International Journal of Agricultural and Food Science

Available online at http://www.urpjournals.com International Journal of Agricultural and Food Science Universal Research Publications. All rights reserved ISSN 2249-8516 Original Article Influence of high molecular weight glutenin subunits on biscuit making quality in wheat ARCHANA VAISHNAVA 1, A. K. GUPTA* AND A. S. JEENA** * Deptt of Molecular Biology and Genetic Engineering ** Deptt. of Genetics and Plant Breeding, G. B. Pant Univ. of Agric. & Tech., Pantnagar, Uttarakhand 263 145 1 Corresponding author: Department of Biotechnology, Dolphin (PG) Institute of Bio-medical and Natural Sciences, Dehradun (Uttarakhand) India Received 18 July 2015; accepted 03 August 2015 Abstract The seed protein of 30 wheat germplasm lines were fractionated using sodium dodecyl sulphate- polyacrylamide gel electrophoresis ( SDS-PAGE) to determine their high molecular weight Glutenin subunits ( HMW-GS) composition in relation to biscuit quality. Amongst the 30 lines, 7 durum cultivars were devoid of D genome and thus fail to show the HMW-GS. Amongst the rest 23 lines, two alleles were identified for the Glu-A1 locus (2*, 1), four alleles for the Glu-B1 locus (7,7+8,7+9, and 17+18) and two for the Glu-D1 locus ( 2+12 and 5+10). On the basis of Glu-1 scoring and baking test, few genotypes were found suitable for biscuit making quality. 2+12 at Glu-D1, 17+18 at Glu-B1 and null at Glu-A1 exhibited relatively low Glu-1 score and larger biscuit spread ratio, appeared to be related with biscuit quality. All these findings suggested the usefulness of identifying and characterizing the different wheat genotypes for end use quality products 2015 Universal Research Publications. All rights reserved Key words: Wheat, HMW-Glutenin Subunits, Biscuit Quality. determinants of end use quality product. Detailed biochemical and biophysical studies are revealing details of the molecular structure and interaction of the individual gluten protein and their roles in determining the functional properties of gluten. The introduction of electrophoresis of polypeptides in the presence of detergent SDS (sodium dodecyl sulphate) opened new opportunities for studying the glutenin complex which is responsible for unique viscoelastic properties of wheat dough on hydration. Greatest initial interest in SDS electrophoresis pattern of gluten protein has focused on the largest polypeptides, the high molecular weight Glutenin subunits (HMW-GS). There is a wide variation among varieties for different quality traits and in the electrophoretic patterns of both HMW-Glutenin subunits [1]. So, to meet out this, the performance of few wheat lines was studied along with the baking test in relation to high molecular weight glutenin subunits. The screening helped in identifying the genotypes having the specific HMW-GS suitable for biscuit making quality purposes. 2. Materials and Methods The experimental material for the investigation comprised of 30 strains of wheat comprising seven durum wheat and 1. Introduction Wheat has been prized since the oldest times, because of its unique properties that permit an array of leavened and unleavened food products to be made from it. End use quality of bread wheat is principle consideration in wheat improvement. So, yield improvement of wheat should be in agreement with superior milling and baking quality characters. During the selection for improved quality, breeder needs quick methods which are easy to put into practice and suitable for serial tests. The end product quality mainly depends on the grain protein content and composition. When wheat flour mixed with water and salt to form dough, most of the proteins are transformed in gluten complex. The gluten comprises glutenins, which confer elasticity of the dough, and gliadins, which give extensibility. Both fractions play an important role in production of quality loaves. There are now known to be about 20 sub-units of glutenin, which differ in their effect on wheat protein quality. So, quality in wheat is a very complex trait, however, the water insoluble gluten proteins responsible for the elasticity and cohesiveness (strength) of dough are important 98

twenty three bread wheat genotypes. Laboratory test and molecular analysis was done in wheat quality lab of Department of Genetics and Plant Breeding, G. B. Pant Univ. of Agric. & Tech., Pantnagar. 2.1 Seed Protein Extraction Total seed protein were extracted from half kernels with sample buffer (200ul) containing 4% (w/v) SDS, 15% (v/v) glycerol, 0.001% (w/v) brmophenol blue, 2% (v/v) 2- mercaptoetanol and 0.06M Tris- 1hr and then centrifuged at 12000 g for 10 min. 10ul of the supernatant were loaded into the sample wells of the gel of SDS-PAGE separation of high molecular glutenin subunits. 2.2 SDS-PAGE The discontinuous SDS-PAGE system used was based on the Laemmli (1970) as modified by [2]. The staking gel had 30% acrylamide 2.7% C (bisacrylamide-to-acrylamide ratio), 0.2% (w/v) SDS and 3.03% (v/v) Tris base, ph 6.8. The separating gel contained 10% acrylamide, 0.2% (w/v) SDS and 9.1% tris base (w/v), ph 8.9. Electrophoresis was performed for 5 h at 40 ma per gel. Staining and destaining were done according to Singh and Shepherd [3]. The numbers assigned to high molecular glutenin subunits were identified for each genotype according to the numbering system suggested by Payne and Lawrence [4]. 2.3 Baking Test The estimate of biscuit spread ratio provides a basic baking method for evaluating the quality of biscuit flour. The procedure involved as suggested by Mishra et al. [5] for preparing two sugar snap biscuits is given below: Sugar (24 g.), nonfat dry milk (1.2 g.) and sodium bicarbonate (0.40 g) were sifted together and creamed with shortening (12.0g). 37.6 g of this creamed mass was weighed and put into a mixing bowl. 4.0 ml.of solution A (79.8 g Sodium bicarbonate in distilled water, final volume 1 liter) and appropriate amount of water were added to achieve optimum dough consistency. 40 g flour was added to the mixing bowl and mixed. The dough was scraped from bowl and cut into two equal portion and these were transferred to lightly greased sheet. Using gauge strips, these were rolled to thickness with one forward rolling pin stroke and then return (backward) stroke. The dough thus obtained was cut with cutter, discarding excess dough. It removal from oven the biscuits were allowed to cool down and after that biscuit spread was measured. For calculation, biscuits were laid edge to edge and width was measured. They were rotated one quarter turn and measured. This was repeated twice. The biscuit width is mean of the flour measurements. Finally biscuit spread ratio was obtained by dividing the biscuit diameter with its thickness. Thickness was measured by Vernier Caliper. 3. Results and Discussion 3.1 HMW-Glutenin Subunits for Biscuit Quality The results of this study provide additional evidence for the existence of direct associations between biscuit quality and certain HMW-glutenin subunits. In general, associations between two traits can be caused by pleiotropy, genetic linkage or chance association. Amongst the 30 selected germplasm lines examined (Fig.1), two alleles were identified for the Glu-1A locus (2*, 1). If both these alleles Fig.1 Allelic variation in high molecular weight glutenin subunits in wheat are absent at Glu-1A locus, they are indicative of null phenotype. Four alleles (7, 7 + 8, 7 + 9 and 17 + 18) at Glu- 1B locus and two alleles (2 + 12 and 5 + 10) at Glu-1D locus were identified (Table 1). The identity of individual subunits was determined by comparison with standard cultivar having known subunits e.g. PBW 343 (1, 7 + 9 and 5 +10) and UP 2338 (2, 17 + 18 and 2 + 12). The standardization of allele symbols of these two cultivars are according to Payne and Lawrence (1983) [4] and Sreeramulu and Singh (1994) [6]. Among 30 selected genotypes, seven durum cultivars viz., PDW 215, UPDGP 716, UPDGP 609, UPDGP 616, UPDGP 621, UPDGP 627 and UPDGP 658 were devoid of genome D and thus failed to show the HMW subunits. HMW-GSs of D genome responsible for strength and elasticity of dough ultimately determine the end use quality product. Most significantly, the HMW glutenin 5 + 10 subunits at D locus have been associated with high dough strength and good bread making, whereas, allelic subunit 2 + 12 are associated with poor bread making quality [7]. At locus Glu-1A, 44 percent of 30 selected germplasm lines had null subunit and 40 per cent of the lines carried 2* subunits (Table 1). The rest 16 per cent genotypes observed the presence of subunit 1 at Glu-1A locus. There was allelic variation at Glu-1B locus where 48 per cent of genotypes carried 7 + 9 subunits, 24 per cent genotype contained subunits 17 + 18, 16 per cent genotypes included 7 + 8 and 12 per cent genotypes carried subunit 7. Seventy two per cent of the selected genotypes possessed Glu-1D high molecular glutenin subunit 2 + 12, while 28 per cent of the selected lines had the allelic subunits 5 + 10. So, the 99

Table 1 Allelic composition at Glu-1A, Glu-1B, Glu-D for selected germplasm lines Glutenin Subunit Frequency (%) No. of line Null 44 11 Glu-1A 1 16 4 2* 40 10 7 12 3 Glu-1B 7+8 16 4 7+9 48 12 17+18 24 6 Glu-1D 2+12 72 18 5+10 28 7 Table 2. HMW-GS, Glu-1 score and Biscuit Spread Ratio of wheat genotypes. S. No. Genotypes HMW-GS Glu-1 Biscuit Glu-1D Glu-1A Glu-1B score Spread Ratio 1. UPAGP 6 2+12 2* 17+18 8 5.7 2. UPAGP 15 2+12 Null 17+18 6 7.3 3. UPAGP 24 2+12 2* 7+9 7 5.2 4. UPAGP 97 2+12 Null 7 4 4.5 5. UPAGP 145 2+12 Null 7+8 6 5.4 6. UPAGP 162 2+12 Null 7+9 5 5.4 7. UPAGP 182 2+12 2* 7+9 7 6.1 8. Raj 3765 2+12 Null 7+8 6 5.1 9. UPAGP 265 2+12 2* 17+18 8 6.2 10. UPAGP 301 2+12 Null 7+8 6 5.1 11. UPAGP 362 2+12 Null 7+9 5 5.0 12. UPAGP 2378 2+12 Null 7+9 5 5.9 13. UPAGP 2484 2+12 Null 7+8 6 5.2 14. PDW 215 * * * * 5.1 15. UPDGP 716 * * * * 5.8 16. UPAGP2519 5+10 2* 7+9 9 5.5 17. UPAGP2523 2+12 Null 7+9 5 5.2 18. UPAGP2524 5+10 2* 7+9 9 5.4 19. UPAGP2532 5+10 2* 7+9 9 5.7 20. UPDGP 609 * * * * 4.7 21. UPDGP 616 * * * * 5.1 22. UPDGP 621 * * * * 4.9 23. UPDGP 627 * * * * 4.8 24. UPDGP 658 * * * * 5.6 25. UPAGP 2667 5+10 1 7 8 6.0 26. UPAGP 2673 2+12 * 17+18 8 6.3 27. UPAGP 2677 2+12 Null 7 4 5.5 28. UPAGP 2680 5+10 1 7+9 9 4.8 29. UPAGP 2654 2+12 2* 17+18 8 5.6 30. UPAGP 2662 5+10 1 7+9 9 5.0 31. PBW 343 5+10 1 7+9 9 32. UP 2338 2+12 2* 17+18 8 HMW-GS were found at widely differing frequencies in the 30 promising germplasm lines. These HMW-GS were found to be associated with the characteristics of end use quality [8]. Therefore, the frequency of particular HMW- GS provides the information to predict end use quality. More frequent bands or subunits in the 30 lines will provide the idea to use the cultivar for particular end use quality product. A selection criterion of 30 promising lines was biscuit quality. Subunit 2 + 12 was found most frequently in these 30 lines. The relation of HMW-GS and biscuit spread ratio indicated that genotypes with 6.30, 6.20, 6.10. 6.85 and 7.30 (UPAGP 2673, UPAGP 265, UPAGP 182, UPAGP 2378 and UPAGP 15, respectively) were suitable for biscuit and contained 2 + 12 subunits at Glu-1D. These findings are in complete agreement with the findings of Giansbelli et al. [9]. Among 30 genotypes (Table 2), UPAGP 2519, UPAGP 2524, UPAGP 2532, UPAGP 2680 and UPAGP 2662 possessed 5 + 10 subunits at Glu-1D locus and showed low 100

biscuit spread ratio 5.5, 5.4, 5.7, 4.8 and 5.0, respectively. These were considered to be inferior for biscuit making and superior for bread making. The relationship of 5 +10 subunit and bread making quality is well established [10]. However, UPAGP 2667 had 5 + 10 subunit and high spread ratio (6.5) which is against the findings of earlier workers. This may be attributed to flour texture, poor dough mixing condition and chance association [11]. Some genotypes having 2 + 12 subunits had a poor spread ratio. This may also be due to above reasons. Thus, research has been conclusive about the importance of HMW-GS, particularly those controlled by the D genome. HMW-GS at locus D has major importance on gluten protein which is responsible for the elasticity and cohesiveness (strength) of dough and important determinants of end use quality product [8, 9 and 10]. At locus Glu-1A, the null subunit was found in large frequency in selected 30 genotypes. Null subunits can serve as informatory band for biscuit quality but poor performance for bread quality. Payne et al. [10] reported that null allele of chromosome 1A is associated with poor bread making quality. Contradictory association concerning the effect of Glu-1 on bread making and biscuit making were reported [12]. Subunits 17 + 18 at locus Glu-1B was not very frequent in the selected germplasm lines but surprisingly showed a great association with biscuit spread ratio. Genotypes viz., UPAGP 6, UPAGP 15, UPAGP 2673 and UPAGP 2654 exhibited 17 + 18 subunits at locus Glu- 1B and showed higher spread ratio of 5.7, 7.3, 6.3 and 5.6, respectively. These findings are in complete agreement with the findings of [1 and 13]. Based on analysis of large number of genotypes, a scoring system for HMW-GS has been developed Payne et al. [10] in which individual subunits are graded with numbers based on quality evaluations. A given cultivar can be assigned a Glu-1 score which is the sum of contributions of each of the three HMW-GS loci. The HMW-GS score has more influence in some sets of wheat than in others [14]. The Glu-1 score thus obtained in the present investigation are shown in Table 2. Five strains (UPAGP 2519, UPAGP 2524, UPAGP 2532, UPAGP 2680 and UPAGP 2662) scored maximum Glu-1 score of 9 which would be for any variety best suited for bread making. All these 5 lines possessed 5 + 10 subunits at Glu-1D locus and comparatively less biscuit spread ratio among 30 selected genotypes. Normally the lines with 5 + 10 subunits are supposed to have strong gluten and thus their Glu-1 score have been at higher side [9, 10, and 14]. Among 30 selected lines some genotypes possessed low Glu-1 scores 6, 5 and 4. Lowest Glu-1 score 4 was found in UPAGP 97 and UPAGP 2677, 5 Glu-1 score in UPAGP 162, UPAGP 2523 and 7 Glu-1 score exhibited by UPAGP 15, UPAGP 145, Raj 3765, UPAGP 301 and UPAGP 6. These 9 lines with low Glu-1 score 6, 5, 4 consisted of 2 + 12 subunits. These subunits are supposed to have weak gluten and thus their Glu-1 score should have been on the lower side. Findings of Kumar and Raghavaiah [15] are in support of our findings of Glu-1 score and end use quality product. In contrast to the positive relationship between Glu-1 quality score and bread making quality, these results support the hypothesis that Glu-1 quality score is a guide to potential elastic development (dough strength), desirable for bread but deleterious for biscuit [10]. It is therefore, concluded that the electrophoretic resolution of seed protein in wheat through the use of High molecular weight subunits of glutenin was remarkably successful in cultivar and end use quality product identification. References 1. M.T. Labuschagne, C.S. Deventer, Van, The effect of Glu-B1 high molecular weight glutenin subunits on biscuit-making quality of wheat. Euphytica 83 (1995) 193-197. 2. G.J. 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