Please wait a minute...
浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (2): 157-163    DOI: 10.3785/j.issn.1008-9209.2018.03.261
园艺学     
蛛网萼未成熟种子培养与离体繁殖
曹丽雯1(),支肖1,戴嘉禾1,于宁宁1,陈怡倩1,叶立新2,陈利萍1(),徐礼根1()
1. 浙江大学农业与生物技术学院园艺系，杭州 310058
2. 浙江凤阳山-百山祖国家级自然保护区凤阳山管理处，浙江 丽水 323700
Immature seed culture and in vitro propagation of Platycrater arguta
Liwen CAO1(),Xiao ZHI1,Jiahe DAI1,Ningning YU1,Yiqian CHEN1,Lixin YE2,Liping CHEN1(),Ligen XU1()
1. Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
2. Fengyangshan Administration Office, Fengyangshan-Baishanzu National Nature Reserve, Lishui 323700, Zhejiang, China
 全文: PDF(3050 KB)   HTML ( )  HTML
摘要：

以国家二级濒危种蛛网萼为研究材料，通过未成熟种子离体培养、“芽生芽”离体繁殖、秋水仙素诱导、生根等组织培养技术开展物种的保护与繁殖研究。结果发现：蛛网萼未成熟种子培养在木本植物用培养基（woody plant medium, WPM）+400 mg/L谷氨酰胺+500 mg/L水解酪蛋白+2 000 mg/L活性炭培养基上可被诱导萌发，35 d萌发率达40%。以种子萌发的带腋芽实生苗茎段为外植体，在WPM附加1 mg/L 6-苄氨基嘌呤（6-benzylaminopurine, 6-BA）和0.5 mg/L α-萘乙酸（1-naphthylacetic acid, NAA）的培养基上培养有利于其芽的诱导与扩大繁殖；在附加200 mg/L秋水仙素的液体MS培养基上培养，可诱导出混倍体的芽。再生芽在WPM+0.5 mg/L 吲哚丁酸（indole butyric acid, IBA）培养基上进行生根并获得完整植株，部分植株野外回归至浙江凤阳山-百山祖国家级自然保护区中。本研究为解决蛛网萼种子萌发率差、植株生长缓慢、种群增强困难等问题提供了新途径。
关键词： 蛛网萼;  未成熟种子;  离体繁殖;  多倍体诱导;  野外回归    
Abstract:

Platycrater arguta, which is one of the Class Ⅱ national endangered species in China, was selected to conduct protective and reproductive study by tissue culture techniques including in vitro culture of immature seeds, in vitro propagation of shoot, colchicine induction, and taking root. When the immature seeds were cultured on the woody plant medium (WPM)+400 mg/L glutamine+500 mg/L casein hydrolysate+2 000 mg/L activated carbon, the germination rate of P. arguta could reach 40% after 35 d. As explants, stem segments of seedlings that germinated from immature seeds were cultured on WPM with different concentrations of 6-benzylaminopurine (6-BA) and 1-naphthylacetic acid (NAA), and MS mediums with different concentrations of colchicine, respectively. The results showed that the WPM with 1 mg/L 6-BA and 0.5 mg/L NAA was best for induction and growth of regeneration buds, and the MS medium with 200 mg/L colchicine could induce mixoploids. Finally, the regeneration buds were transplanted on a WPM supplemented with 0.5 g/mL indole butyric acid (IBA) to induce roots. Some of the plants have been returned to Fengyangshan-Baishanzu National Nature Reserve, Zhejiang. This study provides a new approach to solve the problems such as the poor seed germination rate of mature seed, slow plant growth and regeneration difficulty of P. arguta.

Key words: Platycrater arguta    immature seeds    in vitro propagation    polyploid induction    back to wild
收稿日期: 2018-03-26 出版日期: 2019-04-25
CLC:  S 688.9  
基金资助: 农业农村部“引进国际先进农业技术”项目(2011-G31);浙江省科技计划项目(2019C02026);浙江省重大科技专项重点社会发展项目(2015C03012)
通讯作者: 陈利萍,徐礼根     E-mail: 11216054@zju.edu.cn;chenliping@zju.edu.cn;xuligeng@126.com
作者简介: 曹丽雯（https://orcid.org/0000-0002-5589-4289），E-mail： 11216054@zju.edu.cn|陈利萍（https://orcid.org/0000-0002-3102-0640），E-mail： chenliping@zju.edu.cn|徐礼根（https://orcid.org/0000-0002-8195-7212）， E-mail： xuligeng@126.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
曹丽雯
支肖
戴嘉禾
于宁宁
陈怡倩
叶立新
陈利萍
徐礼根

引用本文:

曹丽雯,支肖,戴嘉禾,于宁宁,陈怡倩,叶立新,陈利萍,徐礼根. 蛛网萼未成熟种子培养与离体繁殖[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(2): 157-163.

Liwen CAO,Xiao ZHI,Jiahe DAI,Ningning YU,Yiqian CHEN,Lixin YE,Liping CHEN,Ligen XU. Immature seed culture and in vitro propagation of Platycrater arguta. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(2): 157-163.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.03.261    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I2/157

培养基名称

Medium name

基本成分

Elementary composition

其他成分

Other composition

种子萌发培养基

Medium for seed germination

 WPM 400 mg/L谷氨酰胺+500 mg/L水解酪蛋白+2 000 mg/L活性炭

1号繁育培养基

No. 1 propagation medium

 WPM 1 mg/L 6-BA+0.5 mg/L NAA

2号繁育培养基

No. 2 propagation medium

 WPM 3 mg/L 6-BA+0.5 mg/L NAA

3号繁育培养基

No. 3 propagation medium

 WPM 1 mg/L 6-BA+1 mg/L NAA

4号繁育培养基

No. 4 propagation medium

 WPM 3 mg/L 6-BA+1 mg/L NAA

生根培养基

Rooting medium

 WPM 0.5 mg/L IBA
表1  培养基成分
图1  蛛网萼未成熟种子离体培养

种子类型

Seed types

处理方法

Treatment

发芽率

Germination rate/%

方正汇总行未成熟种子

Immature seeds

种子萌发培养基

Medium for seed germination

 40.00

成熟种子

Mature seeds

 CK 0.00

种子萌发培养基

Medium for seed germination

 0.00
表2  不同处理方法对蛛网萼种子萌发率的影响
图2  蛛网萼扩繁体系的建立

培养基种类

Medium type

存活率

Survival rate/%

平均起始株高

Average starting plant height/cm

平均终株高

Average final plant height/cm

平均生长高度变化率

Average height growth rate/%

1号繁育培养基

No. 1 propagation medium

 100 0.49±0.13Ba 0.78±0.19Aa 61.54±7.54a

2号繁育培养基

No. 2 propagation medium

 100 0.51±0.08Ba 0.70±0.09Aa 38.10±5.62b

3号繁育培养基

No. 3 propagation medium

 100 0.44±0.11Ba 0.57±0.13Aa 33.62±7.65b

4号繁育培养基

No. 4 propagation medium

 100 0.48±0.19Ba 0.58±0.19Aa 24.94±9.65b
表3  不同植物生长调节剂浓度处理对蛛网萼生长的影响
图 3  不同质量浓度的秋水仙素处理后蛛网萼的流式细胞分析图

ρ（秋水仙素）

Concentrations of colchicine/(mg/L)

存活率

Survival rate/%
100 77.89±11.06a
200 55.67±11.33a
300 11.00±11.00b
500 0.00±0.00b
表4  不同质量浓度的秋水仙素对蛛网萼存活率的影响

ρ（秋水仙素）

Concentrations of colchicine/(mg/L)

诱导混倍体芽的个数

Numbers of induced buds of mixoploids
100 2
200 5
300 0
500 0
表5  不同质量浓度的秋水仙素诱导对蛛网萼混倍体芽个数的影响
图4  蛛网萼生根与移栽炼苗
1 中国科学院中国植物志编辑委员会 .中国植物志:第35卷.北京:科学出版社,1995:189.
Chinese Academy of Sciences . Editorial Committee of Flora of China. Flora of China (Volume 35). Beijing: Science Press, 1995:189. (in Chinese)
2 乐新贵,谭策铭,李林海,等 .贵溪市阳际峰自然保护区新发现成片蛛网萼群落.江西林业科技,2008(4):42-43.
LE X G , TAN C M , LI L H , et al . New discovery on flaky community of Platycrater arguta of Yangjifeng Nature Reserve in Guixi City, Jiangxi, China. Jiangxi Forestry Science and Technology, 2008(4):42-43. (in Chinese with English abstract)
3 QI X S , YUAN N , QIU Y X . Development of 12 microsatellite markers for Platycrater arguta (Hydrangeaceae) endemic to East Asia. American Journal of Botany, 2012,99(8):e304-e306.
4 毛夷仙,袁荣斌,程英,等 .江西武夷山国家级自然保护区蛛网萼群落的发现与保护建议.南方林业科学,2016,44(2):67-68,72.
MAO Y X , YUAN R B , CHENG Y , et al . The discovery and conservation recommendations of Platycrater arguta community in Jiangxi Wuyishan National Nature Reserve. South China Forestry Science, 2016,44(2):67-68,72. (in Chinese with English abstract)
5 傅立国 .中国植物红皮书:稀有濒危植物（第1分册）.北京:科学出版社,1992:608．
FU L G. China Plant Red Data Book: Rare and Endangered Plants (Volume 1). Beijing: Science Press, 1992:608. (in Chinese)
6 张丽芳,林昌勇,俞群,等 .珍稀濒危植物蛛网萼的种子形态及萌发特性.江西农业大学学报,2015,37(3):497-503.
ZHANG L F , LIN C Y , YU Q , et al . The morphological structure and germination characters of seed of rare and endangered species Platycrater arguta . Acta Agriculturae Universities Jiangxiensis, 2015,37(3):497-503. (in Chinese with English abstract)
7 张丽芳,裘利洪 .蛛网萼开花物候、花部特征及繁育系统研究.广西植物,2017,37(10):1301-1311.
ZHANG L F , QIU L H . Flowering phenology, floral traits and breeding system of Platycrater arguta . Guihaia, 2017,37(10):1301-1311. (in Chinese with English abstract)
8 LYNCH P T . Tissue culture techniques in in vitro plant conservation//Plant Conservation Biotechnology . Boca Raton, Florida: CRC Press, 2014:67-88.
9 COMAI L . The advantages and disadvantages of being polyploid. Nature Reviews Genetics, 2005,6(11):836-846.
10 EECKHAUT T G R , WERBROUCK S P O , LEUS L W H , et al . Chemically induced polyploidization in Spathiphyllum wallisii Regel through somatic embryogenesis. Plant Cell, Tissue and Organ Culture, 2004,78(3):241-246.
11 PETERSEN K K , HAGBERG P , KRISTIANSEN K . Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis . Plant Cell, Tissue and Organ Culture, 2003,73(2):137-146.
12 CHEN L P , WANG Y J , ZHAN M . In vitro induction and characterization of tetraploid Lychnis senno Siebold et Zucc. HortScience, 2006,41(3):759-761.
13 MCCOWN B H . Woody plant medium (WPM)-a mineral nutrient formulation for microculture for woody plant species. HortScience, 1981,16:453.
14 丁兰,张丽,郭柳,等 .濒危植物佛手参种子的非共生萌发及种苗的快速繁殖.植物生理学报,2014,50(1):77-82.
DING L , ZHANG L , GUO L , et al . Asymbiotic seed germination and rapid seedling regeneration of endangered Gymnadenia conopsea (L.) R. Br. Plant Physiology Journal, 2014,50(1):77-82. (in Chinese with English abstract)
15 YU L , CHEN H W , HONG P P , et al . Adventitious bud induction and plant regeneration from stem nodes of Salvia splendens ‘Cailinghong’. HortScience, 2015,50(6):869-872.
16 GEORGE E F , HALL M A , KLERK G J D . Plant Propagation by Tissue Culture. 3rd ed. Dordrecht, the Netherlands: Springer, 2008:1-28.
17 黄烈健,王鸿 .林木植物组织培养及存在问题的研究进展.林业科学研究,2016,29(3):464-470.
HUANG L J , WANG H . Advances in tissue culture techniques of trees and the problems existed. Forest Research, 2016,29(3):464-470. (in Chinese with English abstract)
18 WAN Y , PETOLINO J F , WIDHOLM J M . Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus. Theoretical and Applied Genetics, 1989,77(6):889-892.
19 ZAHEDI A A , HOSSEINI B , FATTAHI M , et al . Overproduction of valuable methoxylated flavones in induced tetraploid plants of Dracocephalum kotschyi Boiss. Botanical Studies, 2014,55:22.
20 吉仁花,于林清,白淑兰 .秋水仙素对金达苜蓿染色体加倍效果的影响.西北农林科技大学学报(自然科学版),2018,46(1):103-110.
JI R H , YU L Q , BAI S L . Effects of colchicine on chromosome doubling of Medicago sativa L cv. Jinda..Journal of Northwest A & F University (Natural Science Edition), 2018,46(1):103-110. (in Chinese with English abstract)
21 SHI Q H , LIU P , LIU M J , et al . A novel method for rapid in vivo induction of homogeneous polyploids via calluses in a woody fruit tree (Ziziphus jujube Mill.). Plant Cell, Tissue and Organ Culture, 2015,121(2):423-433.
22 KADOTA M , NIIMI Y . In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N. cv. Hosui). Plant Cell Reports, 2002,21(3):282-286.
23 ZHU X Y , ZHAO M , MA S, et al . Induction and origin of adventitious shoots from chimeras of Brassica juncea and Brassica oleracea . Plant Cell Reports, 2007,26(10):1727-1732.
No related articles found!