Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa

doi:10.12302/j.issn.1000-2006.202012029

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2022, Vol. 46 ›› Issue (1): 187-196.doi: 10.12302/j.issn.1000-2006.202012029

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Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa

YU Zicheng(), LING Cong, CHEN Yingnan, LI Shuxian, YIN Tongming, LI Xiaoping^*()

Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry,Nanjing Forestry University, Nanjing 210037, China

Received:2020-12-19 Accepted:2021-04-14 Online:2022-01-30 Published:2022-02-09
Contact: LI Xiaoping E-mail:yuzc1020@163.com;xpli@njfu.edu.cn

Abstract

Abstract:

【Objective】 The regeneration system and the stable genetic transformation method of male diploid Populus tomentosa was established to provide an experimental platform for further research on gene function.【Method】 The infancy stem segments and leaves of male diploid P. tomentosa were selected as explants and the regeneration system was established by optimizing the concentrations of 6-BA, IAA and TDZ, based on 1/2 MS basic medium. Agrobacterium tumefaciens EHA105 was used for genetic transformation via the leaf disc transformation method; it was combined with pre-culture time, bacterial concentration, infection time, co-culture time, and the kanamycin screening concentration. The protoplasts of young leaves were used as receptor cells and transiently transformed with the fluorescent marker gene, EGFP, using the polyethylene glycol-mediated method.【Result】 The regeneration process of male diploid P. tomentosa involves three stages: subculture, bud elongation, and rooting. The medium components were: 1/2 MS + 0.5 mg/L 6-BA + 0.5 mg/L NAA + 0.005 mg/L TDZ 1/2 MS + 0.5 mg/L 6-BA + 0.3 mg/L NAA and 1/2 MS + 0.3 mg/L NAA + 0.5 mg/L IBA; the rooting percentage was 96.7% and the multiplication coefficient was 4.47. The process of genetic transformation involved five stages: pre-culture, agrobacterium infection, co-culture, resistance screening and rooting. The specific plan was pre-culture for 12 h, with an agrobacterium concentration of OD₆₀₀ as 0.4, infection-time of 20 min, co-culture time of 24 h, kanamycin (30 mg/L) screening for 45 d, and resistant seedlings rooting for 20 d. A total of 86 resistant plants were obtained in this study; among them molecular identification of 14 plants were stable. The efficiency of the EGFP gene transformation was 50% under the guidance of 40% PEG4000.【Conclusion】 The male diploid P. tomentosa was rapidly regenerated and exhibited good transformation stability; it was considered an ideal material for basic polar research. This study broadened the genetic transformation system of poplar and provided a new approach for the molecular assisted breeding of poplar.

Key words: diploid Populus tomentosa, regeneration system, genetic transformation, transient expression

CLC Number:

S792.117

YU Zicheng, LING Cong, CHEN Yingnan, LI Shuxian, YIN Tongming, LI Xiaoping. Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(1): 187-196.

Figures/Tables 14

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References 30

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处理 treatment	消毒时间 disinfection time		种植数 planting number	污染数 pollution number	污染率 pollution rate	褐化率 browning rate	死亡率 death rate	显著性分析 sig.
处理 treatment	70%乙醇/s 70% ethanol	次氯酸钠/min sodium hypochlorite	种植数 planting number	污染数 pollution number	污染率 pollution rate	褐化率 browning rate	死亡率 death rate	显著性分析 sig.
1	20	5	50	46	0.92	0.55	0.34	a
2	20	10	50	43	0.86	0.62	0.25	b
3	20	15	50	39	0.78	0.72	0.36	ac
4	30	5	50	22	0.44	0.54	0.37	ac
5	30	10	50	28	0.56	0.55	0.44	d
6	30	15	50	28	0.56	0.48	0.86	e
7	40	5	50	37	0.74	0.50	0.70	f
8	40	10	50	33	0.66	0.52	0.62	g
9	40	15	50	30	0.60	0.53	0.77	h

培养基 culture medium	顶芽生长情况 the growth rate of bud
培养基 culture medium	10 d	20 d
1/4 MS	生长速度与WPM相似	生长速度最慢
1/2 MS	生长速度最快	生长速度最快
MS	生长速度最慢	生长速度与WPM相似
WPM	生长速度与1/4 MS相似	生长速度与MS相似

处理 treatment	外源激素/ (mg·L^-1) exogenous hormone		平均生根率/% average rooting rate	显著性分析 sig.
处理 treatment	NAA	IBA	平均生根率/% average rooting rate	显著性分析 sig.
1	0.3	0.3	53.3	bc
2	0.3	0.5	96.7	a
3	0.3	0.7	70.0	b
4	0.5	0.3	56.7	b
5	0.5	0.5	66.7	b
6	0.5	0.7	63.3	b
7	0.7	0.3	36.7	c
8	0.7	0.5	16.7	d
9	0.7	0.7	3.3	d

处理 treatment	外源激素/ (mg·L^-1) exogenous hormone			平均增殖系数 average multiplication coefficient	显著性分析 sig.
处理 treatment	6-BA	NAA	TDZ	平均增殖系数 average multiplication coefficient	显著性分析 sig.
1	0.3	0.3	0.005	1.60	c
2	0.3	0.5	0.010	2.10	c
3	0.3	0.7	0.015	2.70	c
4	0.5	0.3	0.015	4.47	abc
5	0.5	0.5	0.005	6.63	a
6	0.5	0.7	0.010	3.00	ab
7	0.7	0.3	0.010	3.43	bc
8	0.7	0.5	0.015	4.10	abc
9	0.7	0.7	0.005	4.03	abc

Establishment of regeneration system and study on genetic transformation for male diploid Populus tomentosa

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References 30

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处理 treatment	外源激素/(mg·L^-1) exogenous hormone		30 d芽平均生长长度/mm average growth length of bud in 30 d
处理 treatment	6-BA	NAA	30 d芽平均生长长度/mm average growth length of bud in 30 d
1	0.1	0.1	16.0
2	0.1	0.3	18.0
3	0.1	0.5	19.0
4	0.3	0.1	15.0
5	0.3	0.3	20.0
6	0.3	0.5	24.0
7	0.5	0.1	23.0
8	0.5	0.3	26.3
9	0.5	0.5	23.3

处理 treatment	时间/h time	接种数/ 个 plants number	产生抗性芽叶盘数/ 个resistant buds number	抗性芽获得率/ %resistant buds rate	显著性分析 sig.
预培养 pre-culture	0	100	7	7	a
	12	100	8	9	b
	24	100	2	2	c
	48	100	0	0	d
共培养 co-cultivation	8	50	0	0	a
	16	50	2	4	a
	24	50	3	6	b
	32	50	1	2	a
	40	50	0	0	a
	48	50	1	2	a
	56	50	0	0	a

菌液浓度 OD₆₀₀	侵染时间/ min infection time	叶盘数 plants number	产生抗性芽的叶盘数 resistant buds number	抗性芽获得率/% resistant buds rate	显著性分析 sig.
0.2	10	45	0	0	a
	20	48	1	2	ab
	30	40	2	5	b
0.4	10	48	4	8	c
	20	47	5	11	c
	30	48	2	4	b
0.6	10	48	0	0	a
	20	48	0	0	a
	30	48	0	0	a
0.8	10	44	0	0	a
	20	42	0	0	a
	30	48	0	0	a

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菌液浓度 OD₆₀₀	侵染时间/ min infection time	叶盘数 plants number	产生抗性芽的叶盘数 resistant buds number	抗性芽获得率/% resistant buds rate	显著性分析 sig.
0.2	10	45	0	0	a
	20	48	1	2	ab
	30	40	2	5	b
0.4	10	48	4	8	c
	20	47	5	11	c
	30	48	2	4	b
0.6	10	48	0	0	a
	20	48	0	0	a
	30	48	0	0	a
0.8	10	44	0	0	a
	20	42	0	0	a
	30	48	0	0	a

菌液浓度 OD₆₀₀	侵染时间/ min infection time	叶盘数 plants number	产生抗性芽的叶盘数 resistant buds number	抗性芽获得率/% resistant buds rate	显著性分析 sig.
0.2	10	45	0	0	a
	20	48	1	2	ab
	30	40	2	5	b
0.4	10	48	4	8	c
	20	47	5	11	c
	30	48	2	4	b
0.6	10	48	0	0	a
	20	48	0	0	a
	30	48	0	0	a
0.8	10	44	0	0	a
	20	42	0	0	a
	30	48	0	0	a