Evaluation of Submergence Tolerance of Different Rice Genotypes at Seedling Emergence Stage under Water Direct Seeding

Xia Su¹, Hui Wu^1,2*, Jing Xiang^2*, Jingjing Zhan¹, Juqin Wang¹, Xiaomin Li¹, Yihao Wei¹, Haifang Dai^1*, Huizhe Chen²

¹Faculty of Agriculture, Forest and Food Engineering, Yibin University, Yibin, China.
²State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, Zhejiang.

DOI: 10.4236/oalib.1108706                

Abstract

In this study, 60 direct seeding rice varieties with different genotypes were used as experimental materials. Through long-term submergence treatment (7 d and 14 d), the responses of different genotypes to submergence stress were compared in germination and seedling emergence, stem, leaf and root elongation, starch and other storage material consumption. The results showed that there were significant differences in submergence tolerance of different genotypes at the germination and seedling stage. Through further analysis by statistical methods such as correlation analysis, principal component analysis, weight comprehensive evaluation and cluster analysis, 60 varieties could be divided into four categories: strong submergence tolerance, medium submergence tolerance, weak submergence tolerance and submergence sensitivity, while 6, 22, 4 and 28 varieties were screened respectively. After 14 d and 4 cm deep submergence, more than 78.1% of rice seeds could germinate, and the average plant height and root length could reach more than 11.99 cm and 9.66 cm respectively. The dry matter mass, starch content and soluble sugar content per plant were significantly higher than those of other tolerant types. The average coleoptile of the sensitive type was only 3.17 cm, and the radicle had little elongation. The evaluation and variety screening of submergence tolerance of different genotypes of direct seeding rice at the seedling stage can provide a theoretical basis for further clarifying the mechanism of rice submergence tolerance, screening suitable direct seeding varieties and cultivating special varieties of direct seeding rice.

Keywords

Direct Seeding Rice, Emergence Stage, Submergence, Evaluation, Variety Selection

1. Introduction

Rice (Oryza sativa L.) is the main grain crop in China, and the sowing area accounts for one-third of the national grain crops. Direct seeding is one of the light and simplified cultivation modes widely used all over the world. In the wet direct seeding cultivation mode widely used in China, the primary problems of low yield and instability of direct seeding rice are poor seedling uniformity, low whole seedling rate and difficult weed control [1] [2] [3]. The construction of field water layer at the seedling emergence stage of direct seeding rice can have a good effect on field closure and grass control, but it often leads to long-term submergence of rice seeds. In addition, direct seeding rice has high requirements for rice field leveling, and the height difference is no more than 3 cm [1], which is often difficult to achieve in production, resulting in low-lying ponding in the field with different depths. At the same time, the emergence stage of direct seeding rice often meets a rainy environment. When rice seeds germinate, they are under submerged conditions, and the rates of stewed seeds, rotten seeds and dead seedlings are high, resulting in uneven emergence, uneven population structure, poorly high yield and yield stability. Therefore, direct seeding with water has higher requirements for the ability of submergence tolerance at the seedling emergence stage of different genotypes.

At present, the research on rice submergence treatment mainly focuses on the identification of submergence tolerance index, coleoptile elongation character, physiological mechanism of seed germination under hypoxia, and so on [4] [5] [6] [7]. Compared with normal germination, rice seeds only grow coleoptile and inhibit the growth of leaves and seed roots under a hypoxia environment such as submergence [8]. Strong submergence tolerant rice varieties rapidly elongate the coleoptile during submergence germination, so as to reach the aerobic environment in the upper layer of the water surface, provide an oxygen source for the growth of other organs such as roots and leaves and seed survival, and also provide necessary physiological and metabolic guarantee for the survival of rice [9] [10] [11] [12]. Moreover, in addition to survival rate and plant height, it is also necessary to comprehensively evaluate the waterlogging tolerance of different genotypes of direct seeding rice at the seedling stage in combination with factors such as root development, chlorophyll level and dry matter consumption.

At this stage, the planting of direct seeding rice in China basically and directly misappropriates the existing transplanted rice varieties, and there is no cultivation and application of special varieties for direct seeding rice. The existing rice varieties are numerous and disorderly. As direct seeding rice, it is difficult to ensure seedling emergence, seedling uniformity and stable yield due to uncertain submergence tolerance. Some high-yield and high-quality rice varieties cannot give full play to their yield and quality advantages under direct seeding mode. Therefore, it is one of the urgent problems to be solved in the current cultivation mode of direct seeding rice to further tap the potential of stable yield and yield increase under the direct seeding mode of existing rice varieties, improve the submergence tolerance and seedling rate of direct seeding rice, promote the full seedling and the whole seedling, and then build a reasonable population structure.

Through the methods of principal component analysis and cluster analysis, this study statistically analyzed and evaluated the response differences of 60 different genotypes of direct seeding rice varieties to submergence stress in terms of germination and seedling emergence, stem, leaf and root elongation, starch and other storage material consumption, which can provide a theoretical basis for further clarifying the mechanism of rice submergence tolerance, screening suitable direct seeding varieties and cultivating special varieties of direct seeding rice.

2. Materials and Methods

2.1. Experimental Design and Material Cultivation

The experiment was conducted in a university laboratory from 2020 to 2021. 60 different genotypes of rice were selected as the experimental materials (Table 1),

Table 1. List of tested varieties.

which were provided by the crop institute of Sichuan Academy of Agricultural Sciences and the China Rice Research Institute of Chinese Academy of Agricultural Sciences. The sand is dried and screened and put into the cultivation box (inner diameter L × W × H: 100 mm × 100 mm × 90 mm), arrange the cultivation boxes neatly on the flat ground, and ensure that the sand depth of each box is 4 ± 0.5 cm through a ruler. The soil in the box is flat and 4 cm away from the box mouth, a total of 360 boxes.

Before the test, the sand shall be soaked by water spray. Each variety selects 100 plump grains/share, a total of 6. After soaking for 24 hours, evenly sow them on the wet sand surface, 100 grains per box, a total of 6 boxes for each variety. After sowing, water was injected along the edge of the box, and the submergence depth was 4 cm, so as to simulate the submergence environment of direct seeding rice at seedling stage.

The potted plants are placed in a light incubator at 25˚C, with a light cycle of 12 h/12 h. The water level is observed and replenished every day, so as to form a continuous submergence environment. After 0, 7 and 14 days of submergence treatment, samples were taken to detect relevant indicators.

2.2. Test Items and Methods

2.2.1. Emergence Rate

The germination number of rice seeds in each basin was counted and the seedling emergence rate (%) was calculated at 7 d and 14 d after submergence treatment. Each treatment had three replicates.

2.2.2. Plant Height and Root Length

At 7 and 14 days of submergence treatment respectively, 10 representative plants were selected from each box to keep the root system intact as far as possible, wash the root soil, and measure the plant height and root length of each plant respectively. If the root system is less than 1 mm, it is recorded as 0, that is, the radicle does not protrude. 10 strains per box, 3 boxes, 30 repetitions in total.

2.2.3. Plant Dry Matter Weight

At 0, 7 and 14 days of submergence treatment, 20 plants or rice seeds with the same growth were randomly selected/repeat, a total of 3 repetitions were put into small envelopes, dried to constant weight at 80˚C after killing at 105˚C and weighed (mg), and the dry matter mass of a single plant (mg) was calculated.

2.2.4. Plant Starch and Soluble Sugar Content

Plant dry matter was grinded into fine powder by high-flux grinder (Shanghai Jingxin). The concentration of starch and soluble sugar (%) was determined by anthrone concentrated sulfuric acid colorimetry, repeated three times. The content of starch and soluble sugar per plant and the total amount of non-structural carbohydrate (NSCs) (μg) were calculated according to the dry matter. The basic principle of the anthrone concentrated sulfuric acid colorimetry method: the furfural or carboxymethyl furfural formed by dehydration of the glucose group under the action of concentrated sulfuric acid reacts with anthrone, and the reaction is blue-green. Within a certain concentration range, the color depth is proportional to the glucose content [13].

2.3. Data Statistics and Analysis

Microsoft Excel 2016 software was used for data sorting and analysis; SPSS25.0 software for multivariate analysis such as principal component analysis and cluster analysis. The full name of SPSS is Statistical Product and Service Solutions, which means statistical products and service solutions. SPSS has always stood in the statistical software with its distinctive features, and is one of the most authoritative statistical software today. The basic functions of SPSS include data management, statistical analysis, chart analysis, output management, etc.; SPSS statistical analysis process includes descriptive statistics, mean comparison, general linear model, correlation analysis, regression analysis, cluster analysis, data simplification, multiple response and so on several categories. There are several statistical processes in each category. For example, regression analysis is divided into multiple statistical processes such as linear regression analysis, Logistic regression, Probit regression, weighted estimation, two-stage least squares method, and nonlinear regression. It also allows the user to select different methods and parameters [14]. For calculation of relevant indicators, refer to literature [15].

In order to eliminate the genotypic difference of each variety, the submergence tolerance coefficient of dry matter mass, starch, soluble sugar and non-structural carbohydrate per plant was converted, that is, the ratio of the measured value of 7 and 14 days to the measured value of 0 days.

Membership function values of comprehensive indexes of different rice varieties u(X_j):

$u\left(X_j\right)=\left(X_j-X_{\min }\right)/\left(X_{\max }-X_{\min }\right),j=1,2,\cdots ,n$ (1)

1) The weight of each comprehensive index w_j:

$w_j=p_j/{\displaystyle \underset{j=1}{\overset{n}{\sum }}{p}_j},j=1,2,\cdots ,n$ (2)

2) The comprehensive submergence tolerance of various rice varieties:

$D={\displaystyle \underset{j=1}{\overset{n}{\sum }}\left[u\left(X_j\right)\times w_j\right]},j=1,2,\cdots ,n$ (3)

3) Among them, X_j represents the j^th comprehensive index; X_min represents the minimum value of the j^th comprehensive index; X_max represents the maximum value of the j^th comprehensive index. w_j represents the importance degree or weight of the j^th comprehensive index among all the comprehensive indexes; p_j represents the contribution rate of the j^th comprehensive index of the rice variety; D is the submergence tolerance of the rice variety evaluated by the comprehensive index under submerged conditions Comprehensive evaluation value.

3. Results and Analysis

3.1. Performance and Correlation Analysis of Individual Indexes of Direct Seeding Rice under Submergence Conditions at Seedling Stage

It can be seen from Table 2 that the germination rate (ER), plant height (PH), root length (RL), dry matter mass (DW), soluble sugar (SS), starch (SC) content and non-structural carbohydrate (NSCs) of different varieties of rice were significantly different after 7 d and 14 d submergence treatment. Compared with 0 d, DW, SS, SC, NSC and other indicators of the tested varieties were reduced to varying degrees, but the decline was significantly different and the change was complex, indicating that the waterlogging tolerance of each variety was significantly different under 7 d and 14 d waterlogging treatment. It was difficult to reasonably evaluate the waterlogging tolerance of rice only by comparing the performance of different individual indicators.

From the correlation coefficient matrix of each single indicator (Figure 1), it can be seen that there is a large or small correlation between each single indicator, which will lead to overlapping information provided by them. At the same time, each single index plays a different role in the waterlogging tolerance of rice, which indicates that the waterlogging tolerance of rice is a complex comprehensive performance, and the direct use of each single index cannot accurately and intuitively reflect the waterlogging tolerance of rice. Therefore, in order to make up for the deficiency of classification and evaluation of waterlogging tolerance of single index, it is necessary to further use other multivariate statistical methods for multiple analysis on this basis.

Figure 1. Correlation matrix of each single index of direct-seedling rice under submergence.

Table 2. The submergence tolerance performance of direct seeding rice in seedling stage under stress.

ER is germination rate, PH is plant height, RL is root length, RDW is relative dry matter, RSC is relative starch content, RSS is relative soluble sugar content; RNS is relative NSCs content; suffix 1 and 2 represented 7 d and 14 d submergence treatments, respectively.

3.2. Principal Component Analysis

Principal component analysis was performed on 14 individual indicators. The contribution rates of the first six comprehensive indicators CI₁ - CI₆ were 27.300%, 19.027%, 12.935%, 10.596%, 8.524% and 5.764%, respectively (Table 3). The cumulative contribution rate was 84.146%, which can be ignored. This

Table 3. Coefficients of comprehensive indexes and proportion.

translates the original 14 individual indicators into 6 new independent composite indicators and represents the vast majority of information carried by the original individual indicators.

3.3. Comprehensive Indicators of Varieties

3.3.1. Analysis of Membership Function

According to Formula (1), the membership function values of each comprehensive index of rice varieties were calculated (Table 4). For the same comprehensive index such as CI₁, under waterlogging stress, the μ(X₁) value of Yanjing 1814 was the smallest, indicating that it showed the worst waterlogging tolerance in CI₁. The μ(X₁) of Yongyou 4953 was the largest, indicating that it had the strongest waterlogging tolerance in CI₁.

3.3.2. Weight Determination

According to the contribution rate of each comprehensive index, the weight can be calculated by Formula (2). After calculation, the weights of the six comprehensive index values are 0.324, 0.226, 0.154, 0.126, 0.101 and 0.068, respectively.

3.3.3. Comprehensive Evaluation

Formula (3) was used to calculate the comprehensive waterlogging tolerance of

Table 4. The value of each variety’s μ(X_j), value D and comprehensive valuation.

each direct seeding rice variety (Table 4), and the waterlogging tolerance was sorted according to D value. Among them, the D value of Deyou 4727 is the smallest, indicating that its waterlogging tolerance is the worst; Yongyou 7753 had the highest D value, indicating its strongest waterlogging tolerance. Systematic cluster analysis of D value (Figure 2) can divide 60 varieties into four categories: six rice varieties such as Yongyou 7753 are the first category; zhaoyou 5431 and other 22 varieties belong to category 2; 4 varieties such as Chuanzhongyou 3877 belong to the third category; 28 varieties such as Deyou 4727 belong

Figure 2. Cluster diagram of submergence tolerance of direct seeding rice of different genotypes at seedling emergence stage.

to the fourth category. Among them, according to the strength of submergence tolerance, the order is: Class 1 (Strong) > Class 2 (Medium) > Class 3 (Weak) > Class 4 (Sensitive).

4. Discussion

Compared with conventional seedling raising and transplanting, direct seedling cultivation of rice saves the process of raising seedlings and transplanting, and has the advantages of significant cost saving, labor saving, labor saving, seedling saving, high efficiency, and is conducive to mechanized operation [16] [17] [18] [19]. It has become the first choice for farmers who lack labor [20]. In addition, rice direct seeding also has significant advantages in saving water resources and reducing greenhouse gas emissions in rice fields [21]. It is an environment-friendly cultivation technology, which can solve the shortage of rural labor resources in the process of rice production and promote sustainable rice production. It plays an important role in increasing agricultural efficiency and farmers’ income, and the development of direct seeding rice production is an objective need [22]. Despite this, the production of direct seeding rice still faces problems such as difficulty in cultivating the whole seedlings, serious weed damage, shortened growth period, insufficient utilization of temperature and light resources, easy lodging under shallow root system, and poor high and stable yield [1] [2] [3]. Periodic submergence leads to rotten seedlings and dead seedlings, and poor complete seedlings are the primary problems. Therefore, on the basis of existing rice varieties, screening of genotypes with strong submergence tolerance at the seedling stage can be directly applied to direct seeding rice production, and can also provide a basis for the breeding of special varieties of direct seeding rice and the promotion and control technology of direct seeding emergence.

Under the hypoxic conditions caused by long-term submergence, whether direct-seeded rice can quickly germinate and extend leaves out of the water surface is the basis for the survival of direct-seeded rice plants, as well as seedlings and whole seedlings. By investigating the emergence rate of plants, it can intuitively reflect the differences in waterlogging tolerance of different genotypes of direct seeding rice at the emergence stage. The stems, sheaths and leaves of submergence-tolerant rice will rapidly elongate in a submerged environment. By being exposed to oxygen outside the water, the supply of oxygen and the accumulation of carbohydrates can be maintained to improve plant survival. At the same time, the rapid elongation and development of the rice root system in the submerged environment is conducive to the upright rooting of seedlings. At the same time, the higher root volume forms a larger root surface area, which can promote the absorption of oxygen and nutrients underwater, but submergence often inhibits Radical development of rice seeds. Chlorophyll is an important medium for plants to absorb, transform and transmit light energy. Under adverse conditions, the reduction of photosynthetic function caused by changes in plant photosynthetic pigments often affects the accumulation of carbohydrates and even the survival of plants. Therefore, the level of chlorophyll content can be used to evaluate the potential of dry matter accumulation and plant survival.

Therefore, this study compared and analyzed the seedling emergence rate, stem and leaf elongation, root development and storage capacity of different genotypes of rice under direct seeding and submergence conditions. The results showed that more than 78.1% of the rice seeds of the strongly submergence-tolerant rice genotype could germinate after 14 days of submergence at a depth of 4 cm, and the average plant height and root length were over 11.99 cm and 9.66 cm, respectively. The quality, starch content and soluble sugar content were significantly higher than those of other submergence-tolerant types of direct-seeded rice, indicating that this type of rice has strong adaptability to long-term submergence environment, not only can survive a large proportion, but also has stronger adaptability to stress conditions; The submergence-sensitive rice genotypes were treated by long-term submergence, and only no more than 56.00% of the rice seeds could germinate. The average plant height and root length were only 3.17 cm and 0.38 cm, respectively, and there was almost no radicle protruding. It is not recommended to broadcast live broadcasts in areas with uneven terrain and rain, which may easily lead to production reduction and higher risks.

5. Conclusion

In this study, 60 rice varieties were compared and analyzed in terms of emergence, stem, leaf and root elongation, and storage material consumption under the condition of water direct seeding. Among them, 6 strong submergence-tolerant types and 22 medium submergence-tolerant types were screened. There were 4 rice genotypes with weak submergence tolerance and 28 submergence sensitive genotypes. The strong submergence tolerant rice genotypes could resist adversity through rapid elongation of stems, leaves and roots.

Funding

This research was financially supported by the Applied Basic Research Project of Sichuan Provincial Department of Science and Technology (2020YJ0414) and Open Fund of State Key Laboratory of Rice Biology (20200403).

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Sun, J.Q., Yin, C.Y. and Wang, S.Y. (2019) Discussion on the Key Techniques of Direct Seeding Rice in North China. China Rice, 25, 125-128.
[2]	Chen, F.X., Tang, Y.P. and Xie, Y.J. (2018) Research Advances of Rice Mechanical Direct-Seeding Technology in China. China Rice, 24, 9-15.
[3]	Sun, K., Li, D.X. and Yang, J. (2019) Genome-Wide Association Analysis for Rice Submergence Seedling Rate. Scientia Agricultura Sinica, 52, 6-19.
[4]	Wang, Y., Wang, Y.Y. and Hong, D.L. (2009) Genetic Variation of Seed Vigor and Tolerance to Anoxia among Rice (Oryza sativa L.) Varieties in Taihu Lake Region. Journal of Nanjing Agricultural University, 32, 1-7.
[5]	Gibbs, J. (2000) Regulation of Alcoholic Fermentation in Coleoptiles of Two Rice Cultivars Differing in Tolerance to Anoxia. Journal of Experimental Botany, 51, 785-796. https://doi.org/10.1093/jexbot/51.345.785
[6]	Li, X. (2016) Resource Mining and Correlation Analysis of Submergence Tolerance of Indica Rice Seeds during Germination. Nanjing Agricultural University, Nanjing.
[7]	Matsushima, K. and Sakagami, J. (2013) Effects of Seed Hydropriming on Germination and Seedling Vigor during Emergence of Rice under Different Soil Moisture Conditions. American Journal of Plant Sciences, 4, 1584-1593. https://doi.org/10.4236/ajps.2013.48191
[8]	Turner, F.T., Cy-Chain, C. and Mccauley, G.N. (1981) Morphological Development of Rice Seedlings in Water at Controlled Oxygen Levels. Agronomy Journal, 73, 566-568. https://doi.org/10.2134/agronj1981.00021962007300030037x
[9]	Ismail, A.M., Ella, E.S. and Vergara, G.V. (2009) Mechanisms Associated with Tolerance to Submergence during Germination and Early Seedling Growth in Rice (Oryza sativa). Annals of Botany, 103, 197-209. https://doi.org/10.1093/aob/mcn211
[10]	Magneschi, L., Kudahettige, R.L. and Alpi, A. (2009) Comparative Analysis of Anoxic Coleoptile Elongation in Rice Varieties: Relationship between Coleoptile Length and Carbohydrate Levels, Fermentative Metabolism and Anaerobic Gene Expression. Plant Biology, 11, 561-573. https://doi.org/10.1111/j.1438-8677.2008.00150.x
[11]	Xiang, J., Wu, H. and Zhang, Y.P. (2017) Transcriptomic Analysis of Gibberellin- and Paclobutrazol-Treated Rice Seedlings under Submergence. International Journal of Molecular Sciences, 18, Article 2225. https://doi.org/10.3390/ijms18102225
[12]	Wu, H., Xiang, J., Chen, H.Z., Zhang, Y.P., Zhang, Y.K. and Zhu, D.F. (2018) Effects of Exogenous Growth Regulators on Plant Elongation and Carbohydrate Consumption of Rice Seedlings under Submergence. Chinese Journal of Applied Ecology, 29, 149-157.
[13]	Li, X.Z. and Guo, J.W. (2017) Chromogenic Condition on Determination of Polysaccharide by Anthrone-Sulfuric Acid. Sugar Crops of China, 39, 15-17+20.
[14]	Li, N.Q. (2021) Prediction of Urban Groundwater Water Consumption Based on SPSS Multiple Regression Analysis. Hebei Water Resources, 33, 42-43.
[15]	Wu, H., Hou, L.L., Zhou, Y.F., Fan, Z.C., Shi, J.Y. and Zhang, J.S. (2012) Analysis of Chilling-Tolerance and Determination of Chilling-Tolerance Evaluation Indicators in Cotton of Different Genotypes. Agricultural Science & Technology, 13, 2338-2346.
[16]	Zhao, L.P., Tao, Y.S., Tang, Y.P. and Tang, Q.Y. (2013) The Evolution History and Development Trend of Rice’s Cultivation Methods. Crop Research, 27, 169-173.
[17]	Qu, Z.G. (1998) A Brief Talk on the Application of Varieties in the Light, Simple and High-Yield Cultivation of Rice. China Rice, 5, 7-8.
[18]	Rao, A.N., Johnson, D.E. and Sivaprasad, B. (2007) Weed Management in Direct-Seeded Rice. Advances in Agronomy, 93, 155-255. https://doi.org/10.1016/S0065-2113(06)93004-1
[19]	Iqbal, M.F., Hussain, M. and Rasheed, A. (2017) Direct Seeded Rice: Purely a Site Specific Technology. International Journal of Advanced Research in Biological Sciences, 4, 53-57.
[20]	Liu, G.C. and Sun, T.Q. (2012) Analysis on Production Status of Direct-Seeded Rice in Jiangsu Province and Discussion on Countermeasures. China Seed Industry, 31, 44-45.
[21]	Ye, T., Qian, C. and Peng, S. (2016) Lower Global Warming Potential and Higher Yield of Wet Direct-Seeded Rice in Central China. Agronomy for Sustainable Development, 36, Article No. 24. https://doi.org/10.1007/s13593-016-0361-2
[22]	Wu, X.Z. and Zeng, Q.S. (2017) Development Status, Existing Problems and Countermeasures of Direct Seeding Rice in Hubei Province. Rural Economy and Science-Technology, 28, 154-155.