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+0.75 mol·L^(-1)蔗糖的液体培养基中25℃下预培养5 d后,放于干硅胶上无菌干燥5 h,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1 d后,茎尖在40℃水浴中化冻2~3 min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+1 mg·L^(-1)6-BA+0.05 mg·L^(-1)NAA+0.75 mol·L^(-1)蔗糖的液体培养基中25℃预培养3 d,用2 mol·L^(-1)甘油+0.4 mol·L^(-1)蔗糖装载液25℃装载90 min并再用PVS2在0℃下处理120 min后直接投入液氮。保存1 d后,取出材料在37℃水浴中化冻2~3 min,并用MS+1 mg·L^(-1)6-BA+0.05 mg·L^(-1)NAA+1.2 mol·L^(-1)蔗糖的液体培养基进行10 min的洗涤后转入MS+1 mg·L^(-1)6-BA+0.05 mg·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。
Shoot tips of A. membranaceus excised from in vitro-grown axillary bud were encapsulated in calcium-algi- nate heads. Subsequently, shoot tips were precultured in liquid MS medium enriched with 0.75 mol. L-1 sucrose for 5 d at 25 ℃ and then desiccated aseptically on dried silica gel for 5 h to a water content of 23.1% （fresh weight ba- sis） prior to immersion in liquid nitrogen （LN） for 1 d. After rewarming at a 40 ℃ water bath for 2-3 min and trans- ferred 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-vitrifi- cation has also been developed. Excised shoot tips were firstly encapsulated into alginate-gel beads and then precul- tured in liquid MS medium containing 1 mg. L-1 6-BA, 0.05 mg. L-1 NAA and 0. 75 mol, L -1 sucrose at 25 ℃ for 3 d. After loading for 90 min with a mixture of 2 mol.L-1 glycerol and 0. 4 mol. L-1 sucrose at 25 ℃, shoot tips were de- hydrated with PVS2 for 120 min at 0 ℃ prior to direct immersion in liquid nitrogen for 1 d. After rapidly thawing at a 37 ℃ water bath for 2-3 min, shoot tips were washed for 10 min with liquid MS medium supplemented with 1 mg. L -1 6-BA, 0. 05 mg. L-1 NAA and 1.2 mol. L-1 sucrose at 25 % and then post-cultured on solid MS medium supplemen-ted with 2 mg- L-1 6-BA, 0.05 mg. 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 con- trol plants. Thus, this encapsulation-dehydration and encapsulation-vitrification technique appears promising as a routine method for the cryopreservation of shoot-tips of A. membranaceus.
出处
《植物分类与资源学报》
CAS
CSCD
北大核心
2015年第6期767-778,共12页
Plant Diversity
基金
The National Natural Science Fund in China(31360072)
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
Cryopreservation
Encapsulation-dehydration
Encapsulation-vitrification
Astragalus membranaceus （ Fisch. ） Bge.
Shoot tips
Germplasm conservation
