Characterization of Bread Wheat (Triticum aestivum L.) Genotypes through Chemical Tests

The investigation was undertaken at the Department of Seed Science and Technology, Junagadh Agricultural University, Junagadh, during rabi 2017-18 to characterize 30 bread wheat genotypes released for general cultivation in Gujarat at state level as well as at the National level in central India based on the chemical tests. The seeds were subjected to phenol, peroxidase, NaOH and KOH test for differentiating the genotypes. Based on the seed colouration with phenol, genotypes were grouped into dark brown (12 genotypes), light brown (11 genotypes) and brown (7 genotypes) in colour. Based on the colour of the solution due to peroxidase activity, genotypes were grouped into four categories viz., brown (15 genotypes), light brown (9 genotypes), no change (4 genotypes) and dark brown (2 genotypes) coloured types. The KOH and NaOH test did not differentiate any wheat genotypes studied.


INTRODUCTION
Wheat is a type of grass grown all over the world for its highly nutritious and useful grain. It is one of the annual or biennial grass having erect flower spikes and light brown grains. It is the world's largest cereal crop. It has been described as the 'King of cereals' because of the acreage it occupies, high productivity and the prominent position it holds in the international food grain trade.
Maintenance of genetic purity of varieties is of primary importance for preventing varietal deterioration during successive regeneration cycles and for ensuring varietal performance at an expected level. The aspects of Distinctness, Uniformity and Stability (DUS) are fundamental for characterization of varieties. In countries having Plant Breeder's Right (PBR) in operation, a new variety is registered only, if it is distinct from other varieties, uniform in its characteristics and genetically stable Laboratory procedures furnish several additional characteristics useful for genotype identification. These chemical tests are very quick, easy to do, reproducible and can be undertaken throughout the year under controlled conditions. In chemical tests, the chemical agents react with the seed and help in genotype identification. Some of the sensitive analytical techniques employed in the laboratory are phenol test, potassium hydroxide (KOH) test, sodium hydroxide (NaOH) test and peroxidise enzyme activity test. and Sodium hydroxide (NaOH) test following procedure as given below: Phenol Test Two hundred (50 x 4) seeds were presoaked in distilled water for 16 hours at 25± 1º C. Then they were transferred on two layer filter paper saturated with two per cent phenol solution. The petri dishes were covered and incubated at 25±1ºC and the colour reactions were noted after four hours. Based on the development of seed coat colour, the genotypes were classified into four categories viz., no change in colour, light brown, brown and dark brown or black colour of the seed coat (Jaiswal & Agrawal, 1995).

Peroxidase Test
The Peroxidase test was carried out as per the procedure given by Agrawal and Pawar (1990) with slight modification. Twenty seeds were soaked in distilled water for two hours and decanted. Seeds of each of genotypes were soaked in 10 mL of 0.5 per cent guaicol solution for one hour. Then 0.5 mL of 0.1 per cent hydrogen peroxide solution was added. The change in colour of the solution was observed within two minute and the genotypes were classified on the basis of no change, light brown, brown and dark brown or black colour of solution.

Potassium hydroxide (KOH) test
Hundred seeds in four repetitions were soaked in five per cent KOH solution for three hours at room temperature. Change in colour of the solution and seeds were observed after three hours. Based on the intensity of the colour, the genotypes were classified into two group's viz., no change in colour and reddish brown (Mckee, 1973).

Sodium hydroxide (NaOH) test
Hundreds seeds in four repetitions were soaked in five per cent NaOH solution for one hour at room temperature. Changes in colour of the seeds were observed after one hour. Based on the colour intensity of the seed, the genotypes were classified into three group's viz., orange, brown and straw types (Agrawal, 1987).

RESULTS AND DISCUSSION
Varietal identification by morphological characters is laborious, time consuming, tedious, cumbersome and costly affair. A number of chemical tests have been developed for varietal identification such as phenol test, sodium hydroxide test and potassium hydroxide test, these chemical tests are very quick, easy and reproducible (Agrawal, 1987), very often these tests provide supportive evidence for the morphological evaluation of the seedling (Vanderburg & Vanzwol, 1991).
The seeds were subjected to phenol, peroxidase, NaOH and KOH test for differentiating the genotypes (Table 1). Based on the seed colouration with phenol, genotypes were grouped into dark brown (12 genotypes), light brown (11 genotypes) and brown (7 genotypes) in colour. Based on the colour of the solution due to peroxidase activity, genotypes were grouped into four categories viz., brown (15 genotypes), light brown (9 genotypes), no change (4 genotypes) and dark brown (2 genotypes) coloured types. The KOH and NaOH test did not differentiate any wheat genotypes studied.
On the basis of various chemical tests, genotype identification keys were prepared (Figure 1) Seed colouration with phenol is one of the important qualitative character which is not affected by environmental condition. The result of phenol test is usually distinct and easily interpreted. Gupta et al. (2007) studied that phenol test which is the index of polyphenol oxidase activity is a simple, quick and accurate test for grouping of wheat varieties. Chandusingh et al. (2017) observed that phenol test, which is an index of polyphenol oxidase activity, has been reported to be associated with intra-varietal diversity and have been used in ascertaining varietal purity. This reaction caused melanin formation by oxidizing phenol via orthoquinones and hydroxyquinones. This reaction is controlled by single gene (monogenically), which is localized in seed coat.
The results obtained in the present study for the peroxidase activity are in conformity with the findings of Reddy et al. (2008), he concluded that peroxidase activity is controlled by a major gene (Ep) with complete dominance and produces high activity and its recessive allele (ep) was responsible for low activity. Loverkovich et al. (1968) reported that peroxidase may play a fairly generalized role in resistance of plant to infectious disease. It is possible that, a high level in the seed coat could be involved in resistance for seed infection. Mckee (1973) reported that potassium hydroxide solution could be useful for separating white grain wheat varieties from red grain wheat varieties.
The colour reaction to sodium hydroxide solution was obtained in wheat due to reaction of seeds to secondary metabolites (Vanderburg & Vanzwol, 1991). The difference in colour reaction of seeds seems to be due to difference in genetic background concerning the enzyme system (Chakrabarthy & Agrawal, 1989).

Fig. 1: Wheat genotypes identification keys on the basis of chemical tests
CONCLUSION It can be stated that the assessment of genetic purity is an important criterion in seed production programme. Therefore, simple and reliable techniques need to be developed for genetic purity assessment and variety characterization. The identified morphological characteristics of wheat genotypes could be utilized in DUS testing, seed production programme and genetic purity testing. The result of chemical test is useful in identifying and grouping of wheat genotypes and also in genetic purity testing.