Abstract: The present study was aimed to develop a novel protocol for the in vitro induction of callus for the production of capsaicin from Capsicum annuum L. For callus production young leaves, growing shoots, nodal region from the sterile germinated seedlings and placental regions and pericarp tissue from the fruit pods were used as explants. They were cultured on MS Medium supplemented with the various combinations of GA, IAA, NAA, 2, 4-D and Kin. Of all tried combinations of growth hormones, MS Medium with 2.0 mg L1 2, 4-D and 0.5 mg L1 Kin was producing significant callus induction and proliferation in placental explants. The placental callus extract was taken for the estimation of capsaicin by colorimetric method. Extract had 1.6 mg1 of capsaicin g1 fresh weight of the callus. This could be an efficient protocol for capsaicin production from the placental calli and used for the large scale commercial production of capsaicin.
INTRODUCTION
The genus Capsicum is a member of the Solanaceae family that includes tomato, potato, tobacco and petunia. The genus Capsicum consists of approximately 22 wild species and five domesticated species (Bosland, 1994). The five domesticated species are C. annuum L., C. baccatum L., C. chinense, C. frutescens L. and C. pubescens. Capsicum annuum L. is one of the major vegetable and spice crops grown world wide. It has become indispensable in every Indian home and also used medicinally, chutneys and pickles. Besides being an important food crops, Chilli peppers are used in pharmaceutical industries. Capsaicin, the main alkaloid responsible for pungency in chilies is used as a counter irritant balm form external application and it is also used in creamy to provide enhanced pain relief for arthritis patients.
The first attempt for the industrial production of secondary metabolites in vitro was made during 1950 to 1960 by the Pfizer Company and the first patent was obtained in 1956 by Routien and Nickell. Several kinds of bioreactors have been designed for large-scale cultivation of plant cells. In several cases cell cultures have been shown producing certain metabolites in quantities equal to (Kaul and Staba, 1967) or many fold greater than (Zenk, 1978) the parent plant. The level of pungency of the Capsicum species depends upon the concentration of capsaicinoids, primarily of capsaicin in the fruit. Capsaicin is the common name for 8- methyl N-vanillyl 6 nonamide, the chemical component most well known for its inhabitance in the internal white ribs of chili peppers and its ability to produce the burning hot sensation of chili-laced spicy foods. The present study was aimed at the production of capsaicin because of its reported medicinal effects such as carminative, digestive irritant, stomachic, stimulant, rubefacient and tonic (Simon et al., 1984). The plants have also been used as folk remedies for dropsy, colic, diarrhea, asthma, arthritis, muscle cramps and toothache. The in vitro production of secondary metabolites in plants can be regulated by the growth hormone used. Researchers in disciplines such as genetics and breeding, agriculture and technology have been interested in Capsicum annuum to develop new varieties with combinations of different optimal levels of the stimuli to maximize production of storable products for specific end uses. Physiologists have been intensely studying the action of the highly potent pungency stimuli. The compositional variations, biosynthesis of the functional components, the carotenoids, the volatile and the capsaicinoids are comprehensively reviewed (Govindarajan, 1986). Hence the present study focused on callus induction and production of capsaicin from Capsicum annuum through in vitro culture methods.
MATERIALS AND METHODS
The present research was carried out in the Biotechnology Laboratory at Prince Shri Venkateshwara Arts and Science College, Chennai (India) during August 2006 and January 2007.
Medium and Growth Hormones
The culture medium used for the present study was Murashige and Skoog (1962)
Basal Medium. The medium was prepared by dissolving 4.27 g of MS Basal Medium
which contains all the nutrients in 1 L of distilled water. In addition 0.44
g of calcium chloride, 3% sucrose and 0.8% agar were added to 1 L of the medium
at pH 5.5.
The different growth hormones used were GA, IAA, NAA, 2,4-D, Kin. The stock solution was prepared as shown in Table 1. The growth hormones were filter-sterilized using Whatman filter Paper and a syringe filter.
Plant Material
Fertile seeds were collected from the pods of Capsicum annuum L.
and were surface sterilized using 70% ethanol for 60 sec and sodium hypochlorite
for 2 min followed by washes with sterile distilled water. The sterilized seeds
were dried in a filter paper and inoculated in MS medium containing gibberillic
acid at various concentrations. The explants were collected from the germinated
sterile seedlings. Young leaves, growing shoots, nodal regions, were selected
as explants. The placental regions and pericarp tissue from the pods were also
used as explants.
Culture Initiation
The in vitro cultivation of plant tissues was done in a laminar air
flow chamber. Absolute aseptic conditions were maintained to avoid microbial
contamination.
Germination
The sterilized seeds were carefully taken and placed on the MS Medium slant
in test tubes with GA. The inoculation was done inside the laminar air flow
chamber. The tubes after inoculation were placed in light for 16 h provided
by cool white light at 25±2°C.
| Table 1: | The prepared growth hormones concentrations |
| GA: Gibberellic acid; NAA: a-naphtalene acetic acid; IAA: Indole-3-ethanoic acid; 2, 4-D: 2, 4-dichlorophenoxyethanoic acid; Kin: Kinetin 6-furfurylaminopurine | |
Leaf Explants Preparation
The sterile germinated seedlings were collected from the test tubes inside
the laminar air flow chamber. The seedlings were cut with sterile blade for
leaves and nodal regions of the stem. The explants were washed with 0.2% mercuric
chloride for 2-3 min. The explants were then washed in sterile distilled water
thrice to remove all the residual contents of mercuric chloride.
Inoculation of Leaf and Stem Explants
The leaves were taken and their edges near to midrib were trimmed and cut
into bits of 1 cm. Each bit of leaf explant was then placed into the test tubes
containing the culture media of different concentrations of growth hormones.
The stem was cut into small pieces of about 2-3 cm in length and inoculated
similarly.
Inoculation of Placental Regions
The pods of Capsicum annuum L. were surface sterilized and was cut
longitudinally with the sterile blade to expose the placenta. The placenta was
cut along the inner pericarp and made into small bits of 1 cm length. These
were then placed into the test tubes with media using sterile forceps at 1-3
bits in a single tube.
Inoculation of Pericarp Tissue
The sterilized pods of Capsicum annuum L. were bisected longitudinally
and the seeds and placenta were removed carefully after removing 4 mm portion
at the two ends of the fruits. The pericarp tissue was cut into pieces (1x1
cm) and used for inoculation.
Callus Initiation
The explants were tried for callus induction in different concentrations
and combinations of growth hormones.
Subculture
The responding cultures were sub cultured at 3 week culture period into
fresh set of media and maintained under the same culture conditions.
Observation and Data Recording
The callus induction and proliferation were monitored at weekly intervals.
The growth hormone combinations and the responses were also recorded.
Estimation of Capsaicin from Callus
Capsaicin was quantitatively estimated according to the method of Sadasivam
and Manickam (1996).
RESULTS AND DISCUSSION
Plant cell culture offers a promising approach for large scale production of phytochemicals and has several advantages over whole plant production. Callus initiation involves three major considerations: selection of explant, medium and culture conditions (Hall et al., 1988). Capsaicin (8-methyl-N-vanillyl-6-noneamide), the principal pungent capsaicinoid, found only in the fruits of Capsicum species, is mainly synthesized in placenta and later transported to the other parts of the fruit (Iwai et al., 1979). Hypocotyl explants exhibits significantly better morphogenetic potential than other parts of the fruit (Pandeva and Simeonova, 1992), but their suspension cultures produce less capsaicin than fruits (Holden et al., 1987). Thus, in order to produce capsaicin commercially, it is desirable to get friable callus in sufficient amounts from high capsaicin synthesizing placenta tissue of highly pungent variety to obtain high capsaicin producing cell suspensions.
| Table 2: | Germination and callus induction from leaf, stem, pericarp and placental explants |
Germination of Seeds
Gibberellic Acid (GA) is a naturally occurring plant growth regulator which
may cause a variety of effects including the stimulation of seed germination
in some cases (Kabar, 1998). Germination of seeds was observed in MS Medium
with 0.4 and 0.5 mg L-1 after 5 days of inoculation. But all the
seeds inoculated were germinated in 1.0 mg L-1 GA concentration within
3 days (Table 2).
Callus Induction and Proliferation
Growth regulator concentrations in culture medium were critical for the
control of growth and morphogenesis. Generally high concentrations of auxins
and low concentration of cytokinins in the medium promote abundant cell proliferation
with the formation of callus (Mohammad et al., 2003). Different explants
were cultured in vitro to find out the most suitable explant for callus
induction. Callus induction was observed in tubes with different concentrations
of growth hormones (Table 2). MS Medium with 2 mg L-1,
2-4 D and 0.5 mg L-1 kinetin was found to be optimal for callus induction
and it gave a callus of 2.5 g fresh weight from single placental explants in
about 30 days (Fig. 1). The result of the present work was
similar to Rabindra et al. (2003). Mohammad et al. (2003) was
found out that 2-4 D, IAA, BAP and kin were the most suitable growth hormones
for the callus induction of wheat. Present study was very similar to Mohammad
et al. (2003) except few growth hormone additions such as BAP and IAA.
Estimation Capsaicin
Capsaicin, an alkaloid, was used mainly as a pungent food additive in formulated
foods. It was obtained from fruits of green pepper (Capsicum sp.). Capsaicin
was also used in pharmaceutical preparations as a digestive stimulant and for
rheumatic disorders (Sooch et al., 1977).
In callus cultures derived from pericarp and seedling explants of four varieties of Capsicum annuum viz., Punjab Surkh, Punjab Guccheder and Sweet Chilli that in Punjab Lal, the capsaicin content in seedling derived callus culture (7.28 mg g-1 dry weight) was comparable to that in fruits (7.00 mg g -1 dry weight). Capsaicin content in pericarp derived callus cultures of all the varieties was much higher than that in seedling-derived callus cultures and in fruits (Varindra et al., 2000).
| Fig. 1: | Placental derived callus after 30 days of culturing |
| Fig. 2: | Estimation of capsaicin from placental callus |
Since the placental callus fresh weight was very high when compared to the stem explant, leaf explant and pericarp explant it was further taken for capsaicin estimation. A standard graph was plotted by using known concentration of capsaicin along the X-axis and absorbance along the Y-axis (Fig. 2). The amount of capsaicin present in the sample was calculated by using the standard graph. The amount of capsaicin in the placental extract was found to be 1.6 mg g-1 fresh weight of the callus. Sudha and Ravishankar (2003) found out that salicylic acid and methyl jasmonate individually enhance capsaicin production but when administered in combination there was no further enhancement in capsaicin production in cell suspension cultures of Capsicum frutescence. We have also worked out for the capsaicin production from callus of Capsicum annuum in a different approach.
Plant tissue culture is a noble approach to obtain their substances in large scale. Many companies in India and abroad are showing interest in this direction. Tissue culture is an alternative way for the production of phytochemical of therapeutic importance. The results obtained in present study could also serve as a potential alternate source for the large scale production of capsaicin for pharmaceutical purposes. Further, the vital enzymes in the biosynthetic pathways of capsaicin can be studied.