Plant Cryopreservation: Principles, Applications, and Challenges of Banking Plant Diversity At Ultralow Temperatures.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

In Vitro Culture Studies for the Mitigation of Heavy Metal Stress in Plants.

Heavy metals are among the most common and dangerous contaminants; their action on plants, as well as the possibility for plants to effectively absorb and translocate them, have been studied for several years, mainly for exploitation in phytoremediation, an environmentally friendly and potentially effective technology proposed and studied for the recovery of contaminated soils and waters. In this work, the analysis has focused on the studies developed using in vitro techniques on the possibilities of mitigating, in plants, the stress due to the presence of heavy metals and/or improving their absorption. These objectives can be pursued with the use of different substances and organisms, which have been examined in detail. The following are therefore presented in this review: an analysis of the role of metals and metalloids; the use of several plant growth regulators, with their mechanisms of action in different physiological phases of the plant; the activity of bacteria and fungi; and the role of other effective compounds, such as ascorbic acid and glutathione.

Efficient Protocol for Improving the Development of Cryopreserved Embryonic Axes of Chestnut (Castanea sativa Mill.) by Encapsulation-Vitrification.

An optimized cryopreservation protocol for embryonic axes (EAs) of chestnut (Castanea sativa Mill.) has been developed based on the encapsulation-vitrification procedure. EAs of mature seeds were aseptically dissected and encapsulated in alginate beads with or without 0.3% (w/v) activated charcoal (AC). Embedded EAs were dehydrated with Plant Vitrification Solution 2 for different treatment times up to 120 min, followed by direct immersion in liquid nitrogen. Cryopreserved embryonic axes encapsulated with AC showed higher survival (70%) compared to those encapsulated without AC (50%). Sixty-four percent of embryonic axes, from synthetic seeds with AC, subsequently developed as whole plants. Plantlet regrowth was faster in AC-encapsulated EAs and showed enhanced postcryopreservation shoot and root regrowth over 2 cm after five weeks from rewarming. Results indicate that encapsulation-vitrification with activated charcoal added to the beads is an effective method for the long-term preservation of Castaneasativa embryonic axes.

Large-Scale Plant Production of Lycium barbarum L. by Liquid Culture in Temporary Immersion System and Possible Application to the Synthesis of Bioactive Substance.

Goji (Lycium barbarum L.) has recognized nutritive and antioxidant properties and many products are commercialized for health in food market. Besides its food use, goji has been the subject of more than 2000 years of traditional Chinese medicine, using berries, root bark, and leaves. Here, the potential of the liquid culture in temporary immersion system (TIS) by using the bioreactor PlantformTM was tested for the large-scale production of high-quality goji shoots and the subsequent production of total phenols and flavonoids. The three tested immersion cycles differently influenced the shoot quality in terms of proliferation and hyperhydricity. The best immersion cycle (time and frequency) was proven to have the shortest daily immersion time (6 min every 24 h) which ensured good levels of relative growth and multiplication rate, very limited onset of hyperydricity, and the longest shoots, promoting direct rooting after only 30 days of culture. In comparison with the semisolid culture, the TIS culture resulted in an increase of the total phenolic content (TPC) and in a lower value of the total flavonoid content (TFC). However, considering the higher quantity of biomass produced in the PlantformTM bioreactor, the difference in terms of TFC productivity between semisolid medium and TIS liquid culture was proven to be statistically equivalent.

Impact of two arbuscular mycorrhizal fungi on Arundo donax L. response to salt stress.

MAIN CONCLUSION: AM symbiosis did not strongly affect Arundo donax performances under salt stress, although differences in the plants inoculated with two different fungi were recorded. The mechanisms at the basis of the improved tolerance to abiotic stresses by arbuscular mycorrhizal (AM) fungi have been investigated mainly focusing on food crops. In this work, the potential impact of AM symbiosis on the performance of a bioenergy crop, Arundo donax, under saline conditions was considered. Specifically, we tried to understand whether AM symbiosis helps this fast-growing plant, often widespread in marginal soils, withstand salt. A combined approach, involving eco-physiological, morphometric and biochemical measurements, was used and the effects of two different AM fungal species (Funneliformis mosseae and Rhizophagus irregularis) were compared. Results indicate that potted A. donax plants do not suffer permanent damage induced by salt stress, but photosynthesis and growth are considerably reduced. Since A. donax is a high-yield biomass crop, reduction of biomass might be a serious agronomical problem in saline conditions. At least under the presently experienced growth conditions, and plant-AM combinations, the negative effect of salt on plant performance was not rescued by AM fungal colonization. However, some changes in plant metabolisms were observed following AM-inoculation, including a significant increase in proline accumulation and a trend toward higher isoprene emission and higher H2O2, especially in plants colonized by R. irregularis. This suggests that AM fungal symbiosis influences plant metabolism, and plant-AM fungus combination is an important factor for improving plant performance and productivity, in presence or absence of stress conditions.

Cryotechniques for the Long-Term Conservation of Embryogenic Cultures from Woody Plants.

Since its development in the 1960s, plant cryopreservation is considered an extraordinary method of safe long-term conservation of biological material, as it does not induce genetic alterations and preserve the regeneration potential of the stored material. It is based on the storage of explants at cryogenic temperatures, such as the one of liquid nitrogen (-196 °C), where the metabolism within the cells is suspended; thus, the time for these cells is theoretically "stopped". Cryopreservation is particularly important for embryogenic cultures, as they require periodic subculturing for their maintenance, and this, in turn, increases the risk of losing the material, as well as its embryogenic potential. Periodic re-initiation of embryogenic cultures is possible; however, it is labor intensive, expensive, and particularly difficult when working with species for which embryogenic explants are available only during a limited period of the year. Among various methods of cryopreservation available for embryogenic cultures, slow cooling is still the most common approach, especially in callus cultures from softwood species. This chapter briefly reviews the cryopreservation of embryogenic cultures in conifers and broadleaf trees, and describes as well a complete protocol of embryogenic callus cryopreservation from common ash tree (Fraxinus excelsior L.) by slow cooling.

In vitro propagation of olive (Olea europaea L.) by nodal segmentation of elongated shoots.

Olive (Olea europaea L.), long-living, ever-green fruit tree of the Old World, has been part of a traditional landscape in the Mediterranean area for centuries. Both the fruits consumed after processing and the oil extracted from the fruits are among the main components of the Mediterranean diet, widely used for salads and cooking, as well as for preserving other food. Documentations show that the ancient use of this beautiful tree also includes lamp fuel production, wool treatment, soap production, medicine, and cosmetics. However, unlike the majority of the fruit species, olive propagation is still a laborious practice. As regards traditional propagation, rooting of cuttings and grafting stem segments onto rootstocks are possible, former being achieved only when the cuttings are collected in specific periods (spring or beginning of autumn), and latter only when skilled grafters are available. In both the cases, performance of the cultivars varies considerably. The regeneration of whole plants from ovules, on the other hand, is used only occasionally. Micropropagation of olive is not easy mainly due to explant oxidation, difficulties in explant disinfection, and labor-oriented establishment of in vitro shoot cultures. However today, the progress in micropropagation technology has made available the complete protocols for several Mediterranean cultivars. This chapter describes a micropropagation protocol based on the segmentation of nodal segments obtained from elongated shoots.

In vitro propagation of peanut (Arachis hypogaea L.) by shoot tip culture.

Peanut (Arachis hypogaea L.), also known as groundnut, is the most important species of Arachis genus, originating from Brazil and Peru. Peanut seeds contain high seed oil, proteins, amino acids, and vitamin E, and are consumed worldwide as edible nut, peanut butter, or candy, and peanut oil extracted from the seeds. The meal remaining after oil extraction is also used for animal feed. However, its narrow germplasm base, together with susceptibility to diseases, pathogens, and weeds, decreases yield and seed quality and causes great economic losses annually. Hence, the optimization of efficient in vitro propagation procedures would be highly effective for peanut propagation, as it would raise yield and improve seed quality and flavor. Earlier reports on traditional micropropagation methods, based on axillary bud proliferation which guarantees the multiplication of true-to-type plants, are still limited. This chapter describes a micropropagation protocol to improve multiple shoot formation from shoot-tip explants by using AgNO(3) in combination with plant growth regulators.

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos.

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5M DMSO, 1M sucrose, 0.5M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H(2)O(2) was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.

Cryogenic technologies for the long-term storage of Citrus germplasm.

With its beautiful trees, Citrus species have long been valued by humanity. The tasteful fruits, extensively used for nutrition, are also good for health due to the high content in vitamins, minerals, and dietary fibers. Like majority of the woody fruit plants, Citrus germplasm is conserved mainly as field collections in clonal orchards. However, such a traditional approach presents several difficulties, among which are the high cost, manual labor, and extensive land required to maintain the collections, as well as the necessity of a careful protection of plants from diseases and extreme environmental conditions. As many species in the genus have seeds recalcitrant to desiccation, conservation in seed banks is also inadequate. On the other hand, cryopreservation, i.e., the storage of specimens at ultra-low temperatures (usually in liquid nitrogen, at -196°C) where reactions within the cells are minimized, presents a unique alternative for the safe storage of such germplasm. The present contribution outlines the cryopreservation techniques applied to seeds, zygotic and somatic embryos, embryogenic callus cultures of Citrus spp. and provides sample protocols to be used for Citrus conservation.

In vitro conservation and cryopreservation of ornamental plants.

Today, the conservation of ornamental germplasm can take advantage of innovative techniques which allow preservation in vitro (slow growth storage) or in liquid nitrogen (cryopreservation) of plant material. Slow growth storage refers to the techniques enabling the in vitro conservation of shoot cultures in aseptic conditions by reducing markedly the frequency of periodic subculturing, without affecting the viability and regrowth of shoot cultures. Cryopreservation refers to the storage of explants from tissue culture at ultra-low temperature (-196 degrees C). At such temperature, all the biological reactions within the cells are hampered, hence the technique makes available the storage of plant material for theoretically unlimited periods of time. An exhaustive review of papers dealing with the slow growth storage and the cryopreservation of ornamental species is reported here. Step-by-step protocols for the slow growth storage of rose germplasm, the production of synthetic seeds for the in vitro conservation of ornamentals, and the cryopreservation of Chrysanthemum morifolium are included.

Green energy from Rhodopseudomonas palustris grown at low to high irradiance values, under fed-batch operational conditions.

Rhodopseudomonas palustris was grown under continuous irradiances of 36, 56, 75, 151, 320, 500, and 803 W m(-2), for a co-production of both bio-H(2) and biodiesel (lipids) using fed-batch conditions. The highest overall bio-H(2) produced [4.2 l(H(2)) l(culture) (-1)] was achieved at 320 W m(-2), while the highest dry biomass (3.18 g l(-1)) was attained at 500 W m(-2). Dry biomass contained between 22 and 39% lipid. The total energy conversion efficiency was at its highest (6.9%) at 36 W m(-2).

Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification or one-step freezing.

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at -196 degrees C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45 degrees C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.