Creation of the source material of winter wheat resistant to cadmium accumulation

Abstract

Wheat is an archeophyte crop that has a sown area of over 200 million hectares and a gross harvest is approximately 800 million tons. Wheat cultivation and consumption are prevalent in the cultures of the majority of the world's peoples. In the absence of reserves to expand sown areas, the main direction of stable wheat consumption is to increase yields and increase the share of crops suitable for the production of environmentally friendly food. Realization of this direction is possible due to developing and introduction in production of varieties with the controlled level of accumulation of separate substances and elements. It needs the formation of theoretical base of inheritance feature and genetic control of qualitative breeding traits. Crops with appropriate quality indicators are used in the production of food and animal feed. This is especially true for heavy metals content that can migrate up the food chain. Wheat is a crop species with a high level of cadmium assimilation and accumulation in the seeds - one of the most toxic heavy metals. The urgent task of modern science is to expand theoretical research and solve practical problems in order to create varieties with a controlled level of cadmium accumulation. The dissertation work provides a theoretical foundation and practical solution to the issues of creating source material of winter wheat with the low Cd - uptake for breeding purpose and establishing molecular - genetic basis of plant cadmium accumulation ability. There were studied collection of varieties representing 7 major breeding centers of wheat in Ukraine. As a result of studying the collection samples of wheat from different institution - originators, samples with valuable breeding characteristics were identified. Growth parameters (height, stem weight, leaf area) were analyzed in wheat varieties from the collection. These traits were related to productivity parameters such as 1000 seed weight, grain weight per ear and yield. Genetic differences in the control mechanisms of resistance trait to cadmium accumulation have been established. The range of variation of the cadmium content trait in plants and grains of winter wheat varieties was determined to be 0.91 - 2.02 and 0.06 - 2.56 mg / kg, respectively. Varieties had minimum indicators of cadmium content in grains: Oktava odes’ka, Svitanok myronivskyi, Melody odes’ka, Kubok, Shchedra nyva. The maximum level of cadmium content was noted in varieties of Rozkvit, Sich, Kantata odesk’a, Duma odesk’a. A group of varieties with the lowest and high cadmium content was formed based on cadmium content values and plant productivity parameters. In groups of varieties with different cadmium content, differences in the structure of correlations between the main selection - controlled parameters were established. It was determined that the group of "low cadmium varieties" is characterized by the presence of direct correlations between the cadmium content and parameters of vegetative development of plants, namely plant height (r=0.54) and stem weight (r=0.88). In addition, a reliable negative correlation was noted with the indicators of the seed mass per ear (r= - 0.60) and 1000 seed weight (r= - 0.74). Thus, the variation coefficient of yield in the group of varieties with a low level of cadmium accumulation was 9.24% versus 6.14% for the group with a high ability to accumulate this metal. In the group of varieties with a high content of cadmium, a reliable level of correlation of the cadmium content trait with the LAR indicator (r= - 0.68) and the absence of statistically significant relationships with plant productivity indicators was noted. Significant differences in the correlation structure of cadmium content trait in seeds from high and low cadmium content variety groups indicates a difference in the mechanisms of genetic control of this characteristic. In the conditions of the field experiment, 40 intervarietal hybrids were obtained and studied. The distribution of inheritance frequencies of resistance to cadmium accumulation trait in F1 was calculated. After crossing low - cadmium varieties, the frequency of inheritance according to the type of heterosis was 12%, incomplete positive dominance - 8%, intermediate inheritance - 16%, and according to the type of depression and incomplete negative dominance - 44%. Inheritance according to the type of heterosis was noted in such combinations as Melody odes’ka x Ovidyi, Kubok x Svitanok myronivskyi and Shchedra nyva x Okhtyrchanka juvileyna. It was established that a characteristic feature of hybrids with minimal cadmium content in seeds was inverse and statistically significant correlations between the values of cadmium content and indicators of the seed weight per ear (r= - 0.70), 1000 seed weight (r= - 0.78) and the indicator of estimated yield (r = - 0.74). In the group of hybrids with a high level of cadmium concentration in seeds, a reliable level of correlation between the values of this indicator and the controlled traits was not found. Six samples were allocated for perspective breeding work: 19/1; 19/13; 19/26; 19/40; 19/33; 19/39, obtained in combinations of Melody odesk’a x Svitanok myronivskyi; Melody odesk’a x Shchedra nyva; Kubok x Svitanok myronivskyi; Shchedra nyva x Kubok; Zorepad x Okhtyrchanka juvileyna; Shchedra nyva x Okhtyrchanka juvileyna. The samples exceed the conventional standart in terms of cadmium content in seeds and one of the indicators of yield structure. Molecular biology approach was used to study the molecular mechanism of cadmium resistance in wheat. The role of individual genes in the control of processes and mechanisms of cadmium accumulation in winter wheat plants was determined. Specific role of TaSFT2L in the control of this element uptake was established. TaSFT2L - novel GOT/SFT2s member termed SFT2L was functionally characterized. The gene TaSFT2L plays an important role in the transport of Cd and is mainly expressed in roots and induced by this metal. Cd toxicity caused rapid Cd - induced up - regulation of TaSFT2L in roots, the main site of Cd toxicity and input in plants. This fact suggests a specific role of TaSFT2L in Cd plant tolerance. The TaSFT2L sequence was found to be highly conserved and exhibited a high homology with other plant species. The TaSFT2L - GFP fusion protein was determined to be localized in the cytomembrane of wheat cell protoplasts. It was studied TaSFT2L involved in tolerance to the cadmium in wheat and usually targets cell membranes. Vacuolar separation of Cd - induced TaSFT2L silencing in plants is a Cd tolerance mechanism in wheat. TaSFT2L was determined as negative regulator of Cd uptake. It was determined TaSFT2L gene expression in wheat suppresses the accumulation of Cd. RNAi/OE wheat lines expressing TaSFT2L were prepared and they were exposed to Cd stress. RNA interference (RNAi) of TaSFT2L resulted in improved plant height, biomass, and chlorophyll accumulation. Cd contents in the grain decreased by 29.40 - 74.95% and the proportion of Cd translocated from the roots to grains decreased by nearly 68% in RNAi lines of the TaSFT2L. The overexpression of TaSFT2L due to cadmium toxicity resulted in compromised growth response, with higher cadmium accumulation in wheat tissues. The results proved that TaSFT2L was a key gene regulating Cd translocation in wheat. It was reaveld that silencing the functional gene TaSFT2L to form transgenic wheat can inhibit Cd accumulation in wheat grains. The TaSFT2L gene from wheat was functionally identified, and it demonstrated that preventing Cd from building up in roots can prevent Cd from transferring to grains. TaSFT2L RNAi lines of wheat exhibited decreased Cd levels in roots by 28.13 - 36.04% and reduced proportions of Cd transferred from roots to grains by about 68%. TaSFT2L silencing is potentially useful since it can help to create new genotypes in breeding programs with low Cd accumulation in wheat grains. An original sourse material was created, which is being researched and refined in scientific programs at Sumy National Agrarian University, the Institute of Agriculture of North-East of Ukraine and Henan Institute of Science and Technology (Xinxiang, China). Based on the results of the research, a working collection of winter wheat samples with a low level of cadmium accumulation was transferred to the laboratory of selection and seed production of the Institute of Agriculture of North - East of Ukraine; materials of study are included in educational programs on disciplines of educational level of Bachelor of 201 Agronomy and 204 Ecology specialties at Sumy NAU. The SFT2L gene has been patented in China (application 20039041 dated October 29, 2020). (Appendex E)