Cryopreservation of Dormant Buds from Diverse Fraxinus Species

Ash (Fraxinus) is an economically important tree genus in the landscape industry, as well as a key component of North American forests, especially in the North Central United States and adjacent regions in Canada. In recent years, the Emerald Ash Borer beetle (Agrilus planipennis) has significantly threatened the survival of native North American Fraxinus species. A dormant-bud cryopreservation technique has been developed as a method to conserve specific clones of ash. Dormant buds of three ash species were successfully cryopreserved when desiccated on their stem sections to 30% moisture content (w/v) and then cooled at rates of either −1°C/h or −5°C/day to either −30 or −35°C before immersion in liquid nitrogen vapor (LNV). Stem sections were removed from LNV, warmed, and rehydrated, and their buds grafted onto rootstocks to evaluate survival. Recovery percentages ranged from 34 to 100% after LNV exposure and were dependent upon accession and cooling rate. The cryopreservation methods proposed herein can complement seed-collection efforts aimed at conserving diversity, supplementing ex situ genebank and botanic-garden collections

Cryopreservation of grapevine (Vitis spp.) shoot tips from growth chamber-sourced plants and histological observations

Many genebanks rely on cryopreservation as a method to preserve vulnerable field collections of vegetatively propagated crops. Effective cryopreservation procedures have been identified for Vitis; however, they usually use in vitro plantlets as the shoot tip source materials. It is costly to establish Vitis collections in vitro prior to cryopreservation. We sought to determine if growth chamber derived Vitis plants could serve as the source of shoot tips for cryopreservation. Nodal sections from growth chamber derived plants were surface-disinfected and placed in tissue culture on pre-treatment medium for 2 weeks. Uniform apical shoot tips (1 mm) were first obtained from the nodal sections and then precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), ascorbic acid and plant preservative mixture. Half-strength PVS2 was applied for 30 min at 22 °C, prior to full-strength PVS2 treatment at 0 °C. Cryopreserved shoot tips had the highest average regrowth of 50 and 55 % without and with cold-acclimation followed with a full-strength PVS2 exposure duration of 40 and 30 min at 0 °C, respectively. This cryopreservation protocol achieved high percentages of regrowth in V. vinifera 'Chardonnay' and 'Riesling' and V. hybrid 'Oppenheim'. Histological observations revealed that shoot tips from growth chamber plants had apical as well as multiple lateral meristems that survived LN immersion. The preservation of multiple meristems in each shoot tip may increase the capacity of shoot tip regeneration in cryopreserved Vitis that originates from ex vitro sources. The high percentage of regrowth after shoot tip cryopreservation using Vitis shoot tips derived from growth chamber source plants suggest that it may be possible to cryopreserve Vitis shoot tips without first introducing each accession into tissue culture

Cryopreservation of Dormant Buds from Diverse Fraxinus Species

Ash (Fraxinus) is an economically important tree genus in the landscape industry, as well as a key component of North American forests, especially in the North Central United States and adjacent regions in Canada. In recent years, the Emerald Ash Borer beetle (Agrilus planipennis) has significantly threatened the survival of native North American Fraxinus species. A dormant-bud cryopreservation technique has been developed as a method to conserve specific clones of ash. Dormant buds of three ash species were successfully cryopreserved when desiccated on their stem sections to 30% moisture content (w/v) and then cooled at rates of either −1°C/h or −5°C/day to either −30 or −35°C before immersion in liquid nitrogen vapor (LNV). Stem sections were removed from LNV, warmed, and rehydrated, and their buds grafted onto rootstocks to evaluate survival. Recovery percentages ranged from 34 to 100% after LNV exposure and were dependent upon accession and cooling rate. The cryopreservation methods proposed herein can complement seed-collection efforts aimed at conserving diversity, supplementing ex situ genebank and botanic-garden collections.This article is from CyroLetters 30, no. 3 (2009): 262–267</p

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation.

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticult sure Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at he National Laboratory for Genetic Resources Preservation in For Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water weresufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure usine either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.Made available in DSpace on 2019-06-19T01:14:51Z (GMT). No. of bitstreams: 1 SugarcaneISHS2019.pdf: 270949 bytes, checksum: 1345b038e7e1ac4e2c7539574a648872 (MD5) Previous issue date: 2019bitstream/item/198664/1/SugarcaneISHS2019.pdfApresentado no Acta Horticulturae, 1234. DOI 10.17660/ActaHor

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation.

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticult sure Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at he National Laboratory for Genetic Resources Preservation in For Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water weresufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure usine either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.Apresentado no Acta Horticulturae, 1234. DOI 10.17660/ActaHor

LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum

Nature Publishing Group, Journal of Investigative Dermatology, 124, 2, 2005, 360-366 authorLympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS

LEKTI Fragments Specifically Inhibit KLK5, KLK7, and KLK14 and Control Desquamation through a pH-dependent Interaction

LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8–D11, and D9–D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8–D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum