Cyhalofop-butyl resistance conferred by a novel Trp-2027-Leu mutation of acetyl-CoA carboxylase and enhanced metabolism in Leptochloa chinensis.

BACKGROUND: Chinese sprangletop (Leptochloa chinensis (L.) Nees) is an invasive grass weed severely infesting rice fields across China. In October 2020, a suspected resistant Leptochloa chinensis population HFFD3 that survived the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide cyhalofop-butyl applied at its field-recommended rate was collected from a rice field in Feidong County, Anhui Province, China. This study aimed to determine the resistance profile of HFFD3 to ACCase inhibitors and to investigate its mechanisms of resistance to cyhalofop-butyl. RESULTS: Single-dose testing confirmed that HFFD3 had evolved resistance to cyhalofop-butyl. Two loci encoding plastidic ACCase were each amplified from the susceptible (S) and resistant (R, HFFD3) individual plants. Target gene sequencing and derived cleaved amplified polymorphic sequence assay revealed all the R plants carried a Trp-2027-Leu substitution in their ACCase1,2 copies. Dose-response bioassays revealed that HFFD3 was highly resistant to cyhalofop-butyl and exhibited cross-resistance to metamifop, fenoxaprop-P-ethyl, quizalofop-P-ethyl, and clethodim. Pre-treatment with piperonyl butoxide and 4-chloro-7-nitrobenzoxadiazole considerably reversed the resistance of the R plants to cyhalofop-butyl, by 23% and 43%, respectively. Liquid chromatography-tandem mass spectrometry analysis suggested the metabolic rates of cyhalofop-butyl were significantly faster in the R than in the S plants. CONCLUSION: This study confirmed the first case of an arable weed species featuring cross-resistance to ACCase-inhibiting herbicides due to a novel Trp-2027-Leu mutation of ACCase. Target gene mutation and cytochrome P450s- and glutathione-S-transferases-involved enhanced metabolism may have simultaneously participated in the resistance of HFFD3 population to cyhalofop-butyl.

Increased Tolerance to Furfural by Introduction of Polyhydroxybutyrate Synthetic Genes to Escherichia coli.

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes (bktB, phaB, and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.

Global grass (Poaceae) success underpinned by traits facilitating colonization, persistence and habitat transformation.

Poaceae (the grasses) is arguably the most successful plant family, in terms of its global occurrence in (almost) all ecosystems with angiosperms, its ecological dominance in many ecosystems, and high species richness. We suggest that the success of grasses is best understood in context of their capacity to colonize, persist, and transform environments (the "Viking syndrome"). This results from combining effective long-distance dispersal, efficacious establishment biology, ecological flexibility, resilience to disturbance and the capacity to modify environments by changing the nature of fire and mammalian herbivory. We identify a diverse set of functional traits linked to dispersal, establishment and competitive abilities. Enhanced long-distance dispersal is determined by anemochory, epizoochory and endozoochory and is facilitated via the spikelet (and especially the awned lemma) which functions as the dispersal unit. Establishment success could be a consequence of the precocious embryo and large starch reserves, which may underpin the extremely short generation times in grasses. Post-establishment genetic bottlenecks may be mitigated by wind pollination and the widespread occurrence of polyploidy, in combination with gametic self-incompatibility. The ecological competitiveness of grasses is corroborated by their dominance across the range of environmental extremes tolerated by angiosperms, facilitated by both C3 and C4 photosynthesis, well-developed frost tolerance in several clades, and a sympodial growth form that enabled the evolution of both annual and long-lived life forms. Finally, absence of investment in wood (except in bamboos), and the presence of persistent buds at or below ground level, provides tolerance of repeated defoliation (whether by fire, frost, drought or herbivores). Biotic modification of environments via feedbacks with herbivory or fire reinforce grass dominance leading to open ecosystems. Grasses can be both palatable and productive, fostering high biomass and diversity of mammalian herbivores. Many grasses have a suite of architectural and functional traits that facilitate frequent fire, including a tufted growth form, and tannin-like substances in leaves which slow decomposition. We mapped these traits over the phylogeny of the Poales, spanning the grasses and their relatives, and demonstrated the accumulation of traits since monocots originated in the mid-Cretaceous. Although the sympodial growth form is a monocot trait, tillering resulting in the tufted growth form most likely evolved within the grasses. Similarly, although an ovary apparently constructed of a single carpel evolved in the most recent grass ancestor, spikelets and the awned lemma dispersal units evolved within the grasses. Frost tolerance and C4 photosynthesis evolved relatively late (late Palaeogene), and the last significant trait to evolve was probably the production of tannins, associated with pyrophytic savannas. This fits palaeobotanical data, suggesting several phases in the grass success story: from a late Cretaceous origin, to occasional tropical grassland patches in the later Palaeogene, to extensive C3 grassy woodlands in the early-middle Miocene, to the dramatic expansion of the tropical C4 grass savannas and grasslands in the Pliocene, and the C3 steppe grasslands during the Pleistocene glacial periods. Modern grasslands depend heavily on strongly seasonal climates, making them sensitive to climate change.

Effect of biological soil crusts on seed germination and growth of an exotic and two native plant species in an arid ecosystem.

Biological soil crusts (BSCs) can improve the stability and health of native plant communities in arid ecosystems. However, it is unknown whether BSCs can also inhibit invasions of exotic vascular plants on stabilized reclaimed sand dunes. To answer this question, we conducted a greenhouse experiment to test the effects of cyanobacteria-dominated BSCs on 1) seed germination and biomass of an exotic grass (Stipa glareosa P. Smirn.), and 2) individual biomass of the exotic S. glareosa growing with two native plants, Eragrostis poaeoides Beauv. and Artemisia capillaris Thunb. Our experiment included three BSC treatments (intact crust, disturbed crust, and bare soil) and five species trials (native E. poaeoides alone, E. poaeoides mixed with exotic S. glareosa, native A. capillaris alone, A. capillaris mixed with exotic S. glareosa, and S. glareosa alone). The results showed that cyanobacteria-dominated crusts can significantly reduce the cumulative percent germination of the exotic grass (P<0.001) and native plants (P<0.001). Maximum cumulative percent germinations of the exotic grass and two native plants were found in bare soil, and minimum in intact crusts. The interaction of crust treatment × species trials on shoot biomass of the two native plants was significant (P<0.05). These results indicate that the presence of BSCs on stabilized sand dunes may reduce the germination of the exotic and two native plants. The effect of reducing exotic and native plant seeds germination would maintain more diverse plant communities and contribute to the formation of clumped vegetation patterns. We conclude that BSCs act as a natural regulator for vegetation patterns and thus promote ecosystem stability and sustainability.

A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant.

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain offspring genetically identical to the maternal plant. Apomixis has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. Apomictic Pennisetum/Cenchrus species, members of the Poaceae (grass) family, reproduce by apospory. Apospory is characterized by apomeiosis, the formation of unreduced embryo sacs derived from nucellar cells of the ovary and, by parthenogenesis, the development of the unreduced egg into an embryo without fertilization. In Pennisetum squamulatum (L.) R.Br., apospory segregates as a single dominant locus, the apospory-specific genomic region (ASGR). In this study, we demonstrate that the PsASGR-BABY BOOM-like (PsASGR-BBML) gene is expressed in egg cells before fertilization and can induce parthenogenesis and the production of haploid offspring in transgenic sexual pearl millet. A reduction of PsASGR-BBML expression in apomictic F1 RNAi transgenic plants results in fewer visible parthenogenetic embryos and a reduction of embryo cell number compared with controls. Our results endorse a key role for PsASGR-BBML in parthenogenesis and a newly discovered role for a member of the BBM-like clade of APETALA 2 transcription factors. Induction of parthenogenesis by PsASGR-BBML will be valuable for installing parthenogenesis to synthesize apomixis in crops and will have further application for haploid induction to rapidly obtain homozygous lines for breeding.

Protocol for callus induction and somatic embryogenesis in Moso Bamboo.

Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10-20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5-2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0-7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0-7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse.

Failure of androgenesis in Miscanthus × giganteus in vitro culture of cytologically unbalanced microspores.

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique.

Soil moisture and fungi affect seed survival in California grassland annual plants.

Survival of seeds in the seed bank is important for the population dynamics of many plant species, yet the environmental factors that control seed survival at a landscape level remain poorly understood. These factors may include soil moisture, vegetation cover, soil type, and soil pathogens. Because many soil fungi respond to moisture and host species, fungi may mediate environmental drivers of seed survival. Here, I measure patterns of seed survival in California annual grassland plants across 15 species in three experiments. First, I surveyed seed survival for eight species at 18 grasslands and coastal sage scrub sites ranging across coastal and inland Santa Barbara County, California. Species differed in seed survival, and soil moisture and geographic location had the strongest influence on survival. Grasslands had higher survival than coastal sage scrub sites for some species. Second, I used a fungicide addition and exotic grass thatch removal experiment in the field to tease apart the relative impact of fungi, thatch, and their interaction in an invaded grassland. Seed survival was lower in the winter (wet season) than in the summer (dry season), but fungicide improved winter survival. Seed survival varied between species but did not depend on thatch. Third, I manipulated water and fungicide in the laboratory to directly examine the relationship between water, fungi, and survival. Seed survival declined from dry to single watered to continuously watered treatments. Fungicide slightly improved seed survival when seeds were watered once but not continually. Together, these experiments demonstrate an important role of soil moisture, potentially mediated by fungal pathogens, in driving seed survival.

Anther and pollen development in some species of Poaceae (Poales).

Anther and pollen development were studied in Olyra humilis Nees, Sucrea monophylla Soderstr, (Bambusoideae), Axonopus aureus P. Beauv., Paspalum polyphyllum Nees ex Trin. (Panicoideae), Eragrostis solida Nees, and Chloris elata Desv. (Chloridoideae). The objective of this study was to characterise, embryologically, these species of subfamilies which are considered basal, intermediate and derivate, respectively. The species are similar to each other and to other Poaceae. They present the following characters: tetrasporangiate anthers; monocotyledonous-type anther wall development, endothecium showing annular thickenings, secretory tapetum; successive microsporogenesis; isobilateral tetrads; spheroidal, tricellular, monoporate pollen grains with annulus and operculum. Nevertheless, the exine patterns of the species studied are distinct. Olyra humilis and Sucrea monophylla (Bambusoideae) show a granulose pattern, whereas in the other species, it is insular. In addition, Axonopus aureus and Paspalum polyphyllum (Panicoideae) have a compactly insular spinule pattern, while Chloris elata and Eragrostis solida (Chloridoideae) show a sparsely insular spinule pattern. The exine ornamentation may be considered an important feature at the infrafamiliar level.

Anther and pollen development in some species of Poaceae (Poales) / Desenvolvimento da antera e do grão de pólen em espécies de Poaceae (Poales)

Anther and pollen development were studied in Olyra humilis Nees, Sucrea monophylla Soderstr, (Bambusoideae), Axonopus aureus P. Beauv., Paspalum polyphyllum Nees ex Trin. (Panicoideae), Eragrostis solida Nees, and Chloris elata Desv. (Chloridoideae). The objective of this study was to characterise, embryologically, these species of subfamilies which are considered basal, intermediate and derivate, respectively. The species are similar to each other and to other Poaceae. They present the following characters: tetrasporangiate anthers; monocotyledonous-type anther wall development, endothecium showing annular thickenings, secretory tapetum; successive microsporogenesis; isobilateral tetrads; spheroidal, tricellular, monoporate pollen grains with annulus and operculum. Nevertheless, the exine patterns of the species studied are distinct. Olyra humilis and Sucrea monophylla (Bambusoideae) show a granulose pattern, whereas in the other species, it is insular. In addition, Axonopus aureus and Paspalum polyphyllum (Panicoideae) have a compactly insular spinule pattern, while Chloris elata and Eragrostis solida (Chloridoideae) show a sparsely insular spinule pattern. The exine ornamentation may be considered an important feature at the infrafamiliar level.

O desenvolvimento da antera e do grão de pólen de Olyra humilis Nees, Sucrea monophylla Soderstr. (Bambusoideae), Axonopus aureus P. Beauv., Paspalum polyphyllum Nees ex Trin. (Panicoideae), Eragrostis solida Nees and Chloris elata Desv. (Chloridoideae) foi estudado visando caracterizar embriologicamente essas espécies de subfamílias consideradas basal, intermediária e derivada, respectivamente. As espécies são similares entre si e entre as demais Poaceae. Apresentam os seguintes caracteres: anteras tetrasporangiadas; desenvolvimento da parede da antera do tipo monocotiledôneo, endotécio com espessamento de parede anelar, tapete secretor; microsporogênese sucessiva; tétrades isobilaterais; grãos de pólen esféricos, tricelulares, monoporados, com anel e opérculo. Por outro lado, o padrão de ornamentação da exina do grão de pólen é distinto. Olyra humilis e Sucrea monophylla (Bambusoideae) apresentam padrão granuloso e as demais espécies padrão insular. Axonopus aureus e Paspalum polyphyllum (Panicoideae) apresentam espínulos densamente agrupados, enquanto Chloris elata e Eragrostis solida (Chloridoideae) espínulos esparsamente agrupados. A ornamentação da exina dos grãos de pólen pode ser considerada caráter importante a nível infrafamiliar.

Role of PGrs and inhibitors in induction and control of somatic embryogenesis in Themeda quadrivalvis.

Somatic embryogenesis could be achieved in Themeda quadrivalvis (Linn.) O. Ktze -fodder grass species on MS medium supplemented with 2,4-D. Incorporation of putrescine in the medium stimulated embryogenesis, however its lower concentration stimulated production of non-regenerative callus. Other polyamines such as spermine and spermidine could not evoke similar response. Ascorbic acid used as antioxidant could not prevent browning in embryogenic cultures, however it stimulated embryogenesis. Inhibition of auxin polar transport by use of TIBA and HFCA reduced the embryogenic response significantly and produced distorted or abnormal embryos. Antiethylene substances such as AgNO3 and CoCl2 added in the medium adversely affected the process of embryogenesis and counteracting the stimulatory role of ethylene.

A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21.

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process--from wild-type seeds to transgenic T(1) seeds--takes approximately 8 months to complete.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana).

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Cytological and embryological studies on apospory in Bothriochloa ischaemum L.

Cytological and embryological studies on apomictic species Bothriochloa ischaemum L. were carried out. Our studies revealed that the chromosome number of its root apical cells was 40, indicating that it was a tetraploid cytotype. During the stage of microsporogenesis, meiosis seemed irregular, as the pairing chromosome number of microspore mother cell was more than 20. It was often found that some chromosomes did not assemble in the equatorial plane or moved to the two poles of the cell, a few laggards were seen. Multiporate pollens (22.3%) were often observed. The studies showed that a high frequency (87.8%) of 1-3 or more aposporous embryo sacs developed in one ovule of the species. The mature aposporous sac was usually characterized by an egg cell and one polar nucleus. The egg cell could develop spontaneously into a large proembryo (100-200 microm) mass prior to anthesis. When several aposporous sacs occurred in the same ovule, usually 2 aposporous sacs were involved in pseudogamy and developed into separate endosperm masses in the same ovary. In the low frequency of mature seed, 13.5% twin-embryo seedlings could be obtained after mature seeds germinated.

Seed bank composition of open and overgrown calcareous grassland soils--a case study from Southern Belgium.

The success of calcareous grassland recreation following abandonment depends to a large extent on the composition of the soil seed bank. We studied the species richness and composition of the seed bank along a chronosequence from well-developed calcareous grassland to scrub and forest vegetation, which had developed on calcareous grassland over the last 225 years. The seed bank density was highest in the calcareous grassland soils (930 seeds/m(2)). However, the seed bank was mainly composed of rather common species of nutrient poor grassland, which were poorly represented in the surface vegetation. There were no significant differences in soil seed bank density and species composition between the calcareous grassland and scrub vegetation that was less than 15 years old, largely because several grassland species had persisted in the scrub vegetation and were therefore able to replenish the soil seed bank. In contrast seed density and species richness declined significantly after 40 years of grassland abandonment. Indeed, forest soils had the lowest seed densities (214 seeds/m(2)) with only a few grassland species represented. This reflects the lack of grassland species in the field layer of the forest, and, therefore, the lack of seed production and seed bank replenishment. It is clear that recreation of calcareous grassland on long abandoned sites cannot rely on germination of target species from the seed bank alone. Even in the calcareous grassland soil, seeds of target species are not abundant as the majority produces transient seeds, which decay rapidly if they do not germinate immediately. Successful grassland recreation on such sites therefore may require seeds of target species to be introduced artificially (e.g. as seed mixtures, green hay, etc.). Alternatively, reinstatement of traditional practices, such as grazing or mowing, will increase the natural dispersal potential of these species, allowing population reestablishment in the long term.

Multispecies and monoculture rhizoremediation of polycyclic aromatic hydrocarbons (PAHs) from the soil.

In this study, we investigated the potential of multispecies rhizoremediation and monoculture rhizoremediation in decontaminating polycyclic aromatic hydrocarbon (PAH) contaminated soil Plant-mediated PAH dissipation was evaluated using monoplanted soil microcosms and soil microcosms vegetated with several different grass species (Brachiaria serrata and Eleusine corocana). The dissipation of naphthalene and fluorene was higher in the "multispecies" vegetated soil compared to the monoplanted and nonplanted control soil. The concentration of naphthalene was undetectable in the multispecies vegetated treatment compared to 96% removal efficiencies in the monoplanted treatments and 63% in the nonplanted control after 10 wk of incubation. Similar removal efficiencies were obtained for fluorene. However, there was no significant difference in the dissipation of pyrene in both the mono- and multispecies vegetated treatments. There also was no significant difference between the dissipation of PAHs in the monoplanted treatments with different grass species. Principle component analysis (PCA) and cluster analysis were used to evaluate functional diversity of the different treatments during phytoremediation of PAHs. Both PCA and cluster analysis revealed differences in the metabolic fingerprints of the PAH contaminated and noncontaminated soils. However, the differences in metabolic diversity between the multispecies vegetated and monoplanted treatments were not clearly revealed. The results suggest that multispecies rhizoremediation using tolerant plant species rather than monoculture rhizoremediation have the potential to enhance pollutant removal in moderately contaminated soils.

Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells.

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.

A cryopreservation protocol for embryos of the endangered species Zizania texana.

Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.