In vitro culture for the biodiversity conservation of vegetables

Today, in vitro culture is a strategic tool to support medium and long-term conservation of plant genetic resources by using the slow growth storage of shoot cultures and the cryopreservation of organs and tissues. Over the last 30 years, considerable progresses were made in the development of both techniques that are nowadays considered as ex situ conservation strategies complementary to traditional seed banks and in-field clonal collections. Efficient protocols were developed for the conservation of a large number of crops, including important vegetables of the temperate environment (garlic, artichoke, asparagus, mint, potato, sweet potato, tomato, red chicory, thyme). Conservation in slow growth storage consists in modifying the medium and/or culture conditions to reduce the growth of plant material without affecting the viability and regrowth potential of shoots when moved back to standard culture conditions. The technique allows medium-term crops conservation, with a storage time of vegetables ranging from a few months to two years and more, without recurring subculturing typical of micropropagation. Cryopreservation preserves plant organs and tissues at ultra-low temperature, as liquid nitrogen temperature (-196°C). Currently, various techniques are available, based on cold tolerance induction by cell cytoplasm vitrification during the fast ultra-freezing through in liquid nitrogen immersion of explants. The term "vitrification" refers to the solidification of a liquid without crystallization. If induced in plant cells, it avoids the formation of lethal intra-cellular ice crystals during the ultra-freezing process, keeping tissues at stopped metabolism condition but vital. Working with vegetables, the techniques based on direct immersion of specimens in liquid nitrogen ("one-step freezing", such as the "droplet-method", the "PVS2 vitrification", the "encapsulation-based" and the "cryo plate-based" procedures) are the most used. Further, the "two-step freezing", i.e. the slow explants cooling before immersion in liquid nitrogen, still finds some applications. The experimental activity mainly focused on three economically important species, i.e., Allium sp., potato and sweet potato. Currently, almost 38,000 accessions of garlic, cassava, mint, potato, sweet potato, taro, yam are maintained in 17 genetic resources conservation centers, located in 12 Countries and 5 Continents (Europe, Asia, Africa, North and South America). Approximately 4/5 of these accessions are maintained in vitro by means of slow growth storage of shoot cultures, but more recent cryopreservation is constantly growing, and over 7,500 accessions from vegetables are stored at -196 °C. The germplasm of potato (Solanum sp.) is by far the most collected in the world, with almost 17,700 accessions presently maintained in in vitro banks and cryobanks

Temporary Immersion System for Production of Biomass and Bioactive Compounds from Medicinal Plants

The cultivation of medicinal plants and the production of bioactive compounds derived from them are of fundamental importance and interest, not only at the pharmacological level but also in nutraceutical and cosmetic industries and in functional foods, as well as plant protection in agriculture. In order to respond adequately to the increased demands of the global market from a quantitative and qualitative point of view and to guarantee environmental sustainability of the productions, it is necessary to resort to innovation tools, such as tissue culture in vitro technology. Nowadays, it is well known that the cultivation through the Temporary Immersion System (TIS) in a bioreactor has considerable advantages both for the in vitro mass production of the plants and for the production of secondary metabolites. The present review focuses on the application of TIS during the last two decades to produce biomass and bioactive compounds from medicinal plants. Indeed, almost one hundred papers are discussed, and they particularly focus on the effects of the culture system, vessel design and equipment, immersion time and frequency, and substrate composition for 88 medicinal species in TIS bioreactor culture

Paulownia spp.: a bibliometric trend analysis of a global multi-use tree

The research on Paulownia spp. has increased in the last twenty years thanks to the growing interest in the application modalities of this plant in various sectors such as wood, phytoremediation, environmental protection, paper, biofuel, chemistry and medicine. For the first time, this study analyzed the papers present in the Web of Science Core Collection on “Paulownia” to obtain a set of characteristics in the work carried out from 1971 to 2021. This analysis selected and took into account 820 articles and provided evidence of the scientific production of authors, institutions, and countries. This work showed that the most studied species was Paulownia tomentosa, followed by P. fortunei and P. elongate. The JCR category and research area with the most publications was plant science, with 20.4% of the total. The papers were published in 460 journals and in a book series. The journals with the most publications were Bioresources, Advanced Material Research, Agroforestry Systems, Journal of Wood Science and Industrial Crops and Products. The institutions with the most prolific affiliation with the field of Paulownia spp. research were Henan University, the US Department of Agriculture, Belgrade University, the Chinese Academy, and Georgia University. Finally, the 3842 keywords were divided into nine different clusters and the trends of interest in the last fifteen years were highlighted

Temporary Immersion System for Micropropagation of Tree Species: a Bibliographic and Systematic Review

This paper was characterized by a bibliometric and systematic review on the database ISI Web of Science, aiming to provide an update of the main points addressed regarding the Temporary Immersion Systems (TIS) for micropropagation of tree species. It was pointed out that the frequency and time of immersion were one of the main parameters studied in the papers and 35% these papers worked with eucalyptus species. The main problem reported in the papers was the hyperhydricity, but it was overcome via procedures such as: a) air injection into the system, b) increasing the immersion intervals and decreasing the immersion time and c) decreasing concentration of cytokinin. Most papers reported that TIS produced plants that were more successful in surviving the ex-vitro acclimation stage than those produced on semi-solid media or continuous immersion systems. Few studies compared different types of temporary immersion bioreactors on micropropagation of plants and within the established criteria, papers with TIS tree species represented only 15% of the total. This system has presented promising results for most of the tree species, and although some gaps have been identified and few are the works with tree species, this process has been taking an increasingly larger space in the propagation of plants

Development of an efficient 'one-step freezing' cryopreservation protocol for a Georgian provenance of chestnut (Castanea sativa Mill.) zygotic embryos.

Experiments were performed to determine the influence of various dehydration and vitrification treatment times on the 'one-step freezing' cryopreservation of embryonic axes (EAs), composed of zygotic embryos and cotyledon residuals, from mature seeds of a Georgian provenance of chestnut (Castanea sativa Mill.). Dehydration was carried out in laminar flow hood from 1 to 5 h, and vitrification experiments were carried out by immersion of EAs in PVS2 vitrification solution up to 120 min, both followed by direct immersion in liquid nitrogen. Both systems resulted in inducing specimen tolerance to ultra-rapid freezing, although to a different extent. Full germination of cryo-stored EAs after 5 h of dehydration (reducing moisture content from initial 66% to 21%) has been increased from 0% to 66.7%. A pre-treatment of EAs in PVS2 vitrification solution for 30 min produced fully developed plantlets at a rate of 55.6% in post-cryopreservation. Plantlet regrowth from cryopreservation was faster in EAs that underwent the dehydration/'one-step freezing' procedure. All the plantlet from cryopreserved EAs could be easily acclimatized, producing healthy potted plants. Finally, the TTC test showed to be useful for a fast evaluation of specimen survival after thawing and, as a consequence, to speed up the development of optimized cryo-protocols. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. ********

Strategies for Fast Multiplication and Conservation of Forest Trees by Somatic Embryogenesis and Cryopreservation: a Case Study with Cypress (Cupressus sempervirens L.)

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker, caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone 'Mediterraneo'. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain selected genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the 'Mediterraneo' embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen ("two-step freezing")

Cryopreservation and In Vitro banking: a cool subject – Preface from the editors

Plant breeding depends largely on having access to a wide variety of plant genetic resources, which are vulnerable to losses caused by biotic and abiotic threats when grown in the field or in a greenhouse. Thus, cryopreservation or in vitro banking is a safe strategy for long-term conservation of such genetic resources, which serves as back-up collections for field genebanks and reduces. For many species, encapsulation technologies can be a promising tool for the management of plant material of high quality, the production in nurseries of plants from in vitro culture, or the conservation of plant genetic resources. Such “synthetic seeds” proved to be of great value in the medium- (slow-growth storage) and long-term (cryopreservation) conservation of germplasm of fruit, ornamental, horticultural and forestry species in small spaces. However, more research is still needed. Cryopreservation projects must have clear goals, long-term funding, skilled technical support staff, necessary infrastructure, and well-defined procedures and protocols, so that they can be routinely implemented in plant cryobanks and help to establish backup collections of valuable plant genetic resources

Biochar successfully replaces activated charcoal for in vitro culture of two white poplar clones reducing ethylene concentration

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Cryopreservation at CNR-IVALSA in Florence: reflections upon ten years of good results and some failures with woody plants

The first experiments on cryopreservation at CNR-IVALSA in Florence date back to 1998. They were a response to the necessity to explore new and innovative systems for the ex situ conservation of woody plant germplasm, complementary to the traditional approach of in-field collection (clonal orchards). In Italy, the IVALSA Institute has one of the largest germplasm collections of fruit species (over 1800 accessions of peach, olive, plum, pear, persimmon, cherry, apple and quince) spread over the 60 ha of its experimental farm. Year by year, the maintenance costs of the collections are becoming heavier to bear. Furthermore, in the middle of the 90s the Sharka disease (Plum Pox Virus) spread through the large plum collection, obliging the burning of a clonal orchard of over 150 accessions of Prunus domestica and Prunus salicina. Hence, cryopreservation technology was regarded as a possible complementary strategy for the future, reducing to a minimum the risks of germplasm loss. Taking advantage of the considerable expertise in plant tissue culture, a small cryolaboratory was soon established to begin with. The first experiments were with poplar (Populus spp.) and olive (Olea europaea), two species of great importance in Italy in terms of germplasm preservation. Since then, various cryopreservation techniques, numerous species (mainly, but not exclusively, woody plants) and various kinds of explants have been tested, mostly with the practical aim of developing effective and reproducible protocols for long-term conservation. Among the cryoprotocols which were satisfactorily developed (thanks also to National and International collaborations), the following are particularly worthy of mention: the cryopreservation of shoot tips from white poplar (Populus alba), plum (Prunus domestica) and a non-woody species (Cichorium intybus), of embryogenic lines from olive and horsechestnut (Aesculus hippocastanum), and of seeds from Pistacia and Citrus spp. However, it has not been ten years of only successes, not at all! On the contrary, enthusiasm over some very good results has often been followed by deep frustration deriving from disappointing experiments. One example: after ten years of trials, testing all the different cryotechniques today available (included slow cooling), the cryopreservation of olive shoot tips is still unreliable, a long way from being proposable as a complementary approach to the conservation in field. Other failures have been the shoot-tip cryopreservation of a grape rootstock ( Kober 5BB ) and of redwood (Sequoiadendron giganteum), as well as the cryopreservation of an embryogenic line of ash (Fraxinus excelsior). What have we learnt from our ten-year experience in cryopreservation? What are the possible reasons for species adapting extraordinarily well to -196°C (e.g., white poplar, red chicory), and others being highly reacalcitrant (e.g., olive)? Do we know all the potentialities, as well as all the limits, of the technology? First reflection: before working with plant cryopreservation, I had many years of experience in the field of somatic embryogenesis of woody plants. In my opinion, the two techniques share a practical similarity when approaching the study of a new species, i.e., for both, if after the first preliminary experiments you get nil (i.e., no explant producing an embryogenic callus, as well as no explant surviving to -196°C), there is little hope that it will be possible to develop a succesful protocol, whatever you change in the methodology. If you get at least one positive (i.e., a fully-developed plantlet), it is worthy to go on! That means much more than only a glimmer of hope! Second reflection: working with woody plants, species having structural difficulties in the initial stages of micropropagation (e.g., big problems with the in vitro establishment, low proliferation rates, very slow shoot elongation) will be, with most probability, also difficult in the development of an effective cryopreservation protocol. One example: olive is very difficult and slow during the introduction and the establishment in vitro. So, is the difficulty in developing an effective shoot-tip cryopreservation protocol strictly due to its low tolerance to ultra-rapid freezing? Or, rather, is it due to the accentuation of a basic problem when a sort of second establishment in vitro is required (i.e., the shoot-tip development after thawing and plating)? Third reflection: a big mistake was made in the 80s in the field of micropropagation, i.e., private users were given to believe that every species could be, soon or later, successfully micropropagated, it just required experiments and tests of different medium/growth regulator combinations. In Italy, that idea led to the proliferation of commercial laboratories in the early 90s, which soon found themselves fighting to produce and offer the same relatively-few species which could be actually produced in vitro. The majority of them went broke in a few years! Of course, cryopreservation is a totally different story (first, it is a non-profit activity). However, we must be very careful to avoid the same mistake when promoting cryopreservation. Last but not least: a warm welcome to projects like Crymcept and CryoPlanet !! Certainly, we are all convinced that plant cryopreservation still needs much experimentation and, more important, a continuous confrontation between scientists with different experiences, made with different species and in different scientific and national contexts.vokMyynti MTT, Tietopalvelut 31600 Jokioine