Temporary Immersion Bioreactors Research Articles

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.

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.

Large-Scale Micropropagation of Vanilla (Vanilla planifolia Jacks.) in a Temporary Immersion Bioreactor (TIB).

The cultivation of vanilla (Vanilla planifolia) is of economic interest because vanillin is extracted from the fruits of this species. Vanillin is a natural flavoring highly valued in the food market. However, there is a short supply of propagules available for establishing commercial plantations and good-quality plants with phytosanitary certification. Plant tissue culture represents a viable option to supply large amounts of healthy plants to vanilla producers. In addition, the use of temporary immersion systems will allow commercial scale-up and the establishment of biofactories dedicated to in vitro vanilla propagation. This chapter describes a large-scale micropropagation protocol for vanilla using temporary immersion bioreactors (TIB).

Alstroemeria Micropropagation in a RITA® Temporary Immersion System.

The use of temporary immersion systems (TIS) for plant micropropagation is an efficient technique for plant production, and we have applied it for the production of alstroemerias. This method involves the cultivation of explants such as rhizomes and axillary buds in a nutrient medium to stimulate shoot growth. TIS offer advantages such as accelerated multiplication processes, uniform production, and cost reduction. This process has shown promise in meeting the growing demand for alstroemeria plants in the market. This chapter describes a specific protocol for temporary immersion bioreactor micropropagation of the "Albatroz" cultivar, with the potential for large-scale automation.

Study on rapid multiplication of two hybrid Arabica coffee varieties F1 Centroamericano (H1) and Mundo Maya (H16) through somatic embryogenesis and Bioreactor technology

In this paper, the research focuses on determining some components and culture conditions in order to optimise the 4 main steps of somatic embryogenesis and embryo regeneration by Bioreactor of two Arabica F1 coffee varieties H1 and H16, including: (1) formation of embryogenic callus, (2) multiplication of embryogenic cells, (3) embryonic differentiation, and (4) regeneration of somatic embryo using the temporary immersion Bioreactor RITA 1L. The results showed that on the medium supplemented with Kinetin 2 mg/ll and Phytagel 4 g/l, the callus formation rate reached 94.2 and 92.1% for 2 varieties H1 and H16, respectively. Besides, the addition of Glycine 20 mg/l to the culture medium increased the embryogenic callus rate by 58.1% in both varieties. Adding Trichostatin A 0.03 mg/l in the embryo differentiation medium in the first 7 days of culture showed that the number of torpedo embryos was scored from 1g of initial callus up to 1287 in H1 and 1635 in H16. Finally, using Bioreactor 1L-RITA, soaking for 1 min every 6 hours, the regeneration efficiency reached 75.02% in H1 and 82.03% in H16.

Micropropagation and Acclimatization of Monstera deliciosa Liebm. ‘Thai Constellation’

Monstera deliciosa Liebm. ‘Thai Constellation’ is a variegated variety of M. deliciosa belonging to the family Araceae, which has become a new favorite in the foliage plant market. However, limited studies exist on its propagation, and growers have difficulties in achieving large-scale production. This study aimed at developing an efficient protocol for the micropropagation of M. deliciosa using SETIS™ temporary immersion bioreactors. Furthermore, we aimed at evaluating the role of a novel biostimulant (IQ Forte) in the acclimatization of M. deliciosa. Significant differences were observed among the different treatments, showing higher multiplication rates under TIS conditions as compared to the semi-solid control. Adjusting immersion parameters also showed benefits in improving multiplication rates. The novel biostimulant (IQ Forte) did not provide significant gains in growth of M. deliciosa ‘Thai Constellation’ during acclimatization.

Genetic stability analysis of the temporary immersion bioreactors–derived sugarcane seedlings with simple sequence repeat (SSR) markers

Genetic stability analysis of the temporary immersion bioreactors–derived sugarcane seedlings with simple sequence repeat (SSR) markers

Improving biomass and dendrobine-type total alkaloids (DTTAs) production of Dendrobium nobile through combining Temporary Immersion Bioreactor System (TIBS) with endophyte MD33 elicitation

Improving biomass and dendrobine-type total alkaloids (DTTAs) production of Dendrobium nobile through combining Temporary Immersion Bioreactor System (TIBS) with endophyte MD33 elicitation

Role of Plant Growth Regulators on Multiplication of Stevia rebaudiana Inside Manufactured Temporary Immersion Bioreactor and Induction of Callus from Explant

The experiment was conducted at Plant Tissue Culture Laboratory - Department of Horticulture and landscape gardening, from October 2021 to January 2023. micropropagation experiments were conducted using five levels of Kinetin 0.0, 1.0, 2.0, 3.0, and 4.0 mg L. Callogenesis experiments were performed using four levels of Naphthalenacetic Acid 0.0, 1.0, 2.0, and 3.0 mg L. A temporary immersion bioreactor was used with a 10-minute immersion period every 4 hours, and two levels of Kinetin and two levels of benzyl adenine were added to the Murashige and Skoog medium as a comparative treatment. The results showed that the concentration of 1.0 mg L had a significant effect on the number of shoot and leaves, and the comparative treatment showed a significant effect on shoots height. The results of multiplication in the manufactured bioreactor showed a significant effect on shoots height, reaching 4.8 cm, and the concentration of 1.0 mg L benzyl adenine showed a significant effect on the number of shoots, with 3.2 shoot per plant, and the number of leaves for the same treatment was 17.1 leaves per shoot.

Role of Sucrose and Benzyladenine in Stevioside Content of Stevia Rebaudiana Leaves in Temporary Immersion Bioreactor

The experiment was carried out in the plant tissue culture laboratory of the Department of Horticulture and landscape gardening from October 2021 to January 2023. micropropagation experiments were conducted using concentrations of BA 0.0, 0.5, 1.0, 1.5 and 2.0 mg L, and an experiment with the addition of three levels of sugar (30, 60 and 90 g L) with two levels of BA (0.0 and 1.0 mg L). The results of the micropropagation showed that the concentration 1.0 mg L giving the highest average number of shoots of 3.5 shoots per plant, and giving the highest average number of leaves of 16.8 leaves of the shoot and the superiority of the comparison treatment in giving the highest average height of shoots amounting to 4.5 cm. The results showed that concentration 60 interacted with BA giving the highest average height of the shoots amounting to 4.3 plant shoots. and the concentration 60 interacted with the comparison treatment in giving the highest average shoot height of 4.4 cm and the concentration 90 interacted with BA in giving the highest average number of leaves amounting to 14.1 leaf shoot. The results indicated that the concentration 90 sucrose interacted with 1.0 mg L BA in giving the highest content of stevioside amounting 171.02

Differential response to calcium-labelled (44Ca) uptake and allocation in two peach rootstocks in relation to transpiration under in vitro conditions

Calcium-labelled (44Ca) uptake and transport under in vitro GreenTray® temporary immersion bioreactor conditions have been studied related to aeration conditions. For this aim, Rootpac®-20 (RP-20) and Garnem® (G × N) were selected as two main rootstocks used in peach production. Two transpiration conditions, aerated and unaerated, were established for each plant material. 44Ca location, plant development and foliar stomata surface were measured after the in vitro culture period. The results showed that aeration improved Ca transport within the shoot, but it did not enhance Ca uptake by the roots. Regarding plant material, G × N presented a better Ca uptake capacity and concentration. The findings suggest that Ca uptake in the roots is a precise process that is influenced by transpiration. However, it was observed that transpiration and thus the water flux is not the only force promoting Ca uptake by roots. Furthermore, the transport of Ca to the shoot was primarily determined by transpiration, indicating that water flux plays a crucial role in the aboveground movement of Ca. The study also revealed distinct behaviors in Ca uptake and allocation between the different peach rootstocks, emphasizing the importance of considering these factors in the selection process of rootstocks. These findings contribute to our understanding of the mechanisms involved in Ca uptake and transport in peach rootstocks under in vitro conditions. They provide valuable insights for rootstock selection processes and highlight the need for further research in this area.

Comparison of Temporary Immersion Bioreactor (SETISTM) and Classical Solid Culture in Micropropagation of ‘Grand Naine’ (Musa spp.) Banana Cultivar

Banana is an important nutritional source for millions of people because it contains protein, carbohydrates, vitamins and minerals. Therefore, the need for banana production is increasing day by day. Various methods are being developed to meet this need. Among these methods, biotechnological applications are becoming more prevalent over time. In micropropagation of in vitro plantlets, which forms the basis of banana production and is one of the most important stages, the methods used and banana varieties must be selected correctly. Therefore, it is very important to decide which protocol is suitable for which variety. In TIBs, in vitro micropropagation and rooting were compared with solid culture by adjusting the frequency of immersion of in vitro plantlets into the nutrient medium and the contact time of nutrients to the plantlets in the immersion medium. It was determined that the TIB application was more successful than the solid medium experiment in many aspects, especially the micropropagation rate (4.20) and rooting rate (84%).