The safe management of sewage waste is a current concern due to population growth and waste production. Biosolids, derived from sewage sludge treatment, are globally used as organic fertilizers, aligning with Sustainable Development Goal 6 for resource recycling. However, biosafety concerns arise due to the presence of metals and microplastics in biosolids, potentially impacting soil and water. This study investigated biosolids\' use for in vitro cultivation of Bowdichia virgilioides Kunth. Results indicate that while biosolids can replace traditional nutritional media, balancing their concentration is crucial for optimizing plant growth. The WPM (Wood Plat Medium) remains essential for in vitro cultivation, but substituting it with biosolids at concentrations of up to 2 g L- 1 is feasible, providing similar plant development compared to the WPM medium. However, when combined, there is a complex and challenging interaction between biosolids and the culture medium.

Lavandula stoechas subsp. luisieri and Pterospartum tridentatum are two valuable aromatic and medicinal plants. Their biometric and morphological parameters, such as the number of new shoots, length of the longest shoot, multiplication rate, and fresh weight, were evaluated using the multiplication MS medium protocol. The rooting protocols involved immersing the explants in IBA (1 g L-1) and a commercial IBA (3.3 g L-1) preparation (Clonex®). Slow-growth conservation assays were carried out using two different sucrose concentrations (15 g L-1 and 30 g L-1), and a control, with the cultures kept at 4 °C for 12 months. The multiplication rate for L. stoechas subsp. luisieri was 6.8, and that of P. tridentatum was 13.3, achieved using the MS medium supplemented with 0.2 mg L-1 BAP, 1 mg L-1 BAP, and 0.5 mg L-1 IBA. The application of Clonex® showed the best ex vitro rooting results in L. stoechas subsp. luisieri (77%) and P. tridentatum (90%). In the slow-growth conservation assays, at 4 °C, in darkness for 12 months, an excellent survival rate was achieved in L. stoechas subsp. luisieri (>80%) and P. tridentatum (>90%), even at the reduced sucrose concentration. This study demonstrates the effectiveness of in vitro multiplication and ex vitro rooting protocols for two valuable aromatic and medicinal plants. These findings are significant for the ex situ conservation of these species, as they provide effective long-term preservation and utilization strategies.

Advancements in micropropagation techniques have made it easier to produce large numbers of cannabis clones, but these methods may also introduce genetic instability over successive generations. This instability often manifests as somaclonal variation, characterized by the progressive accumulation of genetic mutations or epigenetic alterations with each subculture. In this study, we examined how mutations accumulate in cannabis clones subjected to 6-11 subcultures. Using genotyping-by-sequencing, we identified 9405 polymorphic variants across 70 clones. The analysis revealed a correlation between the number of subcultures and the frequency of these mutations, revealing that genetic changes accumulate over successive subcultures despite clones sharing the same chronological age. Furthermore, we evaluated the functional impacts of accumulated mutations, with particular attention to implications on gene function and overall plant health. While rare, 14 high-impact variants were identified in genes that are important for plant development. Notably, six variants were also found in genes related to cannabinoid and terpene synthesis pathways, potentially affecting the plant\'s biochemical composition. These findings highlight the need for genetic assessments in micropropagation protocols, impacting plant breeding and conservation. Understanding genetic variations in clonally propagated plants optimizes practices for stability. Crucial for cannabis and horticultural plants, it emphasizes techniques to prevent genetic decay and ensure viability.

BACKGROUND: Kale, a versatile cruciferous crop, valued for its pro-health benefits, stress resistance, and potential applications in forage and cosmetics, holds promise for further enhancement of its bioactive compounds through in vitro cultivation methods. Micropropagation techniques use cytokinins (CKs) which are characterized by various proliferative efficiency. Despite the extensive knowledge regarding CKs, there remains a gap in understanding their role in the physiological mechanisms. That is why, here we investigated the effects of three CKs - kinetin (Kin), 6-benzylaminopurine (BAP), and 2-isopentenyladenine (2iP) - on kale physiology, antioxidant status, steroidal metabolism, and membrane integrity under in vitro cultivation.
RESULTS: Our study revealed that while BAP and 2iP stimulated shoot proliferation, they concurrently diminished pigment levels and photosynthetic efficiency. Heightened metabolic activity in response to all CKs was reflected by increased respiratory rate. Despite the differential burst of ROS, the antioxidant properties of kale were associated with the upregulation of guaiacol peroxidase and the scavenging properties of ascorbate rather than glutathione. Notably, CKs fostered the synthesis of sterols, particularly sitosterol, pivotal for cell proliferation and structure of membranes which are strongly disrupted under the action of BAP and 2iP possibly via pathway related to phospholipase D and lipoxygenase which were upregulated. Intriguingly, both CKs treatment spurred the accumulation of sitostenone, known for its ROS scavenging and therapeutic potential. The differential effects of CKs on brassicasterol levels and brassinosteroid (BRs) receptor suggest potential interactions between CKs and BRs.
CONCLUSIONS: Based on the presented results we conclude that the effect evoked by BAP and 2iP in vitro can improve the industrial significance of kale because this treatment makes possible to control proliferation and/or biosynthesis routes of valuable beneficial compounds. Our work offers significant insights into the nuanced effects of CKs on kale physiology and metabolism, illuminating potential avenues for their application in plant biotechnology and medicinal research.

The endangered plant species Adenophora liliifolia faces threats to its survival in the wild, necessitating the development of effective micropropagation techniques for potential reintroduction efforts. This study demonstrates that Adenophora liliifolia effectively reproduces on MS synthetic medium with diverse plant growth regulators (PGR) and natural extracts, facilitating swift micropropagation for potential future reintroduction endeavors. It highlights the substantial impact of PGR composition and natural extracts on the growth and development of A. liliifolia. The ideal growth medium for A. liliifolia was determined to be ½ MS with specific treatments. Additionally, incorporating silver nitrate (AgNO3) at 5 mg L-1 into the medium led to enhanced root formation and shoot length, albeit excessive concentrations adversely affected root development. Varying concentrations of NAA significantly affected different plant growth parameters, with the 0.1 mg L-1 treatment yielding comparable plant height to the control. Moreover, 50 mL L-1 of coconut water bolstered root formation, while 200 mL L-1 increased shoot formation during in vitro propagation. However, elevated doses of coconut water (CW) impeded root development but stimulated shoot growth. Experiments measuring chlorophyll a + b and carotenoid content indicated higher concentrations in the control group than differing levels of applied coconut water. Optimizing pH levels from 6.8-7 to 7.8-8.0 notably enhanced plant height and root formation, with significant carotenoid accumulation observed at pH 6.8-7. Soil samples from A. liliifolia\'s natural habitat exhibited a pH of 6.65. Ultimately, the refined in vitro propagation protocol effectively propagated A. liliifolia, representing a pioneering effort and setting the stage for future restoration initiatives and conservation endeavors.

Agaves are plants with multiple possibilities of use and are naturally tolerant to low water availability conditions and high temperatures. This makes them species of great interest in the context of the necessary substitution of crops due to climate change. Unfortunately, the overexploitation of wild specimens has endangered many species of the genus that have not been domesticated or cultivated intensively. In vitro mass culture and propagation techniques have emerged as a very efficient option to produce agave plants that can be used without damage to the natural populations. A protocol is presented here for the in vitro micropropagation of agaves in a two-stage process. In the first step, clusters of slightly differentiated shoots are generated from stem segments cultivated on a semisolid medium added with cytokinin. In a second step, these shoot clusters are cultured in temporary immersion bioreactors where they grow and complete their differentiation, and then the shoots are rooted and transferred to soil. This protocol has been successfully applied to several threatened species of the Agave genus.

This chapter presents the methodological approach for the in vitro propagation of Agave angustifolia \"espadin,\" the base material to produce mezcal. The protocol used in each stage of the crop is addressed in detail, considering the changes in the culture medium and the characteristics of the plant material at each stage. The importance of integrated management between the multiplication and growth phase, as part of the in vitro selection strategy, is mentioned.

The method of plant micropropagation is widely used to obtain genetically homogeneous and infection-free plants for the needs of various industries and agriculture. Optimization of plant growth and development conditions plays a key role in economically successful micropropagation. Computer technologies have provided researchers with new approaches for modeling and a better understanding of the role of the factors involved in plant growth in vitro. To develop new models for optimizing growth conditions, we used plants with a high speed of vegetative in vitro reproduction, such as duckweed (Wolffia arrhiza and Lemna minor). Using the development of the optimal modeling of the biological processes, we have obtained the prescriptions for an individually balanced culture medium that enabled us to obtain 1.5-2.0 times more duckweed biomass with a 1.5 times higher protein concentration in the dry mass. Thus, we have demonstrated that the method of optimization modeling of the biological processes based on solving multinomial tasks from the series of quadratic equations can be used for the optimization of trophic needs of plants, specifically for micropropagation of duckweeds in vitro.

The success of in vitro cultivation, particularly for micropropagation purposes, depends on the efficient control of contaminants. In this context, the sterilization of plant material constitutes a fundamental step in initiating cultures. Microbial contaminants can be found either on the surface (epiphyte) or inside plant explants (endophyte). However, the latter is generally challenging to detect and may not always be eradicated through surface sterilization alone. Endophyte contaminants, such as bacteria, can persist within plant material over several cultivation cycles, potentially interfering with or inhibiting in vitro establishment, growth, or recovery of cryopreserved materials. Therefore, microscopy techniques, such as electron microscopy, can yield valuable insights into bacterial endophytes\' localization, tissue colonization patterns, and functions in in vitro plant culture. This information is essential for adopting effective strategies for eliminating, preventing, or harmonious coexistence with contaminants.

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.