大苞鞘石解原球茎玻璃化超低温保存技术的研究

本文研究建立了大苞鞘石斛(Dendrobium wardianum Warner)原球茎玻璃化法超低温保存的技术体系.结果发现,预处理和玻璃化溶液(plant vitrification solution 2,PVS2)装载脱水是影响大苞鞘石斛原球茎相对存活率的两个关键步骤,高渗与低温一高渗两种预处理方法测定的相对存活...     

防风愈伤组织的玻璃化法超低温保存及再生

为避免连续继代造成的愈伤组织变异,探索新的种质资源保存方法,对防风愈伤组织进行了超低温冷冻保存及植株再生研究。以关防风3周龄的愈伤组织为材料,单一变量法研究适宜的玻璃化法超低温保存程序。结果显示:(1)防风愈伤组织超低温保存的最佳方案为:4℃条件下于MS+1.0mg/L 6-BA+1.0mg/L NAA+5%DMSO的继代培养基中预培养3d,60%PVS2常温装载20min,100%PVS2于2℃脱水45min后直接投入液氮。(2)防风愈伤组织经超低温保存后的相对存活率最高为79.24%,其中预培养和脱水是实现超低温冻存的关键环节,且1.0mol/L蔗糖的MS溶液洗涤、暗培养14d以上有助于冻后愈伤组织恢复生长。研究表明,玻璃化超低温冻存可以作为防风愈伤组织的保存方法,冻后愈伤可以恢复生长并再生成完整植株。     

江西铅山红芽芋胚性愈伤组织的包埋玻璃化超低温保存

为长期安全保存江西铅山红芽芋种质资源,本文以江西铅山红芽芋的胚性愈伤组织为对象,研究了包埋玻璃化冻存过程中各因素对细胞活力和愈伤组织成活率的影响,优化建立了江西铅山红芽芋胚性愈伤组织包埋玻璃化超低温保存体系。将约0.2 g胚性愈伤组织块包埋成海藻酸钙凝胶珠后,在25℃下转入MS+2 mg/L TDZ+1 mg/L NAA+0.75 mol/L蔗糖的培养基中于14 h/d光周期下预培养1 d;预培养后的胚性愈伤组织块用2 mol/L甘油和0.4 mol/L蔗糖的混合物在25℃下装载40 min;采用PVS2在25℃下脱水30 min,更换PVS2后直接投入液氮保存1 d;再将胚性愈伤组织块置于37℃恒温水浴中化冻3 min,然后用MS+2 mg/L TDZ+1 mg/L NAA+1.2 mol/L蔗糖的液体培养基洗涤3次,每次10 min;洗涤后的胚性愈伤组块转入MS+2 mg/L TDZ+1mg/L NAA固体培养基上先暗培养7 d再转到14 h/d光周期中培养。7 d后胚性愈伤组织块开始恢复生长,并且在30 d内分化出胚状体;将胚状体再次转入MS+2 mg/L TDZ+1 mg/L NAA固体培养基上,60 d后形成完整的植株。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为60%,并且红芽芋胚性愈伤组织冻后再生苗没有发生形态性状和染色体数目的变异,此结果为长期安全保存江西铅山红芽芋种质资源奠定了良好的基础。     

菊花茎尖的玻璃化超低温保存研究

本文建立了适合中国菊花种质资源长期保存的玻璃化超低温保存技术体系.在4℃下,把1～2mm的菊花茎尖放在含0.4mol/L蔗糖的MS培养基上暗培养2～3d,用预处理液在25℃下处理30min,再用玻璃化试剂PVS2在冰浴条件下处理15min,换新鲜的PVS2试剂并迅速投入液氮.液氮保存24h后,40℃水浴解冻2min,用含蔗糖1.2mol/L的MS液体培养基洗涤20min,滤纸吸干后接种到恢复培养基中,在25℃条件下弱光培养1～3d转入正常光照培养条件下培养,2周后成活率可达86%以上,成活的茎尖均可再生.     

束花石斛种子超低温保存的研究

以濒危兰科物种野生束花石斛（Dendrobium chrysanthum）的成熟种子为材料,研究了PVS2植物玻璃化液对其萌发的作用,以及快速冷冻法和玻璃化法对种子超低温保存的影响。结果表明,种子的萌发率随着PVS2处理时间的延长而下降。PVS2预处理能明显地增加种子的超低温耐性。当预处理时间为15～45min时,种子的超低温耐性随预处理时间的延长而增加;当预处理时间长于60min后,种子的超低温耐性随预处理时间的延长而下降。经液氮保存后,存活的种子能萌发成为正常的幼苗。结论是经PVS2预处理45min后,成熟的束花石斛种子能成功地进行超低温保存。     

小球藻的玻璃化超低温保存法

先用含0.5 m01·L-1甘油和0.4 mol·L-1蔗糖的预处理液处理20min,然后用含30%蔗糖 15%乙二醇 10%二甲基亚砜 BBMG培养液的玻璃化液处理,在0℃下预冻60min后,将小球藻投入液氮.此法存活率较高,可达到60.14%,小球藻种质保存效果较好.通过试验初步建立了小球藻玻璃化法超低温保存的技术程序.     

冰冻保存小新月菱形藻的包埋-玻璃化法

采用包埋-玻璃化法对小新月菱形藻进行冰冻保存,探讨玻璃化溶液(PVS)配方、装载液浓度和装载时间、脱水时间以及洗涤方法对冰冻保存存活率的影响。结果表明:小新月菱形藻在0℃预冷后50%PVS2装载60min,100%PVS2脱水60min,1mol·L-1蔗糖梯度洗涤30min的条件下存活率最高,为74.1%。包埋-玻璃化法不需要特殊的冷冻设备,冰冻程序操作简单,在藻类种质的超低温保存中有较大的应用潜力。     

红芽芋茎尖的包埋玻璃化法超低温保存（英文）

对红芽芋(Colocasia esculenta var.cormosus ‘Hongyayu’)茎尖的包埋玻璃化法超低温保存技术进行了研究。茎尖从培养8周的试管苗上切下并包埋成海藻酸钙凝胶珠,并在MS+3.5 mg·L~(-1)6-BA+0.5 mg·L~(-1)IBA+0.1 mg·L~(-1)GA_3+0.3 mol·L~(-1)蔗糖的液体培养基中预培养24 h,随后用2 mol·L~(-1)甘油+0.4 mol·L~(-1)蔗糖的混合物在25℃下装载30 min,并用PVS2在25℃脱水20 min后将包埋的茎尖直接投入液氮保存。保存1 d后取出材料在40℃水浴快速复温3 min后,吸去冷冻管中PVS2,并用MS+3.5 mg·L~(-1)6-BA+0.5mg·L~(-1)IBA+0.1 mg·L~(-1)GA_3+1.2 mol·L~(-1)蔗糖的液体培养基在25℃洗涤3次,每次10 min。最后将茎尖接种于MS+3.5 mg·L~(-1)6-BA+0.5 mg·L~(-1)IBA+0.1 mg·L~(-1)GA_3的固体培养基上,暗培养3 d后转入正常的光周期中培养。红芽芋茎尖冻后成活率约为80%,其再生植株没有发生形态学的变化。这种包埋玻璃化法程序有望成为红芽芽茎尖超低温保存的常规方法。     

红花石蒜茎尖的玻璃化超低温保存

2～3mm的石蒜茎尖放在MS+0.4mol·L-1蔗糖的培养基上预培养5d,在25℃下用预处理液处理20min,接着用冰浴的玻璃化保护剂PVS2在冰浴中处理80min后,换新鲜PVS2并迅速投入液氮。液氮保存24h后,于40℃水浴中快速解冻2min,用MS+1.2mol·L-1蔗糖的液体培养基洗涤20min,滤纸吸干后接种到恢复培养基中,在25℃下暗培养7d后,转入光照强度为36μmol·m-2·s-1和光暗周期12/12h条件下培养。2周后的成活率最高可达90%,植株再生率达53%。     

包埋-玻璃化法冷冻保存湛江等鞭金藻的研究

采用包埋-玻璃化法冷冻保存湛江等鞭金藻(Isochrysis zhanjiangensis),探讨了装载液成分和浓度、装载时间、脱水时间、洗涤液浓度及洗涤时间对超低温保存后存活率的影响。结果表明在20℃50%PVS(PVS:30%甘油(GLY) 20%乙二醇(EG) 10%二甲基亚砜(DMSO),用f/2培养基定容)装载4.5h,0℃100%PVS脱水50min,冻存24h后取出冻存管并迅速投入40℃恒温水浴中快速化冻约3min,1.0mol/L山梨醇洗涤40min条件下,湛江等鞭金藻的存活率最高,为54%。与常规的两步法和包埋脱水法相比,包埋-玻璃化法简单、快速且存活率高,在藻类种质保存中有广阔的应用前景。     

Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.     

Droplet-vitrification methods for apical bud cryopreservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Rob.]

This study aimed to develop a cryopreservation protocol for the long-term preservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.)], an Andean crop with high fructooligosaccharide content in its tuberous roots. Initially, the cryopreservation protocol was developed using a yacon clone originated from Ecuador classified as ECU 41. Osmotic dehydration of apical buds (2–3 mm long) was carried out by assessing two plant vitrification solutions, PVS2 (15, 30, and 60 min) at 0 °C and PVS3 (30, 45, 60, and 75 min) at 22 °C. After cryopreservation, the apical buds were thawed and placed on MS medium?±?0.1 mg l^?1 N⁶-benzyladenine (BA). The survival rates ranged from 37 to 90% within all treatments, with those subjected to PVS2 and PVS3 for 60 min showing the highest survival rates on MS medium without BA (87 and 90%, respectively). At 12 weeks post cryopreservation, these treatments also provided the highest regrowth rates, both reaching 73% of normally growing (shooting, rooting) plantlets. Survival rates on MS?+?0.1 mg l^?1 BA regrowth medium reached up to 90%; however, regrowth into normally rooted plantlets did not exceed 67% post cryopreservation. The optimized protocols were then applied to 4 additional yacon clones originated from Bolivia and Peru, classified as BOL 22, BOL 23, PER 12, and PER 14. This resulted in survival and regeneration rates ranging between 79.7–94.1% and 66.3–75.4% respectively. Our study shows that optimal cryopreservation protocols for the long-term conservation of yacon can be based on both PVS2 and PVS3 vitrification solutions.

     

Cryopreservation of ex-vitro-grown Rosa chinensis ‘Old Blush’ buds using droplet-vitrification and encapsulation-dehydration

Axillary buds from greenhouse-grown plants of Rosa chinensis ‘Old Blush’ were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20–80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 °C gave the best regrowth rates (82–86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

Validation of cryopreservation protocols for plant germplasm conservation: a pilot study using Ribes L.

Uniformly applicable techniques for germplasm preservation are important to the international genetic resources community and validation of techniques among working genebanks will enable the integration of new technologies into plant genetic resources programs. Apical meristems from micropropagated plants of Ribes nigrum L. cv. Ojebyn and R. aureum cv. Red Lake were used to test three cryopreservation protocols (controlled freezing, plant vitrification solution no. 2 (PVS2) vitrification and encapsulation–dehydration) at the USDA-ARS National Clonal Germplasm Repository (NCGR), Corvallis, OR, USA and the University of Abertay Dundee (UAD), Scotland. Similar results were obtained with PVS2 vitrification at both locations but meristem regrowth varied greatly for the other techniques. Variable results between the locations were noted for controlled freezing and were largely attributed to differences in ice crystal initiation by the controlled rate freezers. Low survival of `Red Lake' at UAD with all three techniques was attributed to poorly performing shoot cultures. Attention to protocol details is important for limiting variation between locations and step by step instructions for procedures and solution preparation aided protocol standardization. These studies suggest that source plant status, cryogenic facilities, and culture conditions may be the most likely causes of variation when validating cryopreservation methodologies in different locations. However, in-house optimization of standard procedures offers considerable potential in ensuring that cryopreservation methodologies can be transferred between international laboratories.     

Cryopreservation of wild Shih (<Emphasis Type="Italic">Artemisia herba</Emphasis>-<Emphasis Type="Italic">alba</Emphasis> Asso.) shoot-tips by encapsulation-dehydration and encapsulation-vitrification

Artemisia herba-alba, called Shih is a medicinal herbal plant found in the wilds. The biodiversity of this plant is heavily subjected to loss because of heavy grazing, land cultivation and collection by people to be used in folk medicine. In the current study, two cryopreservation dependent techniques to conserve the shoot-tips of in vitro grown Shih were evaluated: encapsulation- dehydration and encapsulation- vitrification. Shoot-tips of Shih were encapsulated into sodium-alginate beads. In encapsulation- dehydration, the effect of sucrose concentration (0.5, 0.75 or 1.0 M) and dehydration period (0, 2, 4 or 6 h) under sterile air-flow on survival and regrowth of encapsulated shoot tips were studied. Maximum survival (100%) and regrowth (27%) rates were obtained when encapsulated unfrozen Artemisia herba-alba shoot tips were pretreated with 0.5 M sucrose for 3 days without further air dehydration. After cryopreservation the highest survival (40%) and regrowth (6%) rates were achieved when Artemisia herba-alba shoot tips were pretreated with 1.0 M sucrose for 3 days without further air dehydration. Viability of Artemisia herba-alba shoot tips decreased with increased dehydration period. In encapsulation-vitrification, the effect of dehydration of encapsulated Artemisia herba-alba shoot tips with 100% PVS2 for various dehydration durations (10, 20, 30, 60 or 90 min) prior to freezing was studied. After cryopreservation the dehydration of encapsulated and vitrified shoot tips with 100% PVS2 for 30 min resulted in 68% survival and 12% regrowth rates. Further conservation techniques must be evaluated to increase both survival and regrowth percentages.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.     

Survival of mint shoot tips after exposure to cryoprotectant solution components

Many plant species can be cryopreserved by treating shoot tips with complex cryoprotectant solutions before rapidly cooling them to liquid nitrogen temperatures. Plant vitrification solution 2 (PVS2), a commonly selected cryoprotectant, can be lethal with extended exposure times. To determine potentially toxic combinations, we have exposed mint shoot tips to one-, two-, three-, and four-component solutions of PVS2 chemicals (30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 M sucrose) at 0 and 22 degrees C. Overall, solution exposures at 22 degrees C were more damaging than exposures at 0 degree C. Solutions with glycerol, particularly in combination with ethylene glycol and dimethyl sulfoxide, were also damaging. Cryoprotectant solutions PGluD (10% PEG8000, 10% glucose, and 10% dimethyl sulfoxide) and PVS3 (50% glycerol, 50% sucrose) were less damaging than PVS2 at 22 degrees C. When plant cryoprotectants are characterized on a toxicological and biophysical basis, less damaging cryoprotectant solutions could be developed.