A semi-hydroponic screening method for wheat growth responses to high CaCO₃ soils

Emdadul Haque¹, Eid Mohammad zaheri^1,2, Nao Sato³, Chiho Motokawa³, Noriaki Kojima³, Yuso Kobara⁴, Hiroyuki Tsuji¹, Tomohiro Ban¹

¹: Kihara Institute for Biological Research, Yokohama City University, Japan

²: Ministry of Agriculture, Forestry, Irrigation and Livestock, Afghanistan

³: Yokohama Science Frontier School, Japan

⁴: National Institute for Agro-Environmental Science, Tsukuba, Japan

Corresponding author: Emdadul Haque

E-mail: haque@yokohama-cu.ac.jp

Abstract

High CaCO₃ is a typical problem in the rain-fed region which makes rhizosphere alkaline and imbalance in nutrient status resulting in reduce root growth and poor plant development. With a final goal to look for genetic diversity within Kihara Afghan wheat landraces (KAWLR) collected by Dr. Kihara et al. in the Kyoto University Scientific Expedition (KUSE) to the Karakoram and Hindukush 1955, a screening method was developed to identify genetic variations in plant growth and nutritional adsorption using a semi-hydroponic two-chambered cup culture system having CaCO₃ solutions. One spring type KAWLR, Kabul-501 (KU11201) and Chinese spring (CS) were grown on the soil of upper cup having several holes in the bottom. The bottom cup contains saturated (T-1) and 1/2 strength (T-2) of CaCO₃ and Hyponex solution, thus giving a gradient of them towards the upper cup as will move through holes. T-1 screen inhibited root length by 22.3% in CS and by 12.5% in Kabul-501 and root drymass 12% in CS and by 8.5% in Kabul-501. Chlorophyll contents were much decreased in flag leaf in CS (17%) compared to Kabul-501 (8%). For elements, the unused N⁺ and P^- level were lower, while K⁺ was higher in the T-1 solution in both cultivars. Kabul-501 showed moderately higher tendency in N⁺, P^- and K⁺ uptake. Thus, T-1 was an effective concentration of CaCO₃ and system for differentiating genetic variations in wheat genotypes. This screen can be extended to other species and in response to other abiotic stress such as salinity, osmotic stress.

Keywords: Afghanistan, Wheat, Landraces, CaCO₃, Plant nutrient

Introduction

The presence of excessive CaCO₃ (15-40%)(FAO, 1972) is a typical problem in Afghanistan soil. In areas of low rainfall, there is a tendency to evaporation from the surface and replacement from the water table below. The groundwater which is drawn up often contains large amounts of dissolved CaCO₃ and on evaporation the CaCO₃ is deposited within the soil-body resulting in an accumulation of this substance. Depending on temperature, water content and concentration of CO₂ in soil, CaCO₃ decomposes into calcium ions (Ca²⁺) and hydrogen carbonate ions (HCO₃^–) and hydroxyl radical (OH^-). Thus, the rhizosphere appears in excess Ca²⁺, high pH (~8.5) and HCO₃^– alkalinity.  HCO₃^– alkalization of rhizosphere markedly inhibits the growth of roots, which makes it more difficult to absorb nutrients (Giel and Bojarczuk, 2011). Excessive calcium uptake by plant may lead to disturbances in ion balance, to the disadvantage of other nutrients (such as potassium and magnesium), or to changes in cytosol pH and a decrease in solubility of some ions, e.g. of phosphorous and iron. All changes in environmental factors deviating from optimum growth conditions are more or less stressful. Many vascular plant species are unable to successfully colonize in CaCO₃ sites due to morphological and physiological alterations resulting in lower final yield and yield quality. Soil alkalinity and water limitations are likely to increase in Mediterranean region and worldwide in this century, making the land as marginal for agriculture. Urban spread is forcing agriculture into drier or more marginal lands. On the other hand, the global food requirements are projected to increase 70% by 2050, requiring gains in agricultural productivity with the rain-fed and marginal land. There is an imperative to develop crops with improved/adaptable root and shoot traits that enable higher yields and yield quality by overcoming alkaline and/or imbalanced nutrient conditions prevailing on these rain-fed soils. The Kihara Afghan wheat landraces (KAWLR; also known as local variety), is a variety with a greater capacity to tolerate biotic and abiotic stresses, resulting in yield stability and an intermediate yield level under a low input agricultural system in the marginal area. In order to look for genetic diversity in KAWLR collected by Dr. Kihara et al. through Kyoto University Scientific Expedition (KUSE) to the Karakoram and Hindukush in 955 (Yamashita et al. 1965), a rapid screening method was developed to identify genetic variations in root growth and nutritional flow under CaCO₃ solution.

Calcification and HCO₃^– alkalization are rarely uniform down the soil profile. Once deposited within the soil-body, leaching of soluble CaCO₃ materials from the soil surface downwards is minimal because of the absence of percolating water (e.g. rain water), which, in Afghanistan, is nowhere more than 381 mm annually with much as winter snow and occasional spring rains (FAO, 1972). Therefore, in the crop growing season, CaCO₃ is mostly greater at soil surface. To cultivate wheat, Afghan farmers usually apply life support commercial irrigation that might dissolve CaCO₃ upto 47 mg/L. Here we prepared saturated CaCO₃ solutions into supplied Japanese tap water following a stable methods and developed a semi-hydroponic two-chambered cup culture system. The aim of this research is to learn the sensitivity of spring type wheat and landraces to grow into CaCO₃ solutions, with a future goal to document genotypic differences in KAWLR.

Materials and methods

One spring type KAWLR from Kabul (Kabul-501; KU11201) and Chinese spring (CS) as a check was used in this study. After 2 d of imbibition followed by 4 ºC treatment, seeds were sowed at a 2.5-cm depth in soil-filled (Volcanic soil; Kaneko Seeds, Japan) two-chambered transparent cups (upper cup, height 11 cm x upper diameter 8.5 cm and bottom diameter 5.2 cm; bottom cup, height 12 cm x upper diameter 8.0 cm and bottom diameter 5.2 cm) in a growth chamber. The bottom cup contained water and different concentration of CaCO₃ solutions. The bottom of the upper cup contained four holes (about 4 mm diameter) so that plant growing in the upper cup can absorb the gradient water and nutrients through bottom cup. In this study, we used three treatment solutions: (i) Tap water (control); (ii) Saturated CaCO₃ (T-1) and (iii) 1/2 dilution of CaCO₃ (T-2). Liquid fertilizer (HYPONeX, Japan) was added into the treatment solutions first at initial solution followed by once per 9 days. The solution of the bottom cup were changed in every 3 days where we analyzed the buffering (pH) level and elemental status. While the total chlorophyll contents (SPAD value) were measured in last leaf at 2 weeks, 5 weeks and in flag leaf during grain filling, root and shoot traits were measured only at 5 weeks after harvesting. For elemental analysis, substrate solution were taken from bottom cup from each treatment at 2 weeks and 5 weeks. The total contents of each element were measured according to the procedure described by respective company as follows; Ca²⁺ (Portable Ion Meter, CA-2031, DKK-TOA Cor. Japan), N⁺ (Portable Ion Meter, N-2031, DKK-TOA Cor. Japan), K⁺ (Portable Ion Meter, K-2031, DKK-TOA Cor. Japan) and P- (Spectroquant® Move 100, Merk KGaA, Germany). 

Results and Discussions

Preparation of semi-hydroponic two-chambered cup culture conditions using CaCO₃

It was considered important to generate a stable semi-hydroponic wheat culture system with saturated CaCO₃ solution in the bottom cup that will gradient and/or evaporate towards upper cup containing soils where germinated seeds were sown. This would mimic most field situations of Afghanistan, where CaCO₃ present in the 62 cm ~ below ground (FAO, 1972), evaporate to accumulate over wheat growing season at the surface. As challenging, a stable and saturated CaCO₃ (Ca²⁺ approximately 35 ppm and pH 8.5)(T-1) was achieved after 23d when dissolved in the outside with the supply of air pump and after 26d when dissolved inside the room with shaker machine at 18 ºC (Figure 1). This longer elapse time to get saturated CaCO₃ with its maximum pH was in similar tendency with the former report by Giel and Bojarczuk, (2011). A half strength of this solution (T-2) was prepared with water (Ca²⁺ and pH level were 26.5 ppm and 7.9, respectively)(Figure 1C) to see which concentration provide most distinctive between different genotypes. When the wheat seedlings were two leave stages and longest primary root just began to pass the upper cup towards bottom cup through holes, T-1 and T-2 were applied. As root tip continued to grow, the lower parts of the roots including tip were always into the solution while the bulk of the upper part of the root system is exposed to CaCO₃-evaporated soils (Figure 2). The two parts of root system thus exposing to CaCO₃ materials close to field situations

Learning the sensitivity of wheat landraces to grow into CaCO₃ solutions

The screen highly inhibited the longest root length under T-1 in CS (22.3%) (Figure 2B). The reduction rate in Kabul-501 under T-1 condition was about half (12%) of the CS. The drymass in T-1 was 12% in CS compared to 8.5% in Kabul-501 (Figure 2C). Again chlorophyll contents were much decreased in the flag leaf in CS (17%) compared to Kabul-501 (8%) only under T-1 condition (Figure 3). The inhibition of root growths under CaCO₃ is in agreement with previous report (Giel and Bojarczuk, 2011).

Learning the status of nutrients in the substrate solution and efficiency of T-1 screen

The rate of the treatment materials (CaCO₃) had a very marked influence on the content of exchangeable ions in the substrate solution (Table 1). For example, the relatively high contents of Ca²⁺ in T-1were clearly reflected in the concentrations of this ion in the CaCO₃ amended T-1 soil solution. This observation do appear to support by higher content of Ca²⁺ in CaCO₃ as found under maize treatments compared to control (Robert et al, 2013). The unused N⁺ level at 5 weeks in T-1 was drastically lower (might be much uptake by plant) with the higher value in Kabul-501 (23%), which is in agreement that nitrification rate is greater on calcareous soil or the same as non-calcareous soils (Kishchuk, 2000). The unused P^- was also lower in the T-1 solution (11% for CS and 23% for Kabul-501), while the unused K⁺ level remained higher in the T-1 solution (might me less uptake by plant) compared to control solution (17% for CS and 14% for Kabul-501). These results indicating that T-1 is the effective concentration of CaCO₃ that can make imbalance situation in nutritional uptake by plant root. The moderately higher absorption efficiency shown by Kabul-501 in N⁺ and P^- compared to CS (Table 1) might be one of the adaptation tendency by KAWLR under high CaCO₃ soil. However, at present we don’t have root and shoot content of these elements as well as other elements such as Mg²⁺, Mn²⁺, Zn²⁺ and Fe²⁺ to confirm the mechanism of their inhibition and transportation. Moreover, there is a wide diversity in KAWLR where 65% was classified as spring wheat (Stanikzai et al. 2015). Authors are now validating this screening methodology in many genotypes including spring, winter and facultative wheat. 

The high or moderate reflection of root growth and nutrient status to the alteration in chlorophyll content (Figure 3) and other upper ground traits such as plant height, top dry weight and stomatal conductance (data not shown) indicating the efficiency of the T-1 screening method, however, in more extended studies in acidic plants, shoot growth was more inhibited followed by root growth under CaCO₃ soils (Giel and Bojarczuk, 2002; 2011). This is reasonable, wheat, particularly landraces, is calcareous habitat plants with wide range of diversities (Sohail et al. 2015; Lopes et al. 2015). In addition, in our study, the roots were not experiencing the stress until they had grown nearly 20 cm and the second leaf was halfly developed, unlike standard experiments in hydroponic or sand culture when all the roots are exposed to the same concentration of stress elements. In this study, the roots encountered the CaCO₃ solution in a gradual incremental manner, and the bulk of the root system remained in contact with low alkalinity, and would be taking up water from the low CaCO₃ solution. This complicates the interpretation of results, however, it mimics the real conditions in field.

Future perspective

This screen may help to bridge the gap between controlled environment screens and the field, as hydroponics where solutions are continually washed over entire root systems can give quite different results from pot experiments in soil, in terms of growth response and transport of minerals (Tavakkoli et al. 2010) and nutrients into the shoot. The method could be adapted to represent the chemical composition of particular soils, for example, by adding more sulphate, or increasing the pH with bicarbonate. The method could also be useful to select for tolerance of toxic compounds such as boron or heavy metals. It could be related to soil water deficit caused by drought, if validated with other osmotica such as polyethylene glycol (PEG). A limitation of the screen is that plant can’t produce additional tillers which resulted less number of final seeds to make good conclusion on yield data.

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