INTRODUCTION

Molecular and biochemical markers are widely used in the identification and characterization of genetic resources (Son et al., 2017;Kim et al., 2021). Such markers are highly efficient in identifying inherent genetic variation. However, they are not without their drawbacks and limitations, such as the requirement of sophisticated analytical instruments and skilled personnel.

Explicit identification of variations using a reliable technique in basic laboratory settings at low cost is very crucial for conservation and proper use of germplasm at the grassroots level. The choice of any analytical and chemical test depends on cost, time, ease of performing the test, expertise of the technician, availability of equipment, purpose for which it is intended, as well as available information on characteristics of the varieties.

Numerous research findings highlight that the diverse reactions of the aleurone layer of seeds to chemical reaction are useful in distinguishing or classifying varieties. The aleurone layer is involved in the synthesis of enzyme substrates such as tyrosinase, which catalyzes a color reaction (Cabaj et al., 2010;Fernandes et al., 2005). Variation in color intensity among germplasms is due to differences in enzyme activity and various secondary metabolites present in the seeds (Sivasubramanian and Ramakrishnan, 1974). The color reactions serve as a basis for the grouping of germplasms and development of seed keys. All the chemical reactions taking place in the seed coat are subject to simple genetic control (Jaiswal and Agarwal, 1995).

The exposure of seeds to particular chemicals results in the development of a characteristic color, which is determined by their chemical or metabolite compositions. This color represents a distinctive trait for each crop variety, and its utilization in the characterization and identification of different crop varieties has been widely recognized and documented for safflower (Biradar Patil et al., 2006), for rice (Nethra et al., 2007), for bread wheat (Punia et al., 2002), for cotton (Reddy et al., 2008), for sesame (Suhasini, 2006). In this study, we utilized a combination of seed morphological traits and several simple and rapid chemical tests. These chemical tests are easy to execute, they offer a rapid and consistent results, and they do not necessarily need any specialized technical skill. When used along with other seed morphological characteristics, their varietal discrimination power increases significantly (Bora et al., 2008).

One of the main areas that need immediate rehabilitation in the Ethiopian seed quality control system is lab equipment and trained personnel (Sahlu, 2012). Furthermore, Ethiopia is known for being the second center of origin for durum wheat, and its highlands have a rich biodiversity of this crop relatives (Sall et al., 2019). Proper classification and systemic management of crop genetic resources are crucial for extensive morphological and molecular characterization. Simple chemical tests are useful for the primary classification of breeding materials and germplasm collections, as they allow for rapid and easy categorization based on physiological traits, aiding in the systematic management of these resources. In this experiment, we used a combination of seed morphological characteristics and a variety of simple and rapid chemical tests to differentiate between 20 Ethiopian durum wheat varieties.

MATERIALS AND METHODS

Chemical assays were conducted on twenty durum wheat varieties using various chemical assays such as phenol, modified phenol, potassium hydroxide, ferrous sulphate, and sodium hydroxide. Color reactions of the seeds to these chemical assays were utilized to differentiate between the genotypes. All color identification and comparison has been done using the Munsell Color Company Manual (1957).

The varieties used in this experiment were released by Debrezeit Agricultural Research Center, Ethiopia from 1982 (cv. Boohai) to 2012 (cv. Mukiye). Each test cultivar was grown and seeds harvested in Bishoftu, Ethiopia in 2016. The seed samples were stored at 10°C in a refrigerator and used for the experiments in 2017.

Two seeds per microfuge test tube were used for the color evaluation of the chemical test reaction solution. One hundred grams of seeds were used to evaluate the seed coloration and the color of the reaction solutions of each chemical test. All tests were repeated three times and the color of the reaction solution was visually classified by the two scientists using the Munsell color chart.

1. Chemical tests

All chemical tests were carried out under optimal environmental conditions under room temperature. Furthermore, seed pretreatment was conducted based on the specific requirements of the seed chemical test method. The seed samples were carefully handled and prepared according to the predetermined protocols to ensure that the results of the tests were valid and accurate.

(1) Standard phenol test

The test was carried out according to the method recommended by International Seed Testing Association (1966) and Jaiswal and Agarwal (1995). Briefly, three sets of ten seeds from each variety were immersed in distilled water for 12 hours at room temperature. Two seeds were then transferred to each 2-ml centrifuge tube containing 1% phenol solution. After 24 hours of immersion in the chemical solution, the seeds were assessed for staining and subsequently categorized into distinct classes based on the intensity of their coloration.

(2) Modified phenol test

Modified phenol test resembles the standard phenol test except the addition of 0.5% CuSO₄ to the later solution which further intensify the color of the solution. Varieties were then classified into different classes based on the color reaction of the seed coat according to Jaiswal and Agarwal (1995).

(3) Potassium hydroxide bleach test

Seeds were immersed in 5% KOH solution for a duration of one hour, and the genotypes were subsequently observed for a no color change (absence of tannic acid) and a change to light yellow color (presence of tannic acid) (Vanangamudi et al. 1988).

(4) Sodium hydroxide test

Seeds were immersed in 5% NaOH solution for a duration of one hour and thereafter, a change in the color of the solution was observed. The solution was observed for yellow color staining (Chakrabarty et al., 1989).

(5) Ferrous sulphate test

Seeds were soaked in 1% ferrous sulphate solution and were subsequently kept in an incubator for one hour. The classification of the varieties into distinct groups such as grey, light grey, and dark grey was determined by observing the color development of the seeds (Bora et al. 2008).

(6) Potassium iodide test

The endosperm of the seeds gives a waxy or non-waxy reaction when potassium iodide is applied (Bates et al., 1943). The non-waxy character is completely dominant over the waxy character (Konishi et al., 1985). To identify the waxiness and the staining property, seeds were soaked in a 1% (w/v) KI solution for 5 hours and the change in color was observed. Classification was made based on the staining property of the endosperm part of the seed.

2. Seed morphological traits

The seed samples were analyzed to assess morphological characteristics such as seed shape, seed length, and seed pubescence. Measurements were taken using three replicates, with each replicate consisting of 10 seeds from each genotype.

(1) Seed shape

The seed shape of each genotype was assessed visually and genotypes were grouped as round, narrow-ended, oblong (broader on both sides) or elliptical (elongated and bulged in the center) types.

(2) Seed pubescence

Seed pubescence was observed under the microscope and cultivars were classified as having short, medium, and long trichromes.

(3) Seed length

Seed length was measured to the nearest two digits using digital microcalipper. Ten randomly chosen seeds were taken from each variety for seed length measurement.

(4) Thousand seed weight

A random selection of one thousand seeds were counted and weighed. The mean thousand weight was expressed in grams, and accordingly, the cultivars were classified into different categories.

RESULTS AND DISCUSSION

1. The response of seeds color to chemical tests

This study aimed to explore the color reaction responses of different durum wheat varieties when subjected to various chemical tests. The seed materials exhibited positive responses in all the conducted chemical tests. Among the chemical tests, standard phenol test proved to be the best at discriminating the varieties. This test separated the varieties into four distinct groups: dark-brown (very strong), brown (strong), light-brown (moderately strong), and no color. The results are summarized in Table 1.

Based on the color development of the seeds subjected to the standard phenol test, three varieties (Werer, Yerer, Hitosa) showed dark-brown, six varieties (Foka, Denbi, Mangudo, Candate, Ginchi, Quamy) showed brown, ten varieties (Kilinto, Cocori-7, Tob-66, Assesa, Bichena, Boohai, Gerardo, Oda, Robe, Ude) showed light-brown, and one variety (Mukiye) showed no color reaction(Table 1, Figure 1). The phenol assay results of this study conform to the findings of Gupta et al. (2007) and Mansing (2010), who reported phenol test as an effective method in distinguishing between different varieties of wheat, and concluded that it could be a useful tool for varietal characterization.

The modified phenol test enhanced the chemical reaction response observed in the standard phenol test, providing additional assistance in distinguishing 11 varieties that could not be differentiated by the standard phenol test alone. Copper divalent ions (Cu⁺⁺) served as a catalyst in the color intensification process (Banerjee and Chandra, 1977). Out of the total number of genotypes tested, four exhibited a highly intense dark-brown response, 12 displayed a strong brown response, three showed a lightbrown response, and only one genotype did not exhibit any color reaction (Table 1, Figure 2). These results are consistent with those reported by El-Kalla et al. (2010).

Both NaOH and KOH are bleaching agents, and they were moderately useful in distinguishing the varieties based on color reaction in the seed coat. The color reactions observed in both the KOH and NaOH tests exhibited similarities and classified the varieties into two broad categories. Using the KOH test, out of the 20 varieties, 11 varieties were grouped into a light-yellow color class, and nine varieties were placed in a colorless category. Similarly, the NaOH assay classified the varieties into a light yellow (14 varieties) and no color (six varieties) groups, respectively. Compared to the phenol tests, these two assays were less efficient in discriminating the varieties. In terms of discriminating the varieties, these two assays were found to be less effective compared to the phenol tests.

The principle of the potassium iodide test lies in the staining of the starchy endosperm. When iodine is introduced to a starch solution, it interacts with the amylopectin and results in the formation of a deep purple color. Otherwise, if the grain possesses high quantity of amylose starch, the reaction yields a color that tends towards reddish. The high amylose level is reciprocated with a low amylopectin level. Waxy types produce mostly branched amylopectin. In this experiment, a dark purplish color was observed in all varieties, which indicated the presence of the waxy property in the starch. However, there was a considerable variation in the proportion of seeds that showed staining when exposed to the solution among the different varieties.

The FeSO₄ test is another method used to categorize the varieties into distinct groups based on their chemical properties. In this particular case, the test was able to classify the varieties into three separate groups, where each group contained multiple varieties.

Generally, a comprehensive classification of the varieties was achieved by combining all the aforementioned tests. Figure 4 shows the hierarchical classification of the major chemical tests. The efficiency of the chemical tests in discriminating between the varieties increases as they move away from the center. The chemical tests located at the center are those that broadly classified the varieties and were less efficient.

2. Seed morphological characters

(1) Seed pubescence

The experimental materials varied with regard to seed pubescence. Based on the presence or absence of hairs on the seed, it was possible to classify the varieties into three groups: short hair, medium hair, and long hair (see Table 2 and Figure 5). The density of hair was not taken into account. Seed pubescence was observed under the microscope at a magnification of 10x. Out of the 20 varieties, five genotypes had short hair (Mukiye, Cocori-7, Boohai, Mangudo, Denbi), ten genotypes had medium hair (Oda, Robe, Bichena, Assesa, Tob-66, Foka, Kilinto, Hitosa, Werer, Yerer), and five genotypes had long brush hair (Quamy, Ginchi, Gerardo, Ude, Candate), respectively.

(2) Seed shape

Twenty varieties showed four types of seed ends. Out of the 20 varieties, one variety (Assasa) had round, six varieties (Ginchi, Cocori-7, Gerardo, Werer, Mukiye, Bichena) oblong, seven varieties (Candate, Foka, Mangudo, Oda, Hitosa, Ude, Tob-66) narrow and six varieties (Denbi, Robe, Quamy, Kilinto, Yerer, Boohai) elliptical end shape, respectively.

(3) Thousand seeds weight

Based on the mean values of the thousand seed weight data of the genotypes, the varieties were classified into three broad classes: low (less than 40 g), intermediate (40- 45 g), and high (greater than 45 g) seed weight groups. Three varieties (Denbi, Bichena, Robe) were grouped into the low group, eight varieties (Hitosa, Gerardo, Yerer, Oda, Mukiye, Candate, Boohai, Tob-66) into the intermediate group, and nine varieties (Ude, Ginchi, Assesa, Quamy, Foka, Werer, Kilinto, Mangudo, Cocori-7) into the high seed weight group, respectively. The genotype Cocori-7 had the highest seed weight, while the genotype Denbi had the lowest.

(4) Seed color

The majority of the durum wheat varieties included in this experiment exhibited amber color and yellow endosperm, which is an indicator for a high-quality trait for pasta making. However, one variety (Kilinto) had white color that is of inferior quality for pasta making. Color of seed is controlled by a diterpenoid alkane content also known as phytan (Nagalaxmi et al. 2009).

(5) Seed length

Table 2 shows that there were differences in seed length within the durum wheat genotypes. The average seed length for all genotypes was 0.94 cm. Five genotypes (Cocori-7, Candate-utuba, Werer, Assasa, Hitossa) had short seeds, measuring less than 0.80 cm while eleven genotypes (Denbi, Mangudo, Kilinto, Mukiye, Bichena, Boohai, Foka, Gerardo, Oda, Quamy, Robe) had medium-length seeds, and four genotypes (Ude, Ginchi, Yerer, Tob-66) had longer seeds.

Generally, no major positive correlation was observed between seed chemical test reactions and seed morphological traits variation. The seed morphological traits have been useful in the characterization and phenotypic identification of the varieties (Table 2). For example, based on the characterization, the Werer variety had amber-colored and ovate-shaped seeds, with a lower 1000 seed weight, short seed length, and medium brush hair length. Similar characterization can be done for other varieties too. However, one of the main limitations of using morphological traits for identification of varieties is their susceptibility to environmental influence (Pascual et al. 1993), thus making it less effective in some cases.

적 요

본 연구에서는 고도의 분자생물학적 분석기법을 활용할 수 없는 기초실험실 조건에서 종자의 화학검정에 대한 반응 특성 과 형태적 특성을 종합적으로 이용하여 에티오피아 듀럼밀 품 종을 구분하는 방법을 개발하였다. 공시재료로는 에티오피아 듀럼밀 개량보급종 20개 품종을 이용하였다. 종자의 화학검정 은 표준페놀검정, 개량페놀검정, 수산화칼륨표백검정, 수산화 나트륨검정, 황산철검정 및 요오드화칼륨검정을 각각 실시하 였다. 종자의 특성은 형태, 길이, 모용 및 천립중 등을 조사하 였다. 주요 결과를 요약하면 다음과 같다.