Impact of Gamma Irradiation on Biochemical and Physiological Characteristics of Black Rice

Rice ( Oryza sativa L .) is a fruit caryopsis, well known for its high calorific value and presence of high antioxidant phytochemicals such as flavonoids and anthocyanins. Further exposure of crops to different doses of physical mutagens is useful to give insight into development of quality traits for plant breeding. So, the objective of the present study was to analyse the effects of gamma irradiation on black rice seeds that were exposed to different radiation doses and to evaluate the irradiation effect on physiological and biochemical characteristics of plants. Variations were observed among different doses from non-enzymatic antioxidant activities that showed the increasing flavonoids, phenolics and carotenoids accumulation from low to high doses. A high dose irradiation releases, high amount of H 2 O 2 as compare to O 2-indicating the involvement of enzymatic antioxidants as a defence mechanism. An inverse relation was shown between peroxidase and catalase enzyme and concentration of Superoxide dismutase also increased with increasing doses. To know the level of stress on cellular metabolism, total proline and malon dialdehyde accumulation was estimated and both showed positive results for increasing doses. In case of measurement of photosynthetic parameters, chlorophyll concentration increased from 50 Gy to 100 Gy and decreased from 150 Gy to 200 Gy due to peroxidation of membrane. Significant effect was not observed in the concentration of soluble sugar among various doses. Thus, the present study, was useful to isolating effective dose (50Gy, 100 Gy) that may contribute towards advancement of qualitative traits for future breeding purposes.


Introduction
Rice (Oryza sativa L.) is a primary source of food for one third of the world's population and cited as a most important cereal crop 1 belongs to family Poaceae having chromosome no 2n= 24. 2 Two main cultivated species of Oryza genus are Oryza sativa DASH & KUJUR, Curr.Agri.Res., Vol.11(3) 813-825 (2023) (Asian rice) and Oryza glaberrima (African rice). 3s a diploid crop and a relatively small genome 430 Mb, 4 rice holds a great potential for understanding genetic mechanisms of crop domestication and improvement.This crop is also riched in amino acids like lysine, tryptophan and vitamins such as vitamin B1, vitamin B2 and folic acid and also act as a good source of minerals iron, zinc, calcium, phosphorus and selenium present in black rice that makes it highly nutritious for health.Phytochemical constituents include carotenoids, phenolics, alkaloids, nitrogen and organosulfur containing compounds and among antioxidants, Phenolics plays a significant role which further categorized as phenolic acids, flavonoids, coumarins and tannins. 5,6,7dia is enriched with a greatest number of short and slender aromatic rice varieties that are famous in traditional rice growing regions.These unique rice germplasms have immense potential of containing valuable genes that can be effectively utilized in present day breeding program not only to explore high yield potential and quality but also resistant to biotic and abiotic stresses 8 As compared to other Asian countries, aroma qualities of Indian aromatic rice varieties have a special feature that enhances the value of rice in international market. 9s a consequence, rice needs a special attention with respect to its cooking quality as well as some biochemical and morphological characteristics. 10lthough coloured rice is less consumed, there are many special rice varieties that contain coloured pigments, such as black, brown and red owing to functional effects.Among them black rice' belongs to species Oryza sativa, and is predominantly famous for its purple colour of grain due to presence of high amount of anthocyanins in the aleurone layer. 11n particular, presence of anthocyanins is associated with health effects such as dietary antioxidants, anti-inflammatory compounds and hypoglycemic activities. 12ilization of Ionizing radiations through induced mutagenesis has played an important role in the crop improvement.Determination of optimum dose, radio-sensitivity and treatment conditions are most essential for genetic manipulation through induced mutation.Gamma rays belong to ionizing radiation and interact with atoms or molecules to produce free radicals in cells that can damage or modify important components of plant cells and have been reported to effect differentially the morphology, anatomy, biochemistry and physiology of plants depending on the irradiation level.These effects include changes in plant cellular structure and metabolism e.g.dilation of thylakoid membranes, alteration of photosynthesis, modulation of antioxidant system and accumulation of phenolics. 13,14,15evious studies reported, that the exposure of gamma irradiation of 0.1 KGy act against biotic stress and exposure to low doses of 0.5 KGy to suitable for enhancement of aroma in aromatic rice. 16Exposure to gamma rays from 200 Gy to 400 Gy induces chlorophyll concentration in normal rice. 17,18Exposure to 2.5 KGy and 5 KGy of gamma irradiation responsible for increment in sugar and antioxidant activities in rice.So, the purpose of the present study was to subject the seeds of black rice to different doses of gamma rays and search for desirable variations in biochemical and physiological characteristics in plants that may be helpful in further insight into the development of quality traits for future plant breeding programs.

Plant Material
The present study was conducted using gamma irradiated seeds of black rice collected from a local farmer which were cultivated in the fields of The Agricultural and Food Engineering Department (Plot size = 5m 2 ), Indian Institute of Technology, Kharagpur, West Bengal, India.Seeds without any treatment were sowed as control.Rice seeds were irradiated using gamma radiation facility in gamma irradiation chamber GC 1200 (Board of Radiation and Isotope Technology, BRIT) with Co 60 isotope.Samples were exposed to gamma irradiation doses of 50 Gy, 100 Gy, 150 Gy and 200 Gy with a dose rate of 2.65 KGy (kilo gray) per hour at UGC-DAE consortium for scientific research council, Kolkata, West Bengal, India.After 23 days of seeding, transplantation was done following a random block design pattern with plant to plant and row to row distance of 0.25 m.Third leaf from each irradiated plants were collected after the reproductive stage and three replications from each doses used for biochemical and physiological analysis.

Biochemical and Physiological Analysis
The leaves collected from cultivated plants were used for non-enzymatic antioxidant estimation.For estimation of total phenolics methods of 19 were followed.Total flavonoid content was measured by AlCl3 method of 20 as earlier described. 21Methods of 22 was followed for estimation of total carotenoids.Lipid peroxidation was measured by the method of 23 to see the effect of irradiation on membrane.Total proline was measured by the method of 24 to check the water stress that induces metabolic irregularities in plants.For estimation of reactive oxygen species level, Superoxide anion (O 2 -) radical content was estimated using nitro blue tetrazolium (NBT) solution by methods of 20 as described earlier by. 25Hydrogen peroxide (H 2 O 2 ) was measured by methods of. 26Methods of 27 were used to estimation of total chlorophyll from third leaves to know the effect of irradiation in important plant pigments.Total soluble sugar was measured by the method of 28 from third leaves.Specific activity of antioxidative enzymes peroxidase, catalase and superoxide dismutase were measured.
In the present study, enzyme activities were represented as specific activity in microgram (U mg-1) of protein.For all the assays, enzymes were extracted by homogenizing 50 milligram( mg) fresh leaves in 100 (millimolar) mM chilled phosphate buffer (pH 7.0) containing 1 mM EDTA, 1mM phenylmethylsulphonyl fluoride (PMSF) and 1 % (w/v) polyvinylpyrrolidone (PVPP).For all the irradiated samples total soluble protein contents of the plant samples were assayed according to Bradford method. 29Guaiacol oxidation method was followed for estimation of peroxidase activity. 30Catalase activity was measured according to 31 as also described in. 32NBT reduction method was followed for measuring SOD activity exactly the same way as described by. 33

Statistical Analysis
TWO WAY ANNOVA was done followed by POST-HOC Test, to know the variation among different doses at the statistical significance of p<0.05.The Pearson correlation statistics were performed between different biochemical and physiological attributes of irradiated doses to test the level of significance using Statistics SPSS16.0 software.

Estimation of Non-Enzymatic Antioxidants in Leaves from Different doses of Gamma Irradiated Samples
In the present investigation, total phenolics, total flavonoids and total carotenoid content was measured to determine the non-enzymatic antioxidants accumulation in the leaves of plants grown from gamma irradiated seeds.The analysis of variance (TWO WAY ANOVA) followed by POST HOC analysis was done to indicate the effect of ionizing radiation among different doses with statistical significance at the level p˂ 0.05.Total flavonoid content (TFC) of leaves from different doses of gamma irradiated samples was expressed as mg quercetin equivalent per gram fresh weight.
High amount of flavonoid was accumulated from 50 Gy (4.23±0.27) to 200 Gy (11.8±0.35) as compared to control (Figure 1A) and according to ANNOVA and post hoc analysis, a significant difference was seen from low to high doses at P < 0.05.Total phenolic content (TPC) of leaves was expressed in terms of µg (microgram) gallic acid equivalent per gram fresh weight.As compared to control, 50 Gy (56.93±1.9)sample, accumulated high phenolics and same trend was also followed for higher doses indicating , plants released a high amount of antioxidants to cope with exposure to high irradiation doses (Figure 1B).Total carotenoid accumulation has effectively increased with respect to increase in dosage, with higher doses accumulating significantly higher carotenoids (p˂ 0.05) (Figure 1C).

Discussion
There are many earlier reports available on the effect of radiation on the biochemical and physiological changes in crop plants.In the present investigation, gamma irradiated seeds (50 Gy, 100 Gy,150 Gy,200 Gy) compared with plants without any treatments and act as reference plants .The third leaf collected from each irradiated dose was used for biochemical and physiological characterization.
When plants are exposed to ionizing radiation, they produced ROS such as H 2 O 2 , O 2 -. , and OH-due to disruption of cellular homeostasis 34 and in response to these adverse condition plants produce enzymatic and non-enzymatic antioxidants 35 to detoxify ROS.An important compound flavonoids complexes with chelating ions, iron or copper has capacity to prevent generation of ROS. 36In the present investigation, it was shown that effective dose (50 Gy, 100Gy) of irradiation accumulates a high concentration of phenolics and flavonoids which were also observed from the study of effect of gamma radiation on phenolic compounds in rice. 37,38Carotenoids have a strong antioxidant capacity to scavenging peroxy-acyl free radicals more efficiently because of their conjugated double bonds. 38Concentration of carotenoids, flavonoids and phenolics also increased to scavenge ROS during stress causing oxidative stress shown a positive correlation with total proline, an important stress indicator( correlation at r =0.906, 0.967,0.886,p< 0.05,0.01),. 39Some of the parameters commonly used as oxidative stress biomarkers, are levels of H 2 O 2 and O 2 -radical accumulation, lipid peroxidation. 40,41The current study reveals, at higher doses (200Gy) concentration of H 2 O 2 is higher as compare O 2 -(correlation statistics r= -0.718).This is because of enzyme superoxide dismutase (SOD) dismutase O 2 -to H 2 O 2 and molecular oxygen and acts as first line of defence against ROS generation. 42Peroxidation of lipid membranes is a reflection of stress induced damage at cellular level. 43Further confirmation was done by measuring MDA content that shown to be increased from lower to higher radiation doses indicating the level of cellular damage due to rapid peroxidation, also reported by. 44Accumulation of proline as a stress indicator is also effectively higher with respect to high doses and acts as an important cellular osmoticum against exposure to high doses of irradiation. 45In the present context, activity of peroxidase was increased from in higher doses (p< 0.05) but reverse was happened in catalase show negative correlation at r = -0.925,p< 0.01).Thus, peroxidase might take important role in detoxification process by activating non-enzymatic antioxidative mechanism, shown positive correlation for flavonoids, phenols and carotenoids(p< 0.05).Significant change was observed in superoxide dismutase for rapid dismutation of O 2 -to H 2 O 2 and to accumulation of antioxidants at high concentration (significant at p< 0.05), also enhancing accumulation of high amount of proline as cellular osmolytes (significant at p<0.01).Earlier studies also reported by 46 that, the concentration of peroxidase (POX) and Super oxide dismutase (SOD) increases when exposes to gamma radiation.How the irradiation affects photosynthetic precursors was measured by estimation of total chlorophyll content.Total chlorophyll content was increased in upto effective dose (100 Gy) may be because of accumulation of O 2 -during water hydrolysis. 20,47Accumulation of total chlorophyll decreased when exposed to high doses (200Gy), may be disturbances in its biosynthetic precursor which was also observed by. 48Concentration of chlorophyll a was higher as compare to chlorophyll b, may be because of disturbances in its metabolic pathways during biosynthesis. 49Hormesis is a biphasic phenomenon of excitation or stimulatory effect of any agent by a small dose in any system and has modulatory effect on plants, whereas higher doses have inhibitory effect on system of plants. 50hus, from the above investigation, it was studied that, effective dose (50Gy,100 Gy) of radiation stimulating the biochemical and physiological changes in a positive way, thus future analysis in this perspective may give quality traits for future breeding programmes.

Conclusion
Biochemical and physiological characterization are widely used in the development of good quality traits and high-performance varieties that are useful for farmer cultural practices.In the present study, black rice seeds were exposed to gamma irradiation (50 Gy, 100 Gy, 150 Gy, 200 Gy) and compared with control i. e without irradiation.Physiological and biochemical characteristics were measured to know the effect of irradiation that may helped to know any metabolic changes in respective crop.Exposure to physical mutagen accumulates more amount of antioxidants to avoid harmful effect of free radicals on cellular metabolites.Plants releases antioxidative compounds in response to avoid damage through reactive oxygen species (ROS) including superoxide radicals (O 2 -.), hydrogen peroxide (H 2 O 2 ).Catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD) are some of the important enzymes that plays a principal role in scavenging these free radicals.In conclusion, effective irradiation doses might be useful to screening of qualitative traits that may helped to further analysis in molecular dimensions of selected doses and also explore their role in future plant breeding programs in black rice.

Fig. 1 :
Fig. 1: A Variation in flavonoid level in irradiated rice ; B Variation in phenolics level; C Variation in carotenoids level; Differenct doses of mean with variation are significantly different at p<0.05 level

Fig. 2 :Fig. 3 :Fig. 4 A
Fig. 2: A Variation in MDA levelsat different doses; B Concentration of total prolne from different doses.Means with variations are significantly different at p< 0.05 level of significance Estimation of Reactive Oxygen Species In this study concentration of H 2 O 2 and O 2 -were measured in terms of mg per gram of FW and µM per gram of FW.An inverse correlation was found between O 2 -(0.24± 0.014, Fig 3B) and H 2 O 2 (6.44±1.99,Fig 3A) levels (Correlation significant at r = -0.718).In higher doses (200 Gy) accumulation of H 2 O 2 (31.68±1.4) was higher as compared to O2 -(0.10±0.005),(p< -0.718) indicates a possible regulation of ROS status in gamma irradiated plants by superoxide dismutase (SOD) enzyme.

Fig. 5 A
Fig. 5 A: Effect of irradiation in content of chlorophyll a, chlorophyll b and total chlorophyll for different doses; B: Concentration of total soluble sugar from leaves from different dose.Means with variation significantly different at P˂ 0.05 level of significance.significantly different at P˂ 0.05 level of significance.