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Plant Diversity ›› 2015, Vol. 37 ›› Issue (06): 767-778.DOI: 10.7677/ynzwyj201515091

• 研究论文 • 上一篇    下一篇


 尹明华, 洪森荣   

  1. 上饶师范学院生命科学学院,江西 上饶334001
  • 收稿日期:2015-05-26 出版日期:2015-11-25 发布日期:2015-09-23
  • 基金资助:

    The National Natural Science Fund in China (31360072)

Encapsulation-Dehydration and Encapsulation-VitrificationBased Cryopreservation of in vitro-Grown Shoot-Tips of Medicinal Plant Astragalus membranaceus

 YIN  Ming-Hua, HONG  Sen-Rong   

  1. College of Life Sciences, Shangrao Normal University, Shangrao, Jiangxi 334001, China
  • Received:2015-05-26 Online:2015-11-25 Published:2015-09-23
  • Supported by:

    The National Natural Science Fund in China (31360072)


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


 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