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植物种质的玻璃化超低温保存 总被引:19,自引:0,他引:19
植物种质的玻璃化超低温保存技术已受到广泛重视。玻璃化法主要由装载、玻璃化保护液脱水、降温、复温、洗涤这5个环节构成。目前已对百余种植物进行过玻璃化冻存研究,但主要应用于高等植物,而用该法保存藻类获得成功的报道很少。将玻璃化法用于某些藻类种质的冻存将会有广阔的应用前景。 相似文献
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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。 相似文献
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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。 相似文献
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拟南芥悬浮细胞系的玻璃化法超低温保存 总被引:6,自引:1,他引:5
悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。 相似文献
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切花百合离体茎尖玻璃化法超低温保存研究 总被引:3,自引:0,他引:3
以切花百合西伯利亚试管苗离体茎尖为试材,通过正交设计试验对预培养培养基中蔗糖浓度、预培养时间和PVS2处理时间等影响超低温保存存活率的主要因素进行了分析,初步建立了切花百合种质玻璃化法超低温保存的技术方案。通过形态观察、可溶性蛋白和同工酶检测,冻存前后材料的遗传稳定性没有发生改变,表明该方法对切花百合的种质保存具有较强的实用意义。 相似文献
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衣藻细胞玻璃化超低温保存技术的研究 总被引:5,自引:1,他引:4
本研究以衣藻为材料,探讨其玻璃化超低温保存的条件和方法,结果表明,衣藻经含0.25mol/L蔗糖溶液的TAP培养基预培养一天后,在玻璃化冷冻保护剂中脱水5分钟,直接投稿液氮,48小时后快速化冻,去保护剂并用含0.5mol/L蔗糖溶液的TAP培养基境培养一天,再转到ATP培养基暗培养一天,最后置光照条件下恢复培养,其存活率可达31.45%,恢复培养后衣藻细胞的生长规律与未冻存的衣藻相一致。 相似文献
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以江西铅山红芽芋胚性愈伤组织为材料,研究各种因素对其玻璃化法超低温保存的影响。结果表明:江西铅山红芽芋胚性愈伤组织玻璃化法超低温保存较佳的预培养条件为0.3mol·L-1蔗糖预培养3d,较佳的60%PVS2装载时间为20min,较佳的100%PVS2脱水条件为25℃脱水30min,较佳的化冻温度为40℃,较佳的洗涤液蔗糖浓度为1.2mol·L-1,较佳的冻后培养条件为暗培养7d再转到光周期中培养。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70%。红芽芋胚性愈伤组织冻后再生苗没有发生形态学、生理学和细胞学的变异。 相似文献
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该研究通过对脱水时间和化冻温度的探索,检验了包埋玻璃化法在超低温保存湿润生境中苔藓的可能性。结果表明:卵叶泥炭藓无菌苗在4℃条件下预培养3d后,在0℃用60% PVS_2装载30min,PVS_2脱水60min后迅速投入液氮保存,24h后用40℃水浴快速化冻2min再培养,成活率可达42.41%,且再生植株与常温状态下的植株形态指标没有显著性差异。研究认为,包埋玻璃化法超低温保存湿润环境中生长的苔藓植物是可行的。 相似文献
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Ex situ conservation of plant germplasm using biotechnology 总被引:6,自引:0,他引:6
Conservation of plant genetic resources attracts more and more public interest as the only way to guarantee adequate food supplies for future human generations. However, the conservation and subsequent use of such resources are complicated by cultural, economical, technical and political issues. Over the last 30 years, there have been significant increases in the number of plant collections and in accessions in ex situ storage centres throughout the World. The present review is of these ex situ collections and the contribution biotechnology has made and can make to conservation of plant germplasm. The applications and limitations of the new, molecular approaches to germplasm characterization are discussed.
In vitro slow growth is used routinely for conserving germplasm of plants such as banana, plantain, cassava and potato. More recently, cryopreservation procedures have become more accessible for long-term storage. New cryopreservation techniques, such as encapsulation-dehydration, vitrification and desiccation, lengthen the list of plant species that can not only tolerate low temperatures but also give normal growth on recovery. Extensive research is still needed if these techniques are to be fully exploited.V.M. Villalobos is with the Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalia, 00100 Rome, Italy. F. Engelmann is with the International Plant Genetic Resources Institute (IPGRI), Via delle Sette Chiese 142, 00145 Rome, Italy. 相似文献
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Natacha Coelho María Elena González-Benito Anabela Romano 《Acta Physiologiae Plantarum》2014,36(12):3333-3336
Tuberaria major is an endangered endemic species from the Algarve, in the south of Portugal. We investigated two techniques for the cryopreservation of T. major shoot tips, namely vitrification and encapsulation-dehydration. Before the cryopreservation trials, shoot tips were precultured for 1 day on liquid Murashige and Skoog (MS) medium containing 0.3 M sucrose. For the vitrification method, shoots tips were exposed for 0, 30, 60, 90 and 120 min to plant vitrification solution 2 (PVS2). As for the encapsulation-dehydration method, shoot tips were dried inside a laminar air flow cabinet for 0, 1, 2, 3, 4, 5 and 6 h at room temperature. The highest regrowth percentages were approximately 60 and 67 % for vitrification and encapsulation-dehydration, respectively. The best times were 60 min exposure to PVS2 for vitrification and 3 h desiccation for encapsulation-dehydration. Though these are preliminary results, the use of the cryopreservation techniques tested here proved to be an important asset in the conservation of this endangered species and will complement the conservation strategies previously developed. 相似文献
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The aim of this study was to compare the genetic stability of chrysanthemum (cv. Pasodoble) apices cryopreserved using two different methods: encapsulation-dehydration and vitrification. The assessment of the genetic stability was developed using RAPDs markers. Assessment of stability was evaluated in pot-cultivated mother plants (from which buds were excised for micropropagation), in shoots (leave tissue) from which apices were extracted for cryopreservation, and in shoots regenerated from cryopreserved apices 30 days after recovery and after further 3 months in culture. Throughout the process the origin of the apices (in vitro-shoot from which they were excised) was recorded. Twenty one regenerants cryopreserved by vitrification and 25 by encapsulation-dehydration were assessed. Only one cryopreserved regenerant from the encapsulation-dehydration method showed a different band pattern. These results support the necessity of monitoring the genetic stability of the regenerants obtained after cryopreservation, as this is a very useful technique for the conservation of plant genetic resources. 相似文献
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Maria Teresa Gonzalez-Arnao Ana Panta William M. Roca Roosevelt H. Escobar Florent Engelmann 《Plant Cell, Tissue and Organ Culture》2008,92(1):1-13
Shoot-tips and somatic embryos are the explants of choice for the in vitro long-term storage of ex situ plant genetic resources
in liquid nitrogen. Cryopreservation of organized structures has significantly progressed, especially for species of tropical
origin, with the development of several vitrification-based procedures such as encapsulation-dehydration, vitrification and
droplet-vitrification approaches. They have allowed improvements in survival and recovery after cryopreservation compared
with conventional crystallization-based protocols, proving their effectiveness for large scale application with embryos and
shoot-tips of different plants. This review addresses the main physical and technological aspects involved in plant cryopreservation
methods, illustrating the development of research with three cases: citrus, cassava and potato. These studies demonstrate
how cryopreservation strategies are increasingly applied for their successful employment in the genebanks. 相似文献
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Maria Dolores Arias Padrò Andrea Frattarelli Alessandra Sgueglia Emiliano Condello Carmine Damiano Emilia Caboni 《Plant Cell, Tissue and Organ Culture》2012,108(1):167-172
Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration,
alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations
(0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification,
apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration,
treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid
nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification
and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant
re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day
sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel. 相似文献
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包埋脱水法是植物材料超低温保存的新技术,从1990年至今,已有30多篇文献报道。本文介绍了包埋脱水法的研究历史、技术要点和主要优点。 相似文献
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Camille Le Bras Pierre-Henri Le Besnerais Latifa Hamama Agnès Grapin 《Plant Cell, Tissue and Organ Culture》2014,116(2):235-242
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. 相似文献