**5. Challenges and perspectives**

allergen from the initial somatic embryos through the third generation homozygous soy‐ beans. Suppression of the allergen did not introduce any changes in the pattern of growth and development of the plant or seed at both the gross and subcellular level. In order to compare the P34-suppressed soybeans with the wild type, large-scale proteomic analysis was performed. Imaging of the 2D gels identified over 1400 individual elements. Mass spec‐ trometry analysis of about 140 of these spots confirmed that the only overt changes in com‐ position in the transgenic soybeans was the suppression of the P34/Gly m Bd 30k protein with no other proteins induced or suppressed [40]. Further analysis with sera samples from soybean-sensitive people confirmed a loss of the P34 allergen and no induction of any new allergens. The proteome and immunological analysis together confirms that it is feasible to suppress an endogenous allergen without introducing adverse effects on the plant or chang‐ ing the composition of the soybean seed in any way other than the removal of the targeted protein. This result meets the test of `substantial equivalence' where the GM soybean seed is essentially identical except for the change in the single desired characteristic. Suppressing P34/Gly m Bd 30k in GM soybeans is a first step and a demonstration in addressing the growing concerns about food allergies and its relationship to the development of GM crops. More detailed studies and approaches should provide the tests needed to gain regulatory approval in nations that are currently cautious about this technology. Natarajan *et al*. [41] have compared the profiles of allergen and anti-nutritional proteins both in wild and GM soybean seeds. 2D-PAGE was used for the separation of proteins at two different pH ranges and applied a combined MALDI-TOF-MS and LC-MS analysis for the identification of pro‐ teins. Although overall distribution patterns of the allergen and anti-nutritional proteins Gly m Bd 60K (conglycinin), Gly m Bd 30K, Gly m Bd 28K, trypsin inhibitors, and lectin ap‐ peared similar, there was remarkable variation in the number and intensity of the protein spots between wild and GM soybean. The wild soybean showed fifteen polypeptides of Gly m Bd 60K and three polypeptides of trypsin inhibitors. GM soybean showed twelve poly‐ peptides of Gly m Bd 60K and two polypeptides of trypsin inhibitors. In contrast, the GM soybean showed two polypeptides of Gly m Bd 30K and three polypeptides of lectin and the wild type showed two and one polypeptides of Gly m Bd 30K and lectin, respectively. The same number of Gly m Bd 28K spots was observed in both wild and GM soybean [41].

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

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The fear of allergic reactions has produced much of the concern about the risks of GM crops. In order to broadly apply genetic modification to crops, there is an urgent need for better biochemical and molecular methods, including animal models, to test for food allergens ex‐ perimentally so that the supporting data can be provided to evaluate newly proposed and actual GM products. In order to design transgenes, it would be useful to predict allergenici‐ ty but, currently, there are no models that would permit accurate assessment of allergenic potential of proteins unrelated to known allergens. Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Pre‐ vious studies on hepatocytes from young female mice fed on GM soybean demonstrated nu‐ clear modifications involving transcription and splicing pathways [42, 43]. The morphofunctional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to Soybean is a species of great agronomic and economic interest. It is one of the most recalci‐ trant plant species to be used as experimental material in proteomic analysis. Furthermore, there are several difficulties in the study of proteins (irrespective of source) with respect to DNA and RNA. The foremost important thing is the maintenance of secondary and tertiary structure during their analysis. They have problems with easy denaturation on exposure to high temperature, extremes of pH, oxidation, specific chemicals etc. There are some classes of proteins which are difficult to analyze due to their poor solubility. Proteins cannot be am‐ plified like DNA, therefore less abundant species are very difficult to detect. However, many potentially important proteins (in scarce) are lost due to non-specific binding or the co-removal of proteins/peptides intrinsically bound to the high abundant carrier proteins. Following are two methods developed recently to resolve detection of less abundant plant proteins [44]:


The current depth of knowledge regarding the soybean proteome is significantly less than that for some other plants. The soybean proteome map which is available in the database (http://proteome.dc.affrc.go.jp/soybean/) corresponds to various types of stresses, allergenic‐ ity, and studies on natural product biosynthesis in soybean. The other challenges in plant proteomics including soybean are standardization of methodologies, dissemination of pro‐ teomics data into publicly available databases and most importantly its cost expensiveness. Furthermore, most proteomics technologies use complex instrumentation and critical com‐ puting power. Currently, there is no expertise available for functional interpretation of data obtained from integration of proteomics with genomics and metabolomics.

The significance of proteomics over genomics and transcriptomics has been debated since the field has emerged. The importance of the proteome cannot be overstated as it is the pro‐ teins within the cell that provide structure, produce energy, as well as allow communica‐

tion, movement, and reproduction. Basically, proteins provide structural and functional framework for cellular life. Genetic information is static while the protein complement of a cell is dynamic. Differential proteomics is a scientific discipline that detects the proteins as‐ sociated with a diseased state (either due to abiotic or biotic stress, toxicity due to allergenic‐ ity, genetic modifications etc.) by means of their altered levels of expression between the control and diseased states. Extensive research towards the development of a soybean pro‐ teome map would permit the rapid comparison of soybean cultivars, mutants, and transgen‐ ic lines. Moreover, studies of soybean physiology will also benefit from the existence of a detailed and quantitative proteome reference map of the soybean plant. The information ob‐ tained from soybean proteomics will be helpful in predicting the function of plant proteins and will aid in molecular cloning of the corresponding genes in the future. The identifica‐ tion of novel genes, the determination of their expression patterns in response to stress, and an understanding of their functions in stress adaptation will provide us with the basis for effective strategies for engineering improved stress tolerance in soybean. With the advance‐ ment of new technologies in proteomics combined with advanced bioinformatics, we are currently identifying molecular signatures of diseases based on protein pathways and sig‐ naling cascades. Applying these findings will improve our understanding of the roles of in‐ dividual proteins or the entire cellular pathways in the initiation and development of disease. The abundance of information provided by proteomics research is entirely comple‐ mentary with the genetic information being generated by genomics research. Proteomics makes a key contribution to the development of functional genomics. The combination of genomics and proteomics will play a major role in understanding molecular mechanisms in plant pathology, and it will have a significant impact on the development of high yield vari‐ eties, with better resistance towards adverse environmental factors as well as various patho‐ genic diseases caused by bacteria, viruses and fungi in the future.
