Temporary Immersion System Research Articles

Effects of pretreatment in a temporary immersion bioreactor on organogenesis efficacy of <i>Lilium candidum</i> L. bulbscales

Our experiment was conducted in two stages, i.e., pretreatment (first stage) and regeneration (second stage). The first stage was carried out in a liquid Murashige and Skoog basal medium (5 µM BAP and 0.05 µM NAA) in a bioreactor with a RITA temporary immersion system under the light of a fluorescent lamp. Explants (bulbscales) were immersed in the medium once a day for 15 minutes (RITA 1×15) or three times a day for 1 (RITA 3×1), 5 (RITA 3×5), and 15 minutes (RITA 3×15) for one to six weeks. For regeneration, the explants were transferred onto a solid medium of the same composition for another six weeks. The bulbscales not exposed to the liquid medium were used as a control. Biomass weight, biomass growth index, number and percentage of dry matter of bulblets, and the content of soluble sugars in the bulblets and in the liquid medium were examined. The bulblets were formed in all combinations from the third week of the culture, and their number increased in the RITA 3×15 combination for both the first and the second stages of the experiment. After the longest, 6-week pretreatment, more bulblets were obtained than in the control. Their fresh weight after six weeks of regeneration was positively associated with extended pretreatment time. This was in contrast with the dry weight of the bulblets, which decreased in the second stage of the experiment along with the extension of its first stage. Prolonged contact of the explants with the liquid medium during the pretreatment resulted in a higher content of soluble sugars in the bulblets at both stages of the experiment. The content of soluble sugars in the liquid medium decreased over time in all tested combinations. After six weeks of bioreactor culture, the lowest level of soluble sugars was observed in the RITA 3×15 combination.

Use of temporary immersion systems for the micropropagation of Puya alpestris (Poepp.) Gay as a source to produce proteolytic enzymes

Use of temporary immersion systems for the micropropagation of Puya alpestris (Poepp.) Gay as a source to produce proteolytic enzymes

Biomass and Phenolic Acid Accumulation in Salvia austriaca Hairy Roots Grown in Temporary Immersion and Mist-Trickling Bioreactors

Transformed roots of Salvia austriaca were cultivated for 45 days in various systems, including Erlenmeyer flasks, a temporary immersion system (TIS) bioreactor, and a mist-trickling bioreactor, under controlled light conditions. The mist-trickling bioreactor yielded the highest biomass, with fresh and dry weights of 155.4 g/L and 10.2 g/L, respectively. Quantitative UHPLC analysis of hydromethanolic extracts revealed the biosynthesis of significant phenolic acids: caffeic acid, rosmarinic acid, and salvianolic acid A. Among these, rosmarinic acid was the most abundant, with its concentration varying based on the cultivation system. The highest total phenolic acid content, 165 mg/L, was obtained in the mist-trickling bioreactor, demonstrating its superiority in both biomass production and phenolic acid biosynthesis. This study highlights the potential of mist-trickling bioreactors for optimizing growth and metabolite production in S. austriaca transformed root cultures.

UV-B light (radiation) affects the metabolism of pigments and fatty acids in green algae Edaphochlorella mirabilis and Klebsormidium flaccidum in vitro

Algae offer a rich source of bioactive compounds suitable for food products and bioenergy. Environmental challenges such as nutrient scarcity, extreme pH and temperature, high light intensity, and UV radiation usually trigger algae to produce excess of lipids, antioxidants, and other bioprotective molecules as part of their adaptations for survival. Algal cultivation provides proteins, lipids, carbohydrates, vitamins, antioxidants, and trace elements. This study focused on understanding how UV-B irradiance, as an abiotic stressor, can influence the growth and metabolite production of two green algal species, Edaphochlorella mirabilis (Chlorophyta) and Klebsormidium flaccidum (Charophyta). Using a temporary immersion system bioreactor for in-vitro algal growth, the results showed no significant difference in biomass for both algal species after the exposure to UV-B rays. However, the assessment of malondialdehyde levels revealed a significantly higher tendency towards lipid peroxidation in treated E. mirabilis (+ 90 %) compared to control. Conversely, K. flaccidum did not display significant differences, thereby highlighting a more advanced adaptive capacity against UV-B radiation. Overall, both algal species treated with UV-B showed increased pigment accumulation. K. flaccidum exhibited an average pigment increase of over 53 %, while E. mirabilis showed a lower increase, over 30 % on average. The notable rise in antioxidant compounds (lutein, β-carotene, and chlorophyll a) in UV-B exposed K. flaccidum samples also suggested a more suitable adaptive strategy to mitigate oxidative stress in Charophyta. In K. flaccidum, the increase in polyunsaturated fatty acids can be associated with increased production of antioxidant compounds. Conversely, E. mirabilis appeared to protect itself by decreasing polyunsaturated fatty acids in favor of saturated ones. In both algal species, the increase in secondary metabolites under UV stress highlighted potential as a novel food source for human consumption, deserving further investigation.

The Impact of the Growth Regulators and Cultivation Conditions of Temporary Immersion Systems (TISs) on the Morphological Characteristics of Potato Explants and Microtubers

Potatoes (Solanum tuberosum L.) constitute one of the most economically important annual crops. In terms of tissue culture, potato microtubers (MTs) have a number of advantages over conventional plants. These advantages include their small size, which greatly facilitates storage, transport, and germplasm exchange compared to in vitro plants. One effective solution for the production and mass propagation of healthy MTs is the use of temporary immersion systems (TISs). In this study, in a SETISTM system containing kinetin/gibberellic acid (GA)/indole-3-butyric acid (IBA) hormones, we investigated the effects of different nutrient media on the morphological characteristics of potato explants and MTs. We determined the optimal cycling duration (3 h) with an immersion frequency of 2 min. The results revealed that the optimal nutrient medium for culturing single-node potato explants in a SETISTM bioreactor was the M7 medium containing kinetin (2 mg/L), GA (0.5 mg/L), and IBA (0.5 mg/L). The optimal nutrient medium for obtaining potato MTs was the M1 medium (hormone-free) with a high concentration of sucrose (9%) at 18 °C under dark growing conditions. Thus, a universal nutrient medium, employed in a bioreactor, was selected for the mass propagation of potato MTs for both domestic and foreign potato varieties.

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.

A Temporary Immersion System as a Tool for Lowering Planting Material Production Costs Using the Example of Pennisetum × advena ‘Rubrum’

A Temporary Immersion System as a Tool for Lowering Planting Material Production Costs Using the Example of Pennisetum × advena ‘Rubrum’

GreenTray®, a TIS Bioreactor for Plant Micropropagation and Abiotic or Biotic Stress Bioassays

The temporary immersion system bioreactor named GreenTray® presents advantages over the existing ones. Firstly, there is no need to use forceps to move the shoots or plantlets in or out of the recipient, nor is the use of a scalpel necessary to divide them. Secondly, the basis of the shoot can remain in the GreenTray® and can sprout again over several cycles of growth. These two characteristics reduce the costs in the process of micropropagation. In addition, shoot or plantlet development is enhanced by the aeration of the recipient, thus enriching the recipient’s atmospheric CO2. This article describes and provides images and values of interest in (1) the micropropagation process of some commercial Prunus or Pyrus rootstocks; (2) the plantlet growth under autotrophic conditions; and (3) the inoculation of plants with pathogens that reproduce in vitro the symptoms observed in vivo, using the GreenTray® bioreactor.

Saffron In Vitro Propagation: An Innovative Method by Temporary Immersion System (TIS), Integrated with Machine Learning Analysis

The propagation of Crocus sativus L. relies exclusively on corm multiplication. As underground storage organs, corms are susceptible to a wide range of pathogens, environmental stresses, and diseases, making traditional propagation methods often ineffective with the loss of valuable material. In vitro propagation offers an alternative for the saffron culture under controlled conditions. In particular, the innovative application of the Temporary Immersion System (TIS) represents a technological advancement for enhancing biomass production with a reduction in operational costs. The current study utilized the Plantform™ bioreactor to propagate in vitro saffron corms from the ‘Abruzzo’ region (Italy), integrating machine learning models to assess its performance. The evaluation of saffron explants after 30, 60, and 90 days of culture showed a marked improvement in growth and microcorm production compared to conventional in vitro culture on semisolid medium, supported by the machine learning analysis. Indeed, the Random Forest algorithm revealed a predictive accuracy with an R2 value of 0.81 for microcorm number, showcasing the capability of machine learning models to forecast propagation outcomes effectively. These results confirm that applying TIS in saffron culture could lead to economically viable, large biomass production within a controlled environment, irrespective of seasonality. This study represents the first endeavor to use TIS technology to enhance the in vitro propagation of saffron in conjunction with machine learning, suggesting an innovative approach for cultivating high-value crops like saffron.

Comparison of two subspecies of a halophytic multi-use plant Mertensia maritima in vitro and ex vitro: propagation, salinity tolerance and mineral nutrition

The aim of the present study was to compare morphological and physiological responses to increasing salinity of the two subspecies of Mertensia maritima during in vitro propagation in two different systems, and subsequent ex vitro cultivation. Plants were brought into culture using seeds and further propagated on Murashige and Skoog medium supplemented with thidiazuron and naphthaleneacetic acid either on agar-solidified or liquid medium with a temporary immersion system (Plantform bioreactors). Salinity tolerance were tested both in tissue culture and with ex vitro greenhouse-cultivated plants. Low concentration of NaCl increased explant biomass in agar-solidified medium, with no negative consequences even at high concentration. However, the effect of salinity on liquid medium was genotype-specific. Explants in Plantform bioreactors showed several-fold higher biomass in comparison to that on agar-solidified medium, but the multiplication potential was not significantly affected. Ex vitro plants were negatively influenced by increasing NaCl concentration in substrate. In contrast to in vitro plants, accumulation potential of Na⁺ in ex vitro plants was low. One of adaptations to salinity at the whole plant level was maintenance of adequate tissue mineral nutrient homeostasis, as mineral nutrient concentrations were not negatively affected by increasing salinity.

New paradigm shifts in micropropagation of fruit crops through bioreactors - a review

within relatively shorter periods, as well as significant reductions in hyperhydricity in plants as a result of efficient gas exchange, oxygen supplementation and automation, bioreactors, specifically temporary immersion systems (TIS), are being utilized for mass multiplication of forestry and horticultural crops. In tissue culture of banana, date palm, strawberrys, papaya, citrus, grape, pineapple, apple, pear, plum, chestnut, pistachio nut, apricot, sweet cherry, and almond, a variety of TIS bioreactors were used, including RITA, Plantform, SETIS and twin glass airlifts. TIS Bioreactors need to be improved in terms of space utilization. The space utilization was found to be highest with the Plantform system (80%) and lowest in the Twin Flask system (26%). Higher head space provides better plant growth and lesser fogging on the walls of the bioreactor. Most bioreactors have not been designed to facilitate better root production in vitro. Roots get coiled and cluttered, which needs improvement in design. The provision of illumination in each tank will facilitate better morphogenesis. This paper describes the micropropagation of fruit crops using different TIS bioreactors.

Comparative Assessment of Lignan Profiling and Biological Activities of Schisandra henryi Leaf and In Vitro PlantForm Bioreactor-Grown Culture Extracts.

This research's scope encompassed biotechnological, phytochemical, and biological studies of Schisandra henryi, including investigations into its in vitro microshoot culture grown in PlantForm bioreactors (temporary immersion systems, TISs), as well as extracts from leaves of the parent plant, focusing on anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. The phytochemical analysis included the isolation and quantification of 17 compounds from dibenzocyclooctadiene, aryltetralin lignans, and neolignans using centrifugal partition chromatography (CPC), HPLC-DAD, and UHPLC-MS/MS tandem mass spectrometry with triple quadrupole mass filter methods. Higher contents of compounds were found in microshoots extracts (max. 543.99 mg/100 g DW). The major compound was schisantherin B both in the extracts from microshoots and the leaves (390.16 and 361.24 mg/100 g DW, respectively). The results of the anti-inflammatory activity in terms of the inhibition of COX-1, COX-2, sPLA2, and LOX-15 enzymes indicated that PlantForm microshoot extracts showed strong activity against COX-1 and COX-2 (for 177 mg/mL the inhibition percentage was 76% and 66%, respectively). The antioxidant potential assessed using FRAP, CUPRAC, and DPPH assays showed that extracts from microshoot cultures had 5.6, 3.8, and 3.3 times higher power compared to extracts from the leaves of the parent plant, respectively. The total polyphenol content (TPC) was 4.1 times higher in extracts from the in vitro culture compared to the leaves. The antiproliferative activity against T-cell lymphoblast line Jurkat, breast adenocarcinoma cultures (MCF-7), colon adenocarcinoma (HT-29), and cervical adenocarcinoma (HeLa), showed that both extracts have considerable effects on the tested cell lines. The antimicrobial activity tested against strains of Gram-positive and Gram-negative bacteria and fungi showed the highest activity towards H. pylori (MIC and MBC 0.625 mg/mL).

Efficiency-cost, micropropagation, bioreactors, temporary immersion systems

Objective: to estimate the production price of strawberry “mother” plant of the CP-Jacona variety in both TIS and TS, and to compare these prices with the price of the imported “mother” plant. Design/Methodology/Approach: micropropagation methods have been used as an effective means for the mass production of pathogen-free plants, in small spaces and relatively short periods of time. In particular, in vitro Temporary Immersion Systems (TIS) applied to the production of strawberry “mother” plants have been shown to offer technological and quantitative benefits, as well as a higher proliferation rate, compared to the in vitro Traditional System (TS). Despite the benefits of TIS, these systems have not been evaluated in terms of the price at which the “mother” plant can be produced and whether it is a profitable option to supply strawberry producers. The traditional method of financial analysis was applied. Results: the price of the CP-Jacona variety plant obtained from the third production period through TIS was lower than the price of the imported Festival or Camarosa varieties. Findings/Conclusions: both the high production rate and the low rate of loss from handling in TIS were fundamental aspects to obtain lower prices than those of imported varieties.

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.

Browning prevention of oil palm somatic embryos in the temporary immersion system with antioxidant mixture and modification of basal media

Browning on in vitro culture is one of the problems in somatic embryo regeneration that leads to growth inhibition and death of explants. This study aimed to prevent and control browning of oil palm somatic embryos (SE) in the Temporary Immersion System (TIS). The research was conducted in two stages; the first stage was applying an antioxidant mixture at concentrations of 20, 50, and 100 mg L-1 on the browning palm oil SE in TIS. The second stage was carried out by modification of the basal media, namely DF (control), MS, SM, and SMT. The explants used in experiment 2 were browning and fresh SE. The results showed that using 50 ppm antioxidants mixture increased the formation of new SE by up to 15 embryos per flask. Applying SMT basal media on browning SE could decrease browning rate sharply after two cycle cultures by up to 60% browning rate reduction. In this media, it was also seen that the number of new SE increased by up to 250 embryos and produced 15 germinants, while in control, only 60 SE and five germinants were formed. Whereas using SMT basal medium on fresh embryos could prevent browning and increase the formation of new embryos and germinants. In conclusion, using 50 ppm antioxidants can stimulate the formation of new embryos. Using SMT basal media could prevent browning and overcome the occurrence of browning in embryos by increasing the formation of new embryos and germinants.

Enhancing the regeneration of TIS-derived somatic embryos of oil palm (Elaeis guineensis Jacq.) with CaCl2 and GA3

Micropropagation of oil palm through somatic embryogenesis (SE) technique provides clonal and uniform seedlings as high-quality planting materials for plantation. However, a problem associated with oil palm SE is the low regeneration rate of the somatic embryos. It was revealed that nutrient balance played a vital role in the success of SE regeneration. Therefore, a study was conducted to determine the balance between a macronutrient, calcium, and the germination-inducing hormone, Gibberellin (GA3) in enhancing the regeneration of the oil palm somatic embryos derived from the Temporary Immersion System (TIS). Treatments consist of various concentrations of CaCl2 (440, 880, or 1320 mg L-1) and enrichment of GA3 (2 or 4 mg L-1) applied into SE regeneration media, followed by the observation of regeneration rate and shoot tip necrosis after five weeks of culture. The study revealed that CaCl2 in the concentration of 880 mg L-1 was the best in stimulating oil palm TIS-derived SE regeneration. A higher concentration of GA3 (4 mg L-1) was needed in the first transition of SE from TIS into SE regeneration media in solid culture, while further SE-derived shoots development needed a lower concentration of GA3.

Plant Micropropagation and Temporary Immersion Systems.

Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.

Temporary Immersion Systems in Plant Micropropagation.

Temporary immersion systems (TIS) are technological tools that support plant micropropagation. Given their high efficiency in the in vitro propagation of shoots, a current goal is to update the protocols addressing micropropagation in semisolid culture systems to protocols involving TIS. To this end, different parameters have been evaluated, including TIS types and designs, immersion times, immersion frequencies, and volume of medium per explant, among other characteristics. This has resulted in the improved production of propagules of plants of economic interest and the production of physiologically upgraded plants with high percent survival during acclimatization. TIS are specialized culture flasks that provide countless advantages during the commercial micropropagation of plants.

Multiplication of Cupressus guadalupensis Using the RITA® Temporary Immersion System.

The Guadalupe cypress (Cupressus guadalupensis S. Watson) is an endangered species included in the list of the NOM-059-SEMARNAT-2010. The presence of wild goats in the habitat has been the greatest threat to the propagation and survival of this species. Therefore, there is a need to generate propagation protocols that facilitate the regeneration of the species. Plant tissue culture offers various possibilities that can facilitate the regeneration of species under some risk. Temporary immersion systems have proven to be an option with various advantages in plant tissue culture, such as increasing the number of seedlings generated and reducing production times, compared to semisolid media. The objective of this chapter is to describe a protocol to propagate Guadalupe cypress tissues in a RITA® temporary immersion system.