Although previous results have conï¬rmed physiological and morphological effects of light quality, responses vary according to plant species and one cannot determine certain effects of light quality. However in case of Capsicum annum no prior reports have published on effect of LED on Capsaicinoid content in fruit. The effects of light and temperature on capsaicinoid have accumulation have also been studied and they reported that chili pepper plants accumulated more capsaicinoids under continuous fluorescent light and temperature than pepper plants kept 18 h at 28C/6 h at 16C (light/dark) cycles Murakami et al. (2006). The major objective of this study was, to examine the effects of blue, red, blue plus red LEDs and normal light on morphology, primary metabolites (total sugar, reducing sugar, starch, total protein, total amino acid, phenol, chlorophyll and carotenoids) and concentration of capsacinoids.
2.1. Materials
Seeds of chilli pepper (Capsicum annuum L. cv. Manna, not released variety) were surface sterilized with 4% (v/v) sodium hypochlorite then washed with 70% ethanol after that again washed thoroughly with sterile distilled water. The Capsicum seeds first germinated on petridish, with water to induce germination. Germinated seeds were then grown in a growth chamber in pots containing sterilized soil. The seedlings were irrigated with water once a day. At the same time, seedlings were also irrigated with Hoagland's solution (pH 6.7) once to prevent mineral deficiency. At the eight-leaf stage, the plantlets were transferred to different chambers containing different LED light at growth chamber room facility in Konkuk University Seoul, South Korea.
2.2. Lighting system and culture conditions
8 leaf stage plantlets were transferred in growth room at 25oC and 70% relative humidity. There were four types of LED light were maintained in: (1) Normal light (provided by white cool florescent lamps; Phillips, Fluotone 40 W), (2) red LED (peak wavelength: 660 nm), (3) blue LED (peak wave- length: 450 nm) and red plus blue (1:1 photon flux density) LED. The LED system was installed by ( ….), Seoul, Korea. The LED system consisted of LED panel, and a time controller for maintaining the photoperiod and light intensity. The spectral distributions of each LEDs were set with the help of spectroradiometer (Li-1800, LI-COR, Lincoln, Nebraska, USA). The photoperiod times of LEDs were 16 h light and 9 h Dark.
Data collection
For each LEDs treatment there were 15 plants were maintained around 90 days. From each light, 3 plants were harvested at the after 2, 4 and 6 weeks for morphological and biochemical analysis. All morphological characteristic like plant height, number of leaf and size, petiole length, root length, were recorded. The dry weight was determined after drying for 48 h at 70C.
Extraction and estimation of chlorophyll
The amounts of chlorophyll under different LEDs were estimated using the method reported by Arnon (1949). About 100 mg of leaf sample were grind in 10 ml of 80% acetone, mixed well and kept at 4°C overnight in dark. Supernatant was withdrawn after centrifugation at 5000 rpm for 10 min. The supernatants were collected and absorbance was recorded at 663.8 and 646.8 nm in Spectrophotometer ( ) for estimation of Chl a, Chl b and total Chl in Capsicum.
Chlorophyll content: (20.2 x OD645nm + 8.02 x OD663nm) x Dil. Factor
Extraction and estimation of total leaf protein
The total leaf protein was extracted according to Parida et al. (2002). The chilli fruit was extracted for protein by using 0.5mM Phosphate buffer pH of 7.2 and estimated following the method of Lowry et al. (1951). The estimation of total protein was determined by a comparison of the values obtained with a standard curve of Bovine serum albumin (Fraction V, Sigma).
Total Carbohydrate - Phenol Sulphuric acid method
The total amounts of carbohydrate were estimated by using hot acid medium. In this method young matured leaves and Chilli pods were grind and digested directly with 2.5N HCL. The carbohydrate dehydrated into hydroxyl methyl furfural and produced a colour was estimated by spectrophotometer (Shimadzu UV 160A) at 490 nm wavelength as described by Sadasivam and Manickam (1992).
Reducing Sugars (Nelson-Somogyi method)
The reducing sugar from young leaf samples of Capsicum were estimated by previously described method (Nelson-Somogyi method as described by Sadasivam and Manickam 1992). The blue colour developed is read at 620 nm against glucose standard.
Starch by Anthrone reagent method
Starch from youngest matured fresh leaves and homogenized fruit samples were estimated by the Anthrone method as described by Sadasivam and Manickam (1992). To remove sugars, the chilli/paprika was treated with 80% alcohol. Then starch is extracted with perchloric acid. In hot acid medium, starch is hydrolyzed to glucose and dehydrated to hydroxyl methyl furfural. This compound forms a green coloured product with Anthrone and has the maximum absorption at 630nm.
Total free amino acids estimation
100mg of fresh leaf samples were first extracted with 80% of alcohol then treated with ninhydrin reagent (Moore and Stein 1948). The produced bluish purple color which was developed by oxidizing agent decarboxylates was read at 570 nm (Sadasivam and Manickam., 1992). The total amino acids of different LED treated plants were estimated by a using a standard curve of Methionine.
Estimation of total polyphenols
Total phenolics were extracted from leaf of Capsicum by 80% ethanol and estimated, according to previously described method by Parida et al. (2002) using Folin-Ciocalteau reagent. The total phenol was measured at 660nm and calculated by comparison with a standard curve value of Gallic acid. Results were expressed as grams of Gallic acid per gram of fresh weight.
Sugar determination
After 50 days of flowering Capsicum fruits were harvested from different LEDs treatment and sugars (glucose, fructose) and sucrose concentrations determined in the water-soluble fraction, by HPLC (Shimadzu). Samples were passed through a Sep-Pak C18 cartridge, preconditioned with methanol (4 ml) and water (10 ml), to remove interfering compounds. Before use, the residual water in the cartridge was expelled with air. The first 2 ml of sample was discarded and the next 1 ml was used for analysis, after filtration through a 0.45-μm Millipore filter. HPLC analysis was performed using Eclipse 250x4.6 mm column, coupled with a UV detector at the 254nm. The mobile phase was acetonitrile: water (85:15), with a flow rate of 1.0 ml min−1.
Statistical analysis
The experiment was conducted in a simple randomized block design (RBD). A total of three replicates for each treatment were taken. Treatment means were compared by analysis of variance (ANOVA) using SPSS.10 (SPSS, Chicago, IL, USA). Least significance difference (LSD) was calculated at the 1% level of probability. The treatment means were separated by different letters.
Result and discussion
Effect of LEDs on Capsicum morphology
Different light irradiations signiï¬cantly affect the growth
of in vitro cultured Doritaenopsis plants. Leaf and root
fresh weight/dry weight, leaf area and leaf length values
are presented in Tables 1 and 2. Fresh weight/dry weight
of leaf as well as root was increased in plants grown under red and blue combined light (Table 1). Under this
light treatment, maximum leaf and root dry weights were
54 mg g-1
FW and 70 mg g-1
FW, respectively, after
8 weeks of in vitro culture, whereas these values were
comparatively low in the plants grown under other treat-
ments. Root growth was optimum in plants grown under
blue light and roots continuously grew with lapse of time.
However, root growth was hindered in plants grown under
red LED treatment. Gradual increase in leaf area was
observed in the plants cultivated under all light treatments
during the culture period (Table 2). Greatest leaf area was
in the plants cultured under red plus blue LED (10 cm2
);
it was 1.4 times more than in the plants grown under
fluorescent light. The leaf length was optimum (6.1 cm) in
plants that were grown under red LED (Table 2)as
compared to those with other light treatments. In contrast blue LED inhibited leaf expansion. Our results show that
the biomass and leaf growth of Doritaenopsis plants were
signiï¬cantly increased under red plus blue LED treat-
ments as compared with fluorescent and monochromic
light (red or blue LED) treatments. This may be due to the
maximum photosynthetic efï¬ciency of plants grown under
red and blue LED, and red plus blue LED wave-
lengths closely coincide with the absorption peaks of
chlorophylls.
The results of analysis of total Chlorophyll, Chl-a, Chl-b
and carotenoids are presented in Table 3. It was observed
that the amount of total Chl, Chl-a, Chl-b and carotenoids
was highest in the plants grown under red plus blue LED
treatments, followed by blue LED and fluorescent light
treatments. 3.1 mg g-1
FWof Chl-a, 1.8 mg g-1
FWChl-b,
0.5 mg g-1
FW carotenoids was measured in the plants grown under the red plus blue LED after 8 weeks of culture
(Table 3). Red LED treatment led to a signiï¬cant reduction
in chlorophyll contents and it was 1.5 times lower compared
with red plus blue LED treatment. However, the Chl a/b
ratios did not vary signiï¬cantly with all light treatments.
Similar to the present results, lowest chlorophyll accumu-
lation was reported in lettuce, spinach, mustard and wheat
plants grown under red light sources by Tibbitts et al. (1983)
and Tanaka et al. (1998). A higher starch content of 11.2 ± 0.5 and 30.2 ±
0.6 mg g-1
FW was found in both leaves and roots,
respectively, after 8 weeks of culture in the plants grown
under red plus blue LED treatments (Fig. 1), followed by
blue LED and fluorescent light treatments, while the lowest
amount was observed in the plants grown under red LED.
Sucrose concentration was also highest in leaves of the
plants grown under red plus blue LED than in the leaves of
plants that had other treatments. Sucrose concentration of
roots decreased signiï¬cantly in plants grown under all LED
treatments. Glucose and fructose contents gradually
decreased in leaves grown under all light treatments
(Fig. 2). Glucose concentration was gradually increased in
roots up to 5 weeks and reduced there afterwards. Fructose
concentration was more in the roots of plants grown under
fluorescent light treatment, compared with other light
treatments.
Our results clearly demonstrate that spectral quality of
light influences the morphogenesis and diverse physiolog-
ical responses of Doritaenopsis plants. Growth,
carbohydrate accumulation and pigment biosynthesis of
plants were signiï¬cantly increased in plants grown under
red plus blue LEDs as compared with the fluorescent light.
Additionally, in these plants blue LED effectively
