The ingress of pesticides into the soil occurs when they are directly applied to the arable layer or when processing the soil surface in order to combat soil pests and weeds; sowing treated seeds; when spraying and dusting the aerial parts of plants; during aerial processing of agricultural and forest land; during storage and transportation of pesticides. There are also facts of unauthorized burials, obsolete and unusable banned pesticides.
Various methods are known to restore disturbed ecosystems. Recently, economically developed and developing countries of the world are increasingly resorting to biological purification of the environment with the help of plants, which not only themselves actively participate in phytoremediation processes, but also in many cases have a beneficial effect on the soil micro flora, increasing the efficiency of the restoration of natural conditions. World experience shows that with the help of genetic engineering it is possible to create plants that accumulate pesticides and destroy them to a harmless state.
Genetic engineering of alfalfa plants (Medicago Sativa L.) to improve the efficiency of phytoremediation of soils contaminated with pesticides.
Tussіpkan D., PhD, H-index 4. Scopus author ID: 57218999913; https://orcid.org/0000-0003-1337-2834.
Manabaeva Shuga Askarovna, PhD
Akhmetollayeva Ainash Serikovna, Master of Biological Sciences
Rakhimzhanova Aizhan Oserbaevna, Master of Engineering and Technology
Ramazanova Malika Baglanovna, Master of Biological Sciences
Gubaidullin Nurtay Nurlanuly, Bachelor
2021
Main Results: The rabbit liver cyp2E1 cytochrome P450 gene was selected as a target gene to improve the efficiency of phytoremediation of pesticide-contaminated soils, the design and synthesis of oligonucleotides for synthesizing the target gene from cDNA was carried out. The complementary DNA was synthesized on a matrix of RNA extracted from rabbit liver and the expression vector pSRB-CYP450 was created for introduction into competent and regenerative alfalfa cells. The features of callusogenesis and morphogenesis of domestic alfalfa varieties were studied. It was found that the embryogenic potential of alfalfa varieties does not depend on explant types. The maximum frequency of callusogenesis was observed in explants of Shortandinskaya 2 variety (93%) when cultivated on a variant of B5K II medium containing growth regulators in the following concentrations: 2,4-D-5.0 mg/l, kinetin-5.0 mg/l and NUC-0.1 mg/l. A variant of B5P I medium with BAP content of 0.2 mg/l allowed to obtain a high frequency of regeneration in the variety Shortanda 2. An effective protocol for regeneration of shoots from alfalfa seeds of domestic breeding varieties for genetic transformation was developed.
2022
Main Results: Agrobacterium-mediated transformation of heterogeneous callus of sown alfalfa was optimized using a reporter gene encoded by the binary vector pSRB-CYP450. Monitoring of transient expression of the reporter gene revealed low competence of the cells of the studied alfalfa cultivar for Agrobacterium-mediated transformation; however, it was found that acetosyringone at a concentration of 100 μM, bacterial suspension at O.P.=0.3, and 48 h co-cultivation with the bacterium were optimal for Agrobacterium-mediated transformation of alfalfa callus. Selection of transformed cells and study of plant regeneration features from transformed alfalfa callus cells are carried out in in vitro culture. Phosphinothricin (PTT) is used as a selective agent, resistance to which is provided by the bar gene, which is part of the pSRB-CYP450 construct. Optimization of B5 medium for hormonal composition was carried out. A total of 126 Petri dishes (more than 3000 pcs) of callus were transformed, of which 235 underwent necrosis on selective medium with RRT. Morphogenic transformed calluses were passaged on 4 variants of nutrient medium (B5-1, B5-2, B5-3, B5-4) for plant regeneration. In the course of the experiments it was determined that variant B5-2 is the most optimal nutrient medium for regeneration of transformed alfalfa callus.
2023
Main results: The CYP2E1 gene from the liver of rabbits was chosen as the target gene for improving the efficacy of phytoremediation in pesticide-contaminated soil. Oligonucleotides were synthesized for the synthesizing target gene from cDNA. Complementary DNA was synthesized in an RNA matrix from rabbit liver and inserted into regenerative alfalfa cells using a pSRB-CYP450 expression vector. Complementary DNA was synthesized in an RNA matrix from rabbit liver and introduced into alfalfa cells using the pSRB-CYP450 expression vector to facilitate regeneration. The characteristics of callus formation and morphogenesis in domestic varieties of alfalfa have been studied. Agrobacterium-mediated transformation of heterogeneous alfalfa calli was carried out using the binary vector pSRB-CYP450. In total, more than 3000 calli and 520 somatic embryos of alfalfa were transformed, resulting in the generation of transgenic plants through direct and indirect regeneration pathways. The expression level of the target gene cyp2E1, a cytochrome P450, was convincingly confirmed using real-time reverse transcription PCR (RT-qPCR). Under conditions simulating soil contamination with pesticides, a correlation between seed germination and the degree of soil pollution was observed. Analysis of the content of persistent organic pollutants (POPs) in the genetically modified alfalfa plants revealed that the root system of transformed plants accumulates a significantly higher amount of POPs compared to the aboveground parts of the transgenic plants. The results of the phytoremediation assessment of transgenic alfalfa suggest that the expression of the cyp2E1 gene in these plants, transformed using the binary vector pSRB-CYP450, enhances the efficiency of transgenic plants in accumulating pesticides. Furthermore, these plants may facilitate the transport of pesticides through the root system of the plants.