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.

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.

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).

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).

Orchid Micropropagation Using Temporary Immersion Systems: A Review.

Temporary immersion systems (TIS) have been used for orchid micropropagation. The main advantage of TIS use for micropropagation is that the explant is periodically immersed in nutrient media, and then, the nutrient solution is drained, which allows the explant tissue to stay in air. The current review resumes the application of TIS in orchid propagation. Fifty-three papers are discussed considering: explant, culture media, TIS bioreactor type, frequency and immersion time, and the TIS effects in acclimatization phase.

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.

Overview of Somatic Embryogenesis.

Somatic embryogenesis is a natural phenomenon through which somatic embryos are produced from somatic cells although. It is considered the most efficient morphogenic pathways for plant multiplication. One of the key features of somatic embryogenesis is the use of cellular totipotency, where dedifferentiation is induced to foster cell proliferation, followed by the induction of differentiation using plant growth regulators to produce new plants. There is a cell group with the potential to undergo the somatic embryogenesis pathway through adequate stimulation (plant growth regulators, incubation conditions, and supplementation of the culture medium). There are two somatic embryogenesis pathways in plants: direct and indirect embryogenesis. Direct somatic embryogenesis consists of the formation of embryos directly from isolated cells, without the formation of "callous" tissue. Indirect somatic embryogenesis is characterized by the formation of a callus as a stage that precedes the formation of somatic embryos. It should be stressed that not all plant cells have this morphogenic capacity; consequently, determining the type of factors that drive this type of response has been challenging. This book provides the reader with updated available information on the techniques, relevant protocols, and tools to perform somatic embryogenesis in different plant species for economic purposes.

Advances in Somatic Embryogenesis in Vanilla (Vanilla planifolia Jacks.).

Somatic embryogenesis is an in vitro plant morphogenetic process due to cell totipotentiality to induce shoot regeneration. To induce this proliferation pathway, we used auxins such as 2,4-dichlorophenoxyacetic acid in combination with cytokinins. There are numerous somatic embryogenesis protocols for a great diversity of plants, including orchids, but none has been yet reported in Vanilla planifolia. Vanilla (V. planifolia) is propagated mainly asexually through cuttings. Under in vitro conditions, it is propagated asexually through direct and indirect organogenesis involving the use of various plant growth regulators in different concentrations. The cell response depends on explant type, culture medium used, and incubation conditions. Direct organogenesis involves de novo formation from differentiated cells; the indirect pathway develops from cell dedifferentiation that produces an explant called "callus." In most cases, this type of cell regeneration uses Benzyladenine. The explants most used in this pathway are shoots, roots, and protocorms, although some studies report the use of other types of explants, including leaves and seeds. Somatic embryogenesis in vanilla has been poorly studied partly because of the recalcitrance of this species. This work mentioned the advances in the in vitro morphogenesis of V. planifolia, mentioning the advantages and disadvantages of each morphogenetic pathway and its characteristics.

Perspectives of Somatic Embryogenesis: Concluding Remarks.

One of the main objectives to achieve in plant tissue culture is the multiplication of the available plant material, taking full advantage of the regenerative capacities of plant cells. Somatic embryogenesis leverages cell totipotency to produce new explants from a cell, thus obtaining many propagules for scientific research, industrial, or exploitation purposes. Somatic embryogenesis (ES) characterizes by being one of the most efficient techniques in plant micropropagation. However, developing an efficient plant ES protocol requires several key factors to consider, as demonstrated throughout the chapters of this book. These chapters highlight the major drivers of the success of ES in different plant species: plant growth regulators, concentration of auxins and cytokines, water deficit, photoperiod, and type of culture medium; techniques such as the use of bioreactors and Thin Cell Layer (TCL); and the influence of stress on the formation of somatic embryos. Research has been conducted to address each phase of somatic embryogenesis, either individually or for all phases. The chapters of this book cover in detail the techniques used and provide guidance that will allow readers to successfully develop all the somatic embryogenesis phases in different cultures, from cell dedifferentiation to differentiation.

Micropropagation of Guarianthe skinneri (Bateman) Dressler et W. E. Higging in Temporary Immersion Systems.

This study's aim was to establish a protocol for the micropropagation of G. skinneri using temporary immersion system (TIS). Different concentrations of 6-Benzylaminopurine (0, 1, 2, and 3 mg L-1), three different systems of cultivate semi-solid (SS) and liquid media under partial (PI) and temporary immersion systems (TIS), different compositions of the inorganic salts, and the number of subcultures were evaluated. The results showed a maximum of 16.56 shoots per explant obtained through TIS, adjusting all the parameters evaluated in our study. One higher number of shoots per explant was observed in the micropropagation of G. skinneri TIS compared to SS and PI. While the use of 3 mg L-1 of BAP + MS (Murashige and Skoog) media was better than 3 mg L-1 of BAP VW (Vacint and Went) for the generation of a greater number of shoots per explant, 6.33 and 2.72, respectively. The immersion frequency of 2 min every 4 h allowed the production to be scaled to 8.54 shoots per explant. While it was necessary to perform three subcultures every 30 days, to obtain 16.56 shoots per explant, a rooting phase was not required due to the generation of adventitious roots during the different subcultures. However, a phase of elongation of the regenerated plants with ½ MS + GA3 (gibberellic acid) was needed to guarantee 100% survival in the process of acclimatization. In conclusion, this plant production system can be applied for the commercial micropropagation of this species for ornamental purposes, as well as for its reintroduction in protected natural areas.

Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andreanum).

Anthurium has been micropropagated mainly through conventional techniques in semisolid culture medium. However, this culture system involves constraints due to the low number of shoots produced and the high costs of the gelling agent and labor. Temporary immersion systems (TIS) are an alternative for increasing biological performance, reducing costs, and facilitating a semi-automated micropropagation process. The objective of this study was to compare the efficiency of different types of TIS during the in vitro propagation of anthurium. We used 2-cm-long nodal segments from in vitro plants. Explants were cultured in different TIS: temporary immersion bioreactors (TIB®), Ebb-and-Flow bioreactor, and recipient for automated temporary immersion (RITA®), with a 2-min immersion frequency at 12-h intervals. We used Murashige and Skoog (MS) medium supplemented with 3% (w/v) of sucrose and 8.88 µM benzylaminopurine. After 60 days of culture, we evaluated various physiological variables and the percent survival in the different TIS. The largest numbers of shoots per explant were observed in TIB® and Ebb-and-Flow, with 50.83 and 43.16 shoots per explant, respectively; the lowest number of shoots per explant was observed in RITA®, with 30.66. TIB® yielded the highest content of photosynthetic pigments (chlorophyll a, b, and total chlorophyll), stomatal index, and percentage of closed stomata relative to both Ebb-and-Flow and RITA®. The TIB® and RITA® systems showed a 99% shoot survival, while Ebb-and-Flow yielded 86% survival. In conclusion, TIS design and type affect a number of physiological processes and in vitro development, with TIB® as a feasible option for the commercial micropropagation of anthurium.

Preliminary molecular detection of the somatic embryogenesis receptor-like kinase (VpSERK) and knotted-like homeobox (VpKNOX1) genes during in vitro morphogenesis of Vanilla planifolia Jacks.

This work aimed to evaluate the embryogenic competence of different tissues from different stages (friable callus, bud-regenerating callus, and whole buds) of Vanilla planifolia, through the molecular detection of the somatic embryogenesis receptor-like kinase (VpSERK) and knotted-like homeobox (VpKNOX1) genes. RNA was extracted with Trizol®, cDNA was obtained, and the studied transcripts were amplified. Using non-specific primers, VpSERK and VpSTM gene expression was detected in the three stages evaluated. This study might contribute to providing an explanation for the recalcitrance of this Vanilla species to somatic embryogenesis.

Vanilla (Vanilla planifolia Jacks.) cell suspension cultures: establishment, characterization, and applications.

The establishment and characterization of cell suspension cultures are an in vitro culture technique very useful for various plant biotechnological applications (production of secondary metabolites, mass micropropagation, protoplast isolation and fusion, gene transfer and the investigation of cell pathways). The objective of this study was to establish and characterization of cell suspension cultures of V. planifolia by inducing friable calluses. For that, friable calluses were obtained from immature seeds cultivated in MS medium supplemented with 0.45 µM thidiazuron (TDZ). The effect of benzyladenine (BA) in different concentrations was evaluated. Cultures were incubated under photoperiod at continuous stirring at 120 rpm on an orbital shaker. The optimal condition found for biomass growth in suspension cultures was 0.5 g of inoculum density (fresh weight) in MS liquid, supplemented with 8.88 µM BA. The growth kinetics of the cell suspension culture revealed a maximum cell growth (exponential growth phase) at day 16 and an 80% cell viability. The establishment and characterization of cell suspension cultures of V. planifolia constitute the bases of future studies and above all a better biotechnological use of this crop.