Hydroponic screening and characterization of aluminium tolerance on finger millet ( Eleusine coracana ( L . ) Gaertn ) accessions

Biotic and abiotic stress combined with the use of less productive local cultivars cause low production of finger millet in Ethiopia. This research was conducted to investigate acidity tolerance of finger millet accessions. Preliminary screening was done on 288 accessions and six improved national cultivars of finger millet. Twenty randomly selected and surface-sterilized seeds of each germplasm were wrapped and germinated in a tissue paper in Petri dishes. Thirty six hours-old seedlings of uniform size were transferred to the nutrient solutions having 500 μM KNO3, 500 μM CaCl2, 500 μM NH4NO3, 150 μM MgSO4.7H2O, 10 μM KH2PO4, 2 μM FeCl3 (III) and 112.5 μM Al2 (SO4)3.18H2O and allowed to grow for a further 8 days along with tolerant and susceptible references. Characterization with (112.5 μM) and without (0 μM) Al conditions was also done on 80 accessions. After eight days root and shoot length of seedlings were measured using a ruler, while fresh weight of these seedlings was taken using a digital balance. Mean separation and analysis of variance on each treatment was conducted using SPSS software. Relative total root length (RTRL) and root growth inhibition (RGI) were also estimated. From screening of 288 accessions, 75 (26.04%) of them were Al tolerant, while 213 (73.95%) of them were medium to susceptible. From characterization, 63 (78.75%) showed significant Al stress in root length, 23 (28.75%) in fresh weight, while no distinct and visible symptom were observed in shoot growth. The study clearly showed the possibility of developing lines and genotypes that can tolerate acidity in Ethiopian context and support agricultural development in acidic soils in the country and in the world.


INTRODUCTION
Acid soils (with a pH of 5.5 or lower) are among the most important limitations to agricultural production.It has been estimated that 15% of the world's soil is acidic and that over 50% of the world's potentially arable lands are acidic (von Uexküii and Mutert, 1995).Aluminium (Al 3+ ) ranks third in abundance among the earth's crust *Corresponding author.E-mail: haftom1@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License elements, after oxygen and silicon, and is the most abundant metallic element.A large amount of Al is incorporated into aluminosilicate soil minerals and very small quantities appear in the soluble form, capable of influencing biological systems (Silva et al., 2012).When pH drops below 5.5, aluminosilicate clays and aluminium hydroxide minerals begin to dissolve, releasing aluminium-hydroxyl cations and Al 3+ then it exchanges with other cations.The chemistry of Al 3+ in soil solution is complicated by the fact that soluble inorganic (such as sulfate and fluoride) and organic ligands form complexes with Al 3+ .Whether a ligand increases or decrease aluminium solubility depends on the particular aluminiumligand complex and its tendency to remain in solution or precipitate.The mononuclear Al 3+ species is considered as the most toxic form of aluminium (Kochian, 1995).Aluminium bioavailability and, in consequence toxicity, is mainly restricted to acidic environment.
At high concentrations, Al can be a serious threat to agricultural production because it inhibits growth of the roots through various mechanisms, inducing oxidative stress (Zheng and Yang, 2005), callose induction, peroxidation of the cellular membrane, aluminium accumulation and nutrient imbalances and that ends with cell death (May and Nordstrom, 1991).There is considerable variability in A1 tolerance within species and this has been useful to breeders in developing Al-tolerant cultivars of various crops.Generally, there are two main types of Al tolerance mechanisms: (a) those that exclude Al from the root cells and (b) those that allow Al to be tolerated once it has entered the plant cells (Barceló and Poschenrieder, 2002;Kochian et al., 2005).
Eleusine coracana commonly known as finger millet or Ragi is cultivated for its grain in many parts of Africa and India (Hilu and Johason, 1992).Archeological studies confirmed domestication of E. coracana started around 5000 B.C. in Western Uganda and highlands of Ethiopia; and it arrived in India much earlier, probably more than 3000 years ago (Hilu et al., 1979).It is a versatile grain that can be used in many different types of food.It is eaten by grinding the grains up for porridge and other food items.Sometimes it is ground into flour and used for bread or various baked products like 'injera' in Ethiopia.Finger millet is particularly rich in dietary fiber and minerals such as calcium, proteins, phosphorus, amino acids, and iron (Asrat and Frew, 2001) as compared to major cereals grown in Ethiopia.In addition to being nutritious, millets are also considered as healthy food.The grains of most millets do not contain gluten, a substance that causes celiac disease or other forms of allergies.Babu et al. (1987) reported that some highyielding varieties also contain high protein content (8 to 12%) and also rich in calcium content (294 to 390 mg/100 g).Even though it is an important food security crop, production of the crop is inconsistent due to biotic and abiotic stresses and aluminium toxicity is one of the major factors.Hence, this work was initiated with the aim of screening finger millet accessions and varieties for their Al tolerance in order to enhance the productivity of finger millet in Ethiopia and in the world.

Equipment setup
Dense narrow holes were introduced into as many Eppendorff tubes as required in such a way that the holes did not allow finger millet seeds to pass through but rather allowed in air bubbles for aerating the seedlings in the tube."Rack" like plates to hold the perforated Eppendorff were made from jar plastic plate by introducing wide holes capable of holding and submerging Eppendorff tubes in the nutrient solution (Haftom et al., 2017).White plastic dishes were used as solution container with adjustable lids.

Plant and germination conditions
Three hundred accessions and six improved national cultivars of finger millet (E.coracana) were obtained from Ethiopian Institute of Biodiversity (EIB) and Nekemte Agricultural Finger Millet Research Center, respectively.Twenty randomly selected and surfacesterilized seeds of each germplasms were wrapped and germinated in a tissue paper, moistened with distilled water, in Petri dishes.Thirty 6 h-old seedlings of uniform size were transferred to the nutrient solutions and allowed to grow for a further 8 days along with tolerant (Gute variety) and susceptible (Necho variety) references.

Treatments
Preliminary screen of 300 accessions was carried on the threshold toxicity level of Al (112.5 µM) on six successive groups.Characterization with (112.5 µM) and without (0 µM) Al conditions was also done on 80 Ethiopian finger millet accessions.The control experiment also included all the above nutrients except Al2 (SO4)3.18H2O.The experiment was laid down in Randomized Complete Block Design (RCBD) with three replications.The pH of the nutrient was adjusted to 4.3 by using 1M HCl and the solution was renewed for every 24 h.

Data recording and statistical analysis
After eight days, root and shoot length of seedlings were measured using a ruler, while fresh weight of the seedlings was taken using a digital balance (Version No. 339, capacity 210 AE Adam ® with 0.0001 precision).Mean and analysis of variance (ANOVA) on each treatment was conducted using SPSS software version 20.Tukey HSD was used to make pair wise mean comparison of each germplasm under control and Al-treated conditions.Relative total root length (RTRL) and root growth inhibition (RGI) were also estimated using the following method (Mendes et al., 1984).

× 100
where RTRL is relative total root length and RGI is the root growth inhibition.

3+ trait response
A total of 288 accessions were screened in successive six groups, each group contained 50 accessions including the two references.The average root length screened in group one varied from 0.20 to 2.30 cm, while the references varieties Gute and Necho showed 2.11 and 1.54 cm, respectively.Furthermore, accessions screened in group two had an average root length ranging from 0.1 to 2.76 cm, while Gute and Necho had 1.92 and 0.12 cm, respectively.Similarly in group three it ranges from 0.11 to 2.61 cm, while Gute is 2.30 cm and Necho is 0.93 cm.Accessions screened in group four showed better average root growth as compared to the other batches and varied from 0.67 to 0.31 cm, while Gute and Necho produced 1.61 and 0.31 cm, respectively.The root length in accessions of group five was between 0.32 to 3.00 cm, Gute and Necho were 1.94 and 1.16 cm, respectively.Likewise, the performance of accessions of group six ranged between 0.17 to 0.27 cm, while Gute and Necho were 1.95 and 0.48cm, respectively details displayed in (Appendix 1 and Figure 1).From the screening result on 288 accessions along with the standard checks in six batches, only a few of them were Al tolerant 75 (26.04%),while 213 (73.95%) of the total accessions showed medium to susceptible tolerance.

Characterization of Ethiopian finger millet for aluminium tolerance
Out of a total of 80 accessions characterized for further evaluation with and without Al conditions, 74 were landrace accessions and six improved national varieties.There was significant (P-value of 0.05) Al induced stress among accessions in root and fresh weight measurement (Figures 3 and 4).In root length, 63 accessions (78.75%) showed significant Al induced stress and 23 (28.75%) in fresh weight, while no distinct and visible symptom of aluminium toxicity were observed in the shoot of finger millet genotypes (Figure 2).High root length inhibition was reported in pigeon pea on 20 µM AlCl 3 (Choudhary et al., 2011) and in maize at 20 µM (Wagatsuma et al., 2005).The present study also confirmed the inhibition of root growth at 112.5 µM due to aluminium phytotoxicity.Root growth inhibition is considered to be the primary consequence of aluminium toxicity, resulting in a smaller volume of soil explored by the plant roots, consequently reducing its mineral nutrition and water absorption.Furthermore, it reduces cell membrane permeability and binds to the phosphate groups of the deoxyribonucleic acid decreasing replication and transcription activity and also cell division inhibition (Kochian et al., 2005).
In the present study, no distinct and visible symptoms of aluminium toxicity were observed in the shoot growth of finger millet genotypes, similar with the findings in pigeon pea on 20 µM AlCl 3 (Choudhary et al., 2011).Long term exposure might affect nutrient uptake, which can lead to nutritional deficiencies in shoots and leaves (Jiang et al., 2008).The overall effect of aluminium toxicity was expressed on the reduction of yield and its total biomass.Fresh weight reduction in 23 (28.75%) accessions was also observed in this study.The decreased root growth could be the main cause for reduction in fresh weight.

Al tolerance in finger millet genotype as revealed by relative total root length
Accessions collected from Western Ethiopia, Gojam (100033and 213035), Awi (100036 and 243642) and Wellega (100095, 100097, and 245084) were the best seven tolerant accessions, while accessions (219815, 219818, 219819, 219820, and 219821) collected from Northern Ethiopia showed least tolerance levels (Appendix 2).According to Abdenna et al. (2007), acidity affected soils are prevalent in the Western and Southern parts of Ethiopia, areas such as Nedjo, Diga, Gimibi and Bedi in Oromiya, Chencha and Sodo in SNNP, and Gozamin and Senan Woreda in Eastern Gojam and Awi zone in West Amhara region.In the Western and Eastern Wellega zones in particular, the large proportion of exchangeable acidity was due to exchangeable aluminium while at West Showa zone, it was due to exchangeable hydrogen.Moreover, accessions collected from these areas were found to be Al 3+ tolerant, this is mainly due to enhanced tolerance against Al concentration that were developed due to long term exposure to soil acidity in this region.This may also be due to the fact that they exclude Al from the root cells and allow Al to be tolerated once it has entered the plant cells (Barceló and Poschenrieder, 2002;Kochian et al., 2005).

Conclusions
Among national varieties, Necho and Wama as was relatively Al sensitive as revealed by root growth inhibition compared to other varieties of finger millet.Thus, these varieties should not be recommended in areas where soil acidity is predominant.However, Gute and Degu varieties were relatively Al tolerant as revealed by root growth performance and can be promoted in areas where soil acidity is a challenge.Root length (RL) was affected more by Al toxicity than shoot length (SL).The impact of Al toxicity on finger millet germplasm became intense upon toxicity level increments.This study is the first of its kind to evaluate the performance of Ethiopian finger millet to Al-toxicity.The study clearly showed the possibility of developing lines and genotypes that can tolerate acidity in Ethiopian context and support agricultural development in acidic soil areas in the country.
Appendix 1A.Mean root length ± standard error on 300 accessions and two standard checks grown under hydroponics at 112.5 µM Al concentration.

Figure 1 .
Figure 1.Sample pictures of 8-day old seedlings showing root length difference between different varieties grown at 0 M (control) above 112.5Mbelow Al 3+ concentrations.

Figure 2 .
Figure 2. Effect of Al toxicity on root length on 80 finger millet accessions grown under treated; 112.5 µM and control; 0 µM Al 3+ under hydroponics.

Figure 3 .
Figure 3.Effect of Al toxicity on shoot length on 80 finger millet accessions grown under treated; 112.5 µM and control; 0 µM Al 3+ on hydroponics.