Preparation and Regeneration Method of Phytophthora infestans Protoplast in Potato

doi:10.13560/j.cnki.biotech.bull.1985.2018-0151

Abstract

Abstract: For better understanding the pathogenicity of Phytophthora infestans,a highly efficient system of preparing and regenerating protoplast should be established. In this study,two lyases,Driselase and Cellulase R-10,were used to prepare protoplasts. The effects of different hyphal age,lyase,lyase concentration,and enzymolysis time on the protoplast preparation were discussed. The optimized method was established with the 18-day-old mycelia,the yield of protoplast reached 9.5 × 104 cells/g after 60 min treatment with 10 mg/mL Driselase. The protoplast regeneration rate was up to 3.1% in rye V8 culture medium supplemented with 0.1 mol/L calcium chloride and 0.4 mol/L mannitol as the osmotic stabilizer.

Key words: potato, Phytophthora infestans, protoplast, preparation and regeneration

WANG Wei-wei, XIAO Yan, ZHANG Yi-xi, LIU Jing, TANG Wei, LI Can-hui. Preparation and Regeneration Method of Phytophthora infestans Protoplast in Potato[J]. Biotechnology Bulletin, 2018, 34(4): 77-82.

References

[1] 谢丛华. 马铃薯产业的现状与发展[J]. 华中农业大学学报:社会科学版, 2012, 97(1):1-4.
[2] 胡同乐, 曹克强. 马铃薯晚疫病预警技术发展历史与现状[J]. 中国马铃薯, 2010, 24(2):114-119.
[3] Judelson HS, Tyler BM, Michelmore RW.Transformation of the oomycete pathogen Phytophthora infestans involves chromatin alterations[J]. Molecular Plant-Microbe Interactions, 1991, 4(6):602-607.
[4] Judelson HS, Coffey MD, Arredondo FR, et al.Transformation of the oomycete pathogen Phytophthora megasperma f. sp. glycinea occurs by DNA integration into single or multiple chromosomes[J]. Current Genetics, 1993, 23(3):211-218.
[5] Mort-Bontemps M, Fevre M.Transformation of the oomycete Saprolegnia monoica to hygromycin-B resistance[J]. Current Genetics, 1997, 31(3):272-275.
[6] Van West P, Kamoun S, Van’t Klooster J, et al.Internuclear Gene Sliencing in Phytophthora infestans[J]. Molecular Cell, 1999, 3(3):339-348.
[7] Bottin A, Larche L, Villalba F, et al.Green fluorescent protein(GFP)as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae[J]. FEMS Microbiol Lett, 1999, 176(1):51-56.
[8] Siammour A, Mauchmani B, Mauch F.Quantification of induced resistance against Phytophthora species expressing GFP as a vital marker:β-aminobutyric acid but not BTH protects potato and Arabidopsis from infection[J]. Molecular Plant Pathology, 2003, 4(4):237-248.
[9] Riedel M, Calmin G, Belbahri L, et al.Green Fluorescent Protein(GFP)as a Reporter Gene for the Plant Pathogenic Oomycete Phytophthora ramorum[J]. Journal of Eukaryotic Microbiology, 2009, 56(2):130-135.
[10] Weiland JJ.Transformation of Pythium aphanidermatum to geneticin resistance[J]. Current Genetics, 2003, 2(2):191-199.
[11] 陈孝仁, 张正光, 等. 大豆疫霉菌原生质体制备及再生菌株的生物学性状[J]. 南京农业大学学报, 2000, 28(4):45-49.
[12] 付丽. 辣椒疫霉果胶裂解酶基因克隆及功能研究[D]. 泰安:山东农业大学, 2012.
[13] 刘士旺, 王政逸, 郭泽建. 绿色木霉的原生质体制备与转化条件[J]. 农业生物技术学报, 2004, 12(1):96-101.
[14] 郑小波. 疫霉菌及其研究技术[M]. 北京:中国农业出版社, 1997:81-83.
[15] Mcleod A, Fry BA, et al.Toward Improvements of Oomycete Transformation Protocols[J]. Journal of Eukaryotic Microbiology, 2008, 55(2):103-109.
[16] Yi SY, Kim YJ, Hwang BK.Protoplast Formation and Regeneration from Mycelia of Phytophthora capsici[J]. The Korean Journal of Mycology, 1993, 21(1):1-8.
[17] 龙昊, 汪天虹, 刘萱, 等. 瑞氏木霉pyrG基因缺陷型菌株筛选以及GnTI和VHb基因表达研究[C]. 中国青年学者微生物遗传学学术研讨会文集, 2006.
[18] 张建萍, 朱凯, 杨爽, 等. 稗草生防潜力菌(Helminthosporium gramineum f. sp. echinochloae)的原生质体制备和再生[J]. 浙江农业学报, 2010, 22(1):14-19.
[19] 黄玉茜, 梁春浩, 陈捷. 绿色木霉菌T23原生质体的制备与再生[J]. 吉林农业大学学报, 2007, 29(1):24-27.
[20] 董磊. 致病疫霉Pi-PIPK-D8功能的研究与分析[D]. 呼和浩特:内蒙古农业大学, 2015.
[21] 赵竟男, 江梅, 苏晓庆. 灭蚊真菌贵阳腐霉原生质体的制备和再生研究[J]. 贵阳医学院学报, 2008, 33(2):111-114.
[22] 江涛. 大豆疫霉营养生理及慢性生长菌株生物学特性研究[D]. 合肥:安徽农业大学, 2012.
[23] 李立凤, 李小梅, 张景涛. 辣椒疫霉菌生长和产孢条件的研究[J]. 东北农业大学学报, 2010, 41(10):139-142.
[24] 雷虹, 曾伟民, 金忠斌, 等. 小白链霉菌(Streptomyces albulus)的原生质体制备研究[J]. 黑龙江大学:自然科学学报, 2007, 24(3):306-309.