Effect of Accelerated and Natural ageing on Total Soluble Seed Protein Profile of Wheat (Triticum aestivum)
D. S. Chauhan & D. P. Deswal
Department of Seed Science and Technology, CCS, Haryana Agriculture University, Hisar- 125004
ABSTRACT: Study was conducted to compare fresh, natural and accelerated seed lots of wheat with germination and vigor index varied from 98.67 to 44.00 and 2960 to 524.92 respectively. Germination loss became more in accelerated ageing as compared to fresh and natural aged lot. Total soluble seed protein banding pattern of different aged seed revealed that there has been decline in band intensity, band numbers or disappearance of some bands with ageing; it is more in accelerated aged lot as compared to natural aged seed lot. Thus, seed lot with slight variation either in germination or vigor in germination or vigor could also be used for varietal characterization by SDS-PAGE to differentiate the cultivars or even for genetic purity testing, but not the seed lots which are severely aged that lost threshold limit of 50 per cent.
Key Words: Wheat seed, accelerated ageing, SDS-PAGE, protein profiles
Recent findings in biochemistry and molecular biology have enabled seed scientists to utilize new techniques for cultivar identification to augment existing tradition methods (Grow-Out-Test). Proteins are direct products of structural genes and are independent of environmental factors; these markers have been used to characterize varieties and to test the hybrid purity of many important crops [1, 2, 3, 4&5]. Seed is the best material to extract protein because examination can be carried out immediately after or even before harvest.
It is very essential to know the effect of ageing on seed protein profile so that an appropriate age of seed can be considered for electrophoretic analysis of seed proteins to characterize and differentiate the cultivars. Hence, an attempt was made to find out threshold limit for seed ageing, up to which these can be used for electrophoretic analysis proteins.
MATERIALS AND METHODS
Seed material comprised of six varieties of wheat Viz. C-306, PBW-502, WH-542, WH-711, WH-283 and RAJ-3765 having germination above minimum seed certification standard (MSCS) was collected at the time of sowing of crop and stored in ambient conditions and present research work was carried out in the laboratories of Department of Seed Science and Technology CCS Haryana Agricultural University, Hisar from 2006 to 2010. For defining the variables for artificial ageing, seed of all six varieties were artificially aged (40±1ºC/72 hrs.) and observation were recorded after ageing. In case of natural ageing, observation were recorded quarterly on the stored wheat seed in cotton bags in ambient conditions up to one year till germination was fall below as compared to fresh seed lot. Aged as well as non aged seed lots were evaluated for germination as per ISTA  and seed vigor index . Further, SDS-PAGE of total soluble proteins of all the seed lots was carried out by using 12 per cent acrylamide gel according to the methods prescribed by Dadlani and Varier  with slight modifications. 0.1 gm seed powder was taken in eppendorf’s tube to which 0.5 ml of extraction buffer (Tris-Glycine buffer, pH 8.3) was added. The contents were thoroughly mixed and kept overnight in refrigerator. The eppendorf’s tubes were taken out, mixed the contents well and subjected to centrifugation at 10,000 rpm for 10 minutes. The supernatant was decanted and from this supernatant 0.1 ml was taken into a separate eppendorf’s tube to which 0.3 ml of working solution (0.06 M Tris-Hcl, pH 6.8, 2% SDS, 10% glycerol, 0.025% bromophenol blue) was added and incubated at 60-70ºC for 10 minutes on dry bath, cooled immediately for 5 minutes and finally it was used as protein source for electrophoresis. The comb and tygon tube were carefully removed after polymerization. The gel cassette was fixed into the electrophoresis apparatus. Then the wells were properly washed with running buffer. In each well 10-15ml sample was loaded. Electrophoresis was carried out at 1.5 mA per well till the sample migrated to the resolving gel. Later 2 mA per well was applied until the tracking dye reached the bottom of the gel.
RESULTS AND DISCUSSION
Seeds attain maximum quality at physiological maturity. Starting from this point, there is a series of degenerative events that reduce the survival capacity of seeds and lead to loss of vigor and germination . Main sites of ageing at cellular level are mitochondria, ribosome and membranes. Ageing process is mainly due to reduction in enzymatic activity; increased respiration and macromolecule synthesis, which are associated with initial deterioration of membrane system. A considerable amount of work has been done on the change in protein content [10,11,12&13] and changes in activity of proteolytic enzymes [14,15,16&17] related to seed deterioration. However, there is a need to know whether the protein profiles, the qualitative aspect of protein is going to alter due to ageing that would help to select the right age of seed lot for varietal characterization since protein profiles are being used to identify the off type at seed level.
In present study, natural as well as accelerated ageing affects germination and vigor of seeds. Germination loss become more accentuated in accelerated ageing and in natural ageing it progressively increased with time of ageing [Table 1&2], it varied from 98.67 to 44.00 from fresh to aged seed lots, accordingly vigor index also varied from 2960 to 524.92.
1. Effect of natural and artificial ageing on Germination of wheat
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When the total soluble seed protein banding pattern of different aged seed lots were compared, in general there was decline in band intensity, band numbers or disappearance of some band as period of ageing advanced, it is observed higher in accelerated aged lot as compared to natural aged lot. This is due to degradation of protein in aged seed lots resulting in reduction of band intensity. Several researchers also reported such degradation of proteins in term of reduction in number and intensity of bands with increased ageing [18, 19&11].