Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for th... more Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.
The United States Department of Agriculture, Agricultural Research Service, National Clonal Germp... more The United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository at Corvallis, Oregon preserves genetic resources for Rubus. The in vitro collection includes about 200 accessions. In vitro cold storage of these accessions is at 4°C with 12 h of low light. Storage facilities for germplasm collections vary, but one week of cold acclimation followed by 4°C storage in the dark or with a photoperiod is acceptable for most Rubus germplasm when quarterly evaluation inventories are used to determine timing of repropagation. A reduced-nitrogen medium extends room temperature storage to nine months and is a useful alternative for cold sensitive and tropical genotypes which typically only survive for a short time in cold storage. Meristems of 34 cold-acclimated genotypes of Rubus (blackberry and raspberry) were successfully cryopreserved by slow cooling through optimization of cryoprotectants, cooling rates and cold acclimation. Alternating low temperatures as a cold acclimation (CA) treatment improved recovery of shoot tips cryopreserved by slow freezing. The length of the CA required varied from 1 to 10 weeks and was genotype dependent. Cryopreserved Rubus shoot tips produced shoots directly from either apical meristems or axillary buds, but not from callus. Shooting increased and callus formation decreased when IBA was eliminated from the recovery medium. Shoot tips of 25 genotypes in 9 Rubus species were successfully cryopreserved using encapsulation-dehydration with recovery of 60-100%. Four genotypes of 3 species were tested using PVS2 vitrification with 71% average regrowth. A protocol for cryopreservation of Rubus germplasm should include a CA period of 2-10 weeks and recovery on auxin-free medium. These studies confirm that all three cryopreservation protocols may be used for cryopreservation of a wide range of Rubus genetic resources.
in Vitro Cellular & Developmental Biology-plant, 2010
Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to exce... more Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.
Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for th... more Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.
The United States Department of Agriculture, Agricultural Research Service, National Clonal Germp... more The United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository at Corvallis, Oregon preserves genetic resources for Rubus. The in vitro collection includes about 200 accessions. In vitro cold storage of these accessions is at 4°C with 12 h of low light. Storage facilities for germplasm collections vary, but one week of cold acclimation followed by 4°C storage in the dark or with a photoperiod is acceptable for most Rubus germplasm when quarterly evaluation inventories are used to determine timing of repropagation. A reduced-nitrogen medium extends room temperature storage to nine months and is a useful alternative for cold sensitive and tropical genotypes which typically only survive for a short time in cold storage. Meristems of 34 cold-acclimated genotypes of Rubus (blackberry and raspberry) were successfully cryopreserved by slow cooling through optimization of cryoprotectants, cooling rates and cold acclimation. Alternating low temperatures as a cold acclimation (CA) treatment improved recovery of shoot tips cryopreserved by slow freezing. The length of the CA required varied from 1 to 10 weeks and was genotype dependent. Cryopreserved Rubus shoot tips produced shoots directly from either apical meristems or axillary buds, but not from callus. Shooting increased and callus formation decreased when IBA was eliminated from the recovery medium. Shoot tips of 25 genotypes in 9 Rubus species were successfully cryopreserved using encapsulation-dehydration with recovery of 60-100%. Four genotypes of 3 species were tested using PVS2 vitrification with 71% average regrowth. A protocol for cryopreservation of Rubus germplasm should include a CA period of 2-10 weeks and recovery on auxin-free medium. These studies confirm that all three cryopreservation protocols may be used for cryopreservation of a wide range of Rubus genetic resources.
in Vitro Cellular & Developmental Biology-plant, 2010
Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to exce... more Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.
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