Confocal laser microscopic observation of the effects of culture environment on somatic embryo formation

共聚焦激光显微观察培养环境对体细胞胚胎形成的影响

• 批准号: 10460114
• 负责人: KURATA Kenji
• 金额: $ 5.18万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10460114/
• 关键词: somatic embryos; carrot; calcium; pH; confocal laser microscope; pH; fluo-3; Fura Red; レーシオイメージング法; Fura red; 共焦点レーザ顕微鏡

Concentrations of Ca^<2+> during carrot somatic embryogenesis was measured based on the ratiometric analysis of fluo-3 and Fra Red fluorescence using a confocal laser microscope. Similarly, pH was also ratiometrically measured using carboxy-SNARF-1 as a fluorescence probe, because Ca^<2+> -ratio varied with pH. The following points were elucidated.1. Changing the culture medium pH did not alter pH in pro-embryogenic cell masses (PEMs) nor in somatic embryos, but influenced the Ca^<2+>-ratio. Also the temperature affected the Ca^<2+>-ratio.2. Stable pH (about 6.8) was observed after transferring PEMs to embryo differentiation medium up to day 9 except for day 1, on which pH of 6.0 was observed, where day 0 is the transferring day.3. [Ca^<2+>] gradually decreased from 0.1 μmolL^<-1> (day 0) to below 0.04 μmolL^<-1> (day 5-7) and sharply increased to 0.4 μmolL^<-1> on day 9 on which globular embryos were first observed.4. In the case of cell masses which have lost embryogenic potential due to repeated subculturing, pH and [Ca^<2+>] were always below the values observed in the case of embryogenic cell masses. No prompt increase in [Ca^<2+>] was observed.

利用共聚焦激光显微镜，基于荧光素3（fluo-3）和弗拉红（Fra Red）荧光的比值分析，测定了胡萝卜体细胞胚胎发生过程中Ca^<2+>的浓度。类似地，还使用羧基-SNARF-1作为荧光探针以比率法测量pH，因为Ca^<2+> -比率随pH而变化。改变培养基的pH值不会改变原胚发生细胞团（PEM）和体细胞胚的pH值，但会影响Ca^<2+>-比率。温度也影响Ca^<2+>-比率。在将PEM转移到胚胎分化培养基中后，观察到稳定的pH（约6.8），直到第9天，除了第1天，在第1天观察到pH为6.0，其中第0天是转移日。[Ca在球形胚第一次孵化的第9天，[μ mol/L]从0.1 μ mol/<-1>L（第0天）逐渐降低到低于0.04 μ mol/<-1>L（第5-7天），并急剧增加到0.4 μ mol/<-1>L。在由于重复传代培养而失去胚胎发生潜能的细胞团的情况下，pH和[Ca^2+]总是低于在胚胎发生细胞团的情况下观察到的值。未观察到[Ca^&lt;2+&gt;]迅速增加。

项目成果

Ibaraki, Y., Kurata, K.: "Automation of somatic embryo production"Plant Cell Tissue and Organ Culture. 65. 179-199 (2001)

Ibaraki, Y., Kurata, K.：“体细胞胚胎生产的自动化”植物细胞组织和器官培养。

Ibaraki, Y., Hirashima, H., Kurata, K.: "Rario imaging of Ca^<++> in carrot somatic embryos"(発表予定).

Ibaraki, Y.、Hirashima, H.、Kurata, K.：“胡萝卜体细胞胚胎中 Ca^<++> 的 Rario 成像”（待提交）。

Shimazu, T., Kurata, K.: "Relationship between production of carrot somatic embryos and dissolved Oxygen concentration in liquid culture"Plant Cell Tissue and Organ Culture. 57. 29-39 (1999)

Shimazu, T., Kurata, K.：“胡萝卜体细胞胚胎的产生与液体培养物中溶解氧浓度之间的关系”植物细胞组织和器官培养。

Ibaraki Y.Hirashima,H.and Kurata,K.: "In vivo calibration of ratio imaging of calcium ion concentration within carrot somatic embryos using fluo-3 and Fura Red"(未定)(発表予定).

Ibaraki Y. Hirashima, H. 和 Kurata, K.：“使用 Fluo-3 和 Fura Red 对胡萝卜体细胞胚胎内钙离子浓度的比率成像进行体内校准”（待公布）。

Y.Ibaraki,K,Kurata: "Ca^<++> distribution during sonatic embryogenesis"Environ.Control in Biol.. (発表予定).

Y.Ibaraki,K,Kurata：“声波胚胎发生过程中的 Ca^<++> 分布”Environ.Control in Biol..（待提交）。