Screening of Red Rice (Oryza sativa L.) Landraces for Drought Tolerance at Early Stages Using PEG 6000

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INTRODUCTION

Red rice refers to the color of rice kernel which appeared on many different types of rice such as cultivated rice, weedy rice, and their wild relatives. In the United States it is well known that red rice is originally from Oryza sativa sup species indica, but using simple sequence length polymorphism (SSLP) marker, Vaughan et al.

(2001) proved that it is not only from sup species indica but also from sup species japonica. Most importantly red rice genotypes from various places such as Arkansas, Louisiana, Mississippi, and Texas form a distinct group and are closely related to the Oryza nivara and rufipogon group. Red rice is also cultivated in restricted areas where people have cultural, ceremonial, and is also used as traditional medicine. Red rice is consumed as a whole grain without any heavy processing, so this type of rice is rich in protein, lipids, dietary fibers,

minerals, vitamins, and phenolic compounds. The flavonoids are proanthocyanidins that are beneficial as antioxidants and anti-inflammatory, and also have immunomodulatory, anticancer, antithrombotic, and cardioprotective properties (Filho et al. 2020).

Despite having more beneficial properties for human health, this type of rice is quite not popular and widely consumed as regular white rice.

The development of red rice is needed to improve some important traits such as productivity, eating quality, resistance to biotic and abiotic stresses, so that more people will widely use and consume it. The breeding program is the main method to improve and develop new varieties. In Indonesia, the development of red rice through the breeding program was lacking behind regular white rice. Mau et al. (2017) studied the genetic diversity of 46 colored rice accessions local from East Nusa Tenggara and found that there were significant differences among rice accessions ARTICLE INFO

Keywords:

Early screening Local varieties Red pericarp Article History:

Received: March 4, 2022 Accepted: March 14, 2023

*⁾ Corresponding author:

E-mail: m.syafii@trunojoyo.ac.id

ABSTRACT

Despite being one of the most important staple food consumed by more than half of the world population, the development of red rice still lacks behind the regular white rice. In Madura, most farmers still use their local landraces with red pericarp. Since most of the growing season is dependent on rainfall, those landraces are expected to provide some useful genes related to drought tolerance. This research aimed to select drought-tolerant accessions candidates of red rice derived from pure lines selection at early stages using PEG 6000. A randomized complete block design was used with two factors and three replications, the first factor was red rice genotype and the second factor was two levels of PEG 6000 concentration (0% and 25%). The results showed that there were different responses in all genotypes used in terms of their responses to PEG 6000 screening. Seedling length, seminal root length, shoot length, seminal root dry weight, and shoot dry weight were reduced by the application of PEG 6000 compared to non-PEG 6000 media. Two genotypes had a better response to PEG 6000 treatment compared to check tolerant (Salumpikit), and these are expected to become a valuable resources for further breeding activities.

ISSN: 0126-0537

Cite this as: Fatimah, S., Amzeri, A., Syafii, M., & Purwaningsih, Y. (2023). Screening of red rice (Oryza sativa L.) landraces for drought tolerance at early stages using PEG 6000. AGRIVITA Journal of Agricultural Science, 45(2), 199- 208. http://doi.org/10.17503/agrivita.v45i2.3723

Screening of Red Rice (Oryza sativa L.) Landraces for Drought Tolerance at Early Stages Using PEG 6000

Siti Fatimah, Achmad Amzeri, Mohammad Syafii^*), and Yuzy Purwaningsih Universitas Trunojoyo Madura, Bangkalan, East Java, Indonesia

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both in qualitative and quantitative traits. Using SSR markers to study genetic diversity based on palatability traits, Susiyanti et al (2020) found that local accessions from Banten form two distinct groups based on dendrogram analysis.

Madura is one of the dry areas in Indonesia, and rice is one of the main crops for most farmers.

Local varieties are also still cultivated in some areas which had better adaptability in dry conditions and are expected to contain some genes related to drought tolerance. As the global warming becomes an important thread for rice productivity, drought resistance genes are important to combat this future problem. So that collecting, preserving, and using traditional rice in a breeding program are necessary. According to Marone et al. (2021) using landraces in breeding activities will help to improve sustainable agriculture production, particularly enhancing adaptation and production in stress- prone environments to cope with climate changes due to their adaptability to unfavorable conditions.

Genetic diversity is a key factor in developing new varieties. The role of a breeding program is to combine as many as possible positive traits in one plant or population, which decreased the diversity within a population. So that there always be a complex relationship between plant breeding and diversity (Louwaars 2018). The presence of high variability within the population will benefit breeders, which will strengthen the divergence of its population. The importance of genetic differentiation is also to create desirable heterotic groups in their base breeding populations (Aljumaili et al. 2018).

Based on preliminary research, local varieties in Madura also have a wide diversity in morphological characters. There was no information related to their tolerance to drought stresses. Assessing genetic diversity related to drought tolerance is necessary to have a better understanding of their potential to provide useful genes on local varieties.

Drought tolerance screening at early stages can be done using polyethylene glycol (PEG) 6000. PEG was regularly used in studies related to drought stresses at early stages in many different plants. Tabatai et al. (2022) used PEG to evaluate and investigated the response of 169 bread wheat inbreed lines at vegetative stages. The results showed that PEG had a significant impact on all parameters tested including shoot length, root length, ratios of root wet weight to shoot wet weight, ratios of root dry weight of root and shoot, and also on plant length. To study drought tolerance in the seedling stage using transcriptome analysis, Abdel- Ghany et al. (2020) used PEG in their study of drought tolerance screening using drought-tolerant

and drought-sensitive sorghum (Sorghum bicolor) and revealed that about 180 genes are differentially regulated in response to drought induced by PEG treatment in drought-resistant genotypes. Drought stresses simulated by PEG at 20% were reported to delay germination in all tested pepper (Capsicum spp.) genotypes (Bernau et al. 2020). Drought stress simulated by PEG 6000 at germination stages significantly changed soluble protein content, peroxidase activity, and malondialdehyde in Larix olgensis A. Henry. Yousefi et al. (2020) also tested several plants such as Atriplex canescens, Salsola kali, Zygophyllum fabago under drought stress simulated by PEG 6000 at germination and vegetative stages. This research was conducted to evaluate local red rice accessions from Madura againts drought condition at early stages using PEG 6000.

MATERIALS AND METHODS

This research was conducted at Biotechnology Laboratory, Agroecotechnology Study Program, Universitas Trunojoyo Madura from December 2019 to February 2020. Twenty- three red rice local accessions from Madura derived from pure line selection were evaluated (Table 1).

Three check varieties namely Salumpikit (drought tolerance check), Ciherang (mega variety), and IR 20 (drought susceptible check). were used in this experiment.

A factorial randomized complete block design with three replications was used to facilitate the combination of two factors. The first factor was two levels of PEG (0% and 25%), and the second factor was 26 red rice genotypes. The PEG concentration of 25% is equal to -9.9 bar water retention at the field. The germinated seeds were placed on a petri dish containing 10 ml of 25% PEG (treatment) and 10 ml of distillate water (control). Each petri dish contained 20 germinated seeds. The observation were carried out at seminal root length (RL), shoot length (ShL), seedling length (SL), seminal root dry weight (SRD), and shoot dry weight (SD).

Drought sensitivity index (DSI) was used to obtain drought tolerance information among genotypes. DSI was calculated based on the eqn. 1:

DSI = (1-Yg/Yi)/(1-Ymr/ Yml) ...1) Where: Yg = mean of certain genotypes under drought treatment, Yi = mean of all genotypes under control condition (non-PEG treatment), Ymr = mean of all genotypes under drought treatment, Yml =

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mean of all genotypes under control conditions. DSI would separate genotype tested into three groups based on their score, ≤ 0.5 for a tolerant group, 0.5 < ISK ≤ 1.0 for moderate tolerant, and > 1.0 for susceptible.

The collected data analyzed and the differences between treatments were tested using least significant difference (LSD). The DSI score among genotypes was subjected to Pearson correlation analysis to look at the correlation among DSI parameters. Principle component analysis (PCA) was done to identify the DSI parameter(s) contribute greatly to diversity. Dendrogram analysis was performed using agglomerative clustering methods to cluster all genotypes using Statistic Tools for Agriculture Research (STAR) software.

RESULTS AND DISCUSSION

The study related with rice genetic diversity among landrace populations in Madura is still limited compared to other area in Indonesia. One of the old

publications that focused on the local rice Madura was published by Katayama in 1987, since then there was no record related to population structure or genetic study Madura local rice. It was believed that rice in Madura was introduced from Java, Bali, India, Philippines, and others, especially those the cultivated varieties. While, the primitive javanica and indica types are indigenous. A later study, based on grain morphology using tripartite classification revealed that rice cultivars in Madura belong to javanica and indica (Katayama 1987; Katayama 1988). Since the regular white rice predominantly cultivated by farmers came from a single mutation of Rc genes in japonica rice and was transferred to an indica during domestication (Sweeney et al. 2007), red rice become less popular. Breeding activities of this pigmented rice was also lack behind the regular white rice, so using this type of rice as breeding material becomes more important to find important genes that are not present in regular white rice due to the domestication process.

Table 1. Rice genotypes used in this study

No Genotypes Origin

1 SM-39 Local accessions from Madura

24 Ciherang Indonesian Center for Rice Research (BB Padi) 25 Salumpikit Indonesian Center for Rice Research (BB Padi)

26 IR 20 Indonesian Center for Rice Research (BB Padi)

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Table 2. Effect 25% PEG 6000 on seminal root length, shoot length, seedling length, shoot dry weight, and seminal root dry weight of Madura red rice landraces GenotypeRL (cm)ShL (cm)SL (cm)SD (g)SRD (g) PEGControlPEGControlPEGControlPEGControlPEGControl SM-101.17efg5.67g0.23l6.83defg1.98lm12.50ef0.006f0.073c 0.012hi0.067a SM-112.09defg3.82n0.32i6.12ij3.29gh9.94lm0.007f0.068de 0.015fg0.051g SM-122.71cde4.88jk0.33i6.74efg4.23cde11.63ij0.015e0.072c 0.017f0.060cde SM-132.45cdef4.32l0.31i6.32hi3.67fg10.64k0.056a0.071cd 0.011hi0.052g SM-141.85defg6.58c0.32i7.00cdef2.95hi13.58c0.007f0.056kl 0.012ghi0.031i SM-152.21def3.80n0.42ef6.49gh3.49fg10.29kl0.012e0.082a 0.016f0.058ef SM-162.22def3.43o0.40fg5.90j3.55fg9.32n0.014e0.062gh 0.023de0.050g SM-21.65defg2.35p0.45d7.25c2.72ijk9.59mn0.007f0.054l 0.013gh0.034i SM-211.79defg5.91f0.30ij6.56gh2.93hi12.47ef0.007f0.073c 0.012hi0.064b SM-244.79a8.31a0.87a5.98ij7.63a14.28b0.014e0.074c 0.035b0.059de SM-260.92fg5.32i0.44de7.01cde1.63m12.32efg0.006f0.078b 0.004m0.061bcde SM-272.45cdef4.69k0.40f7.15cd3.91def11.84hi0.014e0.080ab 0.012hi0.051g SM-283.94abc6.07ef0.65b8.21b6.27b14.28b0.054a0.082a 0.059a0.060cde SM-312.46cdef4.97j0.20m7.15cd3.80ef12.11fgh0.021d0.058ijk 0.021e0.027j SM-354.38ab6.44cd0.50c8.72a6.48b15.15a0.014e0.067ef 0.035b0.056f SM-361.14efg7.26b0.16n6.79defg1.83m14.04b0.007f0.082a 0.010ij0.061bcde SM-371.49defg6.25de0.32i6.84defg2.40jkl13.08d0.006f0.062gh 0.017f0.050g SM-392.02defg4.35l0.36h6.98cdef2.99hi11.33j0.007f0.082a 0.011hi0.041h SM-421.94defg4.81jk0.32i7.05cde2.97hi11.85ghi0.006f0.059hij 0.005lm0.063bc SM-451.75defg5.65g0.37gh6.54gh2.70ijk12.19fgh0.008f0.072c 0.011hi0.060cde SM-51.44efg5.54gh0.26k7.16cd2.31kl12.70de0.009f0.072c 0.013gh0.061bcde SM-503.10bcd5.31i0.44de6.83defg4.55c12.15fgh0.015e0.065ef 0.021e0.060cde SM-91.71defg4.05m0.30ij5.92j2.80hij9.97lm0.006f0.072c 0.008jk0.062bcd Ciherang2.47cdef5.38hi0.28jk6.09ij3.87def11.47ij0.025c0.057jkl 0.024d0.050g IR 200.56g3.40o0.26k5.46k1.01n8.87o0.007f0.060hi 0.008kl0.053g Salumpikit 2.67cde5.61g0.36h6.62fgh4.34cd12.23fgh0.036b0.064fg 0.031c0.052g Remarks: Numbers followed by the same letter in the same column are not significantly different in the LSD test at 5%; RL: root length, ShL: shoot length, SL: seedling length, SD: shoot dry weight, SRD: seminal root dry weight

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Table 3. Drought sensitivity index (DSI) of some observation variables of Madura local red rice genotypes NoGenotypeRLCategoriesShLCategoriesSLCategoriesSDCategoriesSRDCategories 1SM-390.99M1.00M1.04S1.07S1.17S 2SM-350.18T1.00M0.81M0.60M1.01S 3SM-130.56M1.01S0.92M1.17S0.26T 4SM-150.48T0.99M0.93M1.09S1.08S 5SM-211.40S1.01S1.08S1.21S1.15S 6SM-361.94S1.04S1.23S1.24S1.17S 7SM-451.48S1.00M1.10S1.21S1.14S 8SM-51.58S1.02S1.15S1.17S1.12S 9SM-371.67S1.01S1.15S0.98M1.15S 10SM-120.50M1.01S0.90M1.07S0.96M 11SM-240.47T0.90M0.66M0.60M1.03S 12SM-160.22T0.99M0.87M0.81M0.99M 13SM-310.70M1.03S0.97M0.22M0.81M 14SM-101.74S1.02S1.19S1.22S1.18S 15SM-90.96S1.00M1.02S1.29S1.16S 16SM-261.95S0.99M1.23S1.38S1.17S 17SM-110.56M1.00M0.94M1.06S1.14S 18SM-500.57M0.99M0.88M0.96M0.98M 19SM-20.08T0.99M1.01S0.92M1.11S 20SM-280.18T0.97M0.79M0.03T0.44T 21SM-270.63M1.00M0.94M1.14S1.05S 22SM-141.51S1.01S1.11S0.90M1.11S 23SM-421.12M1.01S1.06S1.37S1.15S 24Ciherang0.83M1.01S0.94M0.79M0.72M 25Salumpikit0.73M1.00M0.91M0.59M0.56M 26IR 201.96S1.01M1.30S1.30S1.12S Remarks: RL= root length, ShL= shoot length, SL = seedling length, SD = shoot dry weight, SRD = seminal root dry weight; M = moderate, T = tolerant, S susceptible

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As one of the dry areas in Indonesia, local rice from Madura is expected to contain genes especially related to drought tolerance. The common feature of all drought stress studies is a reduction of water potential in the substrate. In an aqueous method, PEG with an average molecular weight of 6000 or more becomes a common method to assess drought tolerance in plants (Osmolovskaya et al. 2018).

Using 25% PEG 6000 to screen drought tolerance in local red rice from Madura we found that all the observed parameters decreased compared to non- PEG media (Table 2). According to Akbar et al.

(2018), PEG 6000 treatment at 25% will decrease the water potential so that the seedling could not have the ability to absorb more water in the media. In an in-vitro method to assess drought-tolerant among Kurdish rice genotypes, Rahim et al. (2020) also reported that PEG 6000 had a significant impact on callus growth parameters, and there were different responses in all genotypes tested. Increased PEG concentration from 0 to 200 g/l was also reported to decrease the percentage of rice germination in a seed priming study (Abdallah et al. 2016).

The root is an important trait because it is related to water and nutrient uptake ability (Kim et al. 2020). There were significant differences in responses among genotypes tested on 25% PEG 6000 for seminal root length. Seminal root length on PEG 6000 ranged between 0.65 to 4.79. As a susceptible check, IR 20 had the shortest seminal roots (0.56) and no significant difference with 13 other genotypes namely SM-2, SM-5, SM-9, SM- 10, SM-11, SM-14, SM-21, SM-26, SM-36, SM- 37, SM-39, SM-42, SM-45. The genotype with the highest seminal root length was SM-24 (4.79), but no significant difference between Salumpikit as a tolerant check and two other genotypes namely SM-28 and SM-35. The water uptake ability of genetically diverse genotypes is an important criterion for drought response studies in rice (Khan et al. 2020). In response to severe drought stresses, plants will save energy by avoiding an unnecessary process that is irrelevant to their survival. In rice, one such process is the reduction of adventitious roots and inhibits adventitious root formation through the downregulation of miR160 (Nadarajah & Kumar 2019).

The shoot length of the genotype tested in this experiment ranged between 0.157 to 0.87 cm on PEG 6000 Media (Table 2). Han et al. (2018) in a quantitative traits loci (QTL) associated with drought tolerance study under PEG 6000 20 % treatment was also found that 10 growth parameters were inhibited

including shoot length, both for parents and two RILs populations used. Ten QTLs associated with drought tolerance in PEG 6000 simulation were also detected on chromosomes 1, 3, 5, 9, and 11 which explain 5.2-18.5% of the observed variation. SM-36 had the shortest shoot among the genotype tested, while SM-24 was the highest. Nine genotypes had higher shoot lengths compared to tolerant check (Salumpikit) namely SM-2, SM-15, SM-16, SM- 24, SM-26, SM-27, SM-28, SM-35, and SM-50.

This finding was quite interesting since there were genotypes that perform equally or even better than drought-tolerant check varieties. IR-20 consistently becomes the least genotype performing well on PEG media. SM-35 was the highest genotype in seedling length, while IR20 was the lowest.

Unlike some other crops, rice is extremely susceptible to drought at germination and early seedling growth stages (Panda et al. 2021). Another rice growth parameter that was previously reported affected by drought was biomass including shoot and root dry weight. All genotypes tested in drought stresses showed a significant reduction of the shoot and dry weight compared to the control (Miftahuddin et al. 2020). Similar to that result, our study showed that the shoot dry weight of all genotypes under PEG 6000 25% treatment was lower than the control (Table 2). The range between genotypes tested was 0.0060 to 0.056 g in PEG media, while in control was 0.054 to 0.08 g. Four genotypes namely SM- 2, SM-11, SM-21, and SM-36 had the lowest dry weight, with no significant difference compared to IR-20 (susceptible check), while SM-13 and SM-28 had the highest shoot dry weight compared to other genotypes tested. In root dry weight variables, SM- 28 showed the highest root dry weight compared to the other genotypes, whereas SM-26 showed the lowest among the tested genotypes. The comparison between the check varieties and some tested genotypes was presented in Fig. 1.

The drought sensitivity index (DSI) was used to assess drought sensitivity among genotypes, and widely used among researchers to study the response of certain genotypes under drought stresses. Rahmah et al. (2020) used DSI values to asses bambara groundnut genotypes at early stages using PEG. Based on DSI results, six genotypes were considered as tolerant to drought simulated with PEG 6000 treatment, whereas Salumpikit was moderately tolerant with Ciherang and seven other genotypes on seminal root variable (Table 3). In shoot length, thirteen genotypes were categorized as susceptible including IR-20 and Ciherang,

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whereas ten other genotypes were moderately tolerant. On the seedling length variable, IR-20 and twelve other genotypes were considered as susceptible, while Ciherang and Salumpikit were assigned to moderately tolerant with fourteen other genotypes.

For the root dry weight variable, twelve genotypes were categorized as susceptible including IR-20 as susceptible check and seven genotypes were fallen in to moderately tolerant with Ciherang and drought tolerance check (Salumpikit).

Two genotypes had better responses compared to tolerant checks and were categorized as tolerant to drought. Seventeen genotypes were susceptible, four genotypes were moderately tolerant with Ciherang and Salumpikit, and two other genotypes were tolerant. As a tolerant check, Salumpikit consistently becomes moderately tolerant. A similar result was reported by Akbar et al. (2018) where Salumpikit was considered a moderately tolerant genotype and so was Ciherang. These findings were interesting since some of the local red rice from

Madura showed a better response than the tolerant check. We need to further analyze all promising genotypes that perform similarly and better than check on vegetative and generative stages to better understand their drought tolerance traits. It is because drought stresses affect plants throughout the life cycle, from germination to maturity (Seleiman et al. 2021). The response mechanism includes the morpho-physiological, biochemical, cellular, and molecular processes. These processes include the improvement of the root system, leaf structure, osmotic adjustment, relative water content, and stomata regulation (Ilyas et al. 2020).

Based on Pearson correlation analysis in Table 4 showed that DSI seminal root length was significantly and positively correlated with all other DSI variables observed. This result indicated that the higher the root length would induce higher other DSI variables. DSI shoot length was not correlated with DSI shoot dry weight and seminal root dry weight.

Table 4. Correlation between DSI variables

ShL SL SD SRD

RL 0.4216* 0.8939** 0.5846** 0.4663*

ShL 0.6439** 0.3233^tn 0.0798^tn

SL 0.6511** 0.5062**

SD 0.5602**

Remarks: RL= root length, ShL= shoot length, SL = seedling length, SD = shoot dry weight, SRD = seminal root dry weight

Fig. 1. Comparison between IR 20 as check tolerant (A), Salumpikit as susceptible varieties (B), SM 12 and SM 13 as genotype tolerant (C, D, respectively), and SM 21 as genotype susceptible (E) in PEG 6000

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Principle component analysis (PCA) showed that the first three components explain 89.998%

of the total variance (Table 5). The contribution of each component namely PC1, PC2, and PC3 were 58.55%, 20.57%, and 10.86% respectively. Seminal root length and seedling length were DSI variables with more than 50% on PC1 and shoot length was the highest on PC2 (88.19%). It showed that DSI Seminal root length, DSI seedling length, and DSI shoot length were important characteristics that contribute to the diversity response of genotypes

in PEG 6000 treatment. Using PEG 6000 to assess genotypic variability among indigenous rice landraces of Jeypore tract of Odhisa related to drought tolerance on morpho-physiological traits, Mishra et al. (2019) found that the first three PCs explain 62.46% of the total variance, where germination percentage, shoot length, root length, chlorophyll index, proline content, and vigor index was considered as an important trait as selection parameters for drought tolerance rice under PEG 6000.

Fig. 2. The dendogram generated from the cluster analysis of the tested genotypes Table 5. Principle component analysis of tested genotype based on DSI variable

DSI Variables PC1 PC2 PC3

Seminal root length -0.5282 0.1244 -0.4056

Shoot length -0.1833 0.8819 0.4309

Seedling length -0.5379 0.0619 -0.383

Root dry weight -0.4785 -0.1963 0.1113

Shoot dry weight -0.4112 -0.4055 0.7005

Eigen value 2.9277 1.0284 0.5431

Proportion of Variance 0.5855 0.2057 0.1086

Cumulative Proportion 0.5855 0.7912 0.8998

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For clustering data, hierarchical agglomerative methods stand out as particularly effective and popular approaches (Tokuda et al. 2022). Dendogram analysis using agglomerative clustering methods showed that all genotypes were separated into two major groups (Fig. 2.). It clearly showed that the susceptible group was separated from the tolerant group. The degree of tolerance is based on check variety, where genotypes fallen in the same group with the susceptible check would be considered as susceptible. The genotypes in the same group with tolerant check would be considered as tolerant to drought. There were twelve genotypes in a susceptible group, and 14 other genotypes including Ciherang and Salumpikit were considered tolerant to drought. Transcriptome Analysis related to drought tolerance in Triticum aestivum L using PEG 6000 15%

was reported by Xi et al. (2023) 16 key genes were responsive to drought stress. Four genes related to drought were also verified by mutant analysis and RT-qPCR. Since drought-tolerant traits vary between growth stages, it will need further assessment of drought tolerance at different growth stages.

CONCLUSION

PEG treatment decreased all growth parameters due to the lack of water availability in the media compared to the control. There were different responses in all genotypes tested on PEG media, where the majority of the susceptible or tolerant genotypes performed similarly with the pertinent check varieties. There were 10 genotypes of local red rice accessions from Madura considered as tolerance to drought in the germination stage.

Further analysis is needed to obtain their tolerance mechanism for breeding purposes.

ACKNOWLEDGEMENT

We would like to thank Indonesian Center for Rice Research (BB Padi) for providing us with the seeds that we used in this research.

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