ICP-MS ANALYSIS OF BREAD WHEAT CARRYING THE GPC-B 1 GENE OF Triticum turgidum ssp . dicoccoides

64 The content of trace elements within ISO 3768–2010 in wheat grains is important for nutrition and getting a seed culture with high sowing qualities. There are low levels of Zn, Fe and Mn in soils and crops in all the countries, but it is particularly dangerous in regions of growing crops. Those minerals are the basis of human nutrition. The problem of lack of biologically important trace elements such as Fe and Zn is relevant to more than 2 billion people around the world. Pregnant women and children less than 5 years old suffer shortages most [1–4]. In European countries, grains, especially wheat, provide about 30% of daily calorie intake while in the Central Asia go to an average of 50% [1, 4, 5]. The wheat does not always contain sufficient micronutrients to ensure adequate human diet [6]. Micronutrient content in wheat genetically determined and depends on environmental factors. The level of absorption of trace elements in generative organs of wheat is low. Thus, it is a promising trend of genetic improvement of crops. One of the areas of biofortification of micronutrients is the transfer of genes from wild relatives, including, in particular, as spelt and wild wheat [7, 8]. Gene Gpc (grain protein content) within the whole chromosome 6B was transferred from tetraploid hulled wild emmer Triticum turgidum L. subsp. dicoccoides (Korn. ex Asch. & Graebn.) Thell. in hard tetraploid wheat cultivar Langdon (Triticum turgidum L. var. durum) [7]. The gene is responsible for accelerated aging of leaves and causes the effect of increasing the content of important trace elements in wheat, including Zn, Mn and Fe. The effect locus was detected in a significant increase compared to the starting line in the grain content of zinc (60 to 47.5 mg/kg), iron (44.2 to 35.9 mg/kg), manganese (53.9 to 40.9 mg/kg) and protein (14.4 to 10.8%) [8–10]. Well known that iron, zinc and manganese are some important elements to ensure the redox homeostasis of living organisms. Iron is available to plants in the forms Fe and Fe. This element plays an important role in metabolism, is part of enzymes involved in chlorophyll synthesis, UDC 581.134.1 https://doi.org/ 10.15407/biotech9.05.064

The content of trace elements within ISO 3768-2010 in wheat grains is important for nutrition and getting a seed culture with high sowing qualities.There are low levels of Zn, Fe and Mn in soils and crops in all the countries, but it is particularly dangerous in regions of growing crops.Those minerals are the basis of human nutrition.The problem of lack of biologically important trace elements such as Fe and Zn is relevant to more than 2 billion people around the world.Pregnant women and children less than 5 years old suffer shortages most [1][2][3][4].
In European countries, grains, especially wheat, provide about 30% of daily calorie intake while in the Central Asia go to an average of 50% [1,4,5].The wheat does not always contain sufficient micronutrients to ensure adequate human diet [6].
Micronutrient content in wheat genetically determined and depends on environmental factors.The level of absorption of trace elements in generative organs of wheat is low.Thus, it is a promising trend of genetic improvement of crops.One of the areas of biofortification of micronutrients is the transfer of genes from wild relatives, including, in particular, as spelt and wild wheat [7,8].Gene Gpc (grain protein content) within the whole chromosome 6B was transferred from tetraploid hulled wild emmer Triticum turgidum L. subsp.dicoccoides (Korn.ex Asch.& Graebn.)Thell. in hard tetraploid wheat cultivar Langdon (Triticum turgidum L. var.durum) [7].The gene is responsible for accelerated aging of leaves and causes the effect of increasing the content of important trace elements in wheat, including Zn, Mn and Fe.The effect locus was detected in a significant increase compared to the starting line in the grain content of zinc (60 to 47.5 mg/kg), iron (44.2 to 35.9 mg/kg), manganese (53.9 to 40.9 mg/kg) and protein (14.4 to 10.8%) [8][9][10].
Well known that iron, zinc and manganese are some important elements to ensure the redox homeostasis of living organisms.Iron is available to plants in the forms Fe +2 and Fe +3 .This element plays an important role in metabolism, is part of enzymes involved in chlorophyll synthesis, a component Citric acid cycle.The lack of iron during plant growing causes intercostal chlorosis and consequently disrupts the synthesis of chlorophyll and photosynthesis activity [11].Iron is an essential element in the human body and is a part of many substrates and enzymes responsible for transporting oxygen to cells, functioning respiratory chain mitochondrial redox reactions, the functioning of nervous and immune systems.About 60% of iron in the body is in hemoglobin.The lack of this trace element determines, first of all, the development of hypochromic anemia.The recommended daily dose of iron for adults is 10-15 mg [12][13][14].
Plants often use zinc as the divalent cation Zn +2 .It is one of the most important trace elements necessary for the regulation of carbohydrate and a number of enzymes involved in the growth process.It is a cofactor of more than 80 enzymes and acts as a structural component of many proteins [11].The role of zinc in human life is due mainly to the fact that it is a member of more than 40 important enzymes that catalyze the hydrolysis of peptides, proteins, certain esters and aldehydes.Zinc is involved in carbohydrate metabolism, located in insulin.The presence of zinc only let vitamin A function.This ion required for bone formation.Lack of zinc in the body has pleiotropic effects on mRNA expression of hundreds of genes, altering the biosynthesis of many proteins and zinc transcription factors.The average daily recommended amount of zinc for adults is 12 mg [2,15,16].
Plants absorb manganese in the form of divalent cation Mn +2 .It performs a structural role in the chloroplast membrane system [17].The element deficiency leads to intercostal chlorosis of young and old plant tissues [11].Manganese is a part of the active site of many enzymes involved in blood clotting, regulates conversion of molecular oxygen.Also manganese is involved in the synthesis of vitamins B and affects the synthesis of hemoglobin.The lack of manganese leads to disruption of carbohydrate metabolism, delays the growth of hair and nails, causes dermatitis, abrupt formation of cartilage, osteoporosis.The daily dose of manganese for adults is 2.0-5.0 mg [17].
Given the biological importance of iron, zinc and manganese, the aim of our study was to analyze the wheat hybrid families of generations F 4 and F 5 carrying the gene Gpc-B1 from Triticum turgidum ssp.dicoccoides within the substituted chromosome 6B for the content of micro-and macronutrient in grain.

Materials and Methods
To investigate the content of micro-and macroelements the hybrid wheat families of generations F 4 and F 5 marked as 18.2, 50.5, 51.5, 94.4, 98.3, 104.5, 125.5, 158.5 bearing the native gene Gpc-B1 of T. turgidum ssp.dicoccoides in Kuyalnyk background (been the second genetic parent) have been selected.The samples of generation F 4 were represented by ripe grains while the samples of generation F 5 were grains in the milk stage of maturation.
The determination of elements in the grain samples was carried out with a mass spectrometer with inductively coupled plasma ICP-MS Agilent 7700x (Agilent Technologies) together with ICP-MS MassHunter WorkStation.The samples (0.400 g) were treated with in nitric acid of ICP-grade in the microwave sample preparation system Milestone Start D. After the cooling, water was added to the samples to the dissolution rate 250.All solutions were prepared in class 1 water (18 MOm•cm), obtained from the water purification system Scholar-UV Nex Up 1000 (Human Corporation, South Korea).In 2015 Fluka Multielement standard solution 5 for ICP (Switzerland) was used for calibration whereas in 2016 the ICP-MS Complete Standard IV-ICPMS-71A (Inorganic Ventures, USA) was applied which helped identify selenium (Se) too [18,19].Settings of the mass spectrometer is given in Table 1.
The selected elements in the argon plasma may produce false peaks.For example, equal by weight CaO 56 and Fe 56 or ArN or ArO with other isotopes of iron.ArNH and KO may mask Mn, and Ba ++ -Zn.All 6 Ca isotopes can interact with O, H and Ar, which leads to inaccuracies in the determination of Cu, Fe, Sc, Se.Determination of selenium prevent most isotopes isobaric overlap Kr or Ge (masses of 74, 76, 78, 80 and 82) or polyatomic interferences mainly on Ar 2 masses 76, 78, 80.Although copper ionizes well in argon plasma (90%), the definition isotope 63 Cu is complicated with NaAr interaction and types of P, while 65 Cu is covered with SO 2 / SO 2 H. Besides, copper isotopes interfere with calcium oxide and titanium.Taking in to account the named obstacles the measurement was performed in helium purge mode, which effectively removes the above matrix and element interference.As an internal standard the 1 ppb solution of Sc by Inorganic Ventures, USA was used.The statistical analysis of obtained results was performed with ICP-MS MassHunter Software and MS Excel 2014.

Results and Discussion
As a result of ICP-MS detection of a number of elements contents in mature wheat grains generation F 4 8 homozygous hybrid families where parental control served as a cultivar of Kuyalnyk, have the following (Table 2).
Average zinc content among the prototypes of 23.25 mg/kg dry weight, 83% more than the original control (cultivar Kuyalnyk).Maximum accumulation of zinc occurs in the family 94.4 -33 mg/kg, which is 155% higher than the control cultivar.Minimal accumulation of zinc in the family determined 50.5 -17 mg/kg of dry matter, but even this level of content significantly exceeded the results of the control options.
The increase in the accumulation of iron in the homozygous hybrid families was slightly higher compared to the levels of accumulation of zinc.The average iron content of test samples is 52.3 mg/kg of dry matter, which, in turn, 61% more than the original line.The maximum iron content was observed in the family 51.5 -69 mg/kg, minimum -in the family 158.5 -40 mg/kg.
The average concentration of manganese prototypes of 50.7 mg/kg of dry matter, which is 55% more than the original line.The maximum manganese content was observed in the family 18.2 -65 mg/kg, minimum -in the family of 158.5 index of 45 mg/kg.
Given the biological importance of other divalent cations detected and copper content as well as magnesium and calcium.This essential elements, high content which determines crop seed quality and nutritional value of wheat flour.Copper content in grain increased in all variants, but did not reach the size limit accumulation of 10.0 mg/kg, which is regulated by law in accordance with ISO 3768-2010.It was also noted the high content of magnesium accumulation in grains and calcium -in most lines.The average concentration of magnesium, compared with the original line was over 46%, calcium -24%, copper -64%.
As a result of ICP-MS analysis of the content of Zn, Fe, Mn, Cu, Mg and Ca in all research family is the high content of micronutrients compared to the cultivar of Kuyalnyk.This finding confirms previous studies [9,10,19] that Gpc-B1 gene of Triticum turgidum ssp.dicoccoides positive effect on increasing micronutrient content in its carrier.
In 2016 was analyzed hybrid generation families F 5 .For research took grain maturation phase of the milk.Served as a control parental line (cultivar Kuyalnyk).The task of the research was to determine the degree of accumulation of micro-and macroelement number of hybrid gene carriers family -Gpc-B1 of Triticum turgidum ssp.dicoccoides the initial phase of ripening grain.Analysis was performed on 6 hybrid families that they investigated in 2015.The data given in Table 3.
The content of zinc in hybrid families an average of 27.6 mg/kg of dry matter, which is 28% more than in the starting line.The maximum level was observed in the family 98.3 -33 mg/kg.
The average iron content in samples of 2016 amounted to 28.7 mg/kg of dry matter, while the beans control -27.0 mg/kg (8% difference).The maximum iron content was observed in the family 51.5 -32.0 mg/kg, the minimum content 104.5 -27.0 mg/kg.A small difference can be explained by the fact that the grain is not yet mature and the accumulation of iron in the plant continued until fully ripe grain.
The average concentration of manganese incurred samples was 64.6 mg/kg, which is 31% more than in the starting line Kuyalnyk grade.The maximum level was 125.5 in the family -77 mg/kg and the minimum -in the family 94.4 -59 mg/kg of dry matter.
Analyzing the performance of content generation F 4 and F 5 on the content of Zn, Mn, Fe, one can conclude that the gene Gpc-B1 of Triticum turgidum ssp.dicoccoides significantly increases the content of trace elements in samples.This effect was observed even in the early stages of ripening grain.Also, we have determined the content of Cu and Mg in hybrid families.
Mean values of copper in hybrid families by 35% higher than the content in the starting line, whereas the magnesium content -5%.Elevated levels of elements at this early stage indicates that the gene Gpc-B1 of Triticum turgidum ssp.dicoccoides multifunctional and  One of the important micronutrients for wheat is also selenium.It is a component of over 30 vital biologically active substances of the human body as part of the active centers of antioxidant enzymes involved in the metabolism of nucleic acids, lipids, hormones.Among plant products the main source of selenium in Ukraine is wheat flour, so control of the content of selenium is essential [20,21].
The average content of selenium in experimental families of 0.207 mg/kg of dry matter, which is 117% more than the father.The maximum level of selenium -family 18.2 -0.41 mg/kg (4.5 times more than control) in the dry matter.
According to the research in 2015 and 2016 we found that the presence of the gene Gpc-B1 of Triticum turgidum ssp.dicoccoides bread winter wheat growth leads to accumulation of important biologically significant nutrition elements -iron, zinc, manganese, copper, selenium, and magnesium (which is correlated with observations of other researchers on a number of cereals [5,8,19]).Given the importance of iron, zinc, manganese, copper and selenium in redox homeostasis, we can provide high quality seeds sown grain containing this gene, as well as increased resistance of plants to pathogens destructive diseases -Fusarium spp., etc. Enriching copper ions promote the formation of stairs cultures with high levels of nitrogen use efficiency.
Thus, the presence Gpc-B1 locus of Triticum turgidum ssp.dicoccoides and its functioning is important for a bread winter wheat biofortification and further breeding work.

Table 2 . The content of micro and macroelements in physiologically mature grains of wheat crop 2015 (mg/kg dry matter)
Note: the identical letters mark variations that do not differ on the 0.05 significance level.