General Biotechnology: Lecture Course Part II - Blinov V.A. 2004

Agricultural Biotechnology II

♦ Technology for The production of bacterial entomopathogenic preparations.

♦ Technology for the production of fungal entomopathogenic preparations.

♦ Technology for the production of viral entomopathogenic preparations.

♦ Production of bacterial fertilizers and pesticide biodegradation.

♦ Plant Introduction/32.html">Genetic Engineering.

Crop yields heavily depend not only on adverse weather and climate conditions, soil erosion, weeds, and rodents, but also on The impact of insect pests, nematodes, phytopathogenic Fungi, Bacteria, Viruses, etc. For instance, devastating losses in potato farming are caused by the Colorado potato beetle as well as the fungus Phytophthora, the CAUSATIVE AGENT OF early blight (late blight) of potatoes. GROWTH AND DEVELOPMENT of corn are virtually brought to a complete halt by southern corn leaf blight. Tobacco and cotton mosaic disease, tomato winter disease, and others are caused by viruses. Global losses in agriculture and forestry caused by insect pests are estimated at $100 billion annually.

Biotechnological Approaches to Plant protection include: 1) breeding plant varieties resistant to adverse factors; 2) developing chemical control agents (pesticides) against weeds (herbicides), rodents (rodenticides), phytopathogenic fungi (fungicides), bacteria, and viruses; 3) developing biological pest control Methods. It has been established that many species of insect pests (aphids, the Colorado potato beetle, the codling moth, the turnip moth, etc.) perish under METABOLISM/18.html">The Influence of corresponding entomopathogenic preparations.

Currently, more than 30 microbiological entomopathogenic preparations are produced, manufactured on The basis of pathogenic bacteria, microscopic fungi, and viruses. They specifically affect certain insect species and are virtually harmless to humans, warm-blooded animals, birds, and beneficial insects. These preparations do not cause undesirable changes in biocenoses and do not disrupt the ecological balance.

Domestic industry manufactures groups of entomopathogenic preparations:

♦ bacterial preparations based on Bacillus thuringiensis: entobacterin-3, dendrobacillin, insectin, toxobacterin;

♦ the fungal preparation beauverin based on the fungus Beauveria bassiana;

♦ preparations based on nuclear polyhedrosis viruses: virin-ENSh, virin-EKS, and virin-ABB.

All microbial pathogens are produced in the form of wettable powders or pastes, less commonly as granules, encapsulated powders, or a stabilized emulsion of spores and crystals.

Technology for the production of bacterial entomopathogenic preparations. One of the highly effective microbes, B. thuringiensis, produces two toxins: ß and 8. ß-Endotoxin exhibits a broad spectrum of action against insects. It is an adenine nucleotide that competitively inhibits Enzymes catalyzing the Hydrolysis of ATP. This toxin is lethal to mammals. Therefore, production utilizes strains that do not produce ß-endotoxin but instead synthesize the 8-toxin. This toxin is a protein octahedron. Upon entering the insect gut, the toxin dissolves in an alkaline environment and is partially hydrolyzed by proteases. This modified protein interacts with the gut wall, allowing the Contents of the intestinal tract to enter the insect's bloodstream. Larval death results from septicemia. The 8-toxin is harmless to mammalian animals, humans, and birds.

The technology for producing bacterial entomopathogenic preparations includes the following stages:

♦ testing the stock culture for the absence of free phage, as well as for virulence, spore and crystal productivity;

♦ cultivating the culture in flasks until a spore titer of at least 1.7·10⁹ per 1 ml is reached;

♦ transferring the culture from flasks into the seed tank (0.05% of the medium volume);

♦ introducing the inoculum into the main fermenter (adding 0.0012% of the inoculum relative to the medium volume in the bioreactor).

The cultivation duration in seed and main fermenters is 35–40 hours at 28–30 °C (initial pH value of 6.3) until the required spore density per 1 ml of medium is achieved. During cultivation, the medium becomes alkaline up to pH 8.0–8.5; therefore, prior to Separation, the pH is adjusted to 6.0–6.2 by acidification. After separation, a paste is obtained (on average 100 kg per 1 m³ of culture liquid) with a moisture content of 85% and a spore content of 2·10¹⁰ per 1 g.

The finished product is a stable, viscous liquid of cream or light-gray color, odorless. Crystalline microcellulose or kaolin serves as a filler. The spore concentration in the preparations is 3·10⁹/g. Commercial preparations represent a mixture of spores and protein crystals within the Cells of the producer microbe.

Entobacterin is intended for combating insect pests of garden, vegetable, and park plants. It is effective in controlling more than 60 insect species. The bulk of the pests perish within 2–10 days. The crop yield increase from applying entobacterin is 50 centners for vegetable crops and 5 centners per hectare for orchard crops.

Technology for the production of fungal entomopathogenic preparations. Compared to entomopathogenic bacteria and viruses, fungi possess A number of specific features:

♦ infection occurs not through the digestive tract, but directly through the cuticle;

♦ insects are affected during the pupal and adult developmental stages, which does not occur in interactions with Other types of microorganisms;

♦ fungi are characterized by a high growth rate and immense reproductive capacity; in the form of spores under natural conditions, they maintain their entomopathogenic activity for a long time;

♦ fungi exhibit high Specificity in infecting specific insect species.

The fungal spore penetrates the insect's body cavity, where it grows into a hypha, which then develops into a mycelium that buds off conidia. These circulate within the hemolymph, releasing toxins. The insect dies due to the disruption of hemolymph Circulation and the toxic substances secreted by the fungus. If the toxin is present in small amounts or absent, the mycelium fills the entire body of the insect, primarily the Muscle tissue. Insect death occurs within 2–8 days.

In Russia, the industrial production of the entomopathogenic preparation beauverin has been mastered using submerged and surface cultivation Methods based on fungi of the genus Beauveria. Submerged cultivation is carried out under strictly aseptic conditions. Obtaining the inoculum is a particularly crucial stage in the technology.

The initial strain, stored on Agar slants of brewer's wort or Sabouraud medium, is propagated at 25–28 °C for 3–4 days by cultivation in shake flasks on a liquid nutrient medium. The resulting conidiospores are freeze-dried and can be stored for up to 1 year. The inoculum for seeding the fermenter medium is obtained by propagating the culture first in flasks, then in an inoculator, or directly in an inoculator. Seeding the main apparatus requires an inoculum volume equal to 2–10% of the nutrient medium volume.

The nutrient medium typically includes (in %): unlysed fodder Yeast — 2; starch — 1; sodium chloride — 0.2; manganese chloride — 0.01; calcium chloride — 0.05. pH — 4.5–5.6. The cultivation time for the spore inoculum is 25–28 h at 25–28 °C. Cultivation duration in the main fermenter at the same Temperature reaches 3–4 days. Continuous agitation and forced aeration are required; the air flow rate reaches 2.5 volumes per volume of medium per minute. The optimal concentration of amino nitrogen is 10–15 mg/dL.

The finished culture liquid is subjected to separation or filtration. Filtration yields a paste with a moisture content of 70–80%, which is sent to a spray dryer. The dried spores form a finely dispersed powder with a moisture content of 10% and a titer of up to 8∙10⁹ cells per 1 g. To determine LD50, the powder is standardized with the required amount of kaolin; wetting and sticking agents are sometimes added.

Surface cultivation yields spore-bearing fungal films. However, this method of producing beauverin is the most time-consuming and labor-intensive, and is therefore used less frequently than others. Surface cultivation of the fungus is carried out on both liquid and semi-solid nutrient media.

There are three technological approaches to producing beauverin:

♦ cultivation of the fungus in liquid media without autoclaving, agitation, and aeration;

♦ cultivation on Solid and liquid autoclaved media without agitation and forced aeration;

♦ a combined method for growing the fungal film.

Let us focus exclusively on the most productive combined method of fungal film cultivation. It includes:

♦ obtaining master cultures on grain;

♦ growing the inoculum (spore germination) in flasks on a liquid nutrient medium for 12–17 h;

♦ growing and accumulating the vegetative culture in a fermenter with forced aeration and agitation for 22–28 h;

♦ dispensing the culture liquid into trays and growing spore-bearing films;

♦ harvesting, post-ripening, and drying of the spore-bearing films;

♦ standardizing the preparation with kaolin.

The nutrient medium for cultivating the fungus is a mixture containing (in %): molasses — 6; corn steep liquor — 1; magnesium sulfate — 0.05; monopotassium phosphate — 0.2. Cultivation is carried out at 24–26 °C. Inoculation of the main fermenter requires 2–4% of the inoculum relative to the medium volume. The finished culture liquid has a titer of 50–100 million cells per 1 mL.

The films are grown in trays placed in vertical chambers at 25–26 °C. The entire production cycle takes 11–12 days, including: obtaining the inoculum — 1 day; fermenter cultivation — 1–1.5 days; maintenance in cabinets (mass conidia formation) — 5 days; film post-ripening — 2 days; film drying — 2–3 days. The finished films are harvested and dried at 28 °C in an air stream. The dried spore films are placed in polyethylene bags and stored in a dry place at 18–20 °C.

Before preparing the beauverin formulation, the spore material is ground in a ball mill and sifted through a series of sieves. The finished preparation (with kaolin filler) must have a titer of at least 1.5 billion conidiospores per 1 g.

Beauverin is used against leaf-eating orchard pests, as well as the codling moth, oriental fruit moth, and forest pests. Applying beauverin against Colorado potato beetle larvae on potatoes yields excellent results. When combined with chemical insecticides, the preparation achieves 100% mortality across all larval instars. The application rate for beauverin is 1–2 kg per hectare.

Technology for producing viral entomopathogenic preparations. Such preparations exhibit high specificity toward the host insect, making them practically harmless to humans, flora, and fauna. Viruses are highly resistant to adverse environmental factors (temperature, humidity) and can remain active for 10–15 years outside the insect body.

Viral infection occurs when the insect feeds. In the gut, virions are released under alkaline pH conditions. They penetrate the gut wall, and their Replication takes place in the nuclei of susceptible cells. This ultimately leads to the death of the insect larvae.

The production of any viral preparation begins with breeding the host insect, as viruses can only replicate in living tissue. Typically, insect larvae are infected at the caterpillar stage by adding a viral suspension to their feed. The goal is to maximize virus accumulation within the insect Tissues. After 7–9 days, dead and dying larvae are collected, dried at 33–35 °C, and mechanically crushed to release inclusion bodies from the tissues. Physiological saline or distilled Water is added to the resulting mass at a ratio of 1 ml per caterpillar, and the slurry of crushed tissues is filtered.

The technology for producing each entomopathogenic viral preparation has certain distinct features. For instance, the production of Virin-EX involves precipitating polyhedra by centrifugation. A titer of 1 billion polyhedra per ml is established using sterilized glycerin, among other agents. In the production of Virin-ENSH, lactose is added to the filtrate, followed by thorough mixing and The addition of acetone at a 4:1 volumetric ratio; finely dispersed kaolin or bentonite is used as a filler, and the polyhedron titer is adjusted to 1 billion per gram, and so on.

Entomopathogenic viral preparations are either introduced into dense insect populations to trigger an epizootic, or used to spray or dust plants in infested areas during the hatching period or Cytology/cytology/16.html">Early stages of larval development.

As a rule, combinations of several biological agents prove to be more effective than individual preparations applied separately. For example, the mortality rate of the pine moth increases sharply when the cytoplasmic polyhedrosis virus is used in combination with B. thuringiensis preparations.

Plant protection products against phytopathogenic microorganisms can be classified as follows:

♦ Antibiotics. For instance, trichodermin and trichothecin, produced by the fungi Trichoderma sp. and Trichothecium roseum, are used to control ROOT rots in vegetable, grain, and industrial crops;

♦ phytoalexins. These are synthesized in plant tissues in response to phytopathogen invasion and can serve as highly specific substitutes for pesticides. For example, pepper phytoalexin is successfully used to combat late blight;

♦ antagonist microbes that displace the pathogenic species and suppress its development;

♦ vaccine and immunological preparations applied directly to germinating seeds;

♦ the d-factor, a specific agent that reduces the viability of the pathogen.

Thus, biological agents are a vital component of integrated plant protection programs against various phytopathogenic microorganisms.

Production of bacterial fertilizers and pesticide biodegradation. It is now evident that natural, biological technologies offer a viable alternative to the chemicalization of agriculture. These include biological plant protection methods, the breeding of resistant varieties, The Use of modern agrochemical management systems, and more. In this regard, the agricultural application of specific microbial populations holds particular promise. Many of these have been known for quite some time and are used to combat pests, suppress weed growth and development, and produce silage; they are also effective in livestock and poultry farming or serve as an alternative to nitrogen fertilizers. Detailed information on microbial soil-fertilizing preparations can be found in V. A. Blinov's book Biotechnology (2003). Here, we outline only the production technology for rhizotrophin.

Rhizotrophin consists of a peat base mixed with rhizobacteria (5–8·10⁹ cells per 1 g of peat). The technological process of rhizotrophin production includes preparing root-nodule bacteria and obtaining the inoculum, preparing the peat, and "inoculating" the peat with rhizobacteria. Nutrient media are generally quite simple, containing plant extracts or decoctions, sucrose, glucose, and mannitol.

The inoculum is prepared under conditions of moderate aeration on similar media with the addition of phosphates, sulfates, and carbonates (at a temperature of 28–30 °C); antifoaming agents are necessary. Approximately 5 billion cells per ml are accumulated. This suspension is introduced into "acidic" peat (pH 3.0–6.0) at 10–15 °C. The peat must be homogeneous, dried, milled, mixed with chalk, and sterilized by radiation at 2.5 Mrad. The bacterial suspension is added to the prepared peat so that 1 g contains 0.5–1 billion cells, and the mixture is thoroughly stirred. The product can be stored for up to six months at 5–10 °C for fast-growing rhizobacteria and at 12–15 °C for slow-growing ones. The application rate of rhizotrophin is 200 g per hectare for the seeds of any leguminous plants.

Fusicoccin is among the microbial stimulants and plant growth regulators. This substance is of a hormonal nature. Under its influence, root formation is enhanced in many woody, fruit, and vegetable crops, and the germination of carrot, tomato, sugar beet seeds, and others is stimulated.

Fusicoccin is produced by the fungus Fusicoccus amygdali. The product is obtained via submerged batch Fermentation on media containing glucose or sucrose and soybean meal. Fusicoccin is then extracted from the culture liquid using butyl acetate or chloroform, adsorbed on activated charcoal, repurified, and crystallized from ethyl acetate.

Another plant growth regulator is gibberellic acid. It is synthesized by the micromycete Gibberella fujikuroi and belongs to the group of Plant HORMONES with a complex chemical Structure. The Biosynthesis of gibberellic acid proceeds via acetyl-CoA, mevalonic acid, and cyclized structures. During surface cultivation of the producer, gibberellic acid biosynthesis takes about 15 days at 25 °C, and up to 9–10 days under submerged fermentation. The nutrient media for the producer are complex, containing 4–6% glucose, sucrose, or glycerin, 0.7% ammonium salts, citrate, or peptone, 0.3% inorganic phosphate, 0.05% magnesium sulfate, and various Trace Elements; the medium pH is adjusted to 3.0–4.0 with Hydrochloric acid. Under these conditions, the yield of Gibberellins reaches 200 mg/l of culture liquid (surface method) or 1 g/l under submerged cultivation in fermenters. The bulk gibberellic acid preparation is obtained from the vacuum-concentrated culture liquid after separating the producer mycelium. Gibberellic acid is used as a growth promoter for various plants, achieving maximum effect at a dose of 1 µg per plant.

Pesticide biodegradation is a critical issue. Pesticides possess strong yet insufficiently selective action. They can cause substantial damage to agricultural crops and persist in the soil for a long time. To address these challenges, application technologies are being improved, pesticide-resistant plant varieties are being bred, and pesticide biodegradation in the soil is being initiated. The persistence of a given pesticide can be altered by applying it in combination with another chemical pesticide. Genetic engineering METHODS have been used to construct microbial strains with enhanced pesticide degradation efficiency. For instance, the strain Pseudomonas cepacia degrades 2,4,5-trichlorophenoxyacetate.

Naturally, the biodegradation of pesticides diminishes their beneficial effects. Moreover, biodegradation can yield intermediate products that are highly toxic to plants. For example, the use of the herbicide thiobencarb results in the suppression of rice growth and development. This inhibitory effect is caused not by the herbicide itself, but by its dechlorinated derivative, 3-benzyl-N,N-diethylthiocarbamate. To prevent this, thiobencarb is applied in combination with methoxyphenone, which inhibits the dechlorinating enzyme of microorganisms.

Plant genetic engineering. Breeding based on Hybridization, spontaneous Mutations, and induced mutations makes it possible to obtain A wide variety of crop varieties and hybrids. Genetic and cellular engineering techniques address the challenges of enhancing the resistance of new agricultural plant forms, lines, varieties, and hybrids to pathogens, as well as shortening the breeding cycle for new varieties.

In plant genetic engineering, the MAIN STAGES OF obtaining Transgenic Plants are distinguished as follows: 1) Selection of the Gene and its cloning; 2) selection of the recipient plant genotype; 3) gene introduction and expression in the recipient plant genome; 4) regeneration of transformed cells and selection of transgenic plants.

The production of transgenic plants became possible after the transforming activity of Ti-Plasmids from soil agrobacteria was established. These are circular DNA molecules with a length of approx. 200 kb. The most common are Ti-plasmids that encode the Amino Acids nopaline and octopine. The nucleotide sequences of Ti-plasmids are divided into two groups: 1) those required for the metabolism of the agrobacterium itself; 2) those required for plant Cell transformation.

It has been established that agrobacteria and Ti-plasmids do not enter The plant cell, but a portion of the plasmid can be transferred into The Nucleus of such a cell and integrated into its genome. This fragment of the Ti-plasmids was named T-DNA (Transforming DNA). Within The Genome, T-DNA alters the hormonal status of the plant cell, leading to its dedifferentiation and tumor formation. Furthermore, cell transformation requires another region of the Ti-plasmid (the vir region, or virulence region), which transfers the T-DNA into the plant genome.

Currently, the following Vectors for Plant transformation have been developed based on Ti-plasmids.

Cointegrative vector. It facilitates the introduction of foreign DNA into the plant genome. To obtain a cointegrative vector, vectors based on E. coli plasmids are used. A T-DNA fragment and a selective marker gene (typically a gene conferring resistance to kanamycin, hygromycin, or a herbicide)—which subsequently allows for the selection of transgenic plants—are inserted into these vectors. In addition, another vector (A. tumefaciens) carrying the genes necessary for the Integration of the T-DNA region into the plant genome is utilized. This vector contains the vir region. Thus, a cointegrative vector is generated As a result of recombination between two plasmids.

Binary vector. The technique for obtaining such a vector is simpler. Its essence lies in the fact that plasmids containing the T-DNA border repeat region and the vir region are found within an agrobacterial cell. They are integrated into a single vector, introduced into agrobacteria, and subsequently into plant cells.

Plant cell transformation also utilizes vectors based on Ri plasmids, plant DNA-containing viruses, and Mobile Genetic Elements (Transposons), which are DNA sequences with a specific structure capable of transposition throughout the genome.

Modern Methods of plant cell transformation include:

♦ The co-cultivation method with agrobacteria. This is based on the transformation of plant explants by agrobacteria carrying vectors that contain foreign genes inserted into the T-DNA;

♦ Methods of direct gene transfer into plants. The following techniques are used to deliver DNA vectors into protoplast cells: DNA microinjection, electroporation (increasing biomembrane permeability via a high-voltage pulse of 200–350 V with a pulse duration of 54 ms), liposome encapsulation, and biolistic transformation (vector DNA is coated onto tungsten, platinum, or gold particles 0.6–1.2 µm in size). The particles are placed into a gene gun where the pressure is reduced to 0.1 atm. Upon pressure release, the particles accelerate rapidly toward The Cell suspension, piercing the cell walls and entering the Cytoplasm and nucleus. Cells located within a 0.6–1 cm zone from the center of the blast area become transformed. They are then transferred to a nutrient medium for further cultivation and regeneration.

To achieve the expression (functioning) of foreign genes in a plant genome, the 35S RNA promoter of the cauliflower mosaic virus (CaMV) is most commonly used. Such a promoter is capable of expressing bacterial genes within a eukaryotic (plant) cell. Recently, tissue-specific plant gene promoters have also seen widespread application. The key difference is that genes under their control are expressed exclusively in specific tissues. For instance, a gene driven by the patatin promoter will only be expressed in potato tubers, since patatin is a protein characteristic specifically of potato tubers. Another example arises when it is necessary to protect only the underground parts of plants from soil-borne pathogens; in this case, utilizing a root-specific promoter is preferable. Inducible promoters are also employed, where genes under their control are not expressed constitutively, but only under specific environmental conditions.

To detect the expression of foreign genes, expression markers known as reporter genes are used. These encode an enzyme whose reaction product is easily detectable, or they produce a fluorescent protein that is readily identifiable.

Genetic engineering methods make it significantly possible to improve crop quality and increase plant productivity. For example, high-Lysine varieties of barley, corn, and wheat have been developed through genetic engineering. Tobacco and potato plants capable of synthesizing immunoglobulin A-G, enterotoxin, cholera D-toxin, and the hepatitis B surface antigen protein have already been produced. Notably, the protein derived from these transgenic plants exhibited the exact same antigenic and physiological properties as the protein obtained from the original cells.

Research is currently underway on the genetic engineering Modification of the fatty acid profile in several oilseed crops, most notably rapeseed. Active genetic engineering efforts are also aimed at increasing plant photosynthetic activity and enhancing the Synthesis of specific compounds. Since 1999, in the USA, Canada, and several other countries where the use of transgenic plants is permitted, agricultural production has already adopted seven transgenic corn varieties and one wheat variety characterized by high yields and superior product quality.

Biotechnological approaches enable the generation of transgenic plants resistant to various environmental stresses (such as drought, waterlogging, high and low temperatures, soil salinity, and acidity), tolerant to insect pests, resistant to fungal, bacterial, and viral infections, and tolerant to different herbicides, among others. For instance, in North America and Europe, more than 20 herbicide-tolerant transgenic plant varieties have been approved for use, including corn, cotton, rice, soybean, wheat, potato, tomato, and flax. Field trials are currently underway for transgenic varieties of strawberries, sugar beets, and certain ornamental flower crops. By 2001, a total of 78 transgenic plant varieties were cultivated in countries where the use of genetically modified plants is authorized.

At the same time, numerous challenges in plant genetic engineering remain unresolved. One such issue relates to the difficulty of simultaneously introducing large genes (exceeding 10 kb) or multiple functional genes into plant genomes, primarily due to the limited carrying capacity of transformation vectors. Another problem stems from the fact that, frequently after two to five generations, an actively transcribed transgene ceases to be expressed, and reactivating such a "silenced" transgene remains currently impossible. Furthermore, There is a lack of sufficient research dedicated to identifying effective genes and establishing gene banks, while the scientific foundation of genetic engineering remains quite limited due to inadequate financial support for bioengineering.