Maribel Beltran-Cruz
Department of Biology, College of Arts and Sciences (CAS)
Patrishia Joy F. Reytana
Krizia Marie G. Orbase
James Henry A. Esguerra
Beverly Jane R. Macalay
Sheelnor D.R. Reyes
B.S. Biology Program
College of Arts and Sciences (CAS)
How to Cite:
Beltran-Cruz, M., Reytana, P. J. F., Orbase, K. M. G., Esguerra, J. H. A., Macalay, B. J. R., & Reyes, S. D. R. (2017). Effects of gamma irradiation on the phenotype and microbial load of lettuce (Lactuca sativa Linn.). NEU Knowledge Journal: A Compilation of Researches of New Era University Faculty, Staff, Students, and Administrators, 1(1), 1–16. https://doi.org/10.64303/neu-urc-kj2017-EfGaIrPhe
ABSTRACT
A mutation breeding study was conducted to determine the effect of four doses of gamma radiation (5 Gray, 10 Gray, 50 Gray, and 100 Gray) on the phenotype (color, weight, height, root length, and leaves surface area) and microbial load of lettuce (Lactuca sativa Linn.) grown from irradiated seeds under a controlled environment. Based on large effect size, 80% power, and 5% level of significance, 16 seeds per group/treatment were used in Completely Randomized Design involving five treatments (four doses and a control) and four replications. Normality and homogeneity of variance assumptions were checked and met. The alpha level was adjusted using Bonferroni correction for the five related ANOVA tests performed, excluding assessment for color which was based on color guide.
No apparent color difference among treatment and control group was found. Statistically significant treatment effect was found for mean weight, mean height, mean root length, mean leaves surface area, and mean microbial load. Post hoc Tukey’s HSD test indicated that 100 Gray is the most effective dose in increasing mean weight, mean height, and mean leaves surface area and in decreasing microbial load. Ten Gray is the most effective dose in increasing mean root length.
Keywords: Mutation breeding, gamma radiation, Lactuca Sativa Linn., phenotype, microbial load
INTRODUCTION
Mutation breeding is defined as a means of accelerating the process of developing different traits for selection, such as disease resistance, tolerance to harsh growing conditions, and other valuable agronomic trait (FAO/IAEA, 2016). It is a method of exposing parts of a plant or the seed to chemicalsor radiation in order to generate mutants with good or desirable traits which can be bred and raised with other cultivars. It is also the process by which the genetic information of an organism is changed into a stable manner.
Mutagenesis is practiced in agriculture to improve certain plant traits so it can survive in extreme environments (Forster, 2011). Over the years, researches and studies have shown that induced mutations productively crafted seed varieties with high genetic variability. Through mutation breeding, more than 1200 cultivars have been discovered and distributed to farmers and seed growers (IAEA, 1989). The use of radiation on plants is an expansive and complex field. Researches in plant irradiation showed that radiation affected the size and weight of plants. Gamma rays stimulated plant growth and development by inducing changes in cytology, genetics, biochemistry, physiology, and morphology of plant (Basu et al., 2007). Seed exposed to high doses of gamma rays disturbed the protein synthesis, hormone balance, leaf gas exchange, and enzyme activity. The strength and duration of gamma exposure produced differential morphological, structural, and functional changes (Al-Salhi et. al 2004, Hameed et al., 2008). In other studies, gamma irradiation increased plant productivity. It also induced early maturity of plants and caused them to be resistant to certain diseases (Jan et al., 2012). Lettuce, a popular gourmet dish around the world, is the only member of the Lactuca genus which is grown commercially. Lactuca sativa, also known as garden lettuce, is an annual plant. It is erect, usually simple, smooth, very leafy herb reaching a height of a meter when in flower. Leaves are stalkless, obovate to oblong-obovate, 6 to 20 centimeters long, entire or lobed, toothed, thin, and numerous at the base. Heads are numerous, about a centimeter long, and borne in open panicles; the branches, often much reduced, bear bract like leaves. Flowers are yellow. Dark green lettuce like Romaine is an excellent source of vitamin K (97% DV) and vitamin A (21% DV) with higher concentrations of provitamin compound, betacarotene. With the exception of the iceberg variety, lettuce is also a good source of folate and iron (10-19% DV) (Wolford &Banks, n.d.).
In mutation breeding, plant growth analysis is a standard approach used in studying plant growth and productivity. Some of the parameters used in assessing the phenotype of a vegetable include plant height, mass, root length, and color (Islam, 2013). Microbial load is another important parameter considered particularly by the food industry in its effort to control or eradicate food-borne diseases. Raw fruits and vegetables are known to carry a wide range of microorganisms. Food-borne bacterial pathogens commonly detected in fresh vegetables include coliform bacteria, E. coli, Staphylococcus aureus, and Salmonella sp. (Tambekar & Mundhada, 2006).
This study was conducted to determine the effect of gamma irradiation on the phenotype and microbial load of lettuce plants grown from irradiated seeds. Specifically, the study aimed to (1) determine if there is statistically significant difference on the phenotype of lettuce (color, weight, height, root length, and leaves surface area) across treatments, (2) determine the gamma radiation dose that will most improve phenotype, and (3) determine the dose level that will most reduce microbial load.
MATERIALS AND METHODS
Plant material and Irradiation Process
Based on large effect size, 80% power, and 5% level of significance, 16 seeds per group were recommended for five group One-way ANOVA test (Cohen, 1992). These seeds were bought from the Allied Botanical Corporation, Cubao City. Sixty-four dry seeds underwent gamma irradiation at the Philippine Nuclear Research Institute Multipurpose Irradiation Facility using Cobalt-60 Gammacell-220 as source. Sixteen seeds served as control. Dry seeds were exposed at 5 Gray, 10 Gray, 50 Gray, and 100 Gray (16 seeds per dose). These levels of dose were the same doses used by Al-safadi and Simon (1996) on carrots, Thapa (1999) on Pinus kesiya and Pinus wallichian, and Sikder et al. (2013) on tomato, but with some modifications made by the researchers.
Experimental Plot Design
The irradiated lettuce seeds were grown at a Greenhouse in Rodriguez, Rizal based on Completely Randomized Design (5 treatments x 4 replications). Four seeds from each of the four doses and the control were planted 6 inches apart on five seed beds filled with moist soil with four rows each. Water and temperature requirements were carefully monitored and controlled. The duration between sowing and first sampling is characterized as slow vegetative growth stage (SG), duration between first sampling and second sampling as linear growth (LG), and duration between beginning and end of leafing as leafing stage (LS) (Ozalkan et al., 2010). Plant growth followed a logistic growth curve or sigmoid curve. Leaves of matured lettuce were measured and the whole plant was weighed.
Phenotype Assessment
Irradiated seeds were grown and examined after two months. The color, plant height, weight, root length, and leaves surface area were assessed. The GLOBE Plant Color Guide for Plant Tissues was used in checking changes in color. The plant height was measured from top soil to the top of the main plant stem. The weight was based on the fresh weight of the plants. Root length was measured from the primary root or the radicle and the surface area of leaves was measured by tracing the leaves on a graphing paper and the squares covered by the leaves were counted.
Microbial Load Assessment
Twenty-five grams of lettuce leaves per dose were placed in polyethylene bag and weighed. Two hundred twenty-five mL of Butterfield’s phosphate-buffered water was added to the bag. This mixture was placed in a stomacher/blender for 60 seconds. After blending, 1 mL of the mixture was transferred to a sterile Petri dish using a sterile pipette (1:10). Serial dilution was done by transferring ten mL of the food sample into 90mL dilution blank (Butterfield’s phosphate-buffered water) (1:100 dilution). The dilution blank was shaken for 20 times. One mL of the 1:100 food samples was again transferred in a sterile petri dish. Another 1:1000 dilution was made using another 90 mL dilution blank and was shaken as before. Plate count agar (PCA) was poured on the Petri dishes and was incubated for 24 hours at 35oC in an inverted position. The number of the colonies in the PCA plates was counted after 24 hours. All samples were done in triplicate (Cruz et al., 2009). Quantification of microbial load was done at Soil Microbiology Laboratory at Bureau of Soils and Water Management.
Statistical Analysis
One-way analysis of variance (ANOVA) was used to determine significant treatment effect. Normality and homogeneity of variance assumptions were checked and met. Because five related ANOVA test were performed, the 5% significance level initially set to be used was adjusted using Bonferroni correction to control inflation of Type I error, resulting in the use of 1% significance level. Following a significant ANOVA, post hoc Tukey’s Honest Significant Difference (HSD) test was performed to identify significant pairwise comparison. All statistical tests were done using SPSS Statistics v23.
RESULTS AND DISCUSSION
Plant Color
Plant color for the irradiated samples and the control appear fairly similar [5 Green-Yellow (GY) 6/10]. Mean weight increases as irradiation dose increases (Table 1). Plot of mean weight against dose shows increasing non-linear trend (Figure 1). The control group registered the lowest mean weight.
ANOVA test showed that different doses of gamma radiation induced statistically significant weight differences on lettuce, F(4,75)=540.39, p=0.001. Post hoc Tukey’s HSD test indicated that the control was statistically significantly different from each of the other treatment groups (p<0.001). Pairwise comparisons between treatments were also found statistically significant (p<0.05).
Evidently, 100 Gray was the most effective dose i02258/n increasing plant weight. A study of gamma irradiation of wheat also stated that plant weight increased at 180 Gray. Doses higher than 180 Gray decreased the plant weight (Grover & Khan, 2014). The same observation was revealed by a study on two rice varieties. Doses higher than 100 Gray significantly decreased weight of rice yield (Islam, 2013).
Plant Height
As dose increases, mean height also increases and in accelerated fashion. (Table 2 and Figure 2). ANOVA test showed that plant heights were statistically different among doses or treatments, F(4,75)=459.29, p<0.001. Post hoc Tukey’s HSD test revealed that the control group was statistically significantly different from each of the other treatment groups (p<0.001). Plants exposed to 5 Gray and 10 Gray were found not statistically significantly different (p=0.972). One hundred Gray was found to be the most effective dose in increasing mean plant height.
Root Length
Plot of mean root length at various doses approximates a conic curve with a peak point at 10 Gray and a lowest point at 100 Gray (Table 3 and Figure 5). ANOVA test showed statistically significant effect of gamma radiation on root length, F(4,75)=532.61, p<0.001. Post hoc Tukey’s HSD test showed statistically significant difference between the control and each of the doses (p<0.001).
Among the doses, 10 Gray was found the most effective in increasing the mean root length of lettuce. This result is similar to the findings of a study of gamma ray induced phenotypic mutations in chickpea. Relatively low doses of 50 Gray up to 100 Gray increased the root length of chickpea (Qureshi et al., 2014). In another study, gamma radiation of 5 Gray induced the greatest root length. Plot of mean root length at various doses approximates a pyramidal shape curve with a peak point at 10 Gray and a lowest point at 100 Gray (Singh et al., 2013).
Surface Area of Leaves
As the dose increases, the mean surface area of leaves also increases apparently in linear trend (Table 4 and Figure 4). ANOVA test showed significant treatment effect on surface area of leaves, F(4,75)=642.52, p<0.001. Post hoc Tukey’s HSD test revealed significant difference in
surface area of leaves between the control group and each of the other treatments (p<0.001). The control and the 5 Gray group were not statistically significantly different (p=0.131).
Among the doses, 100 Gray was the most effective dose in increasing the surface area of lettuce leaf. This result agrees with the results of a similar study conducted by Singh et al. (2013) on the effects of gamma irradiation on the growth of wheat plants, where the surface area of leaves was wider than that of the control group. In the study of chickpeas, groups irradiated with 50 Gray, 75 Gray, and 150 Gray produced leaf area significantly larger than that of the control group (Qureshi et al., 2014).
Microbial Load Assessment
Only plates with countable microbial load were included. The 1:10 dilution was not included due to TMTC (Too Many To Count) colony forming units (CFUs). The researchers used 1:1000 dilutions for determination of microbial load of lettuce where colonies are countable as opposed to use of 1:100 dilutions. Among the doses, 100 Gray was found the most effective dose in decreasing the microbial load of lettuce. Microbial load decreases as dose increases (Table 5 and Figure 5).
ANOVA test showed statistically significant treatment effect, F(4,75) =309.26, p<0.001. Generally, the irradiated groups had smaller mean CFUs compared with the control. Among the doses, 100 Gray was found the most effective dose in decreasing the microbial load of lettuce. A study reported that the effect of irradiation insofar as reducing microbial load was concerned was proportional to the dose delivered. Doses of 1kGy and 2kGy of gamma radiation reduced the bacterial load of lotus rhizome. (Khattak et al. 2009) found that doses of 4 and 6kGy induced the initial bacterial load to below detection level when analysis was carried out immediately after radiation treatment. Another study on effect of gamma irradiation on microbial load of Pimpinella anisum observed that all irradiated samples had significantly less total aerobic populations than the control (Al-Bachir, 2007).
CONCLUSION
For the phenotypic characteristics of lettuce grown from irradiated seeds, 100 Gray is the most effective gamma radiation dose for increasing mean height, mean weight, and mean leaves surface area; 10 Gray for increasing mean root length. For microbial load assessment, 100 Gray is the most effective gamma radiation dose for decreasing mean microbial load.
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