The identification of proteins connected with androgenic responsiveness in triticale (× Triticosecale Wittm.)

The main object of this study concerns biochemical aspects of androgenesis process in winter triticale (X Triticosecale Wittm.). Triticale is an intergeneric hybrid derived from a cross between wheat (Triticum) and rye (Secale cereale). It has been developed into a commercially valuable feed grain crop due to a combination of several advantageous features: high yield potential, good grain quality and high tolerance to unfavourable environmental conditions. Thanks to these features, triticale has occupied stable position on the crop market and still getting economic importance in organic and low-cost agriculture. Unfortunately, the high potential of this plant crop is still poorly used, mainly because of agro technical neglect. New varieties with higher feeding value, higher resistance to diseases and pathogens, lower climate and soil requirements are extremely important at present age, in the face of the threats: fast growing world population, global and regional climate changes and the implementation of environmental friendly and balanced agricultural production systems. Utilization of the newest research achievements and biotechnological technologies enables the acceleration of the process of improved crop variety creation. One of such techniques is DH technology which introduced to breeding programs significantly accelerates the obtaining of new varieties important in the economic or quality aspects. What is more, DH lines are used in molecular studies, where allow to fast creation of genetic maps, identification and localization of genes that control complex traits with quantitative inheritance (QTLs).
Method of anther culture basing on the process of androgenesis/microspore embryogenesis that is used in IPP PAS to obtain DH lines, could significantly speed up the progress in triticale breeding. However, utilization of this technique on practical scale must be preceded by elaboration of DH production procedure that is effective for a wide spectrum of genotypes. Moreover,  obtained DH lines should be genetically stable. Successful induction of microspore embryogenesis was documented for more than 250 plant species. However, among all of them only in a few, namely rapeseed (Brassica napus), tobacco (Nicotiana tabacum), wheat (Triticum aestivum) and barley (Hordeum vulgare), the androgenic potential is high enough to be routinely used in DH production for breeding programs. Majority of the valuable crops species - among them triticale – do not respond efficiently to embryogenesis induction. The lack of efficient and reproducible procedures for DH production is probably caused by the complexity of the trait, controlled by several independent nuclear genes with additive and dominance effects which interacted with environmental factors and could be modified by cytoplasm background. As result many of endogenous and environmental factors have significant influence on androgenesis effectiveness. Identification of physiological, biochemical and molecular background of this process would facilitate the production of DH and its utilization in breeding programs. It should create better conditions for exploration of great potential of triticale. The fast production of new varieties, adapted to changing climate conditions, to high quality requirements and also adjusted to needs of low-cost and balanced agriculture will make Polish breeding companies more competitive on European Union market. 
The present project builds on our rich experience with the triticale anther and isolated microspore culture model systems. Protein profiling will be a valuable supplement of our studies on various physiological factors important for efficient microspore reprogramming. The identification of the proteins involved in this process will result in deeper understanding of the mechanisms controlling microspore-derived embryo formation and DH production . What is more, triticale has never before been the object of researches on identification of potentially interesting proteins connected with androgenic responsiveness. Therefore, studies planned in the frame of submitted project concerning proteomic analysis of selected DH lines with extremely various androgenic responsiveness have pioneer character.  The results of planned investigation might contribute to work out effective method of triticale DH production  We also expect that the results obtained for triticale can be useful also for other cereal species, first of all in wheat and rye.
The studies will be conducted on a few lines selected from mapping population of 90 DH lines, derived from hybrid: Saka 3006 x Modus (Ugo). Both parental forms are hexaploid winter triticale. Phenotyping for androgenesis efficiency in this population will allow to select the genotypes at the opposing (high and low) extremes. Selected, extremely different in androgenic responsiveness, DH lines will be used as the models for proteomic analysis. The protein analysis using two dimension electrophoresis in poylacrylamid gels allows to obtain a map of hundreds of proteins and comparison of such maps. Further computer analysis of obtaining results make it possible to characterise quality and quantity changing of proteins. It is impossible without specific software. Proteins with differential expression level indentified in gels could be excised and analyse by using MALDI-TOF method. It allows to determine its molecular weight, amino acid sequence and kind of family protein. Finally, it is possible to set a hypothesis about protein function in androgenic responsiveness.

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