Immersion Systems Research Articles

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

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.

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.

Use of Temporary Immersion Systems in the Establishment of Biofactories.

Companies dedicated to the large-scale propagation of plant species are known as biofactories or agricultural biotechnology companies. Globally, there are a large number of biofactories (large-scale production) or plant tissue culture laboratories (small-scale production) in charge of supplying commercial propagules of plants of economic interest. Each biofactory implements technological developments such as temporary immersion (TIS) systems that allow them to reduce costs. This chapter analyzes some of the biofactories established globally, the main plant species propagated, and whether or not they implement the use of TIS.

Conclusions and Perspectives on Plant Micropropagation in Temporary Immersion Systems.

In vitro propagation protocols that include temporary immersion systems are available for the most economically important plant species. However, these have not been established yet for multiple species. Having protocols validated by the scientific community guarantees the success of the mass production of commercial propagules. Besides, adequate TIS parameters should be established for each plant species to improve the efficiency of micropropagation processes. This book compiles basic and applied aspects of temporal immersion systems used for in vitro plant micropropagation, along with several detailed protocols already established, which may be used as a guide by those interested in this technique, including laboratory technicians, scientists, and other professionals.

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.

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.

In Vitro Multiplication of Agave (A. marmorata and A. potatorum) by Temporary Immersion in SETIS™ Bioreactor.

In Mexico, wild agaves are important for the production of alcoholic beverages known as mezcal and pulque. However, the propagation of agave seeds and pups is not enough to satisfy the national demand. Temporary immersion systems represent an agave micropropagation alternative that reduces the labor force, increases development, and improves the quality of seedlings. The use of the SETIS™ bioreactor in A. marmorata and A. potatorum improves the multiplication rate and allows rooting. Additionally, this bioreactor reduces the culture time, labor force, and reagents needed while maintaining high survival rates during the acclimatization phase.

In Vitro Propagation of Plants via Organogenesis in Bambusa vulgaris Schrad. ex Wendl Using Temporary Immersion Systems.

The low multiplication and ex vitro survival rates during acclimatization in the culture house limit the in vitro mass propagation of B. vulgaris. Several scientific studies have described the development of different protocols for bamboo; however, not all of them address the effects of these systems on plant morphology, physiology, and biochemistry in vitro. In this chapter, a complete and optimized protocol is described for plants propagated via organogenesis in temporary immersion systems. In addition, the morphophysiological and biochemical characterization of the plants as well as the survival rates of the obtained plants under ex vitro conditions are analyzed. The obtained results will be the basis for the development of a technology for in vitro propagation as an alternative for the production of plants of the species.

Types of Temporary Immersion Systems Used in Commercial Plant Micropropagation.

Technological innovation in the design and manufacture of temporary immersion systems (TIS) has increased in the past decade. Innovations have involved the size, fitting, and replacement of components, as well as manufacturing materials. Air replacement by compressor has also been substituted by air replacement by preset tilting/rotation of culture bottles. This design modification aims to increase the biological yield (number of shoots) produced in these bottles and reduce manufacturing costs. However, the operative principle has remained unchanged through time: promote an environment where explant immersions in the culture medium are programmable. The changes in the TIS design involve advantages and disadvantages, generating the efficiency of one type over another. However, validation to identify the most effective type of TIS should be carried out for each plant species. This chapter lists the different types of temporary immersion available on the market, emphasizing the advantages and disadvantages of each when used for plant micropropagation.

Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems.

Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors.

Micropropagation of Stevia (Stevia rebaudiana Bert.) in RITA®.

Stevia rebaudiana Bert. is a plant that contains noncaloric sweeteners highly appreciated in the food industry. However, there is a high demand for propagules to establish commercial plantations, and the conventional reproduction types for this species are inefficient. Micropropagation is a technique that allows obtaining a large number of plants and can be used to meet the demand in the field. However, it requires in vitro propagation techniques such as temporary immersion systems (SIT) to increase yield and reduce production costs. This chapter describes an effective protocol for the large-scale micropropagation of S. rebaudiana using a TIS.

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.

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).

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.

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.

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).