浙江大学学报（农业与生命科学版）2025，Vol.51Issue(1)：80-88,9.DOI:10.3785/j.issn.1008-9209.2024.09.241
郁金香叶肉原生质体的分离纯化与瞬时转化方法
A method for isolation,purification and transient transformation of mesophyll protoplasts from tulip(Tulipa gesneriana)
摘要
Abstract
Protoplast isolation and purification are influenced by plant genotype and extraction conditions.Establishing a system for the isolation,purification,and transient transformation of tulip(Tulipa gesneriana)protoplasts is an effective way to explore the mechanism of tulip growth and development and conduct studies of somatic cell fusion.In the present study,we established a polyethylene glycol-calcium ion(PEG-Ca2+)mediated transient transformation system for tulip protoplasts by the leaves of the tulip variety'Dow Jones',which depended upon comparisons of different pre-treatments of leaves,osmotic pressures,and enzymatic hydrolysis durations.The results showed that the most suitable material for protoplast extraction was 3-week-old leaves.The middle part of each leaf was cut into 1.0 cm×2.0 cm pieces and then the upper and lower epidermis of the leaf pieces were peeled off.The enzymatic mixture was composed of 1.5%cellulase R-10,0.75%macerozyme R-10,10 mmol/L 2-morpholinoethanesulphonic acid(pH=5.7),0.4 mol/L D-mannitol,20 mmol/L KCl,10 mmol/L CaCl2,0.1%bovine serum albumin and 1.5 mmol/L β-mercaptoethanol.The optimal enzymatic hydrolysis duration was 6 h.Under the optimal extraction conditions,there ware certain differences in protoplast yield among different varieties.The protoplast yield of'Dow Jones'was greater than that of'Verandi'and'World's Favorite',with 8.05×105 protoplasts per gram of fresh leaves.The p35S::GFP vector was transfected into'Dow Jones'protoplasts using PEG-Ca2+mediated transient transformation,and the expression of green fluorescent protein was observed using confocal laser scanning microscope,with a transformation efficiency of 33.2%.This study provides new technical support for the functional research on tulip genes and the cultivation of new varieties.关键词
郁金香/原生质体/聚乙二醇-钙离子/瞬时转化Key words
Tulipa gesneriana/protoplasts/polyethylene glycol-calcium ion(PEG-Ca2+)/transient transformation分类
农业科技引用本文复制引用
霍信屹,陈欣晨,赵慧敏,向林,产祝龙,王艳平..郁金香叶肉原生质体的分离纯化与瞬时转化方法[J].浙江大学学报（农业与生命科学版）,2025,51(1):80-88,9.基金项目
国家自然科学基金项目(32170372) （32170372）
湖北省重点研发计划项目(2023BBB139) （2023BBB139）
江西省"双千计划"项目(S2021DQKJ2885). （S2021DQKJ2885）
