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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (03): 463-470.doi: 10.3724/SP.J.1006.2018.00463

• RESEARCH NOTES • Previous Articles    

Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant (Camellia sinensis L. O. Kuntze)

Zhang PENG1,2(), Hua-Rong TONG1, Guo-Lu LIANG3, Yi-Qi SHI1, Lian-Yu YUAN1,*()   

  1. 1 College of Food Science, Southwest University, Chongqing 400715, China
    2National Demonstration Center for Experimental Food Science and Technology Education (Southwest University), Chongqing 400715, China
    3 College of Food Science, Southwest University, Chongqing 400715, China
  • Received:2017-07-28 Accepted:2018-11-21 Online:2018-03-12 Published:2017-12-18
  • Contact: Lian-Yu YUAN E-mail:969979310@qq.com;yuanlianyu88@163.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31400583), National Training Program of Innovation and Entrepreneurship for Undergraduates (201610635041), and the Basic and Frontier Research Program of Chongqing (cstc2014jcyjA80011).

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Abstract:

Plant protoplasts are important materials for studies on plant cell culture, somatic cell fusion, genetics and breeding. This work established a stable and efficient method for getting a large amount of vital and intact protoplasts from young leaves and young roots of Fudingdabai tea plant. For protoplast isolation, the young leaves of tea seedlings grown in the condition of constant temperature (23°C) and dark or shading were the best materials, and the young radicle of tea plant was also the better, While the healthy young leaves of tea plants grown in tea plantation were used as materials, only a small quantity of viable protoplasts mixed with a large number of cell fragments were obtained. The optimal enzyme solution for protoplasts isolation from young leaves of tea seedlings contained 1.5% cellulase + 0.1% macerozyme + 0.5% pectolyase + 0.4 mol L-1 mannitol + 20 mmol L-1 MES, and that from radicle of tea seedlings contained 1.5% cellulase + 0.3% macerozyme + 0.5% pectolyase + 0.4 mol L-1 mannitol + 20 mmol L-1 MES. Protoplasts with high yield and viability were purified when incubated in a shaker with low speed (55 r min-1 and 50 r min-1 respectively) under constant temperature (23°C) for 7 h and 8 h, and then centrifuged at 15×g for 4 min. PEG-6000 was used to induce the fusion of protoplasts from young leaves and radicle of tea plants. The fusion time, the concentration of fusion agent PEG-6000 were discussed. The optimal protoplast infusion conditions were induction with 40% PEG-6000 for 20 min, and the fusion rate was about 10%.

Key words: Camellia sinensis, young leaf, young root, protoplast isolation, protoplast fusion

Fig. 1

Selection and treatment of different materials in tea plant A: young radicle produced by seed germination; B: one bud and three leaves from the tea plant grown in normal environments; C: dark treated young tea seedlings; D: slices of leaves; E: enzymatic digested leaves."

Fig. 2

Protoplast of tea plant isolated from different materials CK: protoplasts isolated from leaves of Arabidopsis; A: protoplasts isolated from dark treated young seedlings leaves of tea plant; B: protoplasts isolated from the young radicle produced by seed germination; C: protoplasts isolated from one bud with three leaves of the tea plant grown in normal environments."

Fig. 3

Number and vitality of protoplast from leaves of tea plants CK: number of protoplasts isolated from the leaves of Arabidopsis thaliana (0.25 mm × 0.25 mm squares); A: number of protoplasts isolated from the leaves of tea plants (0.25 mm × 0.25 mm squares); B: number of protoplasts isolated from the leaves of tea plants (0.25 mm × 0.25 mm squares); C: vitality of tea plant protoplasts detected by trypan blue staining."

Table 1

Effects of the concentration of different enzymes on protoplast isolation from young leaves of tea plant"

处理
Treatment		 酶解液浓度 Concentration of enzyme (%) 产量
Yield (×106 g-1 FW)		 活力
Viability (%)
纤维素酶 Cellulase 离析酶 Macerozyme 果胶酶 Pectolase
1 1.4 0.1 0.5 6.8 70
2 1.5 0.1 0.5 8.8 88
3 1.6 0.1 0.5 7.2 75
4 1.5 0 0.5 2.4 50
5 1.5 0.3 0.5 7.6 80
6 1.5 0.1 0.1 3.6 60
7 1.5 0.1 0.3 8.0 85
8 1.5 0.1 0.7 6.0 65

Table 2

Effects of the concentration of different enzymes on protoplast isolation from radicle of tea plant"

处理
Treatment		 酶解液浓度 Concentration of enzyme (%) 产量
Yield (×106 g-1 FW)		 活力
Viability (%)
纤维素酶 Cellulase 离析酶 Macerozyme 果胶酶 Pectolase
1 1.4 0.3 0.5 2.8 83
2 1.5 0.3 0.5 3.2 89
3 1.6 0.3 0.5 3.0 85
4 1.5 0 0.5 1.0 50
5 1.5 0.1 0.5 2.4 75
6 1.5 0.3 0.3 2.0 70
7 1.5 0.3 0.7 2.4 76

Table 3

Effects of enzymolysis time on protoplast yield and viability of tea leaves"

酶解时间
Enzymolysis time
(h)		 原生质体数
Protoplast count
(×106 g-1 FW)		 活力
Viability
(%)
6 6.8 80
7 8.8 88
8 8.0 75
9 6.0 50

Table 4

Effects of osmotic pressure of enzyme solution on protoplasts isolated from tea plant"

酶解液编号
Enzyme solution number		 甘露醇浓度
Mannitol concentration
(mol L-1)		 原生质体形态
Protoplast morphology
1 0.3 原生质体呈浑圆状态, 约70%以上的是完整原生质体, 但细胞碎片及细胞器较多
Protoplasts were rounded, about 70% of protoplasts were intact, but there were a lot of cell debris and organelles
2 0.4 原生质体呈浑圆状态, 约85%以上是完整原生质体, 且细胞碎片及细胞器较少
Protoplasts were rounded, about 85% of them were intact, and only a small amount of cell debris and organelles existed
3 0.5 较正常原生质体体积缩小, 略有皱褶
Compared with normal protoplasts, the protoplast size was smaller and slightly wrinkled

Fig. 4

Protoplast fusion of tea plantA: The fusion process of protoplasts from young leaves from tea plants; B: The fusion process of protoplasts from young radicle of tea seedlings."

[1] Nanjareddy K, Arthikala M K, Blanco L, Elizabeth S A, Miguel L.Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis. BMC Biotechnol, 2016, 16: 53
[2] Rehman L, Su X F, Guo H M, Qi X L, Cheng H M.Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae. BMC Biotechnol, 2016, 16: 57
[3] Burris K P, Dlugosz E M, Collins A G, Stewart C N, Lenaghan S C.Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.). Plant Cell Rep, 2016, 35: 693-704
[4] Larkin P J, Scowcroft W R.Somaclonal variation: a novel source of variability from cell-cultures for plant improvement. Theor Appl Genet, 1981, 60: 197-214
[5] Cocking E C.Method for the isolation of plant protoplasts and vacuoles.Nature, 1960, 187: 962-963
[6] 彭邵锋, 陆佳, 陈永忠, 王瑞, 陈隆升, 马力, 王湘南. 木本植物原生质体培养体系研究进展. 中国农学通报, 2013, 29(1): 1-6
Peng S F, Lu J, Chen Y Z, Wang R, Chen L S, Ma L, Wang X N.Research progress of protoplast culture in woody plants.Chin Agric Sci Bull, 2013, 29(1): 1-6 (in Chinese with English abstract)
[7] 王善平, 许智宏. 木本植物的原生质体培养. 细胞生物学杂志, 1989, (1): 14-18
Wang S P, Xu Z H.Protoplast culture of woody plants.J Cell Biol, 1989, (1): 14-18 (in Chinese)
[8] Liu J H, Xu X Y, Deng X X.Protoplast isolation, culture and application to genetic improvement of woody plants.J Food Agric Environ, 2003, 1: 112-120
[9] Vardi A, Spiegelroy P, Galun E.Citrus cell culture: isolation of protoplasts, plating densities, effect of mutagens and regeneration of embryos.Plant Sci Letters, 1975, 4: 231-236
[10] Mattoo A, Lieberman M.Localization of the ethylene-synthesizing system in apple tissue.Plant Physiol, 1977, 60: 794-799
[11] Ochatt S J, Caso O H.Shoot regeneration from leaf mesophyll protoplasts of wild pear (Pyrus-communis var. Pyraster L.). J Plant Physiol, 1986, 122: 243-249
[12] 羿德磊. 桑树原生质体分离及融合的研究. 山东农业大学硕士学位论文, 山东泰安, 2012
Yi D L.Study on Protoplast Isolation and Fusion of Mulberry. MS Thesis of Shandong Agricultural University, Tai’an, China, 2012 (in Chinese with English abstract)
[13] 俞长河, 陈振光, 吕柳新. 荔枝原生质体培养再生植株. 福建农业大学学报, 1996, 25(03): 386
Yu C H, Chen Z G, Lyu L X.Regeneration of plantlets from protoplasts culture of Litchi.J Fujian Agric Univ, 1996, 25(03): 386 (in Chinese with English abstract)
[14] 林顺权, 陈振光. 枇杷原生质体培养再生植株. 福建农业大学学报, 1994, 23(1): 125
Lin S Q, Chen Z G.Plant regeneration from protoplast of Loquat.J Fujian Agric Univ, 1994, 23(1): 125 (in Chinese with English abstract)
[15] 马锋旺, 李嘉瑞. 中国李原生质体培养及植株再生. 西北农业大学学报, 1999, 27(03): 64-68
Ma F W, Li J R.Protoplast culture and plant regeneration of Chinese plum.Acta Univ Agric Boreali-Occident, 1999, 27(03): 64-68 (in Chinese with English abstract)
[16] 何业华, 胡芳名, 谢碧霞, 何钢, 胡中沂. 枣树原生质体培养及其植株再生. 中南林学院学报, 1999, (03): 29-31
He Y H, Hu F M, Xie B X, He G, Hu Z Y.Protoplast culture of Zizyphus jujube and plant individual regeneration. J Central South For Univ, 1999, (03): 29-31 (in Chinese with English abstract)
[17] 马锋旺, 李嘉瑞. 山杏原生质体培养再生植株. 园艺学报, 1998, 25: 224-229
Ma F W, Li J R.Plant regeneration from cultured protoplasts of Armeniaca vulgaris var. ansu. Acta Hortic Sin, 1998, 25: 224-229 (in Chinese with English abstract)
[18] 中村顺行, 叶乃兴. 茶树原生质体的分离. 茶叶科学简报, 1985, (1): 46-47
Nakamura A, Ye N X.Isolation of protoplasts from tea plants.Tea Sci Bull, 1985, (1): 46-47 (in Chinese)
[19] 赖钟雄, 何碧珠, 陈振光. 茶树花粉管亚原生质体的分离. 福建农业大学学报, 1998, 27(增刊): 41-44
Lai Z X, He B Z, Chen Z G.Isolation of protoplasts from pollen tubes of tea plant.J Fujian Agric For Univ, 1998, 27(suppl): 41-44 (in Chinese)
[20] 王璐, 钱文俊, 王新超, 李娜娜, 杨亚军. 一种茶树原生质体的提取方法.CN105695391A, 2016-06-22
Wang L, Qian W J, Wang X C, Li N N, Yang Y J A method for extracting protoplast from Tea plant. CN105695391A, 2016-06-22 (in Chinese)
[21] 杨子银, 梅鑫, 周瀛, 傅秀敏. 一种茶树花原生质体及其制备方法和应用. CN105670985A, 2016-06-15
Yang Z Y, Mei X, Zhou Y, Fu X M. The preparation method and application of flower tea protoplast. CN105670985A, 2016-06-15 (in Chinese)
[22] Davey M R, Anthony P, Power J B, Kenneth C L.Plant protoplasts: status and biotechnological perspectives.Biotechnol Adv, 2005, 23: 131-171
[23] Liu J H, Xu X Y, Deng X X.Intergeneric somatic hybridization and its application to crop genetic improvement.Plant Cell Tissue Organ Cult, 2005, 82: 19-44
[24] Guo W W, Zhou C H, Yi H L, Deng X X.Intergeneric somatic hybrid plants between Citrus and Poncirus trifoliata and evaluation of their root rot resistance. Acta Bot Sin, 2000, 42: 668-672
[25] Liu J H, Hu C G, Deng X X.Regeneration of diploid intergeneric somatic hybrid plants between Microcitrus and Citrus via electrofusion. Acta Bot Sin, 1999, 41: 1177-1182
[26] Revilla M A, Ochatt S J, Doughty S, Power J B.A general strategy for the isolation of Mesophyll protoplasts from deciduous fruit and nut tree species.Plant Sci, 1987, 50: 133-137
[27] Dovzhenko A, Dal Bosco C, Meurer J, Koop H U.Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana. Protoplasma, 2003, 222: 107-111
[28] Sinha A, Wetten A C, Caligari P D S. Optimisation of protoplast production in White Lupin.Biol Plant, 2003, 47: 21-25
[29] Yoo S D, Cho Y H, Sheen J.Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc, 2007, 2: 1565-1572
[30] Kuzminsky E, Meschini R, Terzoli S, Pavani L, Silvestri C, Choury Z, Scarascia-Mugnozza G.Isolation of mesophyll protoplasts from Mediterranean woody plants for the study of DNA integrity under abiotic stress.Front Plant Sci, 2016, 7: 1-7
[31] Tan B Y, Xu M, Chen Y, Huang M R.Transient expression for functional gene analysis usingPopulus protoplasts. Plant Cell Tissue Organ Cult, 2013, 114: 11-18
[32] Guo J, Morrell-Falvey J L, Labbe J L, Muchero W, Kalluri U C, Tuskan G A, Chen J G. Highly efficient isolation of Populus Mesophyll protoplasts and its application in transient expression assays. PLoS One, 2012, 7: e44908
[33] 李妮娜, 丁林云, 张志远, 郭旺珍. 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, 2014, 40: 231-239
Li N N, Ding L Y, Zhang Z Y, Guo W Z.Isolation of mesophyll protoplast and establishment of gene transient expression system in Cotton. Acta Agron Sin, 2014, 40: 231-239 (in Chinese with English abstract)
[34] 梁大伟. 拟南芥叶肉原生质体分离及瞬时表达体系的建立. 兰州大学硕士学位论文, 甘肃兰州, 2009
Liang D W.Isolation of Mesphyll Protoplast from Arabidopsis thaliana and the Setup of Transient Expression Assay System. MS Thesis of Lanzhou University, Lanzhou,China, 2009 (in Chinese with English abstract)
[35] 李文彬, 王革娇, Stanchina E, Castiglione S, 孙勇如, Sala F. PEG介导原生质体转化获得水稻转基因植株. 植物学报, 1995, 37: 409-412
Li W B, Wang G J, Stanchina E, Castiglione S, Sun Y R, Sala F.Transgenic rice plants produced by PEG-mediated plasmid uptake into protoplasts.Acta Bot Sin, 1995, 37: 409-412 (in Chinese with English abstract)
[1] HU Qiong and LI Yun-Chang. Induction and Improvement of Cytoplasmic Male Sterility in Oilseed Brassica by Somatic Hybridization [J]. Acta Agron Sin, 2006, 32(01): 138-143.
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