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The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices (Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species. 相似文献
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柿休眠芽茎尖玻璃化法超低温保存及植株再生 总被引:15,自引:2,他引:15
对柿 (DiospyroskakiThunb .)休眠芽茎尖进行了玻璃化法超低温保存及植株再生研究 ,结果表明 ,1.5~2 .0mm茎尖在含有 0 .3、0 .5和 0 .7mol·L-1蔗糖的改良MS培养基 (KNO3 和NH4NO3 减半 )上预培养各 1d ,室温(2 0℃ )下装载液 (2mol·L-1甘油 ,0 .4mol·L-1蔗糖 )停留 2 0min ,0℃下玻璃化液 (PVS2 或PVS3 )处理 30~ 4 0min ,投入液氮保存 1d后 ,4 0℃水浴化冻 1min ,含蔗糖 1.2mol·L-1的改良MS培养基洗涤 2 0min ,接种于含 0 .2mg·L-1CPPU、1mg·L-1BA、0 .0 5mg·L-1IAA、5 0 0mg·L-1可溶性PVP、30g·L-1蔗糖和 7g·L-1琼脂的改良MS培养基 (再生培养基 )表面的滤纸上 ,暗处理 1d后转移到新鲜的上述再生培养基中 ,暗培养 1周后转到正常光下 ,成活率高达 70 .2 % ,再生植株生长和分化正常。本研究结果为柿种质的长期保存提供了新途径。 相似文献
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四倍体刺槐试管苗玻璃化研究 总被引:1,自引:0,他引:1
[目的]探索批量组培快繁中的玻璃化的防止方法。[方法]以四倍体刺槐为材料,通过大量规范化系统试验研究试管苗玻璃化的影响因子。[结果]硝态氮与氨态氮处理间差异不显著。培养基中加入水解乳蛋白后玻璃苗减少,但污染率一直很高。棉塞、纱布封口对减少玻璃苗有明显作用。使用KT时玻璃化有所减轻,但繁殖系数只有1.5;使用6-BA时玻璃化苗比例较高,但未发生玻璃化的瓶苗繁殖系数高于3.5。蔗糖和琼脂浓度都可以改变玻璃化频率,其中蔗糖的作用更为明显,但它降低了生长速度。减少玻璃化的温度和光照的最佳组合是温度23℃,光照时间14 h/d。[结论]该研究为防止四倍体刺槐试管苗的玻璃化提供了一些可行的措施。 相似文献
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香蕉茎尖的玻璃化法超低温保存及其植株再生 总被引:12,自引:0,他引:12
以香蕉(Musa spp. ) 为试材, 对其离体培养茎尖玻璃化法超低温保存影响因素进行研究。结果表明, 不定芽在MS + 3.0~5.0 mg/L 6-BA + 0.1 mg/L NAA的培养基上分化较好。香蕉茎尖超低温保存较佳体系是: 2.0~3.0 cm的茎尖在含0.4 mol/L蔗糖培养基上预培养2 d, 剥取带1~2个叶原基的茎尖(长1.0~1.5 mm) , 室温(25℃) 下装载液(MS + 2 mol/L甘油+ 0.4 mol/L蔗糖) 装载20~30 min, 然后用玻璃化溶液( PVS2 ) 于0℃下处理40 min, 换1次PVS2后迅速投入液氮。保存至少1 h后, 在40℃水浴中化冻90 s, 用1.2 mol/L蔗糖培养液洗涤2次, 每次10 min, 然后转入含0.3 mol /L蔗糖的MS培养基上,暗培养10~15 h后转移到含0.5 mg/L 62BA的MS培养基中, 暗培养1周后转移到正常光下, 3个香蕉品种(巴西蕉、广东香蕉2 号、广东粉蕉1 号) 的成活率分别为75.9%、40.0%和69.6% , 再生率分别为63.4%、35.0%和63.4%。再生植株生长和分化正常, 生根后可移栽成活。 相似文献
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Bruna R. Curcio Melba O. Gastal Gabriel R. Pereira Carine D. Corcini Fernanda C. Landim-Alvarenga Severo S. Barros Carlos E.W. Nogueira João Carlos Deschamps Eduardo L. Gastal 《Journal of Equine Veterinary Science》2014
Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes. 相似文献
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