong>of</strong> Capsicum & Eggplant, 2001, Antalya-Turkey<br />
THE EFFECT OF CARROT EXTRACT AND ACTIVATED CHARCOAL ON THE<br />
ANDROGENESIS OF PEPPER<br />
1 Ellialtıoğlu Ş, 2 Kaplan F., 3 Abak K.<br />
1 Department <strong>of</strong> Horticulture, Faculty <strong>of</strong> Agriculture, University <strong>of</strong> Ankara, Turkey<br />
2 Department <strong>of</strong> Horticulture, Faculty <strong>of</strong> Agriculture, University <strong>of</strong> Adnan Menderes, Aydın-Turkey<br />
3 Department <strong>of</strong> Horticulture, Faculty <strong>of</strong> Agriculture, University <strong>of</strong> Çukurova, Adana-Turkey<br />
Abstract<br />
In an<strong>the</strong>r culture, one <strong>of</strong> <strong>the</strong> ways to improve embryo formation is to apply <strong>the</strong>rmal shocks at<br />
<strong>the</strong> beginning <strong>of</strong> cultures. In pepper, it is induced <strong>the</strong> embryo formation to keep an<strong>the</strong>rs in <strong>the</strong><br />
dark conditions <strong>and</strong> incubate <strong>the</strong>m at 35 o C in <strong>the</strong> first 8 days. As an alternative, 29 o C <strong>and</strong><br />
continuous light incubation regime is stated. It is known, that <strong>activated</strong> <strong>charcoal</strong> has positive<br />
<strong>effect</strong> on <strong>the</strong> an<strong>the</strong>r culture <strong>of</strong> several plant species included peppers. In this study, an<strong>the</strong>r<br />
culture was carried out in Kahramanmaraş local pepper genotype <strong>and</strong> <strong>the</strong> <strong>effect</strong>s <strong>of</strong> two<br />
different incubation conditions <strong>and</strong> two different basal medium formulations supplemented<br />
with <strong>charcoal</strong> <strong>and</strong> <strong>carrot</strong> <strong>extract</strong> were investigated. The highest embryo formation <strong>and</strong> plant<br />
growing were obtained from <strong>the</strong> an<strong>the</strong>rs which incubated at 29 o C In <strong>the</strong> continuous light<br />
conditions <strong>and</strong> cultured on <strong>the</strong> MS medium without 1% <strong>charcoal</strong> <strong>and</strong> <strong>carrot</strong> <strong>extract</strong>.<br />
Introduction<br />
Pepper is an important vegetable crop <strong>the</strong> economic point <strong>of</strong> view <strong>and</strong> it is used all over <strong>the</strong><br />
world. Turkey is <strong>the</strong> biggest pepper producer country after China <strong>and</strong> Mexico on <strong>the</strong> world.<br />
There are some traditional methods for pepper breeding. But <strong>the</strong> an<strong>the</strong>r culture has advantages<br />
to short breeding time. On <strong>the</strong> o<strong>the</strong>r h<strong>and</strong>, complex genotypes are fixed by completely<br />
homozygous plants obtained through dihaploidization <strong>of</strong> haploids (Bajaj, 1983, Heberle-Bors,<br />
1985, Vagera, 1990). Haploids <strong>and</strong> diploids can be successfully applied to study pepper<br />
resistance to viruses (Vaulx et al., 1982, Pochard et al., 1983) <strong>and</strong> transmission <strong>of</strong> resistance to<br />
Phytophthora capsici (Abak et al., 1982). The experiments following <strong>the</strong> work <strong>of</strong> Wang et al.<br />
(1973) did not succeed in obtaining <strong>and</strong>rogenic plants (Vagera, 1990). Sibi et al. (1979)<br />
described <strong>and</strong>rogenesis morphologically <strong>and</strong> cytologically. They found that uninuclear<br />
microspores were most suitable for inducing <strong>and</strong>rogenesis. By <strong>the</strong> study <strong>of</strong> Vaulx et. al.<br />
(1981), <strong>the</strong> yield <strong>of</strong> <strong>and</strong>rogenesis in pepper was increased by heat treatment (+35 o C) at <strong>the</strong> first<br />
stage <strong>of</strong> an<strong>the</strong>r culture. Munyon et al. (1989) reported that <strong>and</strong>rogenesis occurred in C. annuum<br />
an<strong>the</strong>rs in a continuous warm environment (29 o C) with continuous light. In <strong>the</strong> study <strong>of</strong> Vagera<br />
<strong>and</strong> Havranek (1985) application <strong>of</strong> <strong>the</strong> media supplemented with <strong>charcoal</strong> <strong>and</strong> <strong>carrot</strong> <strong>extract</strong><br />
has been successfully used to stimulate <strong>and</strong>rogenesis in Severka <strong>and</strong> Morava pepper cultivars.<br />
The positive <strong>effect</strong> <strong>of</strong> <strong>charcoal</strong> on <strong>the</strong> an<strong>the</strong>r culture <strong>of</strong> pepper was described by Chunling<br />
(1992) too.<br />
In this study we aimed to improve <strong>the</strong> yield <strong>of</strong> haploid embryos in pepper an<strong>the</strong>r culture by<br />
using different incubation conditions <strong>and</strong> by application <strong>of</strong> <strong>the</strong> two different basal media<br />
formulation supplemented with <strong>charcoal</strong> <strong>and</strong> <strong>carrot</strong> <strong>extract</strong>.<br />
Materials <strong>and</strong> methods<br />
As <strong>the</strong> plant material in <strong>the</strong> experiments a local genotype <strong>of</strong> pepper (Capsicum annuum L.)<br />
grown in South Eastern region <strong>of</strong> Turkey (in Kahramanmaraş) was used. Dried dark red <strong>and</strong><br />
hot fruits <strong>of</strong> <strong>the</strong>se peppers are used as spices. The fruits <strong>of</strong> this pepper are shown in Figure 1.
Figure 1. The fruits <strong>of</strong> Kahramanmaraş pepper genotype at <strong>the</strong> harvesting time.<br />
An<strong>the</strong>rs were excised from flower buds <strong>of</strong> greenhouse grown-plant when <strong>the</strong> corolla was<br />
lightly longer than <strong>the</strong> calyx, at this stage <strong>the</strong> majority <strong>of</strong> microspores were at <strong>the</strong> late<br />
mononucleate or early binucleate phase.<br />
Two different media compositions were used as <strong>the</strong> basal medium. One <strong>of</strong> <strong>the</strong>m was medium C<br />
described by Dumas de Vaulx et al. (1981) with additions 5.0 mg/l 2,4-D <strong>and</strong> 5.0 mg/l kinetin.<br />
The second medium was MS basal medium with additions 4.0 mg/l NAA <strong>and</strong> 1.0 mg/l BA.<br />
Except control group 1% <strong>activated</strong> <strong>charcoal</strong> was added into <strong>the</strong> media alone or with 200 ml/l<br />
<strong>carrot</strong> <strong>extract</strong> described by Vagera <strong>and</strong> Havranek (1985). The half <strong>of</strong> <strong>the</strong> number <strong>of</strong> petri dishes<br />
were incubated at 35 o C for 8 days in <strong>the</strong> dark, than an<strong>the</strong>rs were placed at 25 o C, 30 μE m -2 min<br />
–1 for 4 days <strong>and</strong> finally <strong>the</strong> an<strong>the</strong>rs were transferred to medium R containing 0.1 mg/l kinetin.<br />
The o<strong>the</strong>r parts <strong>of</strong> cultures were incubated at 29 o C in <strong>the</strong> continuous light. Embryos appeared<br />
after 4-6 weeks <strong>of</strong> culture on <strong>the</strong> both <strong>of</strong> <strong>the</strong> medium. Well developed embryos were<br />
transferred to MS hormone-free medium for fur<strong>the</strong>r development. Plantlets with normal root<br />
development were transplanted in soil under high humidity.<br />
Results <strong>and</strong> discussions<br />
A low frequency <strong>of</strong> <strong>and</strong>rogenesis occurred in vitro in Kahramanmaraş pepper genotype in all<br />
tested media <strong>and</strong> incubation conditions. The frequency <strong>of</strong> responding an<strong>the</strong>rs ranged from 0%<br />
to 2.26%. The highest embryo formation was obtained from <strong>the</strong> an<strong>the</strong>rs which cultured on <strong>the</strong><br />
MS medium without <strong>charcoal</strong> <strong>and</strong> <strong>carrot</strong> <strong>extract</strong> incubated at 29 o C with continuous light<br />
(2.82%) (Table 1).<br />
In <strong>the</strong> present study, at 29 o C incubation was occurred more <strong>effect</strong>ive than <strong>the</strong> o<strong>the</strong>r incubation<br />
regime to induction <strong>of</strong> embryo formation. The pepper embryos developed from an<strong>the</strong>r cultures<br />
are shown in Fig. 2. Total 37 plants were obtained from <strong>the</strong> MS medium without AC or CE.<br />
One <strong>of</strong> <strong>the</strong>m is shown in Fig. 3. The numbers <strong>of</strong> responding an<strong>the</strong>rs <strong>and</strong> <strong>the</strong> frequency <strong>of</strong><br />
embryo formation were relatively higher in <strong>the</strong> DDV medium supplemented, in addition with<br />
<strong>charcoal</strong> an <strong>carrot</strong> <strong>extract</strong> than control. But <strong>the</strong> embryos in <strong>the</strong> containing <strong>charcoal</strong> did not grow<br />
to complete plants. Globular embryoids gradually differentiated to apical <strong>and</strong> root portions.<br />
The apical portions <strong>of</strong> <strong>the</strong> some embryos which obtained from DDV medium containing<br />
<strong>charcoal</strong> <strong>and</strong> <strong>carrot</strong> <strong>extract</strong> became hypertrophied <strong>and</strong> a long root grew on <strong>the</strong> basal part <strong>of</strong>
plantlets. Some o<strong>the</strong>r embryos had no shoots, <strong>the</strong>y had just hairy roots (Fig. 4). They couldn’t<br />
grow to complete plants.<br />
Table 1. Number <strong>of</strong> cultured an<strong>the</strong>rs, number <strong>and</strong> frequency <strong>of</strong> responding an<strong>the</strong>rs <strong>and</strong><br />
regenerated embryos <strong>and</strong> number <strong>of</strong> mature plants obtained on different media <strong>and</strong> under<br />
different incubation conditions.<br />
Basal<br />
medium<br />
Adding <strong>of</strong><br />
AC <strong>and</strong> CE<br />
Incubation<br />
regime<br />
N o <strong>of</strong> cultured<br />
an<strong>the</strong>r<br />
Responding an<strong>the</strong>r<br />
Embryos<br />
Number Frequency(%) Number Frequency(%)<br />
N o <strong>of</strong><br />
plants<br />
Control<br />
29 o C 1097 5 0.45 5 0.45 0<br />
35 o C/25 o C 1056 0 0 0 0 0<br />
DDV AC<br />
29 o C 1047 11 1.05 11 1.05 0<br />
35 o C/25 o C 1133 0 0 0 0 0<br />
AC + CE<br />
29 o C 1195 10 0.83 14 1.17 6<br />
35 o C/25 o C 1147 0 0 0 0 0<br />
Control<br />
29 o C 1062 24 2.26 30 2.82 30<br />
35 o C/25 o C 1215 8 0.66 10 0.82 7<br />
MS AC<br />
29 o C 986 0 0 0 0 0<br />
35 o C/25 o C 864 0 0 0 0 0<br />
AC + CE<br />
29 o C 372 0 0 0 0 0<br />
35 o C/25 o C 382 0 0 0 0 0<br />
DDV : Basal medium described by Vaulx et al. (1981); MS: Basal medium described by Murashige <strong>and</strong> Skoog (1962);<br />
AC: Activated <strong>charcoal</strong>; CE: Carrot <strong>extract</strong><br />
Figure 2. Pollen embryogenesis In Kahramanmaraş pepper genotype on <strong>the</strong> MS medium<br />
containing 4.0 mg/l NAA <strong>and</strong> 1.0 mg/l BA at 29 o C in <strong>the</strong> continuous light condition.
Figure 3. A normal complete plant obtained from <strong>the</strong> MS medium without AC <strong>and</strong> CE.<br />
Figure 4. Root formation on an<strong>the</strong>rs in <strong>the</strong> DDV medium containing AC <strong>and</strong> CE.<br />
In <strong>the</strong> experiments <strong>of</strong> Vagera <strong>and</strong> Havranek (1985), <strong>the</strong> low frequency <strong>of</strong> <strong>and</strong>rogenesis in<br />
Capsicum was increased by using <strong>activated</strong> <strong>charcoal</strong>, which not only increased <strong>the</strong> number <strong>of</strong><br />
embryoids, but also prolonged <strong>the</strong> embryogenic vitality <strong>of</strong> <strong>the</strong> culture. In our study, <strong>the</strong><br />
concentration <strong>of</strong> <strong>charcoal</strong> might be caused unfavorable <strong>effect</strong> on <strong>the</strong> an<strong>the</strong>r culture <strong>of</strong><br />
Kahramanmaraş pepper genotype. Vagera <strong>and</strong> Havranek (1985) reported that <strong>the</strong> embryoids in<br />
an<strong>the</strong>rs in <strong>the</strong> medium with <strong>activated</strong> <strong>charcoal</strong> grew to plants only occasionally. However, <strong>the</strong>y<br />
continued to grow when <strong>the</strong>y were transferred to a medium without <strong>activated</strong> <strong>charcoal</strong>.<br />
In conclusion, <strong>activated</strong> <strong>charcoal</strong> can improve <strong>the</strong> yield <strong>of</strong> <strong>and</strong>rogenesis in some plant species.<br />
But one <strong>of</strong> <strong>the</strong> most important factors in an<strong>the</strong>r culture is <strong>the</strong> genotype <strong>of</strong> plant material. In our<br />
study, adding <strong>of</strong> 1% <strong>charcoal</strong> had positive <strong>effect</strong> on <strong>the</strong> frequency <strong>of</strong> embryo formation from<br />
microspores, but inhibited <strong>the</strong> growing <strong>of</strong> embryoids to complete plants. Adding <strong>of</strong> <strong>carrot</strong><br />
<strong>extract</strong> to <strong>the</strong> media with <strong>charcoal</strong> had also no markedly <strong>effect</strong> on <strong>the</strong> <strong>and</strong>rogenesis in<br />
Kahramanmaraş Turkish local pepper genotype. The combination <strong>of</strong> both substances with<br />
DDV basal medium gave some positive results on <strong>the</strong> embryo formation, but <strong>the</strong> combination<br />
with MS medium decreased <strong>and</strong>rogenic response.
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