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Long-term in vitro cultures of Tussilago farfara (L.), a traditional medicinal plant in Austria, had been stored at 14°C for over 20 years. The cultures were vigorous and showed no visual signs of bacterial presence. The transfer from growth regulator-free culture medium to medium containing kinetin and the increase of temperature from 14°C to 25°C for fast propagation led to the emergence of latent bacteria in all twelve accessions studied.To investigate latent infections occurring during the development of a cryopreservation protocol of genetically interesting material using droplet-vitrification.Two protocols for droplet-vitrification were tested using plant vitrification solutions (PVS) 2 and 3. The bacteria were isolated and identified using 16S rDNA analysis. Next, non-cryopreserved in vitro plantlets were acclimatized and transferred to the glasshouse. After 6 weeks, shoot tips were harvested from the pot plants, surface-sterilized and initiated into culture. Further, newly acquired achenes of Tussilago were surface-sterilized and germinated in vitro and seedlings checked for bacteria.The bacteria from the long-term cultures were isolated and identified as Luteibacter. Regeneration after cryopreservation using PVS3 was successful despite the continuing presence of Luteibacter. Luteibacter could no longer be detected in the newly-initiated in vitro material in subsequent tests and it was also not detected in the seedlings.Luteibacter withstood the cryopreservation procedure. Re-initiation of infected material may be an efficient alternative to antibiotic treatment to manage bacteria in micropropagation systems.

The potato's great genetic diversity needs to be maintained for future agricultural applications and can be preserved at ultra-low temperatures. To decipher detailed physiological processes, the aim of the study was to analyze the regrowth in 28 gene bank accessions and to reveal metabolite changes in a subset of four accessions that showed pronounced differences after shoot tip cryopreservation using DMSO droplet freezing and PVS3 droplet vitrification. Regrowth varied in all 28 genotypes ranging from 5% ('Kagiri') to 100% ('Karakter') and was higher after PVS3 droplet vitrification (71 ± 19%) than after cryopreservation using DMSO (54 ± 17%). Sucrose, glucose, and fructose were analyzed and showed significant increases after pre-culture in combination with PVS3 or DMSO and liquid nitrogen treatment and were reduced during regeneration. In contrast, adenosine triphosphate (ATP) reached its minimum concentration after cryoprotection and liquid nitrogen treatment and recovered most quickly after PVS3 droplet vitrification. A shortening of the explant pre-culture period reduced dramatically the regrowth after PVS3 vitrification. However, correlations between the shoot tip regrowth and sugar concentration were absent and significant at a low extent with ATP (r = 0.4, P 0.01). Interestingly, several sub-cultivations of the donor plants from the previous stock affected negatively the regrowth. In conclusion, the cryopreservation protocol, genotypes, pre-culture period and number of sub-cultures affect the regrowth ability of explants, which was best estimated by the ATP concentration after low-temperature treatment. Due to the superior performance of PVS3, the routine potato cryopreservation at the Gatersleben gene bank was changed to PVS3 droplet vitrification.

Cryopreservation of shoot tips facilitates long-term storage of plant genetic resources which can otherwise only be propagated vegetatively. The vitrification approach using the cryoprotectant plant vitrification solution 3 (PVS3, 50% sucrose and 50% glycerol) is easy to handle, has shown to produce high regrowth percentages in a number of potato, mint, garlic, and shallot accessions, and is, thus, highly suitable for routine cryopreservation of plant genetic resources. In the current chapter, the vitrification procedure is described for potato, mint, garlic, and shallot and includes details about modifications for the different plant species. Special emphasis is given on the preparation of the different culture media, solutions, the culture conditions prior and post-cryopreservation, and the preparation of the shoot tips from different sources. Furthermore, protocols to introduce plants into in vitro culture and methods to estimate cryopreservation success are provided.

Cryopreservation of shoot tips facilitates long-term storage of plant genetic resources which can otherwise only be propagated vegetatively. The vitrification approach using the cryoprotectant plant vitrification solution 3 (PVS3, 50% sucrose and 50% glycerol) is easy to handle, has shown to produce high regrowth percentages in a number of potato, mint, garlic, and shallot accessions, and is, thus, highly suitable for routine cryopreservation of plant genetic resources. In the current chapter, the vitrification procedure is described for potato, mint, garlic, and shallot and includes details about modifications for the different plant species. Special emphasis is given on the preparation of the different culture media, solutions, the culture conditions prior and post-cryopreservation, and the preparation of the shoot tips from different sources. Furthermore, protocols to introduce plants into in vitro culture and methods to estimate cryopreservation success are provided.

Wheat sheds tricellular short-lived pollen at maturity. The identification of viable pollen required for high seed set is important for breeders and conservators. The present study aims to evaluate and improve pollen viability tests and to identify factors influencing viability of pollen. In fresh wheat pollen, sucrose was the most abundant soluble sugar (90%). Raffinose was present in minor amounts. However, the analyses of pollen tube growth on 112 liquid and 45 solid media revealed that solid medium with 594 mM raffinose, 0.81 mM H3BO3, 2.04 mM CaCl2 at pH5.8 showed highest pollen germination. Partly or complete substitution of raffinose by sucrose, maltose, or sorbitol reduced in vitro germination of the pollen assuming a higher metabolic efficiency or antioxidant activity of raffinose. In vitro pollen germination varied between 26 lines (P < 0.001); between winter (15.3 ± 8.5%) and spring types (30.2 ± 13.3%) and was highest for the spring wheat TRI 2443 (50.1 ± 20.0%). Alexander staining failed to discriminate between viable, fresh pollen, and non-viable pollen inactivated by ambient storage for >60 min. Viability of fresh wheat pollen assessed by fluorescein diacetate (FDA) staining and impedance flow (IF) cytometry was 79.2 ± 4.2% and 88.1 ± 2.7%, respectively; and, when non-viable, stored pollen was additionally tested, it correlated at r = 0.54 (P < 0.05) and r = 0.67 (P < 0.001) with in vitro germination, respectively. When fresh pollen was used to assess the pollen viability of 19 wheat, 25 rye, 11 barley, and 4 maize lines, correlations were absent and in vitro germination was lower for rye (11.7 ± 8.5%), barley (6.8 ± 4.3%), and maize (2.1 ± 1.8%) pollen compared to wheat. Concluding, FDA staining and IF cytometry are used for a range of pollen species, whereas media for in vitro pollen germination require specific adaptations; in wheat, a solid medium with raffinose was chosen. On adapted media, the pollen tube growth can be exactly analyzed whereas results achieved by FDA staining and IF cytometry are higher and may overestimate pollen tube growth. Hence, as the exact viability and fertilization potential of a larger pollen batch remains elusive, a combination of pollen viability tests may provide reasonable indications of the ability of pollen to germinate and grow.

Ants are among the most successful insects in Earth’s evolutionary history. However, there is a lack of knowledge regarding range-limiting factors that may influence their distribution. The goal of this study was to describe the environmental factors (climate and soil types) that likely impact the ranges of five out of the eight most abundant Trachymyrmex species and the most abundant Mycetomoellerius species in the United States. Important environmental factors may allow us to better understand each species’ evolutionary history. We generated habitat suitability maps using MaxEnt for each species and identified associated most important environmental variables. We quantified niche overlap between species and evaluated possible congruence in species distribution. In all but one model, climate variables were more important than soil variables. The distribution of M. turrifex (Wheeler, W.M., 1903) was predicted by temperature, specifically annual mean temperature (BIO1), T. arizonensis (Wheeler, W.M., 1907), T. carinatus, and T. smithi Buren, 1944 were predicted by precipitation seasonality (BIO15), T. septentrionalis (McCook, 1881) were predicted by precipitation of coldest quarter (BIO19), and T. desertorum (Wheeler, W.M., 1911) was predicted by annual flood frequency. Out of 15 possible pair-wise comparisons between each species’ distributions, only one was statistically indistinguishable (T. desertorum vs T. septentrionalis). All other species distribution comparisons show significant differences between species. These models support the hypothesis that climate is a limiting factor in each species distribution and that these species have adapted to temperatures and water availability differently.

Satureja spicigera callus initiated from young leaves was successfully cryopreserved by vitrification. Four protocols desiccation, vitrification with PVS2, vitrification with PVS3 and DMSO freezing were tested for suitability. According to the results of the analysis of variance, significant differences were found among four protocols used for callus cryopreservation. The regrowth percentage of cryopreserved callus was significantly highest after treatment with PVS3 (98.7%). Based on this result, vitrification with PVS3 is recommended as the most suitable protocol for cryopreservation of S. spicigera callus. In this protocol, the callus excised from leaves of in vitro-grown plants was precultured on MS medium supplemented with 0.4 M sucrose, 0.5 mg l−1 2,4-D, 0.6 mg l−1 BAP and 6 g l−1 agar under a 16 h photoperiod at 25 °C for two days. After preculture, the callus was transferred to a Petri dish with 2 ml loading solution and loaded at 25 °C for 25 min. Then, the callus was dehydrated with 2 ml PVS3 at 25 °C for two hours. After transferring to a 1.8 ml cryotube containing 1 ml PVS3, the cryotube was directly immersed into liquid nitrogen. The fast rewarming procedure consisted of plunging the cryotubes into a water-bath at 40 °C for 2 min. After removing PVS3, the contents of the cryotubes were poured into a Petri dish with 4 ml of a 1.2 M sucrose solution. Callus clumps were incubated in this solution at 25 °C for 20 min. After that, the callus was transferred to recovery medium. Results of measurements by differential scanning calorimetry (DSC) have shown that callus treated by desiccation and vitrification no freezing and melting peaks occurred. These cryopreservation protocols would be useful for the ex-situ conservation of Satureja spicigera callus at very low temperatures.

Vitrification approaches are widely used to cryopreserve Mentha spp. genetic resources.Here, we compared the response of 20 different Mentha species and hybrids during cryopreservation and elucidated the efficacy of two cryoprotectants.One hundred and fifty three Mentha spp. accessions were cryopreserved using in vitro plants maintained under slow-growth storage and PVS2 or PVS3 as cryoprotectants.The cryoprotectant PVS2 was effective for all species, except M. requienii and M. villosanervata. The use of PVS3 increased the proportion of explants able to regrow after rewarming. The outbreak of endophytes upon rewarming was both less frequent and less severe when PVS3 replaced PVS2.Both PVS2 and PVS3 can be used as cryoprotectant for all the species and accessions of Mentha spp. surveyed. Since higher regenerations were achieved using PVS3, and since the risk of an endophyte outbreak was reduced, this cryoprotectant should be preferred in future for cryopreserving Mentha spp.

The changes in the reproductive barrier between hexaploid wheat ( Triticum aestivum L.) and rye ( Secale cereale L.) can be induced using in situ embryo rescue of abnormal embryos, yielding stable fertile amphidiploid plants. In intergeneric crosses between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.), postzygotic barriers may occur at different stages of hybrid development. One such mechanism is embryo lethality, which is genetically determined by the interaction and expression of two incompatible genes in wheat (Eml-A1) and rye (Eml-R1). Using in vitro culture methods as stressors, we overcame this hybrid lethality. Normal and abnormal embryos were observed to build embryogenic calli and produce regenerated plantlets in a similar manner. The high regenerative capacity of the abnormal embryos led us to conclude that the reproductive barrier in these intergeneric hybrids may have an epigenetic origin that can be easily overcome by culturing immature embryos via callus induction. After colchicine treatment during callus culture, amphidiploid plants were obtained. However, most of these plants did not produce seeds, due mainly to sterility of the pollen but also of the embryo sacs. These findings demonstrate that hybrid sterility affects both male and female gametophytes in plants obtained from abnormal embryos. The key roles of double fertilization and stress factors in the implementation of the apical meristem formation program in embryos from incompatible intergeneric crosses between hexaploid wheat and rye during in vitro culture are discussed. We also propose a hypothetical model for a wheat–rye lethality system involving differential expression of incompatible wheat Eml-A1 and rye Eml-R1b alleles in an identical genetic background.

The maintenance of plant genetic resources in living plant collections (genebanks) causes costs due to employment of staff, usage of buildings, equipment and consumables. Since this is especially challenging in vegetatively propagated material, studies were performed for the case of garlic, which is one of the major vegetatively maintained crops in the genebank of IPK Gatersleben. Data were recorded to compare various scenarios of the main strategies field maintenance and cryopreservation. A spreadsheet tool was developed to be used for cost assessment and for drawing conclusions concerning the most effective way of maintenance. Field culture is cheaper in the short term, whereas after a break-even point cryopreservation becomes the more efficient storage method in the long term. This break-even point depends on the particular scenario, which is determined by various factors such as field and in vitro multiplication rates of various genotypes, presence of bulbils in a part of the genepool, the sample size of the accessions as well as the number of stored accessions in cryopreservation. The comparative discussion is exemplified for a 1-year field rotation versus cryopreservation using either in vitro plantlets or a combination of bulbils and unripe inflorescence bases as organ sources. For the more expensive use of in vitro plants cryopreservation becomes less costly than field culture only after 13 years, whereas this is the case already after 8-9 years when using a combination of bulbils in winter and inflorescence bases in summer.

At the genebank of IPK (Leibniz Institute of Plant Genetics and Crop Plant Research) in Gatersleben, Germany, three long-term conservation methods (field genebank, in vitro slow-growth and cryopreservation) are used for mint germplasm. The plant material of the field genebank was the source to establish the slow-growth in vitro culture collection, from which the cryopreserved collection was set up, using a droplet-vitrification protocol. The genetic identity of 161 samples of three mint accessions (MEN 198, MEN 166 and MEN 186), stored for several years using those three methods, was studied using RAPD markers. Accession ‘MEN 198’ was the only one with a unique RAPD fragment pattern for all its samples, independently of the conservation procedure employed. The field collections of accessions ‘MEN 166’ and ‘MEN 186’ were made up of different genotypes. None of the genotypes detected in the plots of these accessions was represented in the in vitro and cryopreserved samples analyzed, which showed a unique genotype for each accession. From this work we conclude that, for an appropriate management of a germplasm collection of vegetatively propagated species, the determination of the genetic composition of the donor material and a periodical assessment of the preserved material should be carried out.

Garlic and shallot are important vegetable and spice plants, garlic is also a medicinal crop widely used throughout the world. Both belong to the genus Allium, which has been a main target of the taxonomical research at the IPK for many years. Therefore, the IPK’s living collection of Allium is one of the world’s largest special collections of this genus with garlic and shallot being essential parts of this collection. Furthermore, they are subject to special activities to preserve the material in vitro and in cryopreservation. Both methods help protecting valuable germplasm from abiotic and biotic threats and reduce the maintenance costs in the long term. At present, the garlic collection maintained at the IPK incorporates germplasm of 509 accessions in total including all safety duplicates not offered for exchange. Of shallot, 114 accessions are present as well. Passport data as well as phenotypic data of 159 accessions of garlic, one of great headed garlic and 16 of shallot are included in the Garlic and Shallot Core Collection Database (GSCC). An additional part of the database is a comprehensive collection of images illustrating morphological characteristics of the accessions. Exploration of the information is supported by a web-based application. The GSCC is available at http://www.ipk-gatersleben.de/databases/gscc.

Access to propofol remains a challenge for many emergency physicians. This report examines changes in patient care after the introduction of propofol to an emergency department formulary.The Procedural Sedation in the Community Emergency Department registry is a prospective multicentered database of community emergency physician-directed procedural sedation cases. Medication selection and patient outcome were compared at a single Procedural Sedation in the Community Emergency Department registry study site before and after credentialing of emergency physicians for the use of propofol. Analysis was done through analysis of variance and χ(2) test.Over a 36-month period, 573 patients were entered into the registry from the single study site, 255 before and 318 after propofol introduction. The percentage of propofol use increased from 26% of procedural sedation cases in the first 3 months of availability to 69% in the final 3 months analyzed. Before propofol use, 46% of cases were completed with a single agent compared with after propofol use, in which 66% were completed with a single agent (P.001). Complications decreased from 9% of patients before propofol use to 3% of patients after propofol use (P.05), whereas sedation failures decreased from 5.1% to 4.1% (P.02).Granted access to propofol, emergency physicians will preferentially use this medication over prior procedural sedation agents with fewer procedural sedation complications and greater procedural success.

Intergeneric hybridizations for the transfer ofdisease resistance and cold and drought tolerance were performed between 34 Triticum aestivum L. cultivars or strains and 10 wild Triticeae species (Agropyron J. Gaertn., Elymus L., Elytrigia Desv., Lophopyrum A. Love). The crosses of wheat and A. cristatum, A. pectiniJorme, E. gmelinii ssp. ugamicum, E. scabriglumis, E. rrachycaulus, Elytrigia intermedia ssp. barbulata, Elytrigia repens, and Lophopyrum elongatum succeeded. The plantlets derived were cloned in vitro. in the combinations T. aestivum x Elytrigia intermedia ssp. barbulara and 7’. aestivum x Elytrigia repens, a segregating F, and a BC, generation were obtained. All other combinations have so far been perennial and sterile. By crossing Triticale ‘Lasko’ x Lophopyrum elongafum (2x) a good yielding Agrorrificale was produced.

Background Garlic has lost its ability to form seeds in the course of its domestication. Therefore, the germplasm storage via cryopreservation is increasingly applied. The progression of the various steps within the cryopreservation procedure is accompanied by declining survival rates of the explants. Much of the recent work on cryo-stress has been focussed on osmotic and cold stress components. However, two decades after invention of garlic cryopreservation, the function of metabolites and oxygen in and around the cryopreserved tissues is still largely obscure. Methods In this study, hypoxia was characterized in cryopreservation of garlic with oxygen sensors and amino acid metabolism. Furthermore, malondialdehyde, soluble sugars and ammonium were quantified to demonstrate the influence of cryo-stress in declining survival rates. Results To better understand the possible reasons for a reduction in the survival rate at the subsequent steps of cryopreservation, the concentration of amino acids, ammonium, γ-aminobutyric acid (GABA), soluble sugars, malondialdehyde (MDA), and oxygen were measured in garlic shoot tips undergoing cryopreservation. Using microsensors, a very low oxygen concentration (

In living plant collections, vegetatively propagated accessions are outstanding material with respect to vulnerability and labour amount. This is also true for the main vegetative material, held in the IPK, Gatersleben. A survey of the preservation of potato, garlic and other alliums, mint and yam is given. More than 630 accessions are in slow growth conditions. Amongst them, 99 clones of garlic and 35 of shallot have been tested to be virus-free. Cryopreservation is routinely applied for potato using the droplet method. The cryo-collection contains more than 1000 accessions, a part of which has been integrated from another collection, formerly established at Braunschweig. Cryopreservation of garlic has been used to store the accessions of the European core collection. Cryopreservation is successful in three Dioscorea species using a combination of the original vitrification with the droplet method. Investigations about morphogenesis and ultrastructural cell parameters before, during and after cryopreservation were included in these activities.