萱草原生质体制备体系的优化  

Optimization of Protoplast Preparation System of Hemerocallis fulva

作　　者：牛晓茹 景欢欢 田宁 郑硕理 陈白冰[3] 黄程前[3] 陈己任[2,4] NIU Xiaoru;JING Huanhuan;TIAN Ning;ZHENG Shuoli;CHEN Baibing;HUANG Chengqian;CHEN Jiren(School of Landscape Architecture and Art Design Forest,Hunan Agricultural University,Changsha 410128,China;College of Horticulture,Hunan Agricultural University,Changsha 410128,China;Hunan Botanical Garden and Flower Research institute,Changsha 410116,China;Hunan Province Middle Subtropical High Quality Flower and Tree Breeding and Utilization Engineering Technology Center,Changsha 410128,China)

机构地区：[1]湖南农业大学风景园林与艺术设计学院,湖南长沙410128 [2]湖南农业大学园艺学院,湖南长沙410128 [3]湖南省森林植物园花卉研究所,湖南长沙410116 [4]湖南省中亚热带优质花木繁育与利用工程技术中心,湖南长沙410128

出　　处：《湖南生态科学学报》2022年第4期60-68,共9页Journal of Hunan Ecological Science

基　　金：湖南省林业科技攻关与创新(XLKY202212)。

摘　　要：【目的】萱草是集药用、食用和观赏价值于一身的重要经济作物。通过原生质体制备条件及其参数的优化,建立高效的萱草原生质体制备体系,为通过体细胞杂交获取的优异种质资源奠定基础。【方法】以萱草品种‘贵妇’叶片和花瓣为材料,分别探讨纤维素酶浓度、离析酶浓度、酶解时间、渗透压调节剂种类及浓度、离心转速等因素对原生质体的产量和活力的影响。【结果】萱草叶片在含有0.5%纤维素酶R-10,0.75%离析酶R-10的酶解液中,酶解6 h酶解效果最好,原生质体产量达4.25×10^(5)个/克,活力达82.79%;萱草花瓣在含有1.5%纤维素酶R-10,0.5%离析酶R-10的酶解液中,酶解4 h得到的原生质体产量最高,达4.33×10^(5)个/克,活力为82.53%。叶片和花瓣均为使用0.4 mol/L山梨醇作为渗透压调节剂时,原生质体产量最高。【结论】以萱草叶片为材料的原生质体制备最佳方案为:0.5%纤维素酶R-10+0.75%离析酶R-10+0.4 mol/L山梨醇,酶解时间为6 h;以花瓣为材料的原生质体制备最佳方案为:1.5%纤维素酶R-10+0.5%离析酶R-10+0.4 mol/L山梨醇,酶解时间为4 h。在原生质体纯化时,叶片及花瓣最佳离心转速均为800 r/min。【Objective】Hemerocallis fulva is an important economic crop that combines medicinal value,edible value and ornamental value.Establish an efficient H.fulva protoplast preparation system by optimizing the protoplast preparation conditions and its parameters,and to lay the foundation for the use of protoplast fusion technology to create somatic hybrid superior germplasm resources.【Methods】To explore the optimal conditions for the preparation of H.fulva protoplasts,this study was conducted to investigate the effects of cellulase concentration,dissociative enzyme concentration,enzymatic digestion time,osmotic pressure regulator type and osmotic pressure size,centrifugal speed concentration on the yield and viability of protoplasts with the H.fulva variety‘Guifu’leaves and petals as materials,respectively.【Result】H.fulva leaves were hydrolyzed with 0.5%cellulase R-10 and 0.75%macerozyme R-10 for 6 hours.The yield of protoplasts was 4.25×10^(5) pieces/g and the activity of those was 82.79%.Using H.fulva petals as materials,approximately 4.33×10^(5) pieces/g protoplasts was yield and 82.53%of them kept active in the hydrolysate liquid containing 1.5%cellulase R-10 and 0.5%macerozyme R-10 for 4 hours.Protoplast production was highest when both leaves and petals were in osmoregulator leaves and petals were used as 0.4 mol/L sorbitol as osmoregulator.【Conclusion】The best protocol for protoplasmic preparation of H.fulva leaf was 0.5%cellulase R-10+0.75%macerozyme R-10+0.4 mol/L sorbitol for 6 hours.The optimal protocol for the preparation of protoplasts from petals was 1.5%cellulase R-10+0.5%macerozyme R-10+0.4 mol/L sorbitol,with an enzymatic digestion time of 4 hours.When the protoplasts were purified,the optimal centrifugal speed of leaf and petal was 800 r/min.

关 键 词：萱草 叶片 花瓣 原生质体 山梨醇 

分 类 号：S682[农业科学—观赏园艺]