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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."

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