International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

Hematoxylin Root Stain as an Indicator for Al Toxicity Tolerance in F5 Generation from a Cross Between Local Rainfed Rice “Pendek” and IR 78581 Kiky Nurfitri Sari*, Catur Herison**, Marwanto** *Graduate Student of Agroecotechnology **Department Agroecotechnology, Univerisity of Bengkulu, Indonesia [email protected]

ABSTRACT

Aluminum toxicity caused by low pH is one of the abiotic factors limiting rain fed rice yield in Ultisol. Therefore, breeding rain fed rice hybrids tolerant to aluminum toxicity is important. This study was undertaken to determine the genotypic variation for aluminum toxicity tolerance in F5 generations from a cross between local rain fed rice “Pendek” and IR 78581 based on hematoxylin root stain. Fifteen genotypes of the F5 generations were studied for root hematoxylin staining color under different aluminum concentrations using nutrient solution culture. The results showed that aluminium accumulation were observed for all genotypes tested as indicated by the existence of brown color in their root tips, suggesting that this trait was not able to be used for aluminium tolerance screening. Key words: Al toxicity, hematoxylin, nutrient solution culture, ultisol, rice.

INTRODUCTION Aluminum toxicity is a major factor inhibiting crop growth and yields in Ultisol. A serious impact of Al stress on crop growth and yields led researchers to breednew Al tolerant varieties. Breeding for Al tolerance in rice have been carried in recent years. The use of local rice germplasm becomes the main objective to obtain Al tolerant genotypes. In addition, the use of selection methods also vary from nutrient culture selection in laboratory to test directly against crop in the field (Santika, 2011). Generally the nature of test result directly through the analysis crop yield on the field takes time, effort and cost. Therefore, the method of selection in nutrient culture is an efficient way to obtain genotypes tolerant to Al stress. Selection of genotypes resistant to Al stress in nutrient culture was performed by testing to some extent Al stress concentration through staining of hematoxylin. Hematoxylin coloration test was widely used by researchers to find out the degree of tolerance of crops against Al stress. The test was conducted by Dewi and Miftahudin (2012) to differentiate rice varieties having characteristics tolerant and sensitive to Al stress. In this research the test was utilized to determine the genotypic variation for aluminum toxicity tolerance in F5 generations from a cross between local rain fed rice “Pendek” and IR 78581 based on hematoxylin root stain.

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

MATERIALS AND METHODS The research was carried out in the green house of Laboratory of Agronomy Faculty of Agriculture Universityof Bengkulu. The rice genotypes used in this study consisted of 15 genotypes F5 family resulting from a cross between a local rainfed Padi Pendek x IR 78581. The 15 days old rice seedlings was planted in nutrient culture with different Al concentrations (0 ppm Al, 80 ppm Al and 100 ppm Al) for 15 days. After 15 days incubation the seedlings were removed from nutrient culture and their roots were washed in running tap water. The roots were then soaked in a 0.2% hematoxylin solution for 30 minutes, rinsed again with aquades and their staining pattern was observed under a microscope.

RESULTS AND DISCUSSION Rice is one of cereal crops tolerant to Alstress (Dewi and Miftahudin, 2012). Although rice genotypic variations in rice tolerance to Al stress existed, but these variations apparently was not able to be used to differentiate sensitive and tolerant genotypes. Apparently, the level of Al concentration and duration of Al stress affected the test result on the genotype tolerance. Previous works by Polle et al. (1978) and Delhaize et al. (1995) used hematoxylin to detect Al accumulation in wheat roots. Hematoxylin staining results showed that Al accumulates on the walls of the root cell experiencing Al stress (Miguel et al., 2002). The test results on root samples from Family F5 (G3, G13, G16, and G35 G45) showed that hematoxylin root staining color in the roots treated with Al stress was brown. The dark brown color was observed in these areas of the tip of the root (Figure 1). It indicated that Al was absorbed by the rice roots treated with stress. Roots with no stained color were observed for the genotypes not treated with Al stress in nutrient culture. Meanwhile, the results of this study indicated that significant difference in root staining color was not observed in genotypes treated with Al stress. A different result was reported by Dewi and Miftahudin (2012) in that brown root color as an indicator for Al accumulation in root was only observed on the Al sensitive genotype like Krowal and IR 64.

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

(a)

(b)

(c)

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Figure 1. Rice plants under stress aluminum hematoxylin staining. As mentioned earlier that hematoxylin root stain was not able to be used as an indicator to determine genotypic tolerance to Al stress in rice. This was due to the failure to maintain pH nutrient solution at contstant value of 4.0 – 5.0 throughout the experiment. Therefore, the result of this study was not in agreement with the previous work in that Al mechanism tolerance was internally characterized by the presence of Al that accumulate in the plant root either for sensitive or tolerant genotypes to Al stress (Jixing et al., 2010). For the genotypic tolerance, Al will not enter into vacuoles and penetrate the plasma membrane during Al stress period, or vice versa. Generally the internal and external tolerance mechanism occurred simultaneously with the main difference in the area where Al chelating process took place in simplastor apoplast (Utama 2015). In her previous work Sari (2014) suggested that the hematoxylin colorization varied from brown to colorless or clear color in root tips during Al stress. Colorless hematoxylin stain was considered as a mechanism of crop tolerance to Alstress by releasing organic acid exudate in the root tips (Leon et al. 2004). The organic acids chelated Al present around the root tips in the form of ligand so that Al did not disturb nutrient absorption nad root growth. According to Utama (2015), tolerant crops were capable of producing organic acids 2.0-2.5 times higher than those of sensitive crops under Al stress condition.

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

CONCLUSIONS Aluminium accumulation were observed for all genotypes tested as indicated by the existence of brown color in their root tips, suggesting that this hematoxylin root stain was not able to be used for aluminium tolerance determination.

REFERENCES Dewi, I.R and Miftahudin. 2012. Variation Degrees of Tolerance Against Rice Plants Under Stress Aluminum Hematoxylin Staining. Research Report Institut Pertanian Bogor. Bogor: Institut Pertanian Bogor. Jixing, X., N. Yamaji, T. Kasai, and J.F. Ma. 2010. Plasma membrane-localized transporter for aluminum in rice. Agrie. Sci. 107 (43): 18381-18385. Leon, V., Kochain, A. Owen, and Hoekenga. 2004. How do crop plant tolerance of acid soils? Mechanisms of tolerance aluminum and phosphorous efficiency. U.S. Plant, Soil, and Nutrition Laboratory. USDA-ARS. Cornell University, Ithaca. New York. Miguel, A., Pineros, V. Jurandir, V.M.C. Alves and V.K. Leon. 2002. The Physiology and Biophysics of an aluminum tolerance mechanism based on root exudation citrate in maize. Plant Physio 129: 1194-1206. Polle, E., C.F. Konzak, J.A. Kittrick . 1978. Visual detection levels of aluminum tolerance in wheat by hematoxylin staining. Crop Sci. 18: 823-827. Santika, A. 2011. Mechanical testing of upland rice lines against aluminum toxicity glass house. Agricultural Engineering Bulletin 16: 43-47. Sari, K.N. 2014. Performance of F2 population of crossbred offspring local upland rice "Short" with rice IR 78 581 at some level of stress Al (aluminum). Essay. Bengkulu: Faculty of Agriculture. University of Bengkulu. Utama, M.Z. 2015. Rice Cultivation on Marginal Land Tips Increase Rice Production. Yogyakarta: Andi Offset.

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