Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression

doi:10.16420/j.issn.0513-353x.2022-0264

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (5): 1141-1150.doi: 10.16420/j.issn.0513-353x.2022-0264

• New Technology & New Method • Previous Articles Next Articles

Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression

GAO Chengyu¹^,^*, WANG Yiheng¹^,^*, JIN Jiangzhou¹, LI Tao¹, LI Jindou¹, ZHOU Mengyao¹, ZHANG Haixia¹^,², MA Hui¹^,², ZHANG Yuxing¹^,²^,^**(), QI Baoxiu¹^,³^,^**, XU Jianfeng¹^,²^,^**

¹College of Horticulture，Hebei Agricultural University，Baoding，Hebei 071051，China
²Hebei Pear Technology and Innovation Center，Baoding，Hebei 071051，China
³School of Pharmacy and Biomolecular Sciences，Liverpool John Moores University，Byrom Street，Liverpool L3 3AF，UK

Received:2022-11-07 Revised:2023-03-11 Online:2023-05-25 Published:2023-05-31
Contact: ZHANG Yuxing, QI Baoxiu, XU Jianfeng E-mail:xjf@hebau.edu.cn

Abstract

Abstract:

High quality and quantity of protoplasts were succefully isolated from the mesophyll cells of leaves of tissue cultured‘Xinli 7’pear seedlings. This was achieved by optimizing the composition of the cell wall degrading enzymes and the reaction solution where the best results were obtained when the leaves of 30-days old tissue cultured pear seedlings were treated with 1.0% cellulase，0.1% pectinase and 0.4% macerozyme in a buffer containing 20 mmol · L^-1 MES，0.5 mol · L^-1 of D-mannitol，20 mmol · L^-1 of potassium chloride，10 mmol · L^-1of calcium chloride and 1.0 g · L^-1 of bovine serum albumin（pH 5.7）. Approximately 1.09 × 10⁶ protopl · mL^-1 were recovered with their viability being above 90%. To test their usefulness in genetic transformation in transient expression of proteins 1-2 µg · mL^-1 of pROK2- GFP plasmid DNA was transformed into the prepared protoplasts with 40% PEG-4000. After being cultured for 12 hours GFP was clearly observed in about 40% of the protoplasts，the transformation efficiency reached 40%.

Key words: pear, leaf, protoplast isolation, protoplast transient transformation

CLC Number:

S661.2

GAO Chengyu, WANG Yiheng, JIN Jiangzhou, LI Tao, LI Jindou, ZHOU Mengyao, ZHANG Haixia, MA Hui, ZHANG Yuxing, QI Baoxiu, XU Jianfeng. Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression[J]. Acta Horticulturae Sinica, 2023, 50(5): 1141-1150.

Figures/Tables 4

References 32

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Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression

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筛选对象 Screening object	纤维素酶/% Cellulase	果胶酶/% Pectinase	离析酶/% Macerozyme	甘露醇/ （mol · L^-1） Mannitol	酶解时间/h Enzymatic hydrolysis time	原生质体产量/ （× 10⁵ protopl · mL^-1） Protoplast yield
纤维素酶浓度 Cellulase concentration	0.5	0.1	0.5	0.5	12	3.50 b
	1.0	0.1	0.5	0.5	12	5.00 a
	1.5	0.1	0.5	0.5	12	2.88 b
	2.0	0.1	0.5	0.5	12	2.03 b
	2.5	0.1	0.5	0.5	12	2.00 b
果胶酶浓度 Pectinase concentration	1.0	0.0	0.5	0.5	12	4.85 b
	1.0	0.1	0.5	0.5	12	10.67 a
	1.0	0.3	0.5	0.5	12	5.86 b
	1.0	0.5	0.5	0.5	12	5.32 b
	1.0	0.7	0.5	0.5	12	3.81 c
离析酶浓度 Macerozyme concentration	1.0	0.1	0.2	0.5	12	2.55 c
	1.0	0.1	0.3	0.5	12	3.33 bc
	1.0	0.1	0.4	0.5	12	6.92 a
	1.0	0.1	0.5	0.5	12	4.84 b
	1.0	0.1	0.6	0.5	12	3.45 bc
甘露醇浓度 Mannitol concentration	1.0	0.1	0.4	0.3	12	6.17 cd
	1.0	0.1	0.4	0.4	12	7.17 bc
	1.0	0.1	0.4	0.5	12	10.93 a
	1.0	0.1	0.4	0.6	12	8.50 b
	1.0	0.1	0.4	0.7	12	5.37 d
酶解时间 Reaction time	1.0	0.1	0.4	0.5	6	1.83 c
	1.0	0.1	0.4	0.5	8	2.38 bc
	1.0	0.1	0.4	0.5	10	2.86 b
	1.0	0.1	0.4	0.5	12	3.63 a
	1.0	0.1	0.4	0.5	14	2.17 c
	1.0	0.1	0.4	0.5	16	0.16 d

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