Temporary Immersion Bioreactors Research Articles

Optimization of the Production of Secondary Metabolites from Furanocoumarin and Furoquinoline Alkaloid Groups in In Vitro Ruta corsica Cultures Grown in Temporary Immersion Bioreactors

Optimization of the Production of Secondary Metabolites from Furanocoumarin and Furoquinoline Alkaloid Groups in In Vitro Ruta corsica Cultures Grown in Temporary Immersion Bioreactors

Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50

Conventional in vitro propagation using semisolid Murashige and Skoog (MS) culture systems is costly, labor-intensive, and requires substantial space for large-scale plant production. This study investigated the application of a temporary immersion bioreactor (TIB) system for the micropropagation of the banana cultivar Kluai Numwa Pakchong 50, as a promising platform for economical commercial production. The cultivation parameters affecting plantlet multiplication, including plant growth regulator (PGR) use, explant density, and immersion frequency, were examined. Additionally, the ex vitro acclimatization of well-developed in vitro plantlets was also evaluated. Using liquid MS medium supplemented with 7.5 mg/L 6-benzylaminopurine (BAP) in the TIB system yielded significantly better results than the conventional semisolid MS control system, producing more shoots (5.60 shoots/explant) and leaves (2.80 leaves/explant) with longer shoot length (2.19 cm). Optimal conditions in the TIB system included an inoculum density of five explants per culture vessel and an immersion frequency of once every 6 or 8 h for 2 min. For root induction, 0.5 mg/L indole-3-butyric acid (IBA) proved more effective than 1-naphthaleneacetic acid (NAA). After 30 days of ex vitro acclimatization, plantlets regenerated from the TIB system demonstrated high survival rates, vegetative growth performance, and root formation efficiency comparable to those from the semisolid culture system. These findings establish the TIB system as a promising platform for the mass propagation of the Kluai Numwa Pakchong 50 banana. The protocol developed in this study could potentially be adapted for large-scale production of other banana varieties.

Effect of Agitation and Temporary Immersion on Growth and Synthesis of Antibacterial Phenolic Compounds in Genus Drosera.

Sundews (Drosera sp.) are the source of biologically active secondary metabolites: phenolic acids, flavonoids, and 1,4-naphtoquinones. Because obtaining them from the natural environment is impossible (rare and endangered species), in this study modifications of traditional tissue cultures grown in solid medium (SM), such as agitated cultures (ACs) (cultures in liquid medium with rotary shaking) and temporary immersion bioreactors PlantformTM (TIB), were used for multiplication of four sundew species: Drosera peltata, Drosera indica, Drosera regia, and Drosera binata, with simultaneously effective synthesis of biologically active phenolic compounds. Each species cultivated on SM, AC, and TIB was tested for biomass accumulation, the content of total phenols and selected phenolic derivative concentrations (DAD-HPLC), the productivity on of phenolic compounds, as well as its antibacterial activity against two human pathogens: Staphylococcus aureus and Escherichia coli. The results showed that the type of culture should be selected for each species separately. Phytochemical analyses showed that the synthesis of secondary metabolites from the groups of phenolic acids, flavonoids, and 1,4-naphthoquinones can be increased by modifying the cultivation conditions. D. regia turned out to be the richest in phenolic compounds, including 1,4-naphtoquinones: plumbagin and ramentaceone. Extracts from D. indica and D. regia tissue showed strong antibacterial activity against both pathogens. It has also been shown that the growth conditions of sundews can modify the level of secondary metabolites, and thus, their biological activity.

Agitated and temporary immersion bioreactor cultures of Reynoutria japonica Houtt. as a rich source of phenolic compounds

Reynoutria japonica Houtt. (Japanese knotweed) is an invasive plant belonging to the Polygonaceae family. However, being native to East Asia, it has been used in natural medicine for ages because of its broad range of biological activity. Although R. japonica is known as a rich source of phenolic compounds, plant biomass collected from the field may be contaminated with toxic elements like heavy metals, and the level of metabolite accumulation depends on environmental conditions. Therefore, the aim of this study was to derive Japanese knotweed tissue cultures and investigate biomass production and phenolic compound synthesis in in vitro conditions. Plants were cultivated in a traditional agar-solidified medium, in a liquid medium with rotary shaking (agitated culture), and in a temporary immersion bioreactors Plantform™, as well as in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted to determine the sum of phenolic compounds, as well as the quantity of selected phenolic acids, catechins, and other flavonoids. Results have shown that agitated cultures and temporary immersion bioreactors increased biomass accumulation compared to solid medium cultures. Tissue cultures of R. japonica had increased synthesis of phenolic compounds compared to plants from ex vitro conditions. Shoots and roots from agitated cultures were 2.8- and 3.3-fold richer in catechins, respectively, compared to plants cultivated in soil. Based on the obtained results it can be concluded that agitated and bioreactor cultures are the best source of Japanese knotweed biomass rich in valuable secondary metabolites.

A Comparison of Semi-Solid, Liquid, and Temporary Immersion Bioreactor Systems for Effective Plant Regeneration of Gerbera jamesonii “Shy Pink”

Temporary immersion system (TIS) cultures are reported to be superior when compared to semi-solid (SS) and liquid (LQ) cultures for the in vitro plant regeneration of many plant species. In the present study, we used a TIS for plant regeneration of Gerbera jemesonii “Shy Pink” and compared the results with that of SS and LQ cultures. The number of shoots regenerated in the SS, LQ, and TIS cultures was 6.93, 3.13, and 3.03, respectively. The shoots regenerated with the SS and LQ cultures demonstrated 3.33 and 4.22% hyperhydricity, whereas the shoots regenerated with the TIS were healthy even though the number of shoots regenerated was less. The plantlets regenerated with the TIS demonstrated higher values for the number of roots, root length, biomass of the plantlets, leaf length/width, and area compared to the SS and LQ cultures. When the G. jemesonii “Shy Pink” plants were regenerated using the TIS, their levels of photosynthetic pigments were highest. The number of stomata on the abaxial surface of their leaves was 11.40, and the frequency of closed stomata was 59% for the plants regenerated with the TIS. The number of stomata was 21.4 and 14.5 for the plants regenerated with the SS and LQ cultures, respectively. Meanwhile, the frequency of closed stomata was 13% and 15% for the plants regenerated with SS and LQ cultures. Furthermore, G. jemesonii “Shy Pink” showed the highest survival of plants when regenerated in the TIS compared to SS and LQ cultures. The TIS was found to be the most suitable culture system for plant regeneration of G. jemesonii “Shy Pink” compared to SS and LQ cultures.

In vitro physiological and biochemical response of Stevia rebaudiana exposure to carbon nanotubes: Hormetic and photomixotrophic effect

Multi-walled carbon nanotubes (MWCNTs) are cylindrical rolled sheets with different applications in plant biotechnology due to their physicochemical characteristics. This study aimed to evaluate the in vitro physiological and biochemical responses of stevia (Stevia rebaudiana Bertoni) exposure to different concentrations of MWCNTs during multiplication phase in liquid medium using temporary immersion bioreactors. The MWCNTs were characterized regarding their properties by transmission electron microscope. Different treatments (0, 25, 50 and 100 mg/L) of MWCNTs were added to Murashige and Skoog semisolid culture medium at the proliferation phase. At 30 d of in vitro multiplication, the number of shoots, shoot length, number of leaves, dry matter, and antioxidant capacity were evaluated, as well as total chlorophyll, carotenoid, macro and micronutrient, phenolic, Phosphoenolpyruvate (PEP) and Rubisco contents. The results showed that adding MWCNTs at low doses (25 and 50 mg/L MWCNT) led to an increase in the in vitro multiplication rate and a higher number of leaves, while, at the highest dose (100 mg/L MWCNT), there was an inhibition of these variables. MWCNT treatments had an effect on Mg, Fe and C elements. The accumulation of photosynthetic pigments was associated with a higher PEP content in MWCNT treatments. Phenolic content increased at a concentration of 100 mg/L MWCNT, while antioxidant capacity increased at a concentration of 50 mg/L MWCNT. In conclusion, the usage of low MWCNT doses could be used to induce a hormetic response and to promote photosynthesis during in vitro propagation of stevia.

Enhancing Phalaenopsis orchid production: a comparative study of permanent and temporary immersion bioreactors

Enhancing Phalaenopsis orchid production: a comparative study of permanent and temporary immersion bioreactors

Temporary immersion bioreactor as an efficient method for in vitro propagation of Agave marmorata

Agave marmorata is an important wild species in Mexico for mezcal and pulque production. Propagation by suckers and seeds is not sufficient to restore wild populations. The aim of this study was to develop a protocol for large-scale micropropagation of A. marmorata using a temporary immersion bioreactor (TIB). The effect of liquid medium using a TIB and semi-solid medium as the control treatment was compared. Different immersion frequencies and culture medium volumes per explant were evaluated in the TIB. In all treatments, the percentage of hyperhydricity, number of shoots per explant, shoot length, and total chlorophyll and carotenoid content were evaluated. In addition, the survival rate during acclimatization was evaluated. The results obtained show that the TIB with an immersion of 2 min every 8 h and with a culture medium volume of 50 mL had the best developmental parameters, with 19.60 shoots per explant, a shoot length of 1.74 cm and a percentage of hyperhydricity of 3.33 %. The highest chlorophyll content was observed in temporary immersion every 8 h, with 100, 75 and 50 mL of culture medium per explant. Regarding carotenoid content, the highest content was found at the 8 and 12 h immersion frequencies, with the culture medium volumes of 100, 75 and 50 mL per explant. For acclimatization, no differences were found in the survival rate between the culture systems evaluated. In conclusion, this study demonstrates that the use of a TIB is an alternative for large-scale micropropagation of A. marmorata.

Bulbil initiation: a comprehensive review on resources, development, and utilisation, with emphasis on molecular mechanisms, advanced technologies, and future prospects.

Bulbil is an important asexual reproductive structure of bulbil plants. It mainly grows in leaf axils, leaf forks, tubers and the upper and near ground ends of flower stems of plants. They play a significant role in the reproduction of numerous herbaceous plant species by serving as agents of plant propagation, energy reserves, and survival mechanisms in adverse environmental conditions. Despite extensive research on bulbil-plants regarding their resources, development mechanisms, and utilisation, a comprehensive review of bulbil is lacking, hindering progress in exploiting bulbil resources. This paper provides a systematic overview of bulbil research, including bulbil-plant resources, identification of development stages and maturity of bulbils, cellular and molecular mechanisms of bulbil development, factors influencing bulbil development, gene research related to bulbil development, multi-bulbil phenomenon and its significance, medicinal value of bulbils, breeding value of bulbils, and the application of plant tissue culture technology in bulbil production. The application value of the Temporary Immersion Bioreactor System (TIBS) and Terahertz (THz) in bulbil breeding is also discussed, offering a comprehensive blueprint for further bulbil resource development. Additionally, additive, seven areas that require attention are proposed: (1) Utilization of modern network technologies, such as plant recognition apps or websites, to collect and identify bulbous plant resources efficiently and extensively; (2) Further research on cell and tissue structures that influence bulb cell development; (3) Investigation of the network regulatory relationship between genes, proteins, metabolites, and epigenetics in bulbil development; (4) Exploration of the potential utilization value of multiple sprouts, including medicinal, ecological, and horticultural applications; (5) Innovation and optimization of the plant tissue culture system for bulbils; (6) Comprehensive application research of TIBS for large-scale expansion of bulbil production; (7) To find out the common share genetics between bulbils and flowers.

Growth of in vitro–regenerated plants of Gerbera jamesonii following micropropagation in temporary immersion bioreactors

Growth of in vitro–regenerated plants of Gerbera jamesonii following micropropagation in temporary immersion bioreactors

Temporary immersion systems induce photomixotrophism during in vitro propagation of agave Tobalá.

This study aimed to determine the photomixotrophic and physiological responses using different temporary immersion systems (TIS) during in vitro multiplication of agave Tobalá. The culture systems were SETIS™ bioreactor, Temporary Immersion Bioreactor (TIB), Monobloc Advance Temporary Immersion System, and semisolid culture medium as the control. At six weeks of culture, different physiological variables were evaluated: chlorophyll content, stomatal index, percentage of closed stomata, Phosphoenolpyruvate (PEP) and Rubisco during the multiplication stage, and survival percentage in the acclimatization stage. TIS increased multiplication rate (41%), stomatal index (44%), percentage of closed stomata (11%) and chlorophyll content (45%) with respect to the semisolid culture medium system. The highest PEP content (> 35%) was observed in temporary immersion (TI), whereas, Rubisco content, showed no differences among the culture systems evaluated. Regarding survival percentage during acclimatization, the highest shoot survival was obtained in TI, and all regenerate shoots were rooted ex vitro. This study shows that in vitro photomixotrophism was induced in TIS during the multiplication stage. In conclusion, SETIS™ bioreactor and TIB systems are an alternative for mass multiplication of A. potatorum; however, all TIS evaluated guarantee a high survival rate during acclimatization.

Temporary Immersion System as an Innovative Approach for In Vitro Propagation of Sorbus domestica L.

The genus Sorbus has maintained an extremely relevant role over time from a landscape and environmental perspective in many countries in the Mediterranean and Central Europe. Based on the requirements coming from the environmental policies provided in the European strategy Next Generation EU, Sorbus has been considered a valuable species to be introduced in urban and peri-urban areas. The purpose of this study was to propagate four Sorbus accessions selected in the Sicilian territory, Southern Italy, using the liquid substrate in temporary immersion bioreactors Plantform™. The results obtained showed that the presence of 1 mg L−1 mT in the substrate in combination with IBA 0.05 mg L−1 produced a significant number of shoots (4.7) and a greater length (2.2 cm). Among the accessions, there were statistically significant differences; the accession SN2 and SN1 produced more shoots (respectively, 4.0 and 3.6), and a greater length of the shoots was observed in the selections SN4 and SN3 (respectively, 2.4 cm and 2.3 cm). The relative growth rate (RGR) was significantly influenced by the presence of the culture substrate of the combination of cytokines and auxin; SN4 selection showed the best RGR results of 8.3 mg−1 d−1. The use of the bioreactor Plantform™ in Sorbus domestica L. has favored a better development of plants obtained in vitro, demonstrating that this system is a valid alternative for the micropropagation of Sorbus.

Production of phenolic compounds from in vitro shoots of sugarcane (Saccharum spp.) in temporary immersion bioreactors

An alternative way to obtain molecules of high-value from plant origin for the agricultural and pharmaceutical industries is via the production of secondary metabolites from in vitro systems. Among these molecules are phenolic compounds that are important in plant defense system against biotic and abiotic factors. The objective of this work was to establish the culture conditions for the production and scaling up of phenolic compounds from sugarcane shoots produced in Temporary Immersion Bioreactors (TIBs). Two different sucrose concentrations and three inoculum densities of shoots per flask were tested for two sugarcane cultivars C86-56 and C1015-73. These cultivars were micropropagated in TIBs without ascorbic acid for phenolic compound production during 25 days, and were measured every five days. The best established conditions were evaluated on a pilot scale with ten TIBs for three sugarcane cultivars (C86-56, C1051-73 and C87-51). Although phenolic compounds were produced under all conditions evaluated at the experimental level, the most important production was achieved by using 15 shoots per vessel with 20 g.L-1 of sucrose in the absence of ascorbic acid after 25 days of culture. Cultivar C1051-73 had a higher multiplication coefficient independently of the treatments; this effect was not only significant for the phenolic compound production, but also for biomass increases. With respect to the pilot experiment, significant differences were detected for phenolic compound production among the three cultivars with C1051-73 showing the highest yield (23.05 mg.L-1). The results revealed that TIBs are a useful method to produce phenolic compounds with optimum yields.

Enhancing dendrobine production in Dendrobium nobile through mono-culturing of endophytic fungi, Trichoderma longibrachiatum (MD33) in a temporary immersion bioreactor system.

Dendrobine, a valuable alkaloid found in Dendrobium nobile, possesses significant pharmaceutical potential. In this study, we explored innovative approaches to enhance dendrobine production by utilizing endophytic fungi in a Temporary Immersion Bioreactor System (TIBS, Nanjing BioFunction Co. Ltd., China) and traditional test bottles. Dendrobine was unequivocally identified and characterised in D. nobile co-culture seedlings through UHPLC analysis and LC-MS qTOF analysis, supported by reference standards. The CGTB (control group) and EGTB (experimental group) 12-month-old D. nobile seedlings exhibited similar peak retention times at 7.6±0.1 minutes, with dendrobine identified as C16H25NO2 (molecular weight 264.195). The EGTB, co-cultured with Trichoderma longibrachiatum (MD33), displayed a 2.6-fold dendrobine increase (1804.23 ng/ml) compared to the CGTB (685.95 ng/ml). Furthermore, a bioanalytical approach was applied to investigate the mono-culture of T. longibrachiatum MD33 with or without D. nobile seedlings in test bottles. The newly developed UHPLC-MS method allowed for dendrobine identification at a retention time of 7.6±0.1 minutes for control and 7.6±0.1 minutes for co-culture. Additionally, we explored TIBS to enhance dendrobine production. Co-culturing D. nobile seedlings with Trichoderma longibrachiatum (MD33) in the TIBS system led to a substantial 9.7-fold dendrobine increase (4415.77 ng/ml) compared to the control (454.01 ng/ml) after just 7 days. The comparative analysis of dendrobine concentration between EGTB and EGTIBS highlighted the remarkable potential of TIBS for optimizing dendrobine production. Future research may focus on scaling up the TIBS approach for commercial dendrobine production and investigating the underlying mechanisms for enhanced dendrobine biosynthesis in D. nobile. The structural elucidation of dendrobine was achieved through 1H and 13C NMR spectroscopy, revealing a complex array of proton environments and distinct carbon environments, providing essential insights for the comprehensive characterization of the compound. These findings hold promise for pharmaceutical and industrial applications of dendrobine and underline the role of endophytic fungi in enhancing secondary metabolite production in medicinal plants.

Compuestos bioactivos en plántulas de Laelia speciosa (Orchidaceae) cultivados en biorreactor de inmersión temporal

Laelia speciosa is an epiphytic orchid endemic to Mexico, enjoying special protected status due to illegal sales and clandestine field extraction. Until now, in vitro cultivation of this plant has not been explored using temporary immersion bioreactor (TIB). L. speciosa seedlings were grown in a TIB, and the presence of bioactive compounds released into the liquid culture medium was determined. Bioactive compounds such as antioxidants, total polyphenols, and flavonoids were identified. The maximum concentration of total polyphenols was 2.29 ± 0.10 mg GAE/g, and the antioxidant capacity reached 33.7 %. Colorimetric tests revealed the presence of flavanones and flavonols. Gas chromatography-mass spectrometry identified components of the culture medium as 1,3-dihydroxyacetone dimer and 5-(hydroxymethyl)-2-furancarbaldehyde. Fourier transform infrared spectroscopy confirmed the presence of polyphenols in the culture medium. This work provides an alternative method for obtaining bioactive compounds while conserving L. speciosa seedlings.

Potato microclonal propagation method in a temporary immersion bioreactor

A promising and efficient approach to obtaining a genetically pure and identical plant to a given explant is through microclonal propagation. This method pre-determines the reproduction of plants by managing, controlling, and regulating the parameters of external and internal conditions that influence the accelerated reproduction of cultivated plants. The main methods for implementing the accelerated reproduction of cultivated plants are propagation in a closed external environment using specialized nutrient solutions and the use of bioreactors with temporary immersion of plants. The process of reproducing cultivated plants, specifically potatoes, using temporary immersion bioreactors allows for the use of automated monitoring and control systems based on robust control systems. This increases productivity and ensures the quality of the reproduced explants. Research has led to the development of experienced statisticians who have established the impact of individual parameters on the management of plant reproduction on their quality indicators. The reliability of the study is ensured by a large sample size (over 200 objects), parametric statistics, and the use of group theory to estimate plant reproduction parameters.

Micropropagation of Guarianthe skinneri (Bateman) Dressler & W. E. Higging in Temporary Immersion Bioreactors.

Guarianthe skinneri (Bateman) Dressler & W. E. Higgins is an orchid valued for its ornamental characteristics. However, it is an orchid classified as threatened with extinction due to the illegal extraction from its natural habitat. In addition, its propagation through seed germination is very low, as is the case with most members of the family Orchidaceae. Its asexual propagation through pseudobulb separation is slow and produces a few propagules. For this reason, in vitro propagation techniques are an alternative to increase the number of plants obtained and thus be able to recover this valuable plant genetic resource. Temporary immersion systems (TIS) offer the advantage of mass-propagating plants for different purposes. This chapter describes a large-scale micropropagation protocol for Guarianthe skinneri using temporary immersion bioreactors (TIB).

Temporary Immersion Bioreactors for Sugarcane Multiplication and Rooting.

Sugarcane is used to produce sugar, ethanol, and other by-products, so it is considered one of the most important crops worldwide. Using temporary immersion systems for sugarcane micropropagation represents an alternative to reduce the labor force, increase plant development, and improve plant quality. Temporary immersion systems are semi-automated bioreactors designed for the large-scale propagation of tissues, embryos, and organs. These are temporarily exposed in a liquid culture medium under a specific time and immersion frequency. Using this protocol and a temporary immersion bioreactor, it is possible to achieve multiplication and rooting. The use of temporary immersion bioreactors improves the multiplication rate and the rooting of sugarcane, reducing the culture time, labor force, and reagents needed while maintaining high survival rates during acclimatization.

Secondary Embryogenesis of Linaloe in Temporary Immersion Bioreactor-Type RITA®.

The linaloe [Bursera linanoe (La Llave) Rzed, Calderon and Medina] is an endemic species of Mexico, representative of the low deciduous forest of the states of Guerrero, Puebla, Morelos, and Oaxaca, and has been of great economic importance for the people, mainly for the artisanal use of its aromatic wood that is used to make boxes, trunks, and furniture that are manufactured in Olinala, Guerrero, Mexico; and industrial, thanks to the fine aroma of its essential oil (linalool), which is used in the manufacture of perfumes and pharmaceuticals. Overexploitation has endangered the species in recent years, and propagation by seed and/or cuttings has produced very poor results compared to those obtained with other recalcitrant Bursera species. The protection of endangered species makes urgent the need to propose new alternatives for its propagation. Somatic embryogenesis is a reliable and feasible technique, including induction, maintenance, multiplication, and maturation of embryos, often in semisolid culture media; however, the recent use of liquid media has allowed semi-automation in temporary immersion bioreactors, for example, the RITA® system, which favors both the multiplication rate and the final conversion to seedlings.

In Vitro Propagation of Threatened Agave Species Using a Two-Stage System.

Agaves are plants with multiple possibilities of use and are naturally tolerant to low water availability conditions and high temperatures. This makes them species of great interest in the context of the necessary substitution of crops due to climate change. Unfortunately, the overexploitation of wild specimens has endangered many species of the genus that have not been domesticated or cultivated intensively. In vitro mass culture and propagation techniques have emerged as a very efficient option to produce agave plants that can be used without damage to the natural populations. A protocol is presented here for the in vitro micropropagation of agaves in a two-stage process. In the first step, clusters of slightly differentiated shoots are generated from stem segments cultivated on a semisolid medium added with cytokinin. In a second step, these shoot clusters are cultured in temporary immersion bioreactors where they grow and complete their differentiation, and then the shoots are rooted and transferred to soil. This protocol has been successfully applied to several threatened species of the Agave genus.