从组织培养的葡萄属植物叶片中分离原生质体并产生愈伤组织

Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

机构信息

Department of Horticulture, University of Georgia, 30602, Athens, GA, USA.

出版信息

Plant Cell Rep. 1988 Dec;7(7):531-4. doi: 10.1007/BF00272749.

Abstract

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 μM 6-benzyladenine, 4.5 μM 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.

摘要

从葡萄（Vitis rotundifolia cv. Summit 和 V. vinifera cv. Cabernet Sauvignon）的无菌培养物的叶肉中分离原生质体。用于原生质体分离的有效酶是 Macerozyme R-10（0.5%和0.1%）和 Cellulase Onozuka R-10（1.0%和0.5%），分别用于 V. rotundifolia 和 V. vinifera。聚乙烯吡咯烷酮和 2-（N-吗啉基）乙磺酸是分离培养基中必不可少的。使用浮选/离心法纯化原生质体。在 Gamborg 的 B5 基础培养基中培养的 V. rotundifolia 原生质体，用 2.2 μM 6-苄基腺嘌呤、4.5 μM 2,4-二氯苯氧乙酸和 0.4%琼脂糖，在本研究尝试的条件下给出了最佳的平板效率。细胞分裂发生在 5 到 6 天内，一个月内可见微型愈伤组织形成。培养 6 周后，转移到液体培养基中的微型愈伤组织表现出活跃的愈伤组织生长。在这些条件下培养的 V. vinifera 原生质体也有类似的结果。