八仙花萼片有色原生质体游离及应用

doi:10.13304/j.nykjdb.2021.0372

中国农业科技导报 ›› 2022, Vol. 24 ›› Issue (9): 50-57.DOI: 10.13304/j.nykjdb.2021.0372

八仙花萼片有色原生质体游离及应用

张盖天(), 祁惠, 杨锁宁, 褚志云, 袁素霞(), 刘春()

中国农业科学院蔬菜花卉研究所，北京 100081

收稿日期:2021-05-06 接受日期:2021-09-22 出版日期:2022-09-15 发布日期:2022-10-11
通讯作者: 袁素霞,刘春
作者简介:张盖天 E-mail：gaitianzhang@163.com
基金资助:
中央级公益性科研院所基本科研业务费专项(IVF-BRF2020021);中国农业科学院科技创新工程项目(CAAS-ASTIP-2020-IVFCAAS)

Isolation and Application of Sepal Colored Protoplast in Hydrangea macrophylla

Gaitian ZHANG(), Hui QI, Suoning YANG, Zhiyun CHU, Suxia YUAN(), Chun LIU()

Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2021-05-06 Accepted:2021-09-22 Online:2022-09-15 Published:2022-10-11
Contact: Suxia YUAN,Chun LIU

摘要/Abstract

摘要：

为建立八仙花（Hydrangea macrophylla）有色原生质体游离体系，以八仙花萼片为试验材料，分别对材料的预处理方式、酶液组分、酶解时间、纯化离心力等影响酶解的关键因素进行研究。结果表明，预处理方式选择分离花萼片上下表皮法，纤维素酶和离析酶水平分别为4%和0.4%（质量体积分数），甘露醇0.6 mol·L^-1，酶解液加热冷却后加入 0.1%（质量体积分数）BSA和10 mmol·L^-1 CaCl₂，酶解2 h，低温离心机4 ℃、1 426 r·min^-1离心5 min，为最优的八仙花萼片原生质体游离条件。利用所建的原生质体游离体系对4个不同品种的八仙花萼片进行有色原生质体游离，200× 镜下单视野内可获得13~35个有色的原生质体；对有色原生质体进行液泡的H⁺流测定，发现蓝色液泡较粉色液泡H⁺流的外排趋势明显，说明同一八仙花品种蓝色液泡的pH高于粉色液泡。

关键词: 八仙花, 有色原生质体游离, 液泡离子流

Abstract:

In order to establish isolation system of sepals colored protoplast in Hydrangeamacrophylla， sepals of Hydrangeamacrophylla were used as experimental materials. The factors influencing enzymolysis were analyzed， such as treated methods of sepals， the components of enzymatic hydrolysates， enzymolysis time， centrifugal speeds etc. The results showed that the optimal procedure of enzymolysis was followed with the toring epidermis of sepals， 4% （w/v） cellulase， 0.4% （w/v） macerozyme， 0.6 mol·L^-1 mannitol and 0.1% （w/v） BSA， 10 mmol·L^-1 CaCl₂， enzymolysis for 2 h， and 1 426 r·min^-1 centrifuge at 4 ℃. This system was used to isolate sepal colored protoplast of 4 Hydrangea macrophylla cultivars， and 13~35 protoplasts per microscope vision at 200× were obtained. H⁺ flux measurement showed that the H⁺ effluxing tendency of blue vacuoles was more obvious than that of pink vacuoles in the same cultivar， it indicated that pH in blue vacuoles was higher than that in pink vacuoles of same cultivar.

Key words: Hydrangea macrophylla, colored protoplast isolation, vacuole ion flux

中图分类号:

S682.1+9

张盖天, 祁惠, 杨锁宁, 褚志云, 袁素霞, 刘春. 八仙花萼片有色原生质体游离及应用[J]. 中国农业科技导报, 2022, 24(9): 50-57.

Gaitian ZHANG, Hui QI, Suoning YANG, Zhiyun CHU, Suxia YUAN, Chun LIU. Isolation and Application of Sepal Colored Protoplast in Hydrangea macrophylla[J]. Journal of Agricultural Science and Technology, 2022, 24(9): 50-57.

图/表 3

图1 单因素对有色原生质体酶解的影响A：预处理方式；B：酶水平；C：酶解时长；D：甘露醇浓度；E：酶液组分；F：离心速率；不同小写字母表示不同处理间差异在P<0.05水平显著。

Fig. 1 Effect of single foctor on enzymatic hydrolysisA： Material handling method； B： Enzymes concentration； C： Hydrolysis time； D： Mannitol concentration； E： Enzyme solution component； F： Centrifugal speed； different lowercase letters indicate significant differences between different treatments at P<0.05 level.

图2 不同品种八仙花花器官及原生质体游离示意图（Bar=50 μm）

Fig. 2 Schematic diagram of floral organs and protoplast dissociation of different species of Hydrangea （Bar=50 μm）

图3 ‘Baby blue’离子流注：正值为外排，负值为吸收。

Fig. 3 ‘Baby blue’ ion fluxNote：Positive values are efflux and negative values are influx.

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八仙花萼片有色原生质体游离及应用

Isolation and Application of Sepal Colored Protoplast in Hydrangea macrophylla

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