Phenolic profiling and bioactivity assessment of in vitro propagated Psidium cattleianum Sabine: A promising study.

Psidium cattleianum Sabine (strawberry guava) is an evergreen shrub that is grown as a fruiting hedge and has received significant consideration in the food and pharmaceutical disciplines. This study aims to set a promising protocol for in vitro propagation of P. cattleianum, along with profiling the phenolic content of the original plant (OP), induced callus (IC), and regenerated plantlets (RP) extracts, ultimately, evaluating their anti-inflammatory, antioxidant, and anticancer potential. Seeds were treated with commercial bleaching, HCl, and H2O2 to enhance the germination percentage and minimize the contamination percentage. Culturing sterilized leaf explants onto Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA), 2,4-dichloro phenoxy acetic acid, and kinetin showed the best callus induction, while supplementation of MS media with BA, adenine sulfate, naphthalene acetic acid, and gibberellic acid activated regeneration. Augmentation of MS media with indol-3-butyric acid recorded the maximum rooting percentage. Finally, the obtained rooted shoots were successfully acclimatized in sand and peat moss soil. HPLC-MS/MS profiles of OP, RP, and IC showed a variety of phenolic metabolites. IC extract decreased the viability of MCF-7, HepG2, and K-562 cancer cell lines. Also, OP exhibits strong antioxidant activity. P. cattleianum and its RP are profound sources of phenolic compounds promoted for promising applications in the food and pharmaceutical industries.

Elucidation of the composition, antioxidant, and antimicrobial properties of essential oil and extract from Citrus aurantifolia (Christm.) Swingle peel.

The most effective methodologies for generating Musa spp. explants involve the utilization of plant tissue culture micropropagation techniques. However, the pervasive challenge of microbial contamination significantly impedes the successful micropropagation of Musa spp. This study examined the antioxidant and antibacterial characteristics of the essential oil (LPO) and extract (LPE) obtained from the peel of Citrus aurantifolia. Additionally, we explored their mechanisms against common microbial contaminants in Musa spp. micropropagation. Using gas chromatography-mass spectrometry, we identified 28 components in LPO, with δ-limonene, ß-pinene, citral, trans-citral, ß-bisabolene, geranyl acetate, and α-pinene as the primary constituents. Meanwhile, liquid chromatography-mass spectrometry detected 17 components in LPE, highlighting nobiletin, tangeretin, scoparone, sinensetin, tetramethylscutellarein, 5-demethylnobiletin, and pyropheophorbide A as the predominant compounds. Evaluation using the DPPH and ABTS methods revealed the IC50 values for LPE at 0.66 ± 0.009 and 0.92 ± 0.012 mg/mL, respectively, indicating higher antioxidant activity compared to LPO, with IC50 values of 3.03 ± 0.019 and 4.27 ± 0.023 mg/mL using the same methods. Both LPO and LPE exhibited antimicrobial activities against all tested contaminant microorganisms through in vitro assays. Mechanistic investigations employing time-kill analysis, assessment of cell membrane integrity, and scanning electron microscopy (SEM) revealed changes in the morphological characteristics of the tested microbial contaminants, intensifying with increased concentration and exposure duration of LPO and LPE. These alterations led to substantial damage, including cell wall lysis, leakage of intracellular components, and subsequent cell death. Consequently, LPO and LPE emerge as promising alternatives for addressing microbial contamination in banana tissue cultures.

Cryopreservation of sessile oak (Quercus petraea (Matt.) Liebl.) plumules using aluminium cryo-plates: influence of cryoprotection and drying.

BACKGROUND QUERCUS: seeds that are recalcitrant to desiccation and freezing temperatures cannot be stored in gene banks under conventional conditions. However, the germplasm of some recalcitrant seeded species can be stored in liquid nitrogen (-196 °C). Unfortunately, for many species, among them for almost the whole genus Quercus, an effective cryostorage method is still unknown. In this study, we propose a successful cryostorage protocol for Quercus petraea (Matt.) Liebl. germplasm using plumules (a shoot apical meristem of an embryo) frozen on aluminium cryo-plates. RESULTS: The plumules isolated from the acorns of ten provenances were prestored in 0.5 M sucrose solution (for 18 h). To form alginate beads (one plumule per bead), the plumules were placed in the wells of a cryo-plate and embedded in calcium alginate gel. For cryoprotection, the encapsulated plumules were immersed in cryoprotectant solution containing 2.0 M glycerol and different concentrations of sucrose (0.8-1.2 M) for 40 min at 25 °C and desiccated under a laminar flow cabinet for 1.0-4.0 h. Cryo-plates with plumules were directly immersed in liquid nitrogen and then cryostored for 30 min. For rewarming, cryo-plates with plumules were immersed in 1.0 M sucrose solution and rehydrated for 15 min at 25 °C. Survival rates varied from 25.8 to 83.4 were achieved after cryoprotection in 1.0 M sucrose solution and the drying of plumules for 2 h. The in vitro regrowth rate of cryopreserved plumules varied among provenances and was 26-77%. CONCLUSIONS: This study presents, for the first time, a successful, simple and effective protocol for the cryopreservation of Q. petraea germplasm that could be used in gene banks. The experiment was successfully repeated on seeds from various provenances, each yielding similar, good results. However, seed quality and storage time after harvesting are important factors in plumule regrowth after cryopreservation.

Comprehensive study on in vitro propagation of some imported peach rootstocks: in vitro explant surface sterilization and bud proliferation.

The present study was conducted in the Laboratory of Tissue Culture, Horticulture Department, Faculty of Agriculture, Damietta University, Egypt. The objective of this study was to establish a micropropagation protocol suitable for three imported peach rootstocks: Okinawa (P. persica), Nemared (P. persica × P. davidiana) × P. persica), and Garnem (P. dulcis × P. persica) in vitro. The results showed that soaking the explants in sodium hypochlorite (NaOCl) at 20% for 15 min produced the highest responsiveness (82.81%), survival (96.61%), with the lowest mortality (3.14%) and contamination (0.24%). Explants of the Garnem genotype had the best response (89.12%), survival (90.62%), lowest mortality (0.00%), and highest contamination (9.37%) when compared to the other genotypes. In comparison with axillary buds, the shoot tip displayed the highest responsiveness, survival, and death (100, 87.40, and 12.59%, respectively), as well as the least significant contamination (0.00%). Additionally, the percentages of responsive, survived, dead, and contaminated explants at the various collection dates varied significantly. The 6-benzylaminopurine (BAP) concentrations used (3 to 5.0 mg/L) demonstrated similar behavior in terms of in vitro proliferation, with rates of 3.77 to 6.11, 4.33 to 8.88, and 3.33 to 7.44 shoot numbers per explant for the Okinawa, Nemared, and Garnem peach rootstocks, respectively, indicating that the number of shoot proliferations is genotype-dependent. Additionally, using 5.0 mg/L BAP in combination with 0.2 mg/L IBA significantly increased average shoot proliferation (96.29%), number of shoots per explant (7.48), and average leaf number/explant (16.33) compared to the other treatments. Based on these results, adventitious bud development was enhanced during in vitro multiplication of the Okinawa, Nemared, and Garnem peach rootstocks by the synergistic interaction of indole-butyric acid (IBA) and 6-benzylaminopurine (BAP).

Unveiling the silver lining: examining the effects of biogenic silver nanoparticles on the growth dynamics of in vitro olive shoots.

The current study aimed to evaluate the effects of biogenic silver nanoparticles (AgNPs) on growth behavior and leaf anatomy of in vitro growing shoots of 'Picual' and 'Dolce' olive cultivars. Biosynthesis of AgNPs was carried out using the cell-free filtrate of Fusarium oxysporum. The dimension and shape of the synthesized AgNPs have been analyzed using spectroscopy and topography analysis tools, confirming that the biosynthesis of AgNPs is a crystalline nanostructure with an average particle size of 37 nm. The shoots of the selected olive cultivars were cultured on Rugini olive medium-supplemented AgNPs at 0, 10, 20, and 30mg L- 1. The effect of genotypes on shoot multiplication was significant, 'Picual' recorded higher values of shoot growth parameters compared with 'Dolce' cultivar. Adding AgNPs to the culture medium significantly affected the growth of in vitro olive shoots. AgNPs at 20 and 30mg L- 1 produced higher values of the number of shoots, shoot length, and leaf number of Picual cv. compared with the control treatments, but the higher AgNPs concentration harmed the growth parameters of Dolce cv. and recorded lower growth values compared with the lower concentration (10mg L- 1). AgNPs had a significant effect on leaf morphology and their anatomical structure. The current results showed that the stimulatory effect of AgNPs on shoot growth of in vitro olive shoots is highly dependent on plant genotype and nanoparticle concentration.

Long-term organogenic callus cultivation of Ranunculus illyricus L.: a blueprint for sustainable ex situ conservation of the species in urban greenery.

The growing trend of introducing wild plant species into urban environments necessitates the identification of novel species adapted to prevailing conditions. A promising reservoir of such species may be xerothermic communities where Ranunculus illyricus occurs. This study aimed to establish a micropropagation protocol for R. illyricus using indirect organogenesis. The protocol includes initiation of culture from various explants, callus proliferation, shoot regeneration, multiplication, and concurrent rooting. Callus appeared on most types of vegetative explants tested, but stolons were considered the best due to their good availability, high disinfection (85%), and robust callus production (maximum increase - 363.1%). The growth rate of the callus fresh matter (CFM) obtained from stolons was calculated. Greater CFM was obtained on the medium with the supplemented picloram 8.0 mg L- 1 with kinetin 5.0 mg L- 1 and in second part of experiment on medium with the addition of 2,4-D (2,4-dichlorophenoxyacetic acid) 2.0 mg L- 1 alone or picloram 6.0 mg L- 1 with kinetin 8.0 mg L- 1. Shoot organogenesis was observed on macronutrients B5 (Gamborg medium), micronutrients MS (Murashige and Skoog) medium with the addition of 2.0 mg L- 1 IBA (indole-3-butyric acid) and 4.0 mg L- 1 BAP (6-benzylaminopurine). To document the process of callus differentiation, microscopic preparations were prepared. Subsequently, the regenerated plants underwent acclimatisation and their growth in an ex situ collection was monitored over three growing seasons. In particular, in vitro-origin plants exhibited developmental patterns similar to those of their seed-origin counterparts. The incorporation of R. illyricus into urban landscapes not only increases aesthetic appeal, but also ensures the preservation of valuable genetic resources for this rare species, potentially contributing to effective ex situ conservation in the future. This marks the first scientific report on in vitro cultures of R. illyricus.

In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments.

OBJECTIVE: The high industrial demand for Stevia cultivation (Stevia rebaudiana) has increased due to its high stevioside content derived from the leaves. However, the low germination rate makes the cultivation of the plant become the main obstacle. Therefore, an efficient cultivation technique is required. This present work aims to analyze the effect of five combinations of Kinetin (Kin) and benzyladenine (BA) on stevia micropropagation using nodal segment explants. RESULTS: The micropropagation of stevia was performed using Murashige and Skoog (MS) medium supplemented with BA and Kin. We analyzed different organogenesis and callogenesis responses. In addition, the number of shoots and root formed during in vitro culture were also observed. Our results demonstrated that all treatments with Kin, both alone and in combination with BA, resulted in the development of callus on all nodal segment explants. Explants treated in MS with 1 mg L-1 BA exhibited the best average of shoot number (36.27). In contrast, the treatment without PGR resulted in the best root formation (2.6). The overall results suggested that different combination of BA and Kin resulted in distinct organogenesis responses, where 1 mg L-1 of BA was potentially used for boosting the number of shoots in micropropagation of stevia accession Mini.

Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch.

The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L-1), TDZ (0.02 mg·L-1), and NAA (0.01 mg·L-1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.

Study of the physicochemical characteristics, antimicrobial activity, and in vitro multiplication of wild blackberry species from the Peruvian highlands.

The Peruvian Andes are the natural habitat of several wild blackberry species that are little known and exploited due to the lack of technological and scientific development to support their agricultural potential. In this context, a study was conducted to understand the physicochemical composition, bioactive compounds, antimicrobial activity, and in vitro multiplication of four wild blackberry (Rubus sp.) species from the northern Peruvian highlands. The results indicate that fruits of R. floribundus presented the highest content of total soluble solids (9.58 ± 1.83°Brix) and titratable acidity (1.88 ± 0.07% citric acid). The fruits of R. weberbaueri recorded the highest total phenolic content (415.06 ± 8.69 mg GAE/100 g Ff). The antioxidant capacity determined by the DPPH assay varied significantly among species, with the highest value found in fruits of R. andicola (50.27 ± 0.11 mg TE/100 g Ff). The fruit extracts of R. weberbaueri and R. andicola showed better antimicrobial activity, with Staphylococcus aureus being the most sensitive bacterium. In the in vitro multiplication phase, the results show that BAP (6-Benzylaminopurine) has a significant effect at a dose of 1.5 mg l-1 on shoot number, leaf number, and shoot length. The results may help in the management of genetic resources.

Deciphering the interaction of bacteria inoculants with the recipient endophytic community in grapevine micropropagated plants.

Engineering the plant microbiome with beneficial endophytic bacteria can improve the growth, health, and productivity of the holobiont. Here, we administered two beneficial bacterial strains, Kosakonia VR04 sp. and Rhizobium GR12 sp., to micropropagated grapevine cuttings obtained via somatic embryogenesis. While both strains colonized the plant endosphere, only Rhizobium GR12 sp. increased root biomass under nutritional-deficit conditions, as supported by the plant growth promotion traits detected in its genome. Phylogenetic and co-occurrence analyses revealed that the plant native bacterial community, originally dominated by Streptococcaceae and Micrococcaceae, dramatically changed depending on the inoculation treatments, as invading strains differently affected the relative abundance and the interactions of pre-existing taxa. After 30 days of plantlets' growth, Pantoea became a predominant taxon, and considering untreated plantlets as references, Rhizobium sp. GR12 showed a minor impact on the endophytic bacterial community. On the other hand, Kosakonia sp. VR04 caused a major change in community composition, suggesting an opportunistic colonization pattern. Overall, the results corroborate the importance of preserving the native endophytic community structure and functions during plant microbiome engineering.IMPORTANCEA better comprehension of bacterial colonization processes and outcomes could benefit the use of plant probiotics in the field. In this study, we applied two different beneficial bacteria to grapevine micropropagated plantlets and described how the inoculation of these strains impacts endophytic microbiota assembly. We showed that under nutritional deficit conditions, the response of the receiving endophytic bacterial communities to the invasion of the beneficial strains related to the manifestation of plant growth promotion effects by the inoculated invading strains. Rhizobium sp. GR12 was able to preserve the native microbiome structure despite its effective colonization, highlighting the importance of the plant-endophyte associations for the holobiont performance. Moreover, our approach showed that the use of micropropagated plantlets could be a valuable strategy to study the interplay among the plant, its native microbiota, and the invader on a wider portfolio of species besides model plants, facilitating the application of new knowledge in agriculture.

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.

BioMINT: A Temporary Immersion System for Agave Micropropagation.

Agaves are cultivated in Mexico as a source of industrial products such as fibers, nutritional supplements, and alcoholic beverages. Due to the demand for plant material, its long-life cycle, and the need to avoid predation on its natural populations, in vitro micropropagation represents a good option for agaves. Plant tissue culture has been successfully used to micropropagate selected elite individuals from plants of various Agave species of economic interest. However, it is necessary to implement systems that lower production costs without losing the quality of the plantlets obtained. This chapter describes the BioMINT™ bioreactor as an alternative for the micropropagation of agaves in the different stages of the micropropagation process.

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.

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 Chayote (Sechium edule L.) var. virens levis in RITA®.

Chayote (Sechium edule) belongs to the Cucurbitaceae family, an important family at the nutritional and medicinal levels, that has been covering international markets. Having vigorous and healthy plants is important for producers, who are very interested in cultivating chayote plants obtained from in vitro tissue culture in their orchards. Bioreactors have become an alternative with high potential for plant propagation, showing significant advantages over micropropagation in semisolid medium, by generating more plant material, larger, and more vigorous. In this chapter, a micropropagation protocol of S. edule in RITA® bioreactors is reported.

Increased Multiplication Rates of Vriesea hieroglyphica (Carriere) E. Morren Through a Temporary Immersion System.

The main difficulty for the cultivation and conservation of bromeliad species is the reduced number of propagules and slow growth of many of the species, resulting in a low propagation efficiency. Bromeliad plants are hardy and relatively easy to cultivate, with a high ornamental and ecological importance. Aiming at efficient micropropagation rates of V. hieroglyphica, a highly valued bromeliad, with very low propagation efficiency, a temporary immersion system was used and compared to semisolid and liquid static medium. Cultures obtained from in vitro germinated seeds were used as explants, maintaining their genetic diversity. Micropropagation with this simple temporary immersion system, composed of two autoclavable flasks, each with one opening for the attachment of 22 µm syringe filters, connected by a rubber stopper and an inner glass tube. In the bottom flask, an air valve is attached to the filter, which is subsequently connected to an aquarium pump and a timer and plugged to an outlet. This simple temporary immersion system showed improved micropropagation efficiency and is a method that can also be evaluated for other species.

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.

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).

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.

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.