Ewa Stanisławska-Glubiak, Jolanta Korzeniowska and Wojciech Lipiński
The aim of the study was to develop limit values for low microelement concentration in the soil, determined with the use of Mehlich 3 extractant for assessing their deficits in rapeseed crops. The values were prepared on the basis of 1944 fields with rapeseed, covering the whole Poland. In 2017, the samplers of Polish agro-chemical laboratories took soil samples and corresponding plant samples at the BBCH 30/31 stage. In the plant samples, the concentration of microelements was determined, and in the soil samples, apart from microelements, also pH, texture and the concentration of organic carbon and available phosphorus, were determined. Moreover, for each field, data on rapeseed yield were collected. Limit values were determined by two independent methods: 1) the method of regression equations and 2) the so-called high yield method. In the first case, the limit microelement concentration in the soil was calculated from the equation describing the relationship between the R/G bioaccumulation coefficient and a specific soil feature (n=1944). The bioaccumulation coefficient is a quotient of the concentration of a microelement in a plant (R) and its concentration in the soil determined by the Mehlich 3 (G) method. Limit values were calculated after substituting the critical concentration of microelements in the plant (R) to the equation, and subsequently, an appropriate conversion of the equation. The second method was based on the separation of a group of high yields ≥4.0 t ha−1 (n=755) from the whole data set. Then in this group, the lower quintiles (QU1) were calculated for the concentration of individual microelements in the soil determined in Mehlich 3 extract and adopted as limit values. It was found that QU1 is a good indicator of the lowest microelement concentration in the soil at which a yield of at least 4.0 t ha−1 can be obtained. The final limit values were worked out by averaging the values calculated by the equations and high yield method and their appropriate correction. In the combined soil sample collections for wheat and rapeseed (n=3865), the values were checked by evaluating the percentage of soils with microelement shortage separately for rape and wheat. The results of this evaluation were compared with the evaluation using the old system based on the 1 M HCl, which did not take into account the plant species.
Jolanta Korzeniowska, Ewa Stanisławska-Glubiak and Wojciech Lipiński
To implement the Mehlich 3 method in Polish agro-chemical laboratories, limit values for deficiency of B, Cu, Fe, Mn and Zn in soil for wheat were developed. The values were developed on the basis of 1921 fields with wheat, evenly distributed throughout Poland. Soil samples were collected from these fields in 2016, together with the plants growing on them, at the stage of stem elongation (BBCH 30/31). The concentration of micronutrients was determined in all soil and plant samples. In addition, pH, texture, and the content of organic carbon and available phosphorus were determined in soil samples. Moreover, grain yield after wheat harvest was estimated for all fields. Limit values were developed by two independent methods: 1) the regression equation method and 2) the so-called high yield method. In the first case, the limit microelement concentration in soil was calculated from the equation describing the relationship between the bioaccumulation factor (R/G) and a specific soil feature (n=1921). The bioaccumulation factor is the quotient of the concentration of a micronutrient in a plant (R) and its concentration in the soil (G) determined by the Mehlich 3 method. The equations were constructed using the Stagraphics program. For each micronutrient, 8 models were tested in search for the equation with the highest determination coefficient r2. Limit values were calculated after substituting the critical value of microelements in the plant (R) to the selected model and transforming the equation accordingly. The basis of the second method was to separate the “high yield group” ≥7.0 t ha−1 (n=578) from the entire data set. In this group, lower quintiles for the Mehlich 3-concentration of individual microelements in soil were calculated. The lower quintiles (QU1) were taken as limit values. It was assumed that QU1 is a good indicator of the lowest micronutrient concentration in the soil at which a yield of 7.0 t ha−1 or higher can be obtained. The comparison of the values calculated with the regression equations method and the high yield method showed their similarity, which confirmed the reliability of these values. The proposed values define the limit for low microelements concentration in soil determined with the Mehlich 3 method, below which wheat fertilization with these nutrients is recommended.
Jolanta Opiela, Daniel Lipiński, Joanna Romanek, Wojciech Juzwa, Michał Bochenek and Piotr Wilczek
Mesenchymal stem cell (MSC) differentiation is regulated intrinsically by transcription factors and extrinsically by the extracellular matrix. We tested whether matrix metalloproteinase-2 (MMP-2) and its inhibitor TIMP-2, MEF2a and TAZ transcription factors are involved in porcine MSC differentiation towards adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to investigate the expression levels of multipotent cell surface markers CD73 and CD105. Real- time PCR was performed to detect the osteogenic- and adipogenic-specific markers, osteocalcin and aP2, respectively, and to estimate the MMP-2, TIMP-2, MEF2a and TAZ transcript expression levels in three groups of cell, i.e., undifferentiated MSCs, adipocytes (A) and osteocytes (O). We showed that at the transcript level, the differentiation of MSCs towards adipocyte fate may involve MMP-2, TIMP-2 and TAZ. We also show that the differentiation of MSCs toward osteocyte fate may involve TIMP-2, MEF2a and TAZ. Our research provides preliminary data on the possible role of the MMP-2, TIMP-2 and TAZ transcripts in adipogenic differentiation and of the TIMP-2, TAZ and MEF2a transcripts in the osteogenic differentiation of porcine MSCs. We report for the first time the possible involvement of MEF2a in the osteogenesis of porcine MSCs. Our work may provide additional evidence for the MMP-independent function of TIMP-2 during osteogenesis.
Magdalena Hryhorowicz, Joanna Zeyland, Agnieszka Nowak-Terpiłowska, Jacek Jura, Wojciech Juzwa, Ryszard Słomski, Jan Bocianowski, Zdzisław Smorąg, Anna Woźniak and Daniel Lipiński
The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression.