药用植物黄芪离体培养茎尖的包埋脱水法和包埋玻璃化法超低温保存

doi:10.7677/ynzwyj201515091

摘要/Abstract

摘要：

为找出一条黄芪种质长期包埋脱水法保存和包埋玻璃化法保存的程序，以来源于黄芪离体生长腋芽的黄芪茎尖并包埋成海藻酸钙珠。随后，在MS+075mol·L-1蔗糖的液体培养基中25℃下预培养5d后，放于干硅胶上无菌干燥5h，直至含水量达231% （以鲜重为基础）时将材料投入液氮保存。保存1d后，茎尖在40℃水浴中化冻2~3min并转入固体培养基上进行再生培养，2周后大约50%的茎尖可再生出芽。黄芪茎尖包埋玻璃化法超低温保存程序也被优化，同样包埋成海藻酸钙凝胶珠的茎尖在MS+1mg·L-1 6BA+005mg·L-1 NAA+075mol·L-1蔗糖的液体培养基中25℃预培养3d，用2mol·L-1甘油+04mol·L-1蔗糖装载液25℃装载90min并再用PVS2在0℃下处理120min后直接投入液氮。保存1d后，取出材料在37℃水浴中化冻2~3min，并用MS+1mg·L-1 6BA+005mg·L-1 NAA+12mol·L-1蔗糖的液体培养基进行10min的洗涤后转入MS+1mg·L-1 6BA+005mg·L-1 NAA的固体培养基上进行再生培养。茎尖的再生率接近80%。以上两种超低温保存方式均未造成再生植株形态学上的变化。因此，包埋脱水法和包埋玻璃化法两种常规方法对于黄芪茎尖超低温保存来说均具有重要的意义。

关键词: 超低温保存, 包埋脱水法, 包埋玻璃化法, 黄芪, 茎尖, 种质保存

Abstract:

Shoot tips of Amembranaceus excised from in vitrogrown axillary bud were encapsulated in calciumalginate beads. Subsequently, shoot tips were precultured in liquid MS medium enriched with 075mol·L-1 sucrose for 5d at 25℃ and then desiccated aseptically on dried silica gel for 5h to a water content of 231% (fresh weight basis) prior to immersion in liquid nitrogen (LN) for 1d. After rewarming at a 40℃ water bath for 2-3min and transferred to solid culture medium for shoot tip recovery. About 50% of cryopreserved shoottips grew into shoots within 2 weeks after plating. Cryopreservation of Astragalus membranaceus (Fisch.) Bge. shoot tips by encapsulationvitrification has also been developed. Excised shoot tips were firstly encapsulated into alginategel beads and then precultured in liquid MS medium containing 1mg·L-1 6BA, 005mg·L-1 NAA and 075mol·L-1 sucrose at 25℃ for 3d. After loading for 90min with a mixture of 2mol·L-1 glycerol and 04mol·L-1 sucrose at 25℃, shoot tips were dehydrated with PVS2 for 120min at 0℃ prior to direct immersion in liquid nitrogen for 1d. After rapidly thawing at a 37℃ water bath for 2-3min, shoot tips were washed for 10min with liquid MS medium supplemented with 1mg·L-1 6BA, 005mg·L-1 NAA and 12mol·L-1 sucrose at 25℃ and then postcultured on solid MS medium supplemented with 2mg·L-1 6BA, 005mg·L-1 NAA. The regeneration rate of shoot tips amounted to nearly 80%. Both of plantlets regenerated from cryopreserved shoot tips were morphologically uniform, which both showed as that of control plants. Thus, this encapsulationdehydration and encapsulationvitrification technique appears promising as a routine method for the cryopreservation of shoottips of Amembranaceus.

Key words: Cryopreservation, Encapsulation-dehydration, Encapsulation-vitrification, Astragalus membranaceus (Fisch.) Bge., Shoot tips, Germplasm conservation

中图分类号:

Q 943，S 632.3

尹明华, 洪森荣. 药用植物黄芪离体培养茎尖的包埋脱水法和包埋玻璃化法超低温保存[J]. Plant Diversity, 2015, 37(06): 767-778.

YIN Ming-Hua, HONG Sen-Rong. Encapsulation-Dehydration and Encapsulation-VitrificationBased Cryopreservation of in vitro-Grown Shoot-Tips of Medicinal Plant Astragalus membranaceus[J]. Plant Diversity, 2015, 37(06): 767-778.

参考文献

Ai PF, Lu LP, Song JJ, 2012. Cryopreservation of in vitrogrown shoottips of Rabdosia rubescens by encapsulationdehydration and evaluation of their genetic stability［J］. Plant Cell, Tissue and Organ Culture, 108: 381—387
Benson EE, Harding K, Smith H, 1989. Variation in recovery of cryopreserved shoottips of Solanum tuberosum exposed to different pre and postfreeze light regimes［J］. CryoLetters, 10: 323—344
Burritt DJ, 2008. Efficient cryopreservation of adventitious shoots of Begonia xerythrophylla using encapsulationdehydration requires pretreatment with both ABA and proline［J］. Plant Cell, Tissue and Organ Culture, 95: 209—215
Engelmann F, 2011. Encapsulationdehydration for cryopreservation: past, present and future［J］. Acta Horticulturae (ISHS), 908: 165—171
Erica EB, Keith H, 2012. Cryopreservation of shoot tips and meristems: An overview of contemporary methodologies［J］. Methods in Molecular Biology, 877: 191—226
Florence P, Engelmann F, Glaszmann JC, 1993. Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration［J］. Plant Cell Reports, 12: 525—529
Hong SR, Yin MH, 2012a. A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant Anemarrhena asphodeloides Bunge by vitrification［J］. Plant Cell, Tissue and Organ Culture, 109: 287—296
Hong SR, Yin MH, 2012b. Highefficiency encapsulationvitrification protocols for cryopreservation of embryogenic calli of oriental medicinal plant Anemarrhena asphodeloides Bunge. ［J］. CryoLetters, 33: 190—200
Hong SR, Yin MH, Ye LM et al., 2010. In Vitro germination experiment of seeds of Atractylodes macrocephala Koidz. and Astragalus membranaceus (Fish) Bge. ［J］. Hunan Agricultural Sciences, 5: 7—8, 11
Hu G, Gail BM, Shang L et al., 2012. Polysaccharides from astragali radix restore chemicalinduced blood vessel loss in zebrafish［J］. Vascular Cell, 4: 1—8
Huang CN, Wang JH, Yan QS et al., 1995. Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification［J］. Plant Cell Reports, 14: 730—734
Khoddamzadeh AA, Sinniah UR, Lynch P et al., 2011. Cryopreservation of protocormlike bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulationdehydration［J］. Plant Cell, Tissue and Organ Culture, 107: 471—481
Ma X, Bucalo K, Determann RO et al., 2012. Somatic embryog enesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species［J］. In Vitro Cellular & Developmental BiologyPlant, 48:324—334
MarcoMedina A, Casas JL, Elena GBM, 2010. Comparison of vitrification and encapsulationdehydration for cryopreservation of Thymus moroderi shoot tips［J］.CryoLetters, 31: 301—309
Maria DAP, Andrea F, Alessandra S et al., 2012. Cryopreservation of white mulberry (Morus alba L.) by encapsulationdehydration and vitrification［J］. Plant Cell, Tissue and Organ Culture, 108: 167—172
Martín C, Cervera MT, GonzálezBenito ME, 2011. Genetic stability analysis of chrysanthemum (Chrysanthemum × Morifolium Ramat) after different stages of an encapsulationdehydration cryopreservation protocol［J］. Journal of Plant Physiology, 168: 158—166
Matsumoto T, Sakai A, Yamada K, 1994. Cryopreservation of in vitrogrown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration［J］. Plant Cell Reports, 13:442—446
Mohanty P, Das MC, Kumaria S et al., 2012. Highefficiency cryopreservation of the medicinal orchid Dendrobium nobile Lindl［J］. Plant Cell, Tissue and Organ Culture, 109: 297—305
Murashige T, Skoog F, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures［J］. Plant Physiology, 15: 473—497
Nair DS, Reghunath BR, 2009. Cryoconservation and regeneration of axillary shoot meristems of Indigofera tinctoria (L.) by encapsulationdehydration technique［J］. In Vitro Cellular & Developmental BiologyPlant, 45: 565—573
Padro MDA, Frattarelli A, Sgueglia A et al., 2012. Cryopreservation of white mulberry (Morus alba L.) by encapsulationdehydration and vitrification［J］. Plant Cell, Tissue and Organ Culture, 108: 167—172
Paul H, Daigny G, SangwanNorreel BS, 2000. Cryopreservation of apple (Malus × domestica Borkh.) shoot tips following encapsulationdehydration or encapsulationvitrification［J］. Plant Cell Reports, 19: 768—774
Pawlowska B, Bach A, 2011. Cryopreservation by encapsulationdehydration of in vitro grown shoot buds of Rosa ‘New Dawn’ ［J］. Acta Horticulturae (ISHS), 908: 303—307
Ruzˇic＇, Vujovic＇, 2012. Cryopreservation in vitro of blackberry ‘Cacanska Bestrna’ shoot tips by encapsulation dehydration［J］. Acta Horticulturae (ISHS), 946: 55—60
Sakai A, Engelmann F, 2007. Vitrification, encapsulationvitrification and dropletvitrification: a review［J］. CryoLetters, 28: 151—172
Sakai A, Kobayashi S, Oiyama I, 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification［J］. Plant Cell Reports, 9: 30—33
Sarab AS, Rida AS, Mahmoud AK et al., 2012. Cryopreservation of wild Shih (Artemisia herbaalba Asso.) shoottips by encapsulationdehydration and encapsulationvitrification［J］. Plant Cell, Tissue and Organ Culture, 108: 437—444
SeChan K, Hee JK, MiHyun K, 2013. Effects of astragalus membranaceus with supple mental calcium on bone mineral density and bone metabolism in calciumdeficient ovariectomized rats［J］. Biological Trace Element Research, 151: 68—74
Shibli RA, AlJuboory KH, 2000. Cryopreservation of ‘Nabali’ olive (Olea europea L.) somatic embryos by encapsulationdehydration and encapsulationvitrification［J］. CryoLetters, 21: 357—366
Toshiro S, Sakai E, Koichiro S, 1995. Effect of rapid freezing and thawing on hardseed breaking in Astragalus mongholicus Bunge (Leguminosae) ［J］. Journal of Plant Physiology, 147: 127—131
Wang QC, Laamanen J, Uosukainen M et al., 2005. Cryopreservation of in vitrogrown shoot tips of raspberry (Rubus idaeus L.) by encapsulationvitrification and encapsulationdehydration［J］. Plant Cell Reports, 24: 280—288
Wang QC, zgur B, Li P et al., 2002. A simple and efficient cryopreservation of in vitrogrown shoot tips of‘Troyer’ citrange ［Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.］ by encapsulationvitrification［J］. Euphytica, 128: 135—142
Wang QC, Ron G, Nachman S et al., 2002. Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulationdehydration and subsequent plant regeneration［J］. Plant Science, 162: 551—558
Xue SH, Luo XJ, Wu ZH et al., 2008. Cold storage and cryopreservation of hairy root cultures of medicinal plant Eruca sativa Mill., Astragalus membranaceus and Gentiana macrophylla Pall. ［J］. Plant Cell, Tissue and Organ Culture, 92: 251—260
Yin MH, Cao ZL, 2011. The effects of KT and NAA on proliferation of Astragalus membranceus stem with a bud and its induction of callus and rooting［J］. Subtropical Plant Science, 40: 41—44

[1]	王艾平, 尹明华, 洪森荣. 红芽芋茎尖的包埋玻璃化法超低温保存[J]. Plant Diversity, 2015, 37(06): 801-812.
[2]	计明月, 张楠, 闫启, 文彬. 顽拗性竹柏种子的贮藏特性[J]. Plant Diversity, 2015, 37(01): 63-70.
[3]	洪森荣, 尹明华. 江西铅山红芽芋胚性愈伤组织的包埋干燥法超低温保存[J]. Plant Diversity, 2014, 36(04): 505-512.
[4]	文彬. 植物种质资源超低温保存概述[J]. Plant Diversity, 2011, 33(3): 311-329.
[5]	李德铢1、杨湘云1、Hugh W.Pritchard2. 种质资源保存的战略问题和面临的挑战[J]. Plant Diversity, 2011, 33(01): 11-18.
[6]	何明高1,2, 王瑞霞3, 宋希强2,4, 宋松泉3, 张如莲1. 束花石斛种子超低温保存的研究[J]. Plant Diversity, 2010, 32(4): 334-338.
[7]	李雅丽，张凯. 用农杆菌Ri诱导蒙古黄芪发根培养的研究[J]. Plant Diversity, 2005, 27(02): 204-210.
[8]	钱子刚杨耀文. 云南黄芪属（豆科）新资料[J]. Plant Diversity, 2003, 25(03): 1-3.
[9]	陈宗莲；侯岁稳；俞宏渊. 除虫菊的组织培养[J]. Plant Diversity, 1998, 20(03): 1-3.
[10]	吴玉虎. 青海黄芪属新分类群[J]. Plant Diversity, 1998, 20(01): 1-3.
[11]	钱子刚；陈介. 云南黄芪属二新种[J]. Plant Diversity, 1997, 19(03): 1-3.
[12]	王丽顾志建孙航. 青藏高原几种黄芪和棘豆植物核型的初步研究[J]. Plant Diversity, 1994, 16(01): 1-3.
[13]	钱子刚. 云南黄芪属新变种和新记录种[J]. Plant Diversity, 1992, 14(04): 1-3.
[14]	程治英张风雷兰芹英许再富陶国达. 桫椤的快速繁殖与种质保存技术的研究[J]. Plant Diversity, 1991, 13(02): 1-3.
[15]	李沛琼. 横断山区豆科新分类群和新组合[J]. Plant Diversity, 1989, 11(03): 1-3.