铁线莲‘朱卡’组织培养技术及再生体系的建立

doi:10.12302/j.issn.1000-2006.201912012

国家林草科技领军期刊
中国精品科技期刊
中国高校百佳科技期刊
江苏省新闻出版政府奖期刊奖
RCCSE林学权威期刊（A+）
CSCD核心期刊
Scopus数据库收录期刊
中文核心期刊
SCD核心期刊

作者加群：102861116

微信公众号：南京林业大学学报

高级检索

南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3): 109-116.doi: 10.12302/j.issn.1000-2006.201912012

铁线莲‘朱卡’组织培养技术及再生体系的建立

高燕(), 莫建彬, 付艳茹, 奉树成^*()

上海植物园,上海城市植物资源开发应用工程技术研究中心,上海 200231

收稿日期:2019-12-10 修回日期:2020-09-08 出版日期:2021-05-30 发布日期:2021-05-31
通讯作者: 奉树成
基金资助:
上海市科技兴农引进消化吸收再创新项目(沪农科引字(2017)第1-3号)

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

摘要/Abstract

摘要：

【目的】以铁线莲‘朱卡’(Clematis ‘Julka’)的带芽茎段为起始材料,通过组织培养方法建立再生体系。【方法】利用腋芽诱导—不定芽增殖—生根和愈伤组织诱导—增殖—分化—生根两种途径,建立该品种的组织培养再生体系,形成铁线莲‘朱卡’完整植株。【结果】铁线莲‘朱卡’组织培养最适宜灭菌条件为质量分数10%次氯酸钠溶液灭菌12min;带芽茎段诱导腋芽最适培养基为MS+NAA 0.1 mg/L+6-BA 1.0 mg/L,不定芽增殖最适培养基为MS+IBA 0.20 mg/L+6-BA 1.0 mg/L+GA₃ 0.2 mg/L;带芽茎段诱导愈伤组织最适培养基为MS+NAA 0.05 mg/L+6-BA 2.0 mg/L,愈伤组织增殖最适培养基为MS+NAA 0.20 mg/L +6-BA 2.0 mg/L,愈伤组织分化最适培养基为MS+NAA 0.03 mg/L+6-BA 3.0 mg/L;最佳生根培养基为1/2 MS+IBA 0.3 mg/L。【结论】首次用两种器官发生方法建立了稳定高效的铁线莲‘朱卡’再生体系。直接形成不定芽时增殖倍数可达5.52。诱导愈伤组织途径的器官发生中,愈伤组织分化率可达76.7%,分化不定芽平均数超过3。两种途径诱导生根率都在90%以上。

关键词: 铁线莲‘朱卡’, 腋芽诱导, 愈伤组织诱导, 愈伤组织分化, 再生体系

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

中图分类号:

S682.19

高燕,莫建彬,付艳茹,等. 铁线莲‘朱卡’组织培养技术及再生体系的建立[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 109-116.

GAO Yan, MO Jianbin, FU Yanru, FENG Shucheng. Tissue culture and plant regeneration of Clematis ‘Julka’[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(3): 109-116.DOI: 10.12302/j.issn.1000-2006.201912012.

图/表 9

表1

表2

表3

表4

表5

表6

表7

表8

表9

参考文献 25

[1]	王凯. 弱光和盐碱逆境对两种铁线莲生理特性的影响[D]. 哈尔滨:东北林业大学, 2017.
	WANG K. Effect of weak light and salinity stress on physiological characteristics of two species of Clematis[D]. Harbin:Northeast Forestry University, 2017.
[2]	闫双喜, 王鹏飞, 何松林, 等. 河南铁线莲属植物的观赏特性及开发利用[J]. 河南科学, 2005,23(2):218-220.
	YAN S X, WANG P F, HE S L, et al. Ornamental characters of plants of Clematis L.from Henan and their utilization[J]. Henan Sci, 2005,23(2):218-220.DOI: 10.3969/j.issn.1004-3918.2005.02.018.
[3]	黄鑫. 两个品种铁线莲的组织培养及工厂化生产成本控制研究[D]. 哈尔滨:东北林业大学, 2018.
	HUANG X. Studies on the tissue culture and factory propagation on the cost control of two species of Clematis[D]. Harbin:Northeast Forestry University, 2018.
[4]	张丽琼, 常权记, 廖咸康, 等. 绿花重瓣铁线莲愈伤组织诱导研究[J]. 北方园艺, 2010(20):152-154.
	ZHANG L Q, CHANG Q J, LIAO X K, et al. Inducement of Clematis florida var.plena Callus[J]. North Hortic, 2010(20):152-154.
[5]	马育珠, 李林芳, 马玲玲, 等. 铁线莲品种Clematis‘Bill MacKenzie’愈伤组织的诱导[J].北方园艺, 2015(24):94-97.
	MA Y Z, LI L F, MA L L, et al. Callus induction from Clematis‘Bill MacKenzie’[J]. North Hortic, 2015(24):94-97.DOI: 10.11937/bfyy.201524027.
[6]	宋微, 张虎, 王磊, 等. 铁线莲‘Polish Spirit’组培快繁体系的建立[J]. 分子植物育种, 2020,18(1):261-267.
	SONG W, ZHANG H, WANG L, et al. Clematis ‘Polish spirit’ establishment of tissue culture and rapid propagation system[J]. Mol Plant Breed, 2020,18(1):261-267.DOI: 10.13271/j.mpb.018.000261.
[7]	吴红, 李成忠, 张成霞. 钝齿铁线莲根尖组培再生体系的研究[J]. 云南农业大学学报(自然科学), 2017,32(1):120-124.
	WU H, LI C Z, ZHANG C X. Establishment of micropropagation system from root tip segment of Clematis apiifoliavar[J]. J Yunnan Agric Univ (Nat Sci), 2017,32(1):120-124.DOI: 10.16211/j.issn.1004-390X(n).2017.01.018.
[8]	魏淑云, 陈建华, 张荻, 等. ‘里昂城’铁线莲组培体系的建立[J]. 上海交通大学学报(农业科学版), 2017,35(1):22-28,33.
	WEI S Y, CHEN J H, ZHANG D, et al. Establishment of regeneration system of Clematis ‘Ville de Lyon’[J]. J Shanghai Jiaotong Univ (Agric Sci), 2017,35(1):22-28,33.DOI: 10.3969/J.ISSN.1671-9964.2017.01.004.
[9]	巩振辉, 申书兴. 植物组织培养[M]. 2版.北京: 化学工业出版社, 2007: 14-24.
	GONG Z H, SHEN S X. Plant tissue culture[M]. 2nd Ed, Beijing:Chemical Industry Press, 2007: 14-24.
[10]	茅汝佳, 高燕. 巴迪亚寿的组织培养及育苗技术[J]. 浙江农业科学, 2018,59(3):477-481.
	MAO R J, GAO Y. Tissue and seeding technique of Haworthia mirabilis var badia[J]. J Zhejiang Agric Sci, 2018,59(3):477-481.DOI: 10.16178/j.issn.0528-9017.20180337.
[11]	马燕, 韩瑞超, 臧德奎, 等. 木本观赏植物组织培养研究进展[J]. 安徽农业科学, 2012,40(4):1956-1958,2036.
	MA Y, HAN R C, ZANG D K, et al. Research advances in the tissue culture of woody ornamental plant[J]. J Anhui Agric Sci, 2012,40(4):1956-1958,2036.DOI: 10.13989/j.cnki.0517-6611.2012.04.201.
[12]	高燕, 张婷, 奉树成. 木本植物的无性繁殖方法[J]. 现代农业科技, 2018(4):129-130,132.
	GAO Y, ZHANG T, FENG S C. Asexual propagation of woody plants[J]. Mod Agric Sci Technol, 2018(4):129-130,132.
[13]	邓小梅, 吴乔娜, 李蕊萍, 等. 壳斗科植物组织培养研究进展[J]. 南京林业大学学报(自然科学版), 2018,42(4):171-180.
	DENG X M, WU Q N, LI R P, et al. Research progress on in vitro micropropagation of Fagaceae plants[J]. J Nanjing For Univ (Nat Sci Ed), 2018,42(4):171-180.
[14]	胡蓉. 植物激素在组织培养中的作用[J]. 川北教育学院院刊, 1989(1):44-47.DOI: 10.13974/j.cnki.51-1645/z.1989.01.012.
[15]	吉训志, 秦晓威, 胡丽松, 等. 木本植物组织培养[J]. 热带农业科学, 2019,39(4):33-40.
	JI X Z, QIN X W, HU L S, et al. Current research on tissue culture of woody plants[J]. Chin J Trop Agric, 2019,39(4):33-40.
[16]	MANDEGARAN Z, SIEBERV K. Somatic embryogenesis in Clematis integrifolia× C.viticella[J]. Plant Cell Tissue Organ Cult, 2000,62(2):163-165.DOI: 10.1023/A:1026514707169. doi: 10.1023/A:1026514707169
[17]	黄鑫, 张彦妮. 铁线莲‘BlekitnyAniol’组织培养及再生体系建立[J]. 草业科学, 2018,35(3):542-550.
	HUANG X, ZHANG Y N. In vitro tissue culture and plant regeneration of Clematis florida‘BlekitnyAniol’[J]. Pratacultural Sci, 2018,35(3):542-550.
[18]	NAZ S, ALI A, SIDDIQUE A. Somatic embryogenesis and plantlet formation in different varieties of sugarcane (Saccharum officinarum L.)HSF-243 and HSF-245[J]. Journal of Aapos, 2008,24(4):593-598.
[19]	杨塞, 肖层林. 赤霉素的生物合成及促进水稻茎伸长机理研究进展[J]. 作物研究, 2004,18(5):317-320.
	YANG S, XIAO C L. Research progress of gibberellin biosynjournal and mechanism of promoting rice stem elongation[J]. Crop Res, 2004,18(5):317-320.DOI: 10.16848/j.cnki.issn.1001-5280.2004.s1.006.
[20]	RADHA J, SINGH S N, SOLOMON S, et al. Potential regulatory role of gibberellic acid on sprouting and early stalk growth of sugarcane[J]. Guangxi Agricultural Sciences, 2010. 41(9):1025-1028.
[21]	JEON H W, CHO J S, PARK E J, et al. Developing xylem-preferential expression of PdGA20ox1,a gibberellin 20-oxidase 1 from Pinus densiflora,improves woody biomass production in a hybrid poplar[J]. Plant Biotechnol J, 2016,14(4):1161-1170.DOI: 10.1111/pbi.12484. doi: 10.1111/pbi.12484
[22]	郑绪辰, 葛雨萱, 王丽金, 等. 赤霉素、水杨酸、柠檬酸和蔗糖对灰毛黄栌叶色变化的影响[J]. 园艺学报, 2013(11):2199-2206.
	ZHENG X C, GE Y X, WANG L J, et al. The effects of several chemicals on leaf color changes of Cotinus coggygria var. cianerea[J]. Acta Horticulturae Sinica, 2013(11):2199-2206.DOI: 10.3969/j.issn.0513-353X.2013.11.011.
[23]	张志军. 马铃薯试管薯快繁及其调控机理研究[D]. 杭州:浙江大学, 2004.
	ZHANG Z J. Studies on the microtuber propagation and regulation mechanism in potato[D]. Hangzhou:Zhejiang University, 2004.
[24]	张赫岩, 叶冬梅, 白玉娥, 等. 重瓣榆叶梅茎段组织培养体系的建立[J]. 中南林业科技大学学报, 2019,39(5):119-123,137.
	ZHANG H Y, YE D M, BAI Y E, et al. Establishment of tissue culture system of stem segment in Amygdalus triloba f.multiplex[J]. J Central South Univ For Technol, 2019,39(5):119-123,137.DOI: 10.14067/j.cnki.1673-923x.2019.05.017.
[25]	董玉玲, 陈茂盛, 王秀兰, 等. 木本油料作物美藤果组织培养植株再生体系的建立[J]. 分子植物育种, 2016,14(2):462-470.
	DONG Y L, CHEN M S, WANG X L, et al. Establishment of in vitro regeneration system of woody oil crop Plukenetia volubilis[J]. Mol Plant Breed, 2016,14(2):462-470.DOI: 10.13271/j.mpb.014.000462.

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

铁线莲‘朱卡’组织培养技术及再生体系的建立

Tissue culture and plant regeneration of Clematis ‘Julka’

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 25

相关文章 4

编辑推荐

Metrics

本文评价

作者

读者

审稿

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

[1]	程方, 孙婷玉, 叶建仁. 抗松针褐斑病湿地松未成熟合子胚胚性愈伤组织的诱导[J]. 南京林业大学学报（自然科学版）, 2023, 47(6): 175-182.
[2]	俞子承, 凌聪, 陈赢男, 李淑娴, 尹佟明, 李小平. 雄性二倍体毛白杨再生体系的构建和遗传转化的研究[J]. 南京林业大学学报（自然科学版）, 2022, 46(1): 187-196.
[3]	袁德义，范晓明，谭晓风，曾艳玲，唐静，杨亚. 油茶带芽茎段及叶片离体培养再生体系的建立[J]. 南京林业大学学报（自然科学版）, 2013, 37(05): 35-39.
[4]	范晓明,袁德义*,谭晓风,邹锋,姜志娜，王晓红，曾艳玲. 油茶花药离体培养影响因子研究[J]. 南京林业大学学报（自然科学版）, 2011, 35(05): 125-128.