Use of Thin Cell Layer Technique for Induction of Somatic Embryogenesis of Agave fourcroydes.

Agave fourcroydes (henequén) is a plant used for the extraction of hard fiber from its leaves. Due to its long-life cycle, it is very difficult to genetically improve. Somatic embryogenesis (SE) is a very useful micropropagation technique, that can be used for genetic improvement programs and increase the micropropagation of this species. SE is a morphogenic process by which somatic embryos are generated from somatic cells reprogramming. To initiate the regeneration program, the loss of cell-cell communication is suggested to be important. The Thin Cell Layer (TCL) technique allows for the isolation of specific cell or tissue layers, and in conjunction with strictly controlled growth conditions, may lead to the in vitro induction of specific morphogenic programs. Here, we describe a new protocol for the induction of somatic embryogenesis through TCL culture technique, from stem of elite clonal A. fourcroydes vitroplants previously generated through micropropagation of adventitious shoots.

Current challenges and opportunities in the care of patients with fibrodysplasia ossificans progressiva (FOP): an international, multi-stakeholder perspective.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare, disabling genetic disorder characterized by congenital malformations of the great toes and progressive heterotopic ossification of soft and connective tissues. Assiduous attention to the unmet needs of this patient community is crucial to prevent potential iatrogenic harm and optimize care for individuals with FOP. OBJECTIVE: To gather international expert opinion and real-world experience on the key challenges for individuals with FOP and their families, highlight critical gaps in care, communication, and research, and provide recommendations for improvement. METHODS: An international group of expert clinicians, patients and patient advocates, caregivers and representatives from the international FOP community participated in a virtual, half-day meeting on 22 March 2021 to discuss the key unmet needs of individuals with FOP. RESULTS: Individuals with FOP often face the frustration of long diagnostic journeys, the burden of self-advocacy and the navigation of novel care pathways. Globally, patients with FOP are also confronted with inequities in access to diagnosis and specialist care, and consequently, unequal access to registries, clinical trials, and essential support from patient associations. Organizations such as the International FOP Association, the International Clinical Council on FOP, and national FOP organizations work to provide information, facilitate access to expert clinical guidance, nurture patient empowerment, fund FOP research and/or foster meaningful collaborations with the research community. The non-profit Tin Soldiers Global FOP Patient Search program aims to identify and provide a pathway to diagnosis and care for individuals with FOP, particularly in underserved communities. Such global initiatives and the increasingly widespread use of telemedicine and digital platforms offer opportunities to improve vital access to care and research. CONCLUSIONS: This multi-stakeholder perspective highlights some of the unmet needs of individuals with FOP and their families. Regional and international organizations play an important role in improving the quality of life of those they reach in the global FOP community. However, globally, fundamental issues remain around raising awareness of FOP among healthcare professionals, identifying individuals with FOP, reducing time to diagnosis, and ensuring access to best practice in care, support, and clinical research. Medical writing support was industry-sponsored.

Comparison of conventional and temporary immersion systems on micropropagation (multiplication phase) of Agave angustifolia Haw. 'Bacanora'.

The aim of this study was to improve the quality of the micropropagated A. angustifolia Haw. plants cultured in temporary immersion bioreactors (TIS) comparing them with those produced through conventional semisolid-solid tissue culture system (SS). The Recipient for Automated Temporary Immersion (RITA®) bioreactor was used as TIS in this work. The effect of different culture conditions, such as explants density, genotype, and duration of the incubation stages, were analyzed. The growth and morphological parameters measured for the in vitro cultured plants were: plant height, number of new leaves, number of shoots/explants, growth index (GI), dry mass content, and water content. In all experiments, it was observed that plantlets cultivated in the TIS grew larger than those cultivated in SS. Analyzing all the parameters used in this study, the results showed that RITA bioreactor generates a better shoot production and a better GI when using 20 plantlets per container. The number of shoots increased with time of culture (60 days) in both systems. However, the shoots and plantlets cultivated in TIS grew bigger and showed better quality (did not present necrosis in the leaves) than the ones cultured in SS. This study provides experimental evidence that the application of TIS for micropropagation of A. angustifolia is a viable option for the production of high-quality shoots for reforestation purposes.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Tissue culture methods for the clonal propagation and genetic improvement of Spanish red cedar (Cedrela odorata).

The choice of a method to culture red cedar tissues depends on the final objectives pursued. If homogeneous clonal material is required for experimental purposes, the easiest way is to generate the lines through adventitious shoot induction from seedlings germinated from seeds. If the objective is to generate high yielding material for plantation purposes, the choice will be the same method but starting from mature vegetative tissues from selected elite plants. Most of the process are the same, but the initial steps are less efficient and much more elaborate. If the purpose is to generate lines with new genetic characteristics through somaclonal variation, mutagenesis, or genetic transformation, somatic embryogenesis will be required. No single method in its present form is suitable for all purposes. Eventually, the efficient production of somatic embryos from rejuvenated shoots collected from mature selected plants is the ideal way to culture this species, but for the time being we have to choose one or the other. In this chapter, we present a grafting procedure to rejuvenate and maintain mother plants in the greenhouse and the in vitro culture systems we have developed for the production of Cedrela odorata propagules using explants from both young seedlings and mature tissues from selected old trees. Using a modified TY17 medium and the BioMINT(®) temporary immersion system, we obtained high multiplication and ex vitro transplantation rates for efficient large-scale propagation of this species.

Liquid in vitro culture for the propagation of Arundo donax.

We describe a simple and inexpensive plant micropropagation system for giant reed (Arundo donax L.) that uses axillary buds from the lateral stems of elite plants selected from field- or nursery-grown plants. The buds, attached to the stems are cultured in stationary liquid MS culture medium, supplemented with indole 3-acetic acid and kinetin. This formulation is the only one required for all the stages. Contrary to what happens in semisolid medium where roots are not formed, the plants cultured in liquid medium are whole plants with shoots and roots that develop at the same time. The survival rate of these plants when transferred to soil is close to 100% during acclimatization. A clonal line of 900 plants from a single mother plant can be produced in 4 months.

A new temporary immersion bioreactor system for micropropagation.

A new type of bioreactor system for plant micropropagation is described that incorporates a number of features specifically designed to simplify its operation and reduce production costs. The BioMINT unit is a mid-sized (1.2 L) reactor that operates on the principle of temporary immersion. It is built of polypropylene and is translucent, autoclavable, and reusable. It consists of two vessels, one for the plant tissues and the other one for the liquid culture media coupled together through a perforated adaptor piece that permits the flow of the liquid media from one vessel to the other. This flux is driven by gravity through a see-saw movement provided by equipment (SyB) consisting of electric motor powered platforms that change position. The structural simplicity and the modular and independent nature of the bioreactors simplify their operation and reduce the amount of hand labor required for transfers, thereby reducing the cost of the whole micropropagation process.

An efficient method for the micropropagation of Agave species.

Despite their economic importance, the Agave spp. have not been genetically improved. This is probably owing to the fact that they have very long life cycles and many of them have an inefficient sexual reproduction mechanism. Micropropagation offers an alternative to this problem through the efficient cloning of selected high-yielding "elite" plants. We report here an efficient method to micropropagate agaves and a strategy for the management of large scale production that has been successfully applied to several Agave spp.

Micropropagation and field performance of Yucca valida.

Yucca valida is an important potential source of steroidal saponins closely related to Yucca schidigera, the species that is commercially exploited from the wild as a source of steroidal extracts. Neither of the species has been domesticated mainly because of their slow growth and long life span before harvesting. Here, we report a micropropagation method to generate isogenic or clonal lines for plantation purposes. Seventeen clonal lines were propagated and evaluated over a period of 26 months in an experimental plantation and compared with the performance of plants from seeds. The large variability found between the plants derived from seeds is manifested in the differences observed between the different clonal lines; however, these present a much smaller internal coefficient of variation than the one observed in the population of plants derived from seeds. Some clonal lines perform in a superior manner indicating that a process of selection and cloning can generate lines of fast growing individuals for plantations that can satisfy the demand for these materials without putting a natural resource at risk.