Вiotechnological approaches to reduce biogenic risks in crop production: potato case

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Abstract


The article presents an overview of the biogenic agro-ecological risks in crop production, to reduce which it is possible to use biotechnological approaches. Ways to reduce the negative impact of the two most common harmful objects, the сolorado potato beetle ( Leptinotarsa decemlineata Say) and phytophthoras ( Phytophthora infestans (Mont.) De Bary), which lead to significant losses of potato yield, are considered. It is shown that the currently used methods of plant cell engineering (somatic hybridization and microclonal reproduction) are environmentally sound biotechnological methods of controlling black eye rot potato. The need to develop genetically engineered methods is associated with an exacerbation of biogenic agroecological risks, the reduction of which is an important approach is a proactive introgressive breeding strategy based on cell engineering and molecular methods.


Potato productivity in the Republic of Belarus and Russia Against the background of increasing potential risks due to adverse changes occurring in the biosphere, one of the most important tasks is to obtain high crop yields. Efficiency of all agricultural production directly depends on productivity of crop production. A plant organism that can transform the energy of the sun into the energy of organic compounds is a fundamental link in production of food, feed, raw materials, fuel, and medicines. Intensification of crop production in modern conditions should be based on a reasonable combination of traditional approaches with new technologies. Potato (Solanum tuberosum L.) is the second most important agricultural crop in the Republic of Belarus, only in the last five years slightly inferior to rapeseed. The Brest region steadily ranks second in lands under potato in farms of all categories. However, according to Belstat (Fig. 1), potato yields in the Brest region are reduced in comparison with this indicator for the Republic of Belarus and a number of regions. Problem of productivity in potato industry of the Republic of Belarus and Russia is aggravated against the background of observed tendency to reduce sown areas. Between 1995 and 2015, lands under potatoes in the Republic of Belarus decreased 2.3 fold, in Russia - 1.6 fold and they continue to decline annually (Table 1). Performance of potato production level, defined by the national doctrines of food security of the two countries, is impossible without yield increase. Despite the growth of this indicator in 2015 compared to 1995 (in the Republic of Belarus - by 48%, in Russia - by 34%), the average potato yield in two countries is several times lower than the average value for Western Europe (Table 2). According to statistics of the United Nations Food and Agriculture Organization (FAO), in Germany and the Netherlands - the largest potato producers among European countries - reduction of croplands over 20 years was not so high, and since 2015 there is an inverse trend. The high average potato yield in these countries makes it possible to achieve significant productivity in relatively small lands. t/ha 25 20 15 10 5 0 1995 2000 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 The Republic of Belarus The Brest region Fig. 1. Potato productivity in the Republic of Belarus and the Brest region in 1995-2017, t/ha [1] Potato land dynamics in 1995-2017, thousand ha [2] Table 1 Country 1995 2000 2005 2010 2015 2016 2017 Russia 3 390 2 817 2 273 2 109 2 112 2 031 1 889 Republic of Belarus 725 661 461 367 310 292 276 Germany 315 304 277 254 237 243 251 Netherlands 179 180 156 157 156 156 161 Potato yield dynamics in 1995-2017, t/ha [2] Таблица 2 A country 1995 2000 2005 2010 2015 2016 2017 Russia 11.8 10.5 12.4 10.0 15.9 15.3 15.7 Republic of Belarus 13.1 13.2 17.7 21.4 19.4 20.5 23.2 Germany 31.4 43.3 42.0 39.9 43.8 44.4 46.8 Netherlands 41.0 45.7 43.4 43.6 42.7 42.0 46.0 Western Europe 35.1 42.7 42.1 41.2 42.9 41.3 44.9 The problem of low potato yields in the Republic of Belarus and Russia, apart from technological, organizational and economic factors, is directly related to the biogenic risks of cultivating this crop. The shortage of potato harvest is due to the increasing harmfulness of diseases caused by fungal, bacterial and viral pathogens, as well as insect pests. We consider the methods proposed by modern biotechnology to reduce the negative impact of the two most common harmful objects: the Colorado potato beetle (Leptinotarsa decemlineata Say) and phytophthora (Phytophthora infestans (Mont.) De Bary), which lead to significant losses in potato harvest. Biotechnological approaches in the control of late blight Late blight is one of the most harmful potato diseases. Blight affects aerial part of plants and thereby reduces assimilating surface during tuberization, and also causes rotting of tubers during storage. In the Republic of Belarus, strong outbreaks of the disease with 30...50% yield losses are observed every 2-3 years. The main late blight control methods, actively used for several decades of ХХ century and in the new century include cultivation of resistant potato varieties and fungicide application. The latter method, despite its effectiveness, is unsafe for human health and poses the problem of increasing chemical load on agrocenoses, and, as a consequence, deterioration of ecological state of the environment. Therefore, for sustainable development of potato growing in modern conditions, along with the use of effective agrotechnical measures, the creation of new potato varieties highly resistant to P. Infestans is required, which will significantly reduce chemical treatments during plant vegetation. Resistance to P. infestans of previously developed varieties was determined by the presence of R genes transferred by the method of distant hybridization from wild potato variety Solanum demissum [3]. However, high variability of P. infestans manifested itself in formation of new pathogen forms capable of overcoming protective reactions of resistant varieties based on S. demissum R genes. According to Yu.T. Dyakova, frequency of spontaneous mutations in P. infestans at one locus per 1 ha reaches 1,000. It is a high pathogen mutation rate that can provide the level of variability necessary for all kinds of adaptations [4]. The process of variability has sharply intensified since 1984, when new phytophthora populations with two types of sexual process A1 and A2 migrated from Mexico to the area of intensive potato cultivation [3]. The appearance of the A2 type in the Republic of Belarus significantly aggravated harmfulness of late blight, because racial composition became more complicated, spectrum of virulence expanded, pathogen's aggressivity and its resistance to existing pesticides increased [5]. A promising strategy that opposes the rapid loss of potato resistance to late blight is creation of new varieties that retain high resistance to many races of late blight for a long time. Combination of several resistance genes from wild potato variety in one plant (gene pyramidation) makes resistance long-term, since the totality of transferred genes provides recognition of various pathogen races. A change in the racial composition of the pathogen in agrocenosis will not lead to a significant decrease in the productivity of plants with long-term resistance. Such selection is called proactive [6]. The effectiveness of potato introgressive selection can be improved by developing a new source material with complex resistance to late blight both of the aerial parts of the plant and tubers. A decisive role in increasing potato resistance to late blight is played by a targeted search for sources of valuable genes using marker-associated selection methods. Molecular markers make help to distinguish plant forms resistant to different pathotypes of the same pathogen, which significantly accelerates selection of resistant genotypes and their inclusion in the breeding process. Interspecific hybridization has been used in practical potato selection for several decades. Moreover, the transfer of resistance genes from wild species of the genus Solanum, growing in the Americas, to S. tuberosum during sexual hybridization is limited due to physiological and genetic incompatibility. Overcoming incompatibilities with the distant hybridization of cultured tetraploid potatoes with wild-growing species became possible thanks to the development of a number of biotechnological methods. The method of somatic hybridization is based on the fusion of protoplasts isolated from somatic cells of various plant species under in vitro conditions. Somatic hybridization bypassing sexual process allows developing new source material (complex interspecific hybrids of three or more parent cells) with valuable traits from wild species to expand the genetic potential of potato breeding. The advantages of somatic hybridization include combining in one genome not only cultivated potato genes and phylogenetically distant wild relatives, which have more than 230 potatoes, but also cytoplasmic organelle genes (mitochondria, plastids), which ensures developing of qualitatively new interspecific hybrids [7]. Interspecific somatic potato hybrids are obtained in many countries, among which the Republic of Belarus. Somatic hybrids with 10 wild potato species inaccessible for sexual hybridization have been developed at Scientific and Practical Center for Potato, Vegetable and Fruit Growing (National Academy of Sciences of Belarus) by somatic hybridization. Despite the attractiveness of somatic hybridization, development of plant varieties, including potatoes, obtained on the basis of somatic hybrids, remains a rare phenomenon, since there is a problem with fertility of somatic hybrids and their ability to generate viable offspring when crossed with cultivated potatoes [8]. Another environmentally effective biotechnological method of controlling tuber blight, which reduces quality of planting material during storage, is the method of microclonal propagation of test-tube plants, which allows to obtain in sufficient quantities material free from fungal, and viral pathogens. Microclonal propagation makes it possible to obtain genetically identical clone plants under in vitro conditions. It includes three stages: introduction of a cloned plant into explant culture; plant regeneration and its propagation in the required number of copies; stimulation of root formation in microplants and their adaptation to in vivo conditions. Obtaining healthy potato microplants starts in the first year according to a four-year seed-growing scheme of virus-free potato planting material. Breeding institutions organize this work. At the first stage, tubers are taken from obviously healthy plants and germinated. An apical meristem is isolated from sprouts under aseptic conditions and cultivated on nutrient media in phytotrons with controlled light and temperature conditions. Plants regenerated from the meristem are propagated by multiple cuttings according to the number of internodes to the required number of copies. Diagnosis of pathogen presence is mandatory. Healthy potato test plants are transferred to seed farms for mini-tubers, super-super elites and super-elites. The considered biotechnological approach, based on apical meristem method, is, in fact, a supportive selection and, being widely introduced into practice, makes it possible to annually provide farms with pathogen-free, high-quality potato planting material. Decoding of S. tuberosum and P. infestans genomes increased possibilities of genetic engineering to create genetically modified potato varieties with increased resistance to late blight. To transform potatoes with genes of closely related wild-tuberous forms of Solanum (section Petota Dumort.) a new term “cisgenesis” was proposed, which is designed to distinguish such forms of potato from genetically modified organisms obtained using foreign genes [9]. Biotechnological approaches in controlling Colorado potato beetle Besides phytophthora, Colorado potato beetle, which can damage up to 40% yield in favorable years, is a serious biogenic risk for potatoes. Despite the rapid development of science, Colorado potato beetle still remains a harmful potato pest, and considerable funds are spent on combating it. Colorado potato beetle belongs to the objects of external and internal quarantine in the Republic of Belarus. It is very difficult to control it due to high fecundity, ability to go without food for a long time, fall into diapause for a long time, and travel considerable distances. In addition, Colorado potato beetle has few natural enemies, since eating Solanaceae plants containing poisonous solanine, Colorado beetles become poisonous to them. Complicating the fight against potato pests is the fact that with the beginning of spring, beetles wintering in soil come to soil surface not at the same time, but at intervals. In addition, some females overwinter already fertilized and immediately after reaching soil surface begin to lay eggs. Due to the climatic conditions of Belarusian Polesie, number of generations of Colorado potato beetle can be increased to three, which can further determine the duration of its harmfulness. The traditional approach to reducing potato crop losses from the pest is chemical control methods. However, the use of chemicals against Colorado potato beetle causes the insect to quickly get used to the poison and also contributes to the preservation of more stable individuals that give more viable offspring. Over the past decades in the Republic of Belarus there has been an increase in number of Colorado potato beetle, a change in some bioecological features of pest development due to varying weather conditions of growing season, and formation of resistance to chemical agents. In this situation, microbiological agents and plant extracts for controlling Colorado potato beetle are considered to be the most suitable [10]. Most existing and newly developed microbial agents are based on strains of Bacillus thuringiensis soil bacterium, which is capable of producing Bt-toxin. This toxic protein differs depending on B. thuringiensis subspecies, therefore it has a high selectivity and allows to adjust the number of only certain insect types. When bacteria are consumed by susceptible insects, secreted Bt-toxin becomes active under the action of enzymes in alkaline conditions of digestive tract. In the active form, the toxin specifically binds to the receptors of epithelial cells and causes their destruction. Insects stop feeding and soon die, and Bt-toxin is destroyed in the sunlight. Bt-toxin does not have a negative effect on warm-blooded animals. The development of genetic engineering methodology became it possible to detect and isolate genes encoding Bt-toxins, their modification and transfer to the plant genome. The first publications on practical developments in transferring potato varieties into transgenic status appeared in the early 90s of XX century. In particular, one such publication showed that genetically improved plants of the Russet Burbank potato variety contained a modified bacterial gene, B. thuringiensis var. tenebrionis encoding the control protein L. decemlineata Say, and were not damaged by Colorado potato beetle in the laboratory [11]. In transgenic varieties, the Bt-toxin gene is found in all cells, which means that it is able to express constantly, which allows the plant to protect itself from Colorado potato beetle and its larvae throughout the growing season. In all other respects, the transgenic variety does not differ from the original unmodified variety. In a review of biotechnological achievements of the first decade in crop production, it was noted that among other crops for which field trials and commercialization of transgenic plants were carried out from 1986 to 1995, potatoes accounted for 11% [12]. The profitability of growing genetically modified plants on an industrial scale was immediately evaluated by companies in the USA, Argentina, Brazil, Canada, and India, which since 1996 have remained the undisputed leaders in expanding cultivated areas and a specific assortment of genetically engineered crops. According to the International Service for the Acquisition of Agribiotech Applications (ISAAA), in 2016 the cultivated area under genetically engineered crops in the world reached 185.1 million hectares, an increase of more than 100 times over ten years (for comparison, 1.7 million ha in 1996) [13]. The commercial benefits from genetically modified plants, due to increased yields while reducing chemical means of protection, as well as issues related to development of effective measures to maintain sustainable development, economic stability, encourage scientists from different countries, including the Republic of Belarus, to conduct research on genetic improvement of crop varieties of domestic selection. Such varieties form the basis of varietal resources of the country and compares favorably with their foreign counterparts, especially in terms of their adaptability to growing conditions, diseases resistance, and other characteristics. The directions of research on genetic engineering of potatoes, carried out in scientific institutions of the Republic of Belarus, include the developing of transgenic lines based on Skarb Belarusian variety having resistance to Colorado potato beetle, as well as to late blight. In 2014-2015, the genetically engineered potato line resistant to Colorado potato beetle and developed at the Institute of Genetics and Cytology of the National Academy of Sciences of Belarus, was tested in an experimental field that met safety requirements. The transgenic line was obtained by introducing the Cry3aM gene, whose donor was bacterium B. thuringiensis var. tenebrionis, into the potato plant genome, under the control of the CaMV 35S promoter from cauliflower mosaic virus. The foreign gene expresses the Bt-endotoxin protein, which exhibits insecticidal properties against Colorado potato beetle and does not affect other insects. Assessment of potential risks associated with the use of transgenic organisms in the Republic of Belarus Considering the short global practice of using genetically modified plants, as well as the fact that potatoes are a significant food crop for consumers, safety of its genetically modified varieties should not only be ensured by scientists, but also guaranteed at the state level. In the Republic of Belarus for the period following the accession in May 2002 to the Cartagena Protocol on Biosafety to the Convention on Biological Diversity, a National Security System was created that included legislative and regulatory components to regulate safety of genetic engineering activities and aimed at protecting human health and the environment. The legislative acts are aimed at creating the legal and organizational foundations of safety carried out in research institutions, as well as at an objective assessment of potential risks of genetically engineered organisms that are only released for environmental tests in the field and to the market. Under Belarusian law, the procedure for assessing environmental risks and risks to human health is carried out twice. For the first time, the standard risk assessment procedure [14] is carried out before the genetically modified organism is released into the environment for testing on specially equipped experimental fields that meet world biosafety requirements. For this, the applicant scientific organization submits to the expert council of the Ministry of Natural Resources and Environmental Protection a full study on the risk assessment of the possible harmful effects of the developed genetic engineering organism, which is further evaluated by experts from among scientific institutions competent in this field. After it is considered at a meeting of the expert council of the Ministry of Natural Resources, at which it is decided whether or not the release of such an organism is permissible. Risks are reassessed before releasing genetically modified organisms into agricultural production. Genetically engineered organisms that have undergone a full cycle of research and are admitted to the market should not have negative effects on human health. All information on risk assessment is freely available on the website of the National Biosafety Coordination Center of the Republic of Belarus (http://www.biosafety.by). The National Biosafety Coordination Center was established in 1998 with the aim of ensuring the effective participation of the Republic of Belarus in solving the global problem of preserving biological diversity and coordinating activities related to the safety of using the achievements of modern biotechnology. Conclusions The current global trend in the development of plant protection methods is associated with increasing developing and implementation of transgenic organisms, the genome of which contains foreign genes to ensure their resistance to insect pests and pathogens. The use of genetic engineering methods aimed at reducing biogenic agroecological risks, in turn, raises concerns related to the ability of genetically modified organisms to have an adverse effect on conservation and sustainable use of biological diversity, and human health risks. It should be noted that the gene itself does not carry risks, since it consists of a sequence of nucleotides that are the same in all living organisms. Potential risks may arise due to the non-specific insertion of a new gene into the plant genome, i.e. insertion can occur anywhere in the DNA. The result of the insertion can be both favorable and undesirable consequences. If a foreign gene is inserted into a region of DNA that already encodes a gene, this can lead to its silencing, and, as a result, to termination of synthesis of a certain substance, which should not be observed when a new gene is inserted into a silent part of the genome. If the initial unmodified organism, into which the new gene is transformed, initially has undesirable traits, then with the integration of the gene such properties can be enhanced. That is why it is imperative that when assessing risks to human health, allergological and toxicological specialized tests are carried out, including animal studies. At the same time, the duration of the research will be increased if the parent organism, in which the new gene is inserted, is initially allergenic, has an increased level of anti-nutritional substances (e.g. soy), since after gene inserting, they can increase. It is also imperative to develop a molecular method that can effectively identify and track in the future the distribution on the market of a genetically modified organism and products derived from it. Currently, there are 18 specialized laboratories for detection of genetically modified organisms accredited by state or international standards in the republic. Another group of potential risks is associated with the safety of using genetically engineered organisms. Since transgenic plants begin to multiply in the environment, the risks associated with the consequences of transgene transfer during pollination for closely related cultural and wild species should be evaluated. Therefore, the transgenic organisms being developed undergo mandatory expert assessment, which involves consideration of all potential environmental and human health risks that may appear after insertion of a specific new gene and the new sign that will appear. When transgenic plants are released into the environment, there must be full scientific certainty about absence of threat of serious or irreversible damage. Summing up the issue of potential risks associated with the use of organisms and products of genetic technologies, we would like to note that all transgenic potato lines obtained in the Republic of Belarus are tested in laboratory conditions and on experimental fields that meet safety requirements. Since potato is an introduced species for Belarus, there is no possibility of uncontrolled transfer of its genetic material, including and transgenic, wild plant species growing in natural biocenoses. The need to use genetic engineering methods is associated with an exacerbation of biogenic agroecological risks, which can be reduced through the strategy of proactive introgressive selection based on cell-engineering and molecular methods.

Svetlana Mikhailovna Lenivko

Brest State A.S. Pushkin University

Author for correspondence.
Email: lenivko@brsu.brest.by
Brest, Republic of Belarus

Candidate of Biological Sciences, Associate Professor, Department of Zoology and Genetics

Vladimir Ivanovich Boyko

Brest State A.S. Pushkin University

Email: boikobio@yandex.by
Brest, Republic of Belarus

Candidate of Biological Sciences, Associate Professor, Department of Botany and Ecology

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