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Chrysanthemums are amongst the most economically important flowers in the world. The protection and storage of these valuable genetic resources is of great importance. Today, cryopreservation, or the storage of biological material...
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Chrysanthemums are amongst the most economically important flowers in the world. The protection and storage of these valuable genetic resources is of great importance. Today, cryopreservation, or the storage of biological material at the temperature of liquid nitrogen (-196 degrees C), is believed to be the most promising long-term storage method. To optimise the cryopreservation protocol, the shoot tips of Chrysanthemum x grandiflorum/Ramat./Kitam. 'Lady Orange' and 'Lady Salmon' mutants were cryopreserved using the encapsulation-dehydration technique. During the experiment, the influence of sucrose concentration (2, 3 and 6%) during preculture and the concentration of kinetin (0.25, 0.5, 0.75 and 1.0 mg dm-3) in the regrowth medium were tested. A higher survival rate was observed for 'Lady Salmon'. In general, the media with higher sucrose levels provided the best survival and recovery rates (35-40%). Kinetin had no influence on the survival rate; however, it influenced the morphogenesis of the plants. The lowest number of explants forming multiple shoots was observed on the medium with the lowest sucrose (during preculture) and kinetin (in the recovery medium) concentration. On the other hand, the best rhizogenesis efficiency was observed when 0.25 mg dm-3 kinetin was added. In conclusion, the composition of both preculture and recovery media need to be adjusted to single cultivars. The use of 3% sucrose (preculture) and 0.25 mg dm-3 kinetin (recovery) seems reasonable, since it guarantees a satisfying recovery rate of the explants and at the same time prevents the formation of callus and multiple shoots, stimulating the rooting instead.
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BACKGROUND: Gesneriaceae family contains numerous species endemic to China, and many of them are listed as endangered species. There is a need for a simple and efficient method for long term conservation of these species. OBJECTIV...
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BACKGROUND: Gesneriaceae family contains numerous species endemic to China, and many of them are listed as endangered species. There is a need for a simple and efficient method for long term conservation of these species. OBJECTIVE: The study aimed to establish an efficient procedure for cryopreserving Paraisometrum mileense, a critically endangered species endemic to Yunnan, China. METHODS: Effects of sucrose concentration of preculture solution, duration of sucrose preculture, duration of plant vitrification solution 2 (PVS2) treatment, and cold acclimation on regeneration of cryopreserved adventitious shoot tips (ASTs) were assessed. RESULTS AND CONCLUSION: Among different sucrose preculture regimes tested, preculture with 0.3M sucrose for 24h resulted in best regeneration of cryopreserved ASTs. PVS2 treatment also affected regeneration considerably with the maximum survival of ASTs after incubation in PVS2 for 90 min at 0°C. With the optimised parameters, the level of shoot regeneration from cryopreserved ASTs reached 86%. No morphological abnormalities were observed during one year's growth of the plantlets developing from cryopreserved ASTs. Procedure established in this research is a promising technique for the cryopreservation of ASTs of this species.
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Cryopreservation is the only alternative, safe and cost-effective method for long-term storage of plant genetic resources, particularly for stone fruits ( Prunus spp.). In this study, an efficient cryopreservation protocol was dev...
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Cryopreservation is the only alternative, safe and cost-effective method for long-term storage of plant genetic resources, particularly for stone fruits ( Prunus spp.). In this study, an efficient cryopreservation protocol was developed for sour cherry ( Prunus cerasus L.). In vitro shoot tips of two varieties (Montmorency and Schattenmorelle) were successfully cryopreserved using the vitrification technique. Our study showed the possibility of replacing the 3-week cold hardening treatment of mother-plants with preculture of apices on medium enriched with sucrose and/or glycerol. The highest recovery percentages after liquid nitrogen exposure were obtained after a cold hardening treatment followed by a 3-day preculture on 0.8 M sucrose medium or by replacing cold hardening with a 7-day preculture on the following media: 0.4 M glycerol or sucrose, 0.4 M sucrose +0.4 M glycerol or 0.8 M glycerol. Under these conditions, recovery after cryopreservation ranged between 41 and 63%. These results complement the range of Prunus species successfully cryopreserved using in vitro explants. Our protocol, which is simplified in comparison with the original one, since cold hardening of mother-plants in a cold chamber is replaced by pretreatment of apices on medium with high sugar concentration, may facilitate the application of vitrification for cryopreservation of additional Prunus species.
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A simple and efficient method for cryopreservation of Chrysanthemum cinerariaefolium shoot tips is described. It comprized: treatment of the explanted stem spices for 3 d in proliferation medium enriched with 0.55 M sucrose, incub...
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A simple and efficient method for cryopreservation of Chrysanthemum cinerariaefolium shoot tips is described. It comprized: treatment of the explanted stem spices for 3 d in proliferation medium enriched with 0.55 M sucrose, incubation for 1 h in medium with 7.5 % DMSO, transfer to cryotubes, direct immersion in liquid nitrogen, rewarming at 40 deg C and culture of shoot tips on solidified nutrient medium containing 11 #mu#M ANA and 4.5 #mu#M BAP. The average viability rate of cryopreserved apices was 62 %. Dehydration of tissue cells prior to freezing with sucrose was found to be more effective than dehydration under sterile air flow for stem apices with high moisture content. The morphogenic potential of frozen stem apices and the total chlorophyll and pyrethrin contents of regenerated plants were preserved after the different treatments.
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This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant The effects of type and size of explants, sucrose preculture (duration and concentra...
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This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant The effects of type and size of explants, sucrose preculture (duration and concentration) and vitrification treatment were tested Preliminary experiments with PVS1, 2 and 3 produced shoot growth only for PVS2 When optimizing the PVS2 vitrification of nodal segments, those of 0.31 - 0.39 cm in size were better than other nodal sizes and or apices Sucrose preculture had a positive role in survival and subsequent regrowth of the cryopreserved explants Seven days on 0.5 M sucrose solution significantly improved the viability of nodal segments PVS2 incubation for 45 minutes combined with a 7-day preculture gave the optimum result of 66% Plantlets derived after cryopreservation resumed growth and regenerated normally.
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A simple cryopreservation method is described for olive in vitro grown apical shoot-tips. It involves preculturing the meristematic dome with 1 or 2 pairs of leaf primordia for 2 days in culture medium enriched with 0.75 M sucrose...
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A simple cryopreservation method is described for olive in vitro grown apical shoot-tips. It involves preculturing the meristematic dome with 1 or 2 pairs of leaf primordia for 2 days in culture medium enriched with 0.75 M sucrose, followed by dehydration in the air current of a flow cabinet for 2 h (30 % moisture content), transference to cryovials, plunging into liquid nitrogen for storage and rewarming at room temperature. A progressive decrease in survival was achieved both for control and frozen samples as the dehydration period was increased. Shoot recovery rates were around 30 % for dried and frozen samples which was equivalent to 77 % of those surviving dehydration alone. Thermal transitions in samples cryopreserved by this procedure showed absence of enthalpy changes and glass transition phases occurred between -45 and -55 deg C for cooling and rewarming.
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The report describes the impact of preculture with sucrose and sucrose + ABA on desiccation and cryopreservation tolerance of cell cultures of Ginkgo biloba L., an important landscape and medicinal tree. Callus clumps were incubat...
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The report describes the impact of preculture with sucrose and sucrose + ABA on desiccation and cryopreservation tolerance of cell cultures of Ginkgo biloba L., an important landscape and medicinal tree. Callus clumps were incubated on MS medium supplemented with high sucrose concentrations (up to 24%, w/v), employed alone or with ABA (2-10 mg 1-I) for various durations followed by desiccation for 0-240 min and cryopreservation. The beneficial effect of preculture on regrowth after desiccation without cryopreservation was only observed for the cells with water content of 20% FW and was not influenced by presence of ABA. However, preculture of calli in presence of ABA resulted in a lower desiccation rate as compared with untreated controls and calli pretreated with sucrose alone. In calli precultured with sucrose alone, post-thaw regrowth was occasional regardless of the sugar concentration in the medium, while pretreatment of calli with ABA and sucrose ensured stable regrowth after cryopreservation. The highest post-thaw regrowth of 54% was achieved for calli precultured on medium supplemented with 10% (w/v) sucrose and 2 mg 1-1 ABA for 21 days followed by desiccation for 150 min. The different effects of preculture treatments on post-thaw regrowth were associated with significant changes in content and in composition of endogenous soluble sugars in calli. Sucrose and glucose accumulated preferentially in ABA_precultured calli, while the fructose content was higher in calli precultured in absence of ABA. The possible role of preculture on desiccation and cryopreservation tolerance of G biloba cell cultures is discussed.
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Shoot tips sampled on in vitro cultured apple plantlets of 6 accessions of M.domestica and one accession of M. robusta were successfully cryopreserved using the encapsulation-dehydration technique. Shoot tips were excised from pla...
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Shoot tips sampled on in vitro cultured apple plantlets of 6 accessions of M.domestica and one accession of M. robusta were successfully cryopreserved using the encapsulation-dehydration technique. Shoot tips were excised from plantlets which had been submitted to 3 weeks of cold-acclimation at 5 deg C, 70 d after their last subculture. After preculture at 5 deg C in media with progressively increased sucrose concentration (0.1M, 0.3M and 0.7M), shoot tips were encapsulated and pregrown in medium with 1.0M sucrose for 1 d, dehydrated for 4 h under the air current of the laminar flow cabinet, thus reaching a moisture content of around 30 % (fresh weight basis )and directly immersed in liquid nitrogen. The regeneration rate of cryopreserved apices varied between 70 and 90 %, depending on the accession. Using apices sampled on plantlets which had been maintained on standard medium without subculture for 6 months, sucrose preculture became unnecessary to achieve regrowth after cryopreservation and the dehydration period was shortened. These experiments showed that the physiological state of the plant material directly affects the results and procedures for cryopreservation of apple shoot tips.
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BACKGROUND: Evaluation of the conservation status of Calanthe plants in China indicated that 18 species were endangered or critically endangered. Comprehensive conservation solutions including ex situ methods, are urgently require...
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BACKGROUND: Evaluation of the conservation status of Calanthe plants in China indicated that 18 species were endangered or critically endangered. Comprehensive conservation solutions including ex situ methods, are urgently required to protect Calanthe species in China. OBJECTIVE: The study aims to develop a simple and efficient cryopreservation protocol using droplet-vitrification for Calanthe davidii. METHODS: Protocorm-like bodies (PLBs) were induced from nodal sections of in vitro shoots, and their proliferation was promoted by a thin cell layer culture procedure. Shoot tips excised from three leaf-stage PLBs were used in cryopreservation experiments. Key factors of the droplet-vitrification procedure including sucrose preculture, treatment with PVS2 solution and post- rewarming culture conditions were optimized to achieve a high level of regeneration. RESULTS: When the optimized procedure was applied, 77.8 ± 3.9% of cryopreserved shoot tips withstood liquid nitrogen exposure and regenerated into new PLBs. CONCLUSION: These results highlighted the importance of post-rewarming osmo-conditioning for regeneration of cryopreserved shoot tips.
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Thymus is an important genus of the Lamiaceae family, comprising more than 400 perennial aromatic thyme species, which are used extensively for medicinal and culinary purposes. The present study focused on the development of cryop...
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Thymus is an important genus of the Lamiaceae family, comprising more than 400 perennial aromatic thyme species, which are used extensively for medicinal and culinary purposes. The present study focused on the development of cryopreservation procedures for Thymus vulgaris and T. cariensis, the latter being an endemic and endangered species of Turkey. For cryopreservation of T. vulgaris shoot tips, PVS2-based one-step freezing methods, i.e., PVS2 vitrification, encapsulation-vitrification and droplet-vitrification, were compared. Cold hardening and sucrose preculture were also optimized before the cryopreservation trials. For T. cariensis, a droplet-vitrification method was applied to coldhardened shoot tips, and after sucrose preculture. In all the methods tested, PVS2 was applied for up to 120 min. The best T vulgaris cryopreservation was achieved with a dropletvitrification method, that involved 2-weeks cold hardening of shoot cultures, 48 h preculture of shoot tips on MS medium supplemented with 0.25 M sucrose, and a 90 min PVS2 treatment in droplets. After direct immersion in LN, thawing and plating, 80% of shoot-tips recovered. Post-thaw recovery was significantly lower when the same procedure was applied to T. cariensis shoot tips; however also here 90 min PVS2 treatment produced the highest survival (25%) and recovery (25%) levels.
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