Tissue culture and plant regeneration of Clematis ‘Julka’

doi:10.12302/j.issn.1000-2006.201912012

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (3): 109-116.doi: 10.12302/j.issn.1000-2006.201912012

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Tissue culture and plant regeneration of Clematis ‘Julka’

GAO Yan(), MO Jianbin, FU Yanru, FENG Shucheng^*()

Shanghai Botanical Garden,Shanghai Engineering Research Center of Sustainable Plant Innovation, Shanghai 200231, China

Received:2019-12-10 Revised:2020-09-08 Online:2021-05-30 Published:2021-05-31
Contact: FENG Shucheng E-mail:69262835@qq.com;shbg2009@126.com

Abstract

Abstract:

【Objective】Stem segments having buds of Clematis ‘Julka’ were used as the explants to construct a regeneration system. 【Method】The system was established via two ways: one is initiates from induction of axillary buds, then proliferation of adventitious buds, and rooting induction;the other is from callus induction and proliferation to differentiation, final rooting. 【Result】The method for explants surface sterilization was incubated in 10% (mass fraction) sodium hypochlorite for 12 min. The medium for inducing axillary buds was MS+NAA 0.1 mg/L+6-BA 1.0 mg/L and for adventitious buds proliferation was MS+IBA 0.20 mg/L+6-BA 1.0 mg/L+GA₃ 0.2 mg/L. The medium for callus induction was MS+NAA 0.05 mg/L+6-BA 2.0 mg/L, for callus proliferation was MS+NAA 0.20 mg/L +6-BA 2.0 mg/L, and for callus differentiation was MS+ NAA 0.03 mg/L +6-BA 3.0 mg/L. The Optimal rooting induction medium was 1/2MS+IBA 0.3 mg/L. 【Conclusion】This study established a stable and high-efficient regeneration system in vitro for Clematis ‘Julka’ by using two ways of organogenesis. The multiplication rate was 5.52 when shoot organogenesis were formed directly. In the organogenesis of callus induction pathway, the ratio of callus differentiation was 76.7% and the average number of differentiated adventitious buds was more than 3. The rates of plantlet rooting in two ways were more than 90%.

Key words: Clematis ‘Julka’, axillary buds induction, callus induction, callus differentiation, plant regeneration

CLC Number:

S682.19

GAO Yan, MO Jianbin, FU Yanru, FENG Shucheng. Tissue culture and plant regeneration of Clematis ‘Julka’[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(3): 109-116.

Figures/Tables 9

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References 25

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编号 No.	灭菌时间/ min sterilizing time	污染率/% rate of contamination	出芽率/% rate of germination	出愈率/% rate of callus induction
A1	6	65.5±6.8 a	7.3±3.6 a	16.4±3.6 a
A2	12	14.6±4.5 ab	41.8±4.5 b	56.4±5.7 b
A3	18	3.6±4.5 b	9.1±5.7 ac	23.6±4.4 ab

编号 No.	基本培养基 basic medium	出芽率/ % rate of germination	芽苗生长状况 state of shoots growth
B1	MS	35.3±1.6 a	生长健康,无黄化、玻璃化现象
B2	1/2MS	6.0±2.1 b	生长基本健康,较矮小,生长缓慢
B3	WPM	2.0±1.6 b	出芽后生长极缓慢,叶逐渐发黄后期枯萎

编号 No.	c(NAA)/ (mg·L^-1)	c(6-BA)/ (mg·L^-1)	出芽率/% rate of germination	生长状况 state of shoots growth
C1	0	0	0.0±0.0 a	无法诱导
C2	0.1	0	8.6±1.9 abc	腋芽长速慢
C3	0.3	0	12.4±2.3 abc	腋芽长速慢,叶片黄
C4	0	1.0	10.5±1.9 abc	腋芽生长慢,增殖效率差
C5	0.1	1.0	72.4±3.6 d	腋芽长出于节间生长点, 生长迅速
C6	0.3	1.0	38.1±3.0 c	腋芽长出于节间生长点, 两侧切口处略有膨胀
C7	0	2.0	5.7±1.9 ab	诱导出芽少
C8	0.1	2.0	11.4±2.3 abc	诱导出芽较少
C9	0.3	2.0	15.2±1.9 bc	两侧切口略膨胀

编号 No.	c(IBA)/ (mg·L^-1)	c(6-BA)/ (mg·L^-1)	c(GA₃)/ (mg·L^-1)	增殖倍数 rate of multiplication	生长状况 state of shoots growth
D1	0.05	0.5	0	1.16±0.15 a	茎段细,单节略有伸长,丛生芽少,生长速度慢
D2	0.05	1.0	0.1	2.32±0.30 bc	茎段细,生长速度较快,叶片舒展
D3	0.05	2.0	0.2	2.08±0.32 b	生长速度较快,叶片舒展
D4	0.20	0.5	0.1	3.28±0.20 d	茎段粗,单节伸长,丛生芽较少
D5	0.20	1.0	0.2	5.52±0.30 e	茎段粗,单节略有伸长,分节多,且增殖多,生长迅速
D6	0.20	2.0	0	2.36±0.15 bc	茎段细,生长速度差异大
D7	0.40	0.5	0.2	2.04±0.15 b	单节有伸长,部分分节,生长较慢
D8	0.40	1.0	0	2.48±0.20 c	茎段细,分节较多
D9	0.40	2.0	0.1	2.32±0.24 bc	增殖较低,茎段较细,生长速度较快

编号 No.	基本培养基 basic medium	出愈率/% rate of callus induction	愈伤组织生长状况 state of callus growth
E1	1/2 MS	33.3±2.9 a	16 d左右两端切口处开始膨胀,形成浅黄色松软愈伤组织
E2	MS	56.3±5.1 b	12 d左右两端切口处开始膨胀,形成浅黄色松软愈伤组织
E3	WPM	10.4±2.9 a	愈伤组织黄色,致密

Tissue culture and plant regeneration of Clematis ‘Julka’

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编号 No.	c(NAA)/ (mg·L^-1)	c(6-BA)/ (mg·L^-1)	出愈率/% rate of callus induction	生长状况 state of callus growth
F1	0	1.0	17.1±5.7 a	量少、淡黄色、疏松
F2	0.05	1.0	22.9±7.0 ab	淡黄色、有突起
F3	0.10	1.0	14.3±0.0 a	愈伤组织长速快、疏松
F4	0	2.0	45.7±5.7 abc	淡黄色、疏松
F5	0.05	2.0	71.4±9.0 bc	淡黄色、疏松
F6	0.10	2.0	40.0±5.7 abc	淡黄色、较致密
F7	0	4.0	77.1±7.0 c	黄色、愈伤组织长速快、过度致密
F8	0.05	4.0	54.3±5.7 abc	黄色、致密
F9	0.10	4.0	22.9±7.0 ab	黄褐色、过度致密

编号 No.	c(NAA)/ (mg·L^-1)	c(6-BA)/ (mg·L^-1)	增殖倍数 rate of callus proliferation	生长状况 state of callus growth
G1	0.2	1.0	1.25±0.13 a	增殖很差
G2	0.5	1.0	2.19±0.11 b	淡黄色、疏松
G3	1.0	1.0	2.03±0.10 b	淡黄色、疏松
G4	0.2	2.0	4.09±0.14 f	淡黄色、疏松
G5	0.5	2.0	3.89±0.11 ef	淡黄色、疏松
G6	1.0	2.0	2.63±0.23 c	淡绿色、致密
G7	0.2	4.0	3.35±0.16 d	黄色、致密
G8	0.5	4.0	3.68±0.07 e	黄色、过度致密、硬化
G9	1.0	4.0	3.19±0.17 d	黄褐色、硬化

编号 No.	c(NAA)/ (mg·L^-1)	c(6-BA)/ (mg·L^-1)	分化率/% rate of callus differentiation	不定芽数 number of adventitious buds	分化生长状况 state of callus differentiation
H1	0.01	1.0	0.0±0.0 a	0.0±0.0 a	愈伤组织不分化
H2	0.03	1.0	0.0±0.0 a	0.0±0.0 a	愈伤组织不分化
H3	0.05	1.0	0.0±0.0 a	0.0±0.0 a	愈伤组织不分化
H4	0.01	3.0	19.4±3.9 bc	1.6±0.3 b	愈伤组织少量分化
H5	0.03	3.0	76.7±4.8 d	3.1±0.3 b	愈伤组织黄绿色,分化出1~3个节间的茎段,叶片略小
H6	0.05	3.0	29.2±4.1 c	1.9±0.3 b	愈伤组织黄绿色,可分化茎段
H7	0.01	5.0	15.3±3.1 b	1.3±0.4 b	极少形成不定芽,愈伤组织较致密
H8	0.03	5.0	0.0±0.0 a	0.0±0.0 a	愈伤组织褐化且不分化
H9	0.05	5.0	0.0±0.0 a	0.0±0.0 a	愈伤组织褐化且不分化

编号 No.	c(IBA)/ (mg·L^-1)	c(NAA)/ (mg·L^-1)	生根数 number of rooting	生根率/% rate of rooting	生长状况 state of rooting
I1	0.1	0	1.35±0.28 b	90.0±6.9 c	根系发达,生长快,15 d左右开始长根
I2	0.3	0	4.10±0.42 d	91.7±8.3 c	愈伤组织上生根,15 d左右开始长根;根粗壮洁白,根系发达,生长快
I3	0.5	0	2.65±0.29 c	60.0±6.9 b	根特别粗壮,极个别出现畸形
I4	0	0.1	0.0±0.0 a	0.0±0.0 a	无生根
I5	0	0.3	0.0±0.0 a	0.0±0.0 a	无生根
I6	0	0.5	0.0±0.0 a	0.0±0.0 a	无生根

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