Chin Bull Bot ›› 2017, Vol. 52 ›› Issue (4): 511-519.doi: 10.11983/CBB16155

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Preparation of Protoplast and Establishment of Transient Expression System in Gerbera hybrida

Aihua Song1, Wenbin Zhang1, Shulan Sun1, Lingfei Li1,2*, Xiaojing Wang1   

  1. 1Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China
    2Key Laboratory of Southern Subtropical Plant Diversity, Fairy Lake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen 518004, China;
  • Received:2016-07-22 Accepted:2016-10-16 Online:2017-05-05 Published:2017-07-01
  • Contact: Li Lingfei
  • About author:

    # Co-first authors


Gerbera hybrida has been used as model plant to study the evolution and development of composite inflorescences. To establish an efficient protoplast isolation and transient transformation system in G. hybrida, leaves of cultivar ‘Shenzhen No. 5’ in vitro seedlings were used as materials and the effects of different concentrations of cellulase R10 and macerozyme were tested to screen a suitable preparation method. The best enzyme solution concentration consisted of cellulase R10 2.0% (w/v), macerozyme 0.3% (w/v), and 0.4 mol·L-1 mannitol and more than 2.2×107∙g-1 FW protoplasts with high activity were obtained after 4 h treatment under 25°C condition. Subcellular localization and protein-protein interaction assays were performed using these protoplasts and revealed 80% transformation rate. Collectively, this study established an efficient protoplast isolation and transient assay system that can provide an important platform to facilitate gene function studies in G. hybrida.

Figure 1

Isolation efficiency of protoplast in Gerbera hybrid^(A) Leaves isolated from two-month-old in vitro seedlings (Bar=1 cm); (B) Protoplast yields under different cellulase R10 concentrations with 0.3% macerozyme; (C) Protoplast yields under different macerozyme concentrations with 2.0% cellulase R10; (D) Morphological characteristics of protoplasts, isolated from 2.0% cellulase R10 +0.3% macerozyme, observed by optical microscope (Bar=200 μm)"

Figure 2

Transformation efficiency of protoplast in Gerbera hybrid^Transformation efficiency of protoplast observed by laser confocal scanning microscope. From left to right are GFP fluorescence, chloroplast autofluorescence, and a merge of above. Arrows indicate the protoplasts with unsuccessful transformation. Bar=100 μm"

Figure 3

Subcellular localization of GFP and YFP in protoplasts from Gerbera hybrid^(A) Subcellular localization of GFP by laser confocal scanning microscope, from left to right are GFP fluorescence, chloroplast autofluorescence, bright, and a merge of above; (B) Subcellular localization of YFP by laser confocal scanning microscope, from left to right are YFP fluorescence, chloroplast autofluorescence, bright, and a merge of above. Bar=10 μm"

Figure 4

Analysis of interaction of IBH1 and HBI1 by bimolecular fluorescence complementation (BiFC)^IBH1-nYFP and HBI1-cYFP represent IBH1 and HBI1 connected to the N-terminal fragment and the C-terminal fragment of YFP, respectively. IBH1-nYFP+cYFP and HBI1-cYFP+nYFP were used as negative control. Bar=10 μm"

[1] 陈名红, 熊立, 陈学军 (2005). 烟草叶肉原生质体分离和纯化研究. 云南民族大学学报 14, 326-329.
[2] 崔喜艳, 张继晓, 窦瑶, 孙小杰, 尹悦佳, 刘相国 (2013). 双分子荧光互补技术(BiFC)分析玉米SSI与PPKD1之间的蛋白互作. 西北农林科技大学学报 41, 49-53.
[3] 李丹, 梁丹, 任相亮, 高月, 王火旭 (2009). 正交实验优选非洲菊叶片原生质体分离条件研究. 安徽农学通报 15, 45-50.
[4] 李妮娜, 丁林云, 张志远, 郭旺珍 (2014). 棉花叶肉原生质体分离及目标基因瞬时表达体系的建立. 作物学报 40, 231-239.
[5] 刘敏, 顾志敏 (2014). 烟草叶片原生质体制备及其在蛋白互作研究中的应用. 安徽农业科学 42, 5002-5006.
[6] 马占强, 林良斌, 董娜, 祁永琼 (2005). 紫罗兰下胚轴原生质体分离条件的研究. 云南农业大学学报 27, 155-158.
[7] 舒小娟, 温腾建, 邢佳毅, 卢龙, 胡建芳 (2015). 葡萄原生质体分离及瞬时转化体系的建立. 西北植物学报 35, 1262-1268.
[8] 王欢, 何彦峰, 习洋, 冯玥, 孙宇涵, 李云 (2014). 刺槐愈伤组织原生质体的分离和PEG融合. 东北林业大学学报 10, 28-33.
[9] 于晓玲, 李春强, 彭明 (2009). 植物原生质体技术及其应用. 中国农学通报 25, 22-26.
[10] 张钟仁, 陈鹏 (2016). 苦荞叶肉细胞原生质体的分离纯化及瞬时转化. 西北植物学报 36, 183-189.
[11] 赵苏州, 卢运明, 张占路, 赵杨敏, 王磊 (2014). 玉米和拟南芥的原生质体制备及瞬时表达体系的研究. 安徽农业科学 12, 3479-3482.
[12] 周波, 聂玉哲, 张晓磊, 李玉花 (2008). 芜菁原生质体的分离及绿色荧光蛋白的瞬时表达. 生物技术通讯 19, 542-544.
[13] 朱蠡庆, 王伯初, 周菁, 陈岑曦, 段传人 (2005). 紫锥菊愈伤组织原生质体分离方法初探. 重庆大学学报 28, 92-95.
[14] An Cl, Sawada A, Kawaguchi Y, Fukusaki E, Kobayashi A (2005). Transient RNAi induction against endogenous genes in Arabidopsis protoplasts using in vitro prepared double-stranded RNA. Biosci Biotechnol Biochem 69, 415-418.
[15] Bai MY, Fan M, Oh E, Wang ZY (2012). A triple helix-loop- helix/basic helix-loop-helix cascade controls cell elonga- tion downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.Plant Cell 24, 4917-4929.
[16] Broholm SK, Tahtiharju S, Laitinen RA, Albert VA, Teeri TH, Elomaa P (2008). A TCP domain transcription factor controls flower type specification along the radial axis of theGerbera(Asteraceae) inflorescence. Proc Natl Acad Sci USA 105, 9117-9122.
[17] Cao H, Guo S, Xu Y, Jiang K, Jones AM, Chong K (2011). Reduced expression of a gene encoding a golgi localized monosaccharide transporter (OsGMST1) confers hypersensitivity to salt in rice (Oryza sativa). J Exp Bot 62, 4595-4604.
[18] David H, Laigneau C, David A (1989). Growth and soluble proteins of cell cultures derived from explants and protoplasts ofPinus pinaster cotyledons. Tree Physiol 5, 497-506.
[19] Elomaa P, Uimari A, Mehto M, Albert VA, Laitinen RA, Teeri TH (2003). Activation of anthocyanin biosynthesis in Gerbera hybrida(Asteraceae) suggests conserved protein-protein and protein-promoter interactions between the anciently diverged monocots and eudicots. Plant Physiol 133, 1831-1842.
[20] Fan M, Bai MY, Kim JG, Wang T, Oh E, Chen L, Park CH, Son SH, Kim SK, Mudgett MB, Wang ZY (2014). The bHLH transcription factor HBI1 mediates the trade-off between growth and pathogen-associated molecular pat- tern-triggered immunity in Arabidopsis.Plant Cell 26, 828-841.
[21] Fontes N, Silva R, Vignault C, Lecourieux F, Gerós H, Delrot S (2010). Purification and functional characterization of protoplasts and intact vacuoles from grape cells.BMC Res Notes 3, 19.
[22] Guo J, Morrell-Falvey JL, Labbé JL, Muchero W, Kalluri UC, Tuskan GA, Chen JG (2012). Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS One 7, e44908.
[23] Jones AM, Chattopadhyay A, Shukla M, Zoń J, Saxena PK (2012). Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana). BMC Plant Biol 12, 1-11.
[24] Laitinen RA, Pollanen E, Teeri TH, Elomaa P, Kotilainen M (2007). Transcriptional analysis of petal organogenesis in Gerbera hybrida. Planta 226, 347-360.
[25] Li LF, Zhang WB, Zhang LL, Li N, Peng JZ, Wang YQ, Zhong CM, Yang YP, Sun SL, Liang S, Wang XJ (2015). Transcriptomic insights into antagonistic effects of gibbe- rellin and abscisic acid on petal growth in Gerbera hybrida. Front Plant Sci 6, 168.
[26] Liqing Z, Bochu W, Jing Z, Lingxi C, Chuanyun D, Chu- anren D (2005). Protoplast isolation of callus inEchinacea augustifolia. Colloids Surf B Biointerfaces 44, 1-5.
[27] Lung SC, Smith MD, Chuong SD (2015). Isolation of chloroplasts from plant protoplasts.Cold Spring Harb Pro- toc 2015, 895-899.
[28] Malinovsky FG, Batoux M, Schwessinger B, Youn JH, Stransfeld L, Win J, Kim SK, Zipfel C (2014). Antago- nistic regulation of growth and immunity by the Arabidopsis basic helix-loop-helix transcription factor HOMOLOG OF BRA- SSINOSTEROID ENHANCED EXPRESSION2 INTERACTING WITH INCREASED LEAF INCLINATION1 BINDING bHLH1.Plant Physiol 164, 1443-1455.
[29] Mazarei M, Al-Ahmad H, Rudis MR, Jr Stewart CN (2008). Protoplast isolation and transient gene expression in switchgrass,Panicum virgatum L. Biotech J 3, 354-359.
[30] Sheen J (2001). Signal transduction in maize and Arabidopsis mesophyll protoplasts.Plant Physiol 127, 1466-1475.
[31] Tahtiharju S, Rijpkema AS, Vetterli A, Albert VA, Teeri TH, Elomaa P (2012). Evolution and diversification of the CYC/TB1 gene family in Asteraceae—a comparative study in Gerbera(Mutisieae) and sunflower (Heliantheae). Mol Biol Evol 29, 1155-1166.
[32] Tang Z, Fan X, Li Q, Feng H, Miller AJ, Shen Q, Xu G (2012). Knockdown of a rice stelar nitrate transporter alters long-distance translocation but not root influx.Plant Phy- siol 160, 2052-2063.
[33] Teeri TH, Elomaa P, Kotilainen M, Albert VA (2006). Mining plant diversity:Gerbera as a model system for plant developmental and biosynthetic research. Bioessays 28, 756-767.
[34] Yoo SD, Cho YH, Sheen J (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.Nat Protoc 2, 1565-1572.
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[2] ZHANG Shi-Gong;GAO Ji-Yin and SONG Jing-Zhi. Effects of Betaine on Activities of Membrane Protective Enzymes in Wheat (Triticum aestivum L.) Seedlings Under NaCl Stress[J]. Chin Bull Bot, 1999, 16(04): 429 -432 .
[3] HE Wei-Ming and ZHONG Zhang-Cheng. Effects of Soil Fertility on Gynostemma pentaphyllum Makino Population Behavior[J]. Chin Bull Bot, 1999, 16(04): 425 -428 .
[4] SHE Chao-WenSONG Yun-Chun LIU Li-Hua. Analysis on the G_banded Karyotypes and Its Fluctuation at Different Mitotic Phases and Stages in Triticum tauschii (Aegilops squarrosa)[J]. Chin Bull Bot, 2001, 18(06): 727 -734 .
[5] Guijun Yang, Wenjiang Huang, Jihua Wang, Zhurong Xing. Inversion of Forest Leaf Area Index Calculated from Multi-source and Multi-angle Remote Sensing Data[J]. Chin Bull Bot, 2010, 45(05): 566 -578 .
[6] Man Chen, YishengTu, Linan Ye, Biyun Yang. Effect of Amino Acids on Thallus Growth and Huperzine-A Accumulation in Huperzia serrata[J]. Chin Bull Bot, 2017, 52(2): 218 -224 .
[7] Yefei Shang, Ming Li, Bo Ding, Hao Niu, Zhenning Yang, Xiaoqiang Chen, Gaoyi Cao, Xiaodong Xie. Advances in Auxin Regulation of Plant Stomatal Development[J]. Chin Bull Bot, 2017, 52(2): 235 -240 .
[8] CUI Xiao-Yong, Du Zhan-Chi, Wang Yan-Fen. Photosynthetic Characteristics of a Semi-arid Sandy Grassland Community in Inner Mongolia[J]. Chin J Plan Ecolo, 2000, 24(5): 541 -546 .
[9] LI Wei, ZHANG Ya-Li, HU Yuan-Yuan, YANG Mei-Sen, WU Jie, and ZHANG Wang-Feng. Research on the photoprotection and photosynthesis characteristics of young cotton leaves under field conditions[J]. Chin J Plan Ecolo, 2012, 36(7): 662 -670 .
[10] HU Bao-Zhong, LIU Di, HU Guo-Fu, ZHANG A-Ying, JIANG Shu-Jun. Random Amplified Polymorphic DNA Study of Local Breeds in Chinese lfalfa[J]. Chin J Plan Ecolo, 2000, 24(6): 697 -701 .