Comparison of several physiological and biochemical indexes in germinating seeds of two different acid-Resistant rice (oryza sativa l.) varieties om1490 and cr203 under the influence of the acid environment and acid environment plus KClO3 treated to seeds

169 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2018-0086 Natural Sciences 2018, Volume 63, Issue 11, pp. 169-176 This paper is available online at COMPARISON OF SEVERAL PHYSIOLOGICAL AND BIOCHEMICAL INDEXES IN GERMINATING SEEDS OF TWO DIFFERENT ACID-RESISTANT RICE (Oryza sativa L.) VARIETIES OM1490 AND CR203 UNDER THE INFLUENCE OF THE ACID ENVIRONMENT AND ACID ENVIRONMENT PLUS KClO3 TREATED TO SEEDS Nguyen Nhu Khanh 1 , Nguyen Tan Le 2 and Ngo Thi Hoan 3 1 Faculty of Biology, Hanoi National University of Education 2 University of Education, Da Nang University, 3 Ha Long University Abstract. Comparison of several physiological and biochemical indexes in germinating seeds of two different acid-resistant rice (Oryza sativa L.) varieties OM1490 and CR203 under the influence of the acid environment and acid environment plus KClO3 treated to seeds. It is reported that acid environment decreased water absorption of seeds, germination ratio, and in the 3 days old germination seeds, acid environment inhibit a starch hydrolysis process results in increased nonhydrolysed starch content, but decreased reduced sacharids, total organic acid content, the activity of amylase, protease and catalase enzymes; yet activity of peroxydase was decreased in the germinating seeds of variety CR203, but increased in germinating seeds of variety OM1490. KClO3 has opposite effect on OM1490 and CR203 at germination phase, in which the more positive effects are revealed in the variety CR203. The contrary effects to the one of acid environment with exception of enzyme peroxidase to which KClO3 has same tendency as the acid environment, but with lower effects in variety OM1490, yet for variety CR203, KClO3 effected contrary to the acid environment, i.e. KClO3 increased the activity of peroxidase. Thus, KClO3 has increased effects on almost physiological, biochemical indexes studied of both rice varieties at germinating phase, in which good effects are more clarity in CR203 variety. Keywords: OM1490 and CR203 rice varieties, acid environment, KClO3, physiology, biochemistry indexes. 1. Introduction Regarding acid soil amelioration in agriculture, there are many researches about measures such as irrigation (watering, acid drainage, etc.), farming (appropriate fertilization, intercropping, alternative culture, etc.). Additionally, using acid-resistant rice varieties is considered as the most effective measure [1]. However, since toxicants change complicatedly according to different locations and other factors, other supportive solutions are in need to enhance the adaptation ability of rice in disadvantaged environment conditions, including acid environment. Among these solutions, research of using KClO3 to crops to improve drought (Vu Tuyen Hoang et al, 1995) [2], Received September 4, 2018. Revised November 12, 2018. Accepted November 19, 2018. Contact Nguyen Nhu Khanh, e-mail address: nguyenkhanhsinhtv@gmail.com Nguyen Nhu Khanh, Nguyen Tan Le and Ngo Thi Hoan 170 salinity and acid tolerance brought good results. This has been proved by experiments of Vo Minh Thu (1999) [1] on four different salt resistant rice varieties in Binh Dinh province; Phung Thi Hang and Nguyen Nhu Khanh (2008) [3, 4] on 3 acid resistant rice varieties in Bac Lieu province; Nguyen Thi Thu Thanh and Nguyen Nhu Khanh (2008) [5] on 4 acid resistant rice varieties in An Giang province. In this article, we present our experiments’ outcome about several physiological and biochemical indexes in germinating seeds of two different acid-resistant rice varieties OM1490 and CR203 under the influence of acid environment and acid environment plus KClO3 treated to seeds. 2. Content 2.1. Object and research method * Object - Rice variety OM1490 with average acid resistant ability (crossed from OM606/IR 44592- 62-1-1-3) provided by Cuu Long Delta Rice Research Institute. - Rice variety CR203 with low acid resistant ability (selected from IR8432-132-622) provided by Plant Protection Research Institute. * Research method Acid solution (DDP) and kali chlorate solution, acid solution includes Al 3+ (use Al2(SO4)3 with concentration of 100ppm) combined with Fe 3+ (use FeCl3 with concentration of 800ppm). This is the actual concentration measured on acid soil [6, 7]. The combination of Al 3+ and Fe 3+ in 1:1 volume proportion is the acid environment used in the experiment [7]. KClO3 is added into the acid solution for seed treatment with concentration of 50ppm [3, 8]. Select seeds that are similar, disease-free, no damage and have normal color. Selected seeds are soaked for 24 hours, incubated for 48 hours, cleaned and then placed in Petri plates that have distilled-water blotting paper with different environment: distilled water, acid solution (DDP); DDP + KClO3 place those Petri plates with seeds which have treated at 28 0 C thermostat to germinate seeds within 2 days. Analyze physiological indexes: the seeds’ water absorption capacity (the amount of water absorbed after 12, 24, 36 and 48 hours being immersed into water); germination ratio of seeds (after being incubated within 24, 48 and 72 hours); determination of biochemical indexes: the activity of enzyme amylase, catalase, peroxidase, protease, reduced sugar , starch content and total organic acid. Determine water absorption of seeds by weighing and drying. Determine the original amount of water in air-dry seeds by using electric weight scale and drying at 100°C, weigh again and have this process repeated until reach the stable weight. The original amount of water in the air-dry seeds is the initial index to compare with the amount of water determined after the time phases that seeds are soaked as mentioned above. At each experiment stage of being soaked for 12, 24, 36 and 48 hours, seeds are drained and blotted by filter paper until they become air-dry, then they are weighed to get the weight of seeds and water at that stage. In order to determine the amount of water at each experiment stage, seeds are dried until they reach the stable weight. The difference between these weights and the original weight is the amount of water that is absorbed by seeds in each experiment. These steps would provide us with the information about the extra amount of water that seeds absorb at each time frame. The comparison of the absorbed amount of water after each time frame being drained will give us the information about the water absorbing speed of seeds. Determine the amylase activity according to Nguyen Van Mui [9]. The comparison of several physiological and biochemical indexes in germinating seeds of two different 171 Determine the protease activity according to Pham Thi Tran Chau, et al. [10]. Determine the catalase activity according to A.N. Bath and A.I. Oparin [11]. Determine the peroxidase according to A.N. Boiakin [11]. Determine the reduced sugar and starch content according to Bertrand [10]. Determine the total organic acid based on titration method of Ermakov [11]. Carrying out the three sets of draining and incubating seeds with each set being repeated 3 times. The data is processed according to biostatistics method (deviation of average number: X ± m). 2.2. Outcome and discussion 2.2.1. The water absorption capacity of seeds As we know, water plays a key role in any living organism, including the rice. The early growth and development stage is germination. The germination process needs enough water to make metabolism happen. Thus, the water absorption of seeds plays the decisive role in germination. Under the influence of environmentally adverse conditions, the water absorption capacity of seeds changes. This is the reason why we have to determine the water absorption capacity of seeds from researched rice varieties after 12, 24, 36, 48 hours being soaked. The result of the water absorption capacity of two researched rice varieties under the influence of acid environment and acid environment plus KCLO3, showed in Table 1. Table 1. The water absorption capacity of seeds after each hours being soaked V a ri et y Variant 12-h 24-h 36-h 48-h g/g seed % Con- trol g/g seed % Con- trol g/g seed % Con- trol g/g seed % Con- trol O M 1 4 9 0 Control 0.131 ± 0.008 100 0.157 ± 0.003 100 0.168 ± 0.002 100 0.176 ± 0.001 100 DDP 0.106 ± 0.019 80.92 0.140 ± 0.018 89.65 0.149 ± 0.008 89.07 0.156 ± 0.018 88.53 DDP+ KCLO3 0.119 ± 0.001 90.99 0.156 ± 0.004 99.67 0.168 ± 0.006 100 0.170 ± 0.004 97.06 C R 2 0 3 Control 0.123 ± 0.001 100 0.145 ± 0.006 100 0.154 ± 0.004 100 0.164 ± 0.001 100 DDP 0.116 ± 0.001 94.09 0.139 ± 0.002 96.27 0.144 ± 0. 003 93.46 0.145 ± 0.003 88.02 DDP+ KCLO3 0.114 ± 0.001 92.23 0.142 ± 0.001 97.86 0.146 ± 0.002 94.30 0. 148 ± 0.003 90.26 Nguyen Nhu Khanh, Nguyen Tan Le and Ngo Thi Hoan 172 After 24 hours soaking, under the influence of DDP environment, the amount of water absorbed in rice variety OM1490 is 89.65% and rice variety CR203 is 96.27%, decreased by 10.35% and 3.73% respectively compared to control. After 36 hours soaking, in DDP environment, the amount of water absorbed in rice variety OM1390 went down by 10.93%, approximately equals to the rate at 24-hour time frame (10.35%); rice variety CR203 witnessed the decline by 6.54% compared to control (at the after 24-hour time frame, the rate is 3.37% compared to control). Shortly, the amount of water absorbed of rice variety CR203 at this time frame continues decreasing notably in comparison with the time frame after 24-hour soaking. After 48 hours soaking, in the same environment, the rice variety OM1490 and CR203 experienced a decrease by 11.47% and 11.98% compared to control. To sum up, under the influence of acid environment, the rice variety CR203 maintains its high water absorption capacity within the first 24 hours after being soaked but this capacity decreases more significantly in the next hours. At the same time, the water absorption capacity of rice variety OM1490 falls off within the first 24 hours but shows a slower decreasing rate in the following hours. Until the time frame after 48-hour soaking, the amount of water absorbed are almost equal between two varieties (OM1490 is 88.53% and CR203 is 88.02%). Adding KClO3 into acid environment helps increasing the amount of water absorbed in comparison with the acid environment at all researching moments. In details, after 12, 24, 36, 48 hours soaking, the rate of water absorbed in add-KClO3 acid environment increase 10.07%, 10.22%, 11.71% and 8.51% respectively with OM1490 rice variety while they are 1.86%, 1.59%, 0.84%, 2.24% with CR203 within the same time frame order compared to acid environment. This is possibly because of KClO3 that inhibits the intrusion of toxic ions of aluminium and iron into seeds [7] and this is consistent with researches of Le Huy Ba [12] that said K+ has the reversed correlation with toxic ions of acid environment. Nguyen Nhu Khanh and Vo Minh Thu [8] have also said that KClO3 help increasing the water absorption capacity of rice in salty solution. In short, KClO3 has the effect of decreasing the inhibition of acid environment towards the water absorption capacity of rice. 2.2.2. The germination ratio of seeds After researching the influence of acid environment and acid environment plus KClO3 on the water absorption capacity, this part will analyze these factors’ influence on the germination ratio of the two researched rice varieties with Table 2 indicating experimental data. The data in Table 2 shows that germination ratios of two rice varieties experiencing 24, 48, 72-hour soaking in control variant are consistent. Under the influence of acid environment, the germination ratios in different time frames decrease compared to control. Especially at the time frame of after 24 hours, the germination ratio of rice variety CR203 is inhibited the most with the rate being 68.5%, reduces by 31.5% compared to control. Meanwhile, also at the time frame of 24 hours, the rate of rice variety OM1490 is 94.8%, drops by 5.2% compared to control. At the other time frames, the rate is almost consistent. Thus, the germination ratio of CR203 is more inhibited than the ratio of OM1490 by the acid environment within the first 24 hours. Adding KClO3 into acid environment in general improves the germination ratio of the two rice varieties in comparison with acid environment. Regarding rice variety CR203 specifically, at the time frame of after 24 hours soaking, the germination ratio of soaked seeds in acid environment plus KClO3 achieves 89.2% while it is only 68.5% in acid environment, which shows that KClO3 helps the germination ratio grow by 20.7% compared to acid environment. The comparison of several physiological and biochemical indexes in germinating seeds of two different 173 Table 2. Germination ratio of seeds after time frames (hour - h) V a ri et y Variant 24-h 48-h 72-h Germination ratio % Compared to control Germination ratio % Compared to control Germination ratio % Compared to control O M 1 4 9 0 Control 58.67 ± 0.33 100 95.00 ± 0.58 100 98.67 ± 0.88 100 DDP 55.60 ± 1.08 94.8 93.75 ± 0.63 98.7 96.66 ± 0.33 98.0 DDP+ KCLO3 57.33 ± 0.63 97.7 94.75 ± 0.63 99.7 98.00 ± 0.58 99.3 C R 2 0 3 Control 61. 00 ± 1.64 100 96.00 ± 0.55 100 98.33 ± 0.88 100 DDP 41.80 ± 1.96 68.5 93.67 ± 0.88 97.6 96.33 ± 0.33 98.0 DDP+ KCLO3 54.40 ± 1.20 89.2 94.00 ± 1.00 97.9 95.66 ± 0.67 97.3 2.2.3. Starch, reduced sugar and acid in the 3-day old germinated seeds The germination results from the growth of germ that requires energy from the process of oxidation of sucrose molecule in respiration. The sugar providing source in germination process is from starch hydrolysis; from sugar through metabolism process to organic acid, intermediary substances, contribute mostly to generating acid in cells. Hence, following is the consideration of the correlation between starch, sugar and acid during the germination under the influence of acid environment and acid environment plus KClO3. The analysis data of starch, reduced sugar and total acid in germinated seeds of two varieties OM1490 and CR203 is shown in Table 3. The results show that acid environment inhibits the conversion process of starch to glucose in germinated seeds; consequently, the amount of starch left is not hydrolyzed higher than control in both varieties. Variety CR203 is inhabited more than OM1490, as a consequence, the amount of starch left which is not converted in variety CR203 is higher than OM1490 (120,01 compared to 114,86 ). Acid environment plus KClO3 has made this process improved clearly. However, starch conversion in rice variety CR203 is still inhibited as we can see the amount of starch left is higher than control (104.01%). Out result is consistent with the research outcomes of Phung Thi Hang (2008), Nguyen Thi Thu Thanh and Nguyen Nhu Khanh (2008) which were done in other rice varieties [3, 5]. Nguyen Nhu Khanh, Nguyen Tan Le and Ngo Thi Hoan 174 Table 3. The starch, reduced sugar and total acid content in the 3-day old germinated seeds Studied Variants Rice variety OM1490 Rice variety CR203 X ± m % compare to Control X ± m % compare to Control Starch content (% dry seeds) Control 18.225 ± 0.193 100.00 20.120 ± 0.167 100.00 DDP 20.934 ± 0.229 114.86 21.731 ± 0.152 120.01 DDP + KClO3 18.000 ± 0.135 98.77 20.926 ± 0.930 104.01 Reduced sugar content (% dry seeds) Control 6.079 ± 0.171 100.00 5.723 ± 0.333 100.00 DDP 5.366 ± 0.136 88.27 4.650 ± 0.117 81.25 DDP + KClO3 6.439 ± 0.111 98.77 6.235 ± 0.110 108.95 Total acid content (mle/g (*) fresh seeds) Control 48.30 ± 0.045 100.00 50.000 ± 0.030 100.00 DDP 45.00 ± 0.289 93.17 33.330 ± 0.034 66.66 DDP + KClO3 46.67 ± 0.333 96.63 41.670 ± 0.670 83.34 *Note: mle/g – milli-equivalent/g The total amount of acid in control seeds is approximate in both varieties; decreased under the influence of acid environment with the decrease level in CR203 being higher than OM1490, proving that toxic of acid environment has inhibited the metabolism of intermediary substances in the respiration process of seeds. This inhibition is seen clearer in the low acid resistant rice variety CR203 than the good acid resistant rice variety OM1490. In acid environment plus KClO3, the total acid content in both varieties have the tendency to rise with CR203 increasing more significantly. The above indexes are the results of metabolism process which connect closely with enzyme catalytic activity. Next, how the enzyme activity that is relevant to respiration and converted stored substances in germinated seeds is affected by acid environment and acid environment plus KClO3 will be discussed. 2.2.4. Activities of catalase, peroxidase, amylase and protease enzymes in germinated seeds Activities of catalase, peroxidase, amylase and protease enzymes in 3-day old germinated seeds under the influence of toxic acid and KClO3 are shown in Table 4. Catalase and peroxidase enzymes play important role in the respiration process of germinated seeds. Toxic acid has made activities of these two enzymes declined notably compared to control in both rice varieties. Acid environment plus KClO3 has increased the activities of the two enzymes, especially peroxidase - an enzyme that is closely related to tolerance and has higher activity compared to control. Amylase catalyzes starch hydrolysis to sugar when seeds germinate. The increasing activity of this enzyme shows the environmental stress tolerance ability of rice variety [3, 4]. Under the influence of toxic acid environment, the activity of enzyme amylase drops in both varieties. The acid environment plus KClO3 has increased enzyme activity compared to its activity in acid environment, which means KClO3 has the ability to diminish negative effects of acid environment. The comparison of several physiological and biochemical indexes in germinating seeds of two different 175 Enzyme protease has the effect of hydrolyzing stored proteins in germinating seeds. Toxic acid environment has reduced the enzyme activity. Adding KClO3 into acid environment help increase the enzyme activity, which means KClO3 has diminished effects of acid environment. Our experimental data has clearly shown that acid environment inhibits activities of all 4 researched enzymes and kali chlorate has the effect of diminishing the inhibition of toxic acid environment towards catalase, peroxidase, amylase and protease enzymes. Table 4. Activities of catalase, peroxidase, amylase and protease enzymes in 3-day old germinated seeds Studied formula Rice variety OM1490 Rice variety CR203 X ± m % compare to Control X ± m % compare to Control Catalase (UI) Control 22.50 ± 0.48 100.00 23.33 ± 0.47 100.00 Acid solution 11.54 ± 0.55 51.30 10.21 ± 0.47 43.76 Acid solution + KClO3 15.63 ± 0.82 69.47 16.77 ± 0.52 71.88 Peroxidase (UI) Control 16.79 ± 0.03 100.00 17.03 ± 0.06 100.00 Acid solution 16.72 ± 0.43 93.63 15.69 ± 0.26 92.13 Acid solution + KClO3 17.39 ± 0.54 103.57 19.20 ± 0.71 112.74 Amylase (UI) Control 0.060 ± 0.001 100.00 0.066 ± 0.001 100.00 Acid solution 0.056 ± 0.001 93.30 0.055 ± 0.002 83.30 Acid solution + KClO3 0.057 ± 0.001 95.00 0.057 ± 0.005 86.40 Protease (mg%) Control 3.183 ± 0.074 100.00 2.122 ± 0.049 100.00 Acid solution 2.281 ± 0.048 71.66 1.303 ± 0.100 61.40 Acid solution + KClO3 2.759 ± 0.064 86.68 1.737 ± 0.080 81.86 3. Conclusions In germination phase: In comparison with rice variety CR203, variety OM1490 absorbed more water and faster. The germination ratio is similar in both varieties. The acid environment inhibits the water absorption and germination in both varieties, but the inhibition level is stronger in variety CR203. KClO3 diminishes the negative effects of acid environment in both varieties with good effects are seen clearer in variety CR203. In three-day old rice seedling phase: In comparison with rice variety CR203, the starch conversion in variety OM1490 is faster; as a result, the amount of starch content left is less than variety CR203 while the amount of reduced sugar is more than that. Acid environment decreased the conversion in both varieties, but the effect of inhibition is witnessed stronger in variety CR203. Kali chlorate decreases inhibition effect of acid environment on the two varieties. Nguyen Nhu Khanh, Nguyen Tan Le and Ngo Thi Hoan 176 Compared to CR203, variety OM1490 has slightly lower amount of total organic acid; the catalytic activities of 3 enzymes (catalase, peroxidase and amylase) are equivalent; catalytic activity of protease in variety OM1490 is higher. Acid environment differently inhibits all 4 enzymes in both varieties, in which the inhibition of acid environment stronger affected catalase, protease and CR203. Kali chlorate has the effect of diminishing the inhibition of acid environment towards the both 4 enzymes and 2 varieties. Generally, acid environment inhibits all biochemical and physiological indexes studied in both rice varieties; kali chlorate has the effect of diminishing the inhibition of acid environment. The damage of acid environment towards variety CR203 is stronger than OM1490. 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