Production of Plant Bioactive Compounds Using Different Techniques

Production of Plant Bioactive Compounds Using Plant Organ, Tissue and Cell Culture Techniques

Abstract

Bioactive compounds produced by plants to attract insects for pollination, repel pests, or protect against microorganisms are important components for numerous different industries. The amount of bioactive compounds within the plant's total chemical composition varies according to soil, environment, or the plant's genotype characteristics. Plant organ, tissue and cell culture methods enable the production of metabolites in stable proportions under culture medium conditions while plant cloning is performed under sterile conditions. This study explains the production of plant metabolites under sterile plant propagation conditions.

Introduction

Primary metabolites are essential metabolites for plants to sustain their lives. Secondary metabolites are metabolites that plants produce for purposes such as defense, pheromone production, and waste storage. These products also form the basis for medical and cosmetic use. While WHO's Essential Medicines Model List contains 210 small-molecule drug raw materials, 17 of them are of plant origin (Başer, 2012). Plants have developed aromas that attract pollen-carrying bees, bitter or toxic tastes that repel predators, or allelopathic chemicals. Bioactive products may be chemicals that can accumulate in the coffle to attract another organism as well as biochemicals that can be harmful to cells. All these phytochemical compounds can be used as food supplements, pain relievers, sedatives, cough suppressants, as antioxidants and dyes in the textile industry, as insecticides or in agricultural production to combat pathogens, and as aroma and flavor components in the food industry (Yu et al. 2021). The yield of plant metabolites is unstable under natural conditions and production costs are high (Chandran, 2020). Through tissue culture methods, the intensive labor in vitro conditions, low and unstable product yields can be standardized independently of seasonal conditions throughout the year. Production of bioactive compounds through tissue culture methods in vitro conditions reduces the risk of chemical contamination. The yield of bioactive metabolites can reach a significant level using organ, tissue and cell culture methods (Wawrosch and Sergey, 2021).

Totipotency and Micropropagation

Plants can regenerate different organs from their differentiated tissues thanks to their totipotency characteristics. The totipotency characteristic provides plants with vegetative propagation ability. This method makes clonal propagation possible (Feher, 2019). The tissue culture method is an important example of vegetative propagation in sterile laboratory conditions. Organ, tissue or cell propagation becomes possible in containers created with artificial growing media under sterile conditions free from viruses and other pathogens. Tissue culture methods provide consistent uniform production for twelve months throughout the year. Tissue culture methods provide uninterrupted production free from disease agents, unwanted chemical contamination, pest and virus infections. Initial investment costs and the need for skilled experienced labor are factors that make this method challenging. This situation has created a field of specialization in plant tissue cultures. Instead of harvesting and processing plants cultivated over hectares of land or collected from nature, production of active compounds through tissue culture methods is more advantageous (Espinosa-Leal et al., 2018). The tissue culture method is the propagation of plants in sterile containers in the laboratory environment under artificial lighting and climate control conditions. This method provides year-round uninterrupted disease-free clonal plant production. Since production conditions are independent of climate and seasonal factors, arithmetic propagation speed is achieved. Tissue culture methods serve as a commercial propagation method for economically valuable plants as well as a method through which plant bioactive compounds can be intensively propagated for breeding and ex-situ conservation purposes. Through cell suspension cultures and following bioprocess procedures, bioactive products can be produced. These products can be pharmaceutical raw materials, pharmaceuticals, plant growth regulators, and many other products that can be produced within plant cells and added to the economy (Motolinía-Alcántara et al., 2021).

Establishment of Plant Tissue Cultures

A plant tissue culture laboratory should consist of at least 4 separate rooms (Figure 1) (Mather and Roberts, 2002). These rooms consist of a preparation room where the growth medium is prepared, pre-sterilization is performed and growing medium containers are cleaned, a transfer room where inoculation takes place, a climate room where plants develop, and an acclimatization room or greenhouse where plants are adapted to the external environment. When the addition of bioprocess procedures is intended, it would be beneficial to also have bioreactors, extraction equipment, and chromatography equipment (Mineo, 1990). For plants to be propagated under sterile conditions, both the plants and the containers in which they are propagated must first be sterilized. The most commonly used chemicals for surface sterilization of plants are sodium hypochlorite, calcium hypochlorite, ethanol, mercury chloride, silver nitrate, ozone, biocides and fungicides. Plants to be placed in culture are washed three times with soapy water and rinsed. Treatment is performed with 70% concentration ethyl alcohol for 30 seconds and 20% sodium hypochlorite for 20 minutes. For woody parts, seeds and underground organs, concentration can be increased, while the ratio can be reduced for young shoots and delicate plants. After the time period, rinsing is done at least three times with sterile distilled water under a sterile hood, and planting is performed in growing medium containers with appropriate growing medium at the base. The culture medium protocols used for plant tissue culture methods are formulated according to plant morphological conditions and the nutrients they utilize. The contents are also determined according to antagonistic and synergistic effects of plant nutrients. For example, chloride and iodine were extracted from the medium, and Chee and Pool (1987) developed media for grapevine plants and Quoirin and Lepoivre (1977) for Prunus, Anderson (1980) for Rhododendron and berry groups. N6 media were developed for monocotyledon anther cultures by Chu. To these groups can be added sweet potato (Chee, 1992), sugar beet (De Greef and Jacobs, 1979), walnut (Drive and Kuniyuki, 1984), soybean (Gamborg et al., 1968), orchid (Morel, 1965), carrot or Pinus (Litvay, 1985), media for woody plants (Lloyd and Mc Cown, 1980), and olive (Rugini, 1993). In the development of all these growing media, there is a very important growing medium content for tissue culture applications. Influenced by Murashige and Skoog (1962); White (1939) and Skoog (1944), the MS medium developed for tobacco is still used for many plants. In plant growing media, in addition to macro and micronutrients, certain vitamins, amino acids, sugars, hormones, some organic compounds, plant protection products and solidifying agents may be present. The basic function of the growing medium is to ensure healthy development of plantlets. Propagation at geometric growth rate is quite rapid. Through the meristem culture method, when each explant produces 4 new shoots, approximately 1 million clones are obtained at the end of the year through 30-40 day subculture processes. When 5 new shoots are obtained from a single plant, the approximate number of clones obtained at the end of the year will be 10 million. While plant species have an effect on this increase, the content of the growing medium is also very important. Plant nutrient requirements, compound uptake forms, plant growth regulator types and quantities, and antagonistic and synergistic effects between plant nutrients affect media components. Plants placed in growing medium under sterile conditions are left to develop under artificial lighting. Medium temperature, relative humidity in the container, lighting intensity, light frequency, and photoperiod affect plant development. For example, heliophyte plants prefer intense lighting intensity while xerophyte plants prefer low intensity.

Organ Culture Method

The purposes of production according to organs where bioactive compounds accumulate in plants may differ. Since astragaloside IV accumulates in the root organ in Astragalus membranaceus, regeneration root; in Catharanthus roseus, vinblastine and catharanthine active compounds accumulate in the leaf, while diosgenin accumulates in Dioscorea spp tubers, different organs are preferred. In plant organ culture regenerations, plant growth regulators are preferred while nutrient contents in the medium are also recombined. While cytokinin and gibberellic acids are preferred for shoot regeneration of root development, auxin-type growth regulators are preferred for root regeneration. For tubers, chlorocholine chloride (CCC), paclobutrazol (PBZ), and sugar concentration can be increased (Zeng et al., 2012). For root regeneration, inoculation with Rhizobium rhizogenes bacteria, which creates fibrous roots, is also possible. For fibrous root culture, Catharanthus alkaloids, atropine and hyoscyamine are used (Shakeran, 2017). Plants can be propagated in air-saturated liquid solutions in the tissue culture medium. This method is called the temporary immersion system (Figure 2). In temporary immersion systems (TIS), ideal plant development is provided since regular fresh air flow is supplied to the culture medium. The most important thing to pay attention to in TIS is that contamination factors are not present in the air flow provided to the medium containing sterile plants. TIS works as follows. Two sterile containers that allow fluid transfer between them are used; plant explants are located in the upper one, while nutrient solution is located in the lower one. An air compressor is attached to the lower container. Air supply is provided by passing through a hepafilter. Two conditions exist in TIS. In the first condition, the air compressor operates; in the second condition, the air flow provided by the compressor is cut off. When the air compressor supplies clean fresh air to the lower container, the nutrient solution is transferred to the upper container through a capillary tube due to the positive air pressure effect inside the container. The plant parts present in the upper container are treated with fresh air-saturated nutrient solution. After some time, the compressor is turned off. In this case, the nutrient solution flows to the lower container due to the effect of gravity, while the plants are held in the upper container with the help of a filter. As the nutrient solution flows to the lower container, some air is supplied from the outside, and a hepafilter should also be located over the opening. Through TIS, banana, potato microtubule propagation is possible (Bello-Bello et al., 2019, Jimenez et al., 1999).

Tissue Culture Method

Meristem culture is the most frequently used tissue culture method for clonal micropropagation because it is resistant to somaclonal variations in its maintenance. The best examples of secretory tissues on stems and leaves are glandular trichomes (hair) sacs. Methods in which volatile compounds propagated in secretory tissues are propagated through tissue culture methods can be exemplified. Aromatic compounds such as fragrance components can be propagated with this method. From the glandular hair of Drosera capensis plant, plumbagin, 1,4-naphthoquinones and 7-methyljuglone are obtained (Budzianowski, 2000).

Cell Culture Method

Cell cultures are composed of callus or independent cells with cell walls removed. Cell cultures can be used in suspension medium as well as in solid and semi-solid media. Since cells in cell cultures are independent, signal transmissions are cell-independent and cells are not affected by neighboring cells in terms of gene expression. Cells can be regenerated into organs that produce bioactive substances with the help of growth regulators, and valuable bioactive molecule yields can be increased with heavy metals, elicitors, or measured biochemicals. The tissues from which initial cells are obtained are quite valuable in terms of active compound yield. For example, initial cells obtained from different organs of Morinda citrifolia plant showed differences in anthraquinones yield (Deshmukh et al., 2011). Plant cells, in cell suspension culture medium, can move independently from each other like bacterial cells. In cell suspension cultures, the container is continuously agitated or cells are kept mobile through clean air supply. Otherwise, plant cells will colonize. While the process shows similarity to microorganism cultures, since plants are heterotrophic organisms, some fundamental differences are observed in their growing media. On the other hand, processes can be provided with fully controlled environmental conditions like bioreactors. Through bioreactors, autonomous propagation of living cells in suspension culture medium is possible. A bioreactor is a computer-controlled device that controls nutrient solution feeding within the growing medium, pH, EC, clean air intake, and bioprocess procedures (Figure 3). The concentration of the targeted bioactive substance in the suspension is also important (Espinosa-Leal et al., 2018). The maximum increase amount and duration of cells varies depending on the consumption rate of the growth medium (substrate) present in the bioreactor environment. Besides this, phenolic or toxic compounds secreted by plant cells into the medium also determine the length of the production period. Cell yield stabilization is effective in determining the type of bioreactor (Motolinía-Alcántara et al., 2021).

Bioactive Compound Metabolic Pathways

Plant anabolic activities beginning with the conversion of solar energy into chemical bond energy result in glucose production through photosynthesis reactions. These basic functions are carried out in chlorophyll cells containing the green pigment. Under light, carbon obtained from carbon dioxide molecules and hydrogen and oxygen obtained from water molecules are used in glucose synthesis. Through photosynthesis, plants produce glucose from inorganic materials. Glucose produced through photosynthesis reactions forms an important and basic step in our food chain. Glucose is converted to pyruvate via the glycolysis pathway, and the conversion of pyruvate to acetyl Co-A molecules creates raw material for the citric acid cycle. Glycolysis Pathway and Krebs Cycle (TCA or citric acid cycle) are the fundamental cycles in which primary and secondary metabolites are produced. Through these two important glycolysis and citric acid cycles, pathways of nucleic acids, amino acids, vitamins, and fatty acids as primary and secondary metabolites are formed. For example, phosphoenolpyruvate enters the shikimic acid cycle, and flavonoids are produced at the end of this cycle. The final product of the glycolysis pathway, acetyl CoA, not only forms the raw material of the Krebs cycle but also forms the raw material for reactions that create phenolic compounds through the malonic acid cycle and terpenes and alkaloids through the mevalonic cycle. Using the TCA or Krebs cycles, the concentration ratios of certain plant amino acids and nucleic acids in plant cells can be naturally increased (Figure 4) (Zhang and Fernie, 2018; Alabduladhem and Bordoni, 2021). For the use of plant bioactive substances as plant bioactive compounds, their pharmacological effects must be examined and tested through clinical studies. In repeated trials, they must demonstrate statistical superiority over placebo samples. On the other hand, herbal drug products must be standardized from botanical, physical, chemical and biological perspectives. The herbal product whose active compounds will be utilized must be standardized botanically in terms of color, odor, taste, texture, fracture, qualitative and quantitative aspects; physically in terms of moisture content, solubility, ash, spectroscopic aspects; biologically from microbiological, pharmacological and toxicological perspectives; and chemically in terms of chromatographic aspects (Shivatare, 2013).

Conclusion

For plant drugs to be evaluated as high-value plant bioactive compounds, the biochemical composition contained in them must be standardized. To achieve Good Manufacturing Practice standards (GMP), it is possible to implement plant tissue organ, tissue and cell culture applications in addition to conventional production. Tissue culture applications provide standard raw material supply, production continuity and high quality standards, but high investment costs, the need for trained personnel, and the need to regularly protect and maintain sterile conditions must be observed. With advancing technology, the frequency of application of these practices is increasing day by day. Like all other innovative achievements of Turkey, it is possible for our country to produce plant bioactive compounds by benefiting from biotechnology applications, transferring the richness of plants in our natural flora to laboratory conditions, and evaluating our technical experience in this field. 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