The measurement of growth parameters was done on the plants which are grown in the small pits. The plants were transfer to other pits to study the uniform growth of the plants. The parameters such as plant height, leaf width and number of leaflets per plant were measured and it is observed that the growth of all plants was uniform (Table 1 and Fig. 2) and the same plants were exposed to UV radiations in UV chambers at different time intervals (Fig. 3).
Growth parameters of Capsicum species with different chili genotype
No. of plants
No. of seeds
Effect of Induced Stress
Estimation of Proteins
The amount of protein in control was calculated on the basis of the standard graph (y = 0.0017x + 0.0118
R² = 0.9921) obtained in Capsicum species. Similarly, the least protein content was found in 60 min mutated plant and had 799.997 µg/ml of protein that showed decrease contents compared to the other plants (Fig. 4). This might be because of damage existed in peptide bonds formed between the a-amino group of one amino acid and the a-carboxyl group of other amino acid corrosive that avoided in yielding a dipeptide and then polypeptide. Thus, polypeptide chain unfurls from various conformations in the protein giving distinctive basic structures.
Estimation of Carbohydrates
The amount of carbohydrate in control was calculated on the basis of the standard graph (y = 0.0404x + 0.0142
R² = 0.9847) obtained in Capsicum species. Similarly, the least carbohydrate content was found in 60 min mutated plant and had 39.13 µg/ml of carbohydrate that showed decrease contents compared to the other plants (Fig. 5). This might be because of harm existed in both straight and spread polymer of homopolysaccharides f framed inside the layer bound cell organelles that counteracted in keeping up trademark appearance and shape. Additionally, harms happened in cellulose by detaching together by b (1®b) linkages. These chains are straightforwardly separated to each other by intermolecular and intramolecular hydrogen bonds and Vander Waals force.
Estimation of Lipids
Lipids or free fatty acids activity was calculated by the formula given for each of the sample in the Capsicum species. Minimum free fatty acid percentage was seen in 673.2 µg/ml concentration in 60 min post mutated samples. Hence, the present study revealed that 60 min of the post mutated samples is required for normal growth of the plant with the minimum lipid content (Fig. 6). This might be because of destruction existed in covalent bonds formed in the long-chain saturated and unsaturated fatty acids that capacities as damage to plant from water gain and from rough harm. Along these lines long chain unfurls from various compliances in the lipids giving distinctive straightforward structures.
Estimation of Enzyme Activity
Estimation of Stress Enzymes by Peroxidase activity
The highest percentage of inhibition by the peroxidase activity (0.1685 μg/ml) was found in 50 µl mutated plants of Capsicum species that revealed increase activity compared to the other samples (Fig. 7). In this manner, Ultraviolet-C radiation initiated oxidative worry in Chilli by expanding lipid peroxidation and layer porousness which demonstrates that points of confinement of resistance are substantially less than wounded caused by UV radiation . In this way, the peroxidase activity is additionally a vital part of the antioxidant stress system for scavenging H2O2. However, catalase changes H2O2 into O2, while peroxidase breaks down H2O2 by the oxidation of co-substances .
Estimation of Stress Enzymes by Catalase activity
The highest percentage of inhibition by the catalase activity (0.078 μg/ml) was found in 60 µl mutated plants of Capsicum species that showed increased activity compared to the other samples (Fig. 8). Oxidative stress is supplemented by the amalgamation of hydrogen peroxide, which is typically detoxified by catalase activity in the peroxisomes and by ascorbate peroxidase in the cytosol, mitochondria, and chloroplasts of the Capsicum species .
Estimation of free radical scavenging activity of Diphenyl-1-picrylhydrazyl (DPPH) assay
The highest percentage of inhibition by the DPPH activity (68.57 μg/ml) was found in 50 µl mutated plants of Capsicum species that indicated increased activity compared to the other samples (Fig. 9). After UV exposure, the capacity of marginally higher DPPH radical scavenging capacity was found in UV-C treated plants. UV-C treatments did not cause checked modifications in DPPH radical scavenging capacity. UV-C treated peppers kept up somewhat higher DPPH radical scavenging capacity mainly when the appearance of the disorder was already advanced. In this way, the UV-C treatment expanded aggregate cell antioxidants [30-31].
Likewise, a decrease in biochemical synthesis of the peppers treated with a UV-C radiation was accounted for by Vicente et al. . UV-C treatments diminished the occurrence of Chilling damage and seriousness. UV-C treated leaves also exhibited lower electrolyte discharge, respiration rate and phenolic compound substance recommending lower injury in response to low temperature stockpiling. The UV-C treatments could be a valuable method for decreasing decay and maintaining bell pepper fruit quality. Moreover, Chilling damage occurrence and seriousness could be decreased by short UV-C treatments. They initially evaluated if a higher dose might be even more beneficial. On analysing the DPPH radical scavenging capacity, immediately after UV-C treatment, distinctions were recognized between control and treated plants .
Estimation of free radical scavenging activity of 2, 2’- azino-bis (3-ethylbenzoithioazoline-6-sulphonic acid) (ABTS) assay
The highest percentage of inhibition by the ABTS activity (97.88 μg/ml) was found in 50 µl mutated plants of Capsicum species that exhibited increased activity compared to the normal plants (Fig. 12). Enzymes for example, polyphenol oxidase, ascorbate peroxidase, and glutathione reductase in addition to the peroxidase and catalase demonstrated upgraded action in UV-C treated plants and these enzymes proteins may fill in acclimatization systems to scavenge the harmful free radicals of oxygen created under pressure condition. The results of the present work illustrate that in Capsicum species, UV-C radiation generates antioxidant substances that provide protection against UV-C radiation. Along these lines it very well may be presumed that UV radiation induced both enzymatic and nonenzymatic activities that secure plants against UV radiation in a given dose in Capsicum plant species.
Besides, Ultraviolet-C radiation is a possibly harmful, physical mutagenic agent and shape an important component of terrestrial radiation to which plants have been uncovered since attacking area . From that point forward, plants have advanced components to maintain a strategic distance from and fix from UV radiation injury. Most of the compounds accumulated are directly involved in UV-C protection; they are either efficient in filtering excess radiation or in scavenging radicals. The substance of chlorophyll a, b and carotenoids of pepper leaves were decreased significantly in those plants which were presented to UV-C radiation and contrasted with control plants. Thus, Ultraviolet-C radiation induced oxidative stress in Chilli by increasing lipid peroxidation and membrane permeability which indicates that limits of tolerance are much less than damaged caused by UV-C radiation . Likewise, the impact of UV-C radiation on chlorophyll, flavonoids, anthocyanin, proline, membrane permeability, lipid peroxidation and UV-absorbing compound was analysed with reference to the Capsicum species. The seeds were treated with different concentration of gamma rays and the effect of gamma rays on phytochemical constituents in Chilli was considered on M2 generation. The phytochemical constituents induced chlorophyll, Capsaicin, Oleoresin, Capsanthin and Ascorbic acid .
The impacts on the structure and ultrastructure of Chilli plants when presented to UV radiation under greenhouse conditions. Changes in shoot development demonstrated the critical decline in UV-C exposed plants. Leaf area also diminished altogether in UV-C-exposed plants. Stomata expanded in number and size in UV-C exposed plants. Chloroplast thylakoids were enlarged and starch decrease was seen at the ultra-basic dimension. UV treatment resulted in the formation of crystals in the peroxisomes of mesophyll cells. The development of these crystals was due to an increase in catalase activity, which is an antioxidant enzyme. The Chilli plants were delicate to UV and the discoveries gave the knowledge into the physiological changes during UV exposure, and showed this plant was progressively touchy to UV-C radiation .
Genomic DNA Isolation
In AFLP strategy, a total genomic DNA is processed with two restriction enzymes and adaptors of known sequence are then ligated to the DNA fragments. Primers complementary to the adaptors, with extra 1-3 selective nucleotides on the 3’ end are utilized to intensify the restriction fragments. The PCR amplified parts would then be able to be isolated by gel electrophoresis and the banding patterns visualized. AFLP profiles require no earlier DNA sequence information and the number and nature of amplified fragments are adjusted by the decision of primer pair.
The DNA of Capsicum species is isolated from cells in stepwise manner. DNA was released from the cells and nucleus (Fig. 11). All the chemicals and buffers aids for the release and purification of DNA. After extraction of pure DNA from samples, DNA is quantified using agarose gel electrophoresis and the DNA fragments/ bands are observed under UV light (Fig. 12). The purified DNA fragments are double digestion using the enzyme ECORI and MSEI and the bands are observed under UV light (Fig. 13).
This enzymatic can be used for cleaving DNA molecule at specific sites, ensuring that all DNA fragments that contain a particular sequence at a particular location have same size, furthermore each fragment that contain the desired sequence has the sequence located at exactly the same position within the fragment it). The resulting digested DNA is very selectively amplified using PCR reamplification (Fig. 14).
A dendrogram showing similarity in the DNA bands of Capsicum species based on the scoring of unexposed and exposed plants in six different time intervals of UV-C radiations with three major clusters showed significant negative affinities. 50-60 minutes of UV-C radiations exposed plants accounted for low scoring of DNA bands belonging to the first cluster; 10 and 40 minutes of UV-C radiations exposed plants of DNA bands in addition to the 20-30 minutes of UV-C radiations exposed plants of DNA bands accounted for moderate scoring in the second cluster, whereas normal plants without exposure of UV-C radiations accounted alone for the third cluster with a high scoring of DNA bands (Fig. 15).
Henceforth, there is a little variety in the occurrence of DNA bands of Capsicum plant mutated at 50-60 minutes intervals after the UV-C radiation exposure when contrasted with the other plant mutated samples. Our outcomes uncovered that the perception of incited UV-C mutation in of Capsicum species was maybe recorded by these divided DNA banding designs. Still more than one hour of UV exposure is required to make/create the fake change in the chose plant source.
DNA from plants presented to UV radiation displayed polymorphic bands which were not detectable in DNA of unexposed or controlled plants of Capsicum species. The upgraded development of AFLP polymorphisms was additionally seen in DNA of plant presented in situ to a physical source for mutagenesis. The correlation among "unexposed" and "exposed" genomes demonstrate that AFLP analysis can be utilized to assess how the mutagenic agents change the structure of DNA in living life forms.
The AFLP procedure likewise has the advantage of sampling many loci simultaneously and it is more robust than discretionary preparing strategies, for example, RAPD, in light of the fact that increasingly stringent conditions are utilized. Along these lines, AFLP gives a novel and amazing asset for gene tagging technique of any origin or complexity [24, 35]. It is ordinarily acquired in Mendelian form and in this way be utilized for typing, identification of molecular marker and mapping of genetic loci.
The present study privileges an induced mutation by Ultraviolet-radiation and its impact on the plant cells and DNA structure of Chilli plants (Fig. 16) utilizing the physiological methods and AFLP investigations.