作物杂志,2018, 第6期: 162–167 doi: 10.16035/j.issn.1001-7283.2018.06.026

• 种子科技 • 上一篇    

消毒方法对红花种子发芽率的影响和种子环境细菌研究

马梦雪,赵玲玲,唐思,陈贤军,覃瑞   

  1. 中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北重点实验室,430074,湖北武汉
  • 收稿日期:2018-05-09 修回日期:2018-10-11 出版日期:2018-12-15 发布日期:2018-12-06
  • 作者简介:马梦雪,硕士研究生,研究方向为植物细胞遗传
  • 基金资助:
    湖北省科技条件平台建设专项(2017BE014);湖北省技术创新专项重大项目(2018ABA093)

The Effects of Different Disinfection Methods on Seed Germination and Study on the Environmental Bacteria in Safflower (Carthamus tinctorius L.)

Ma Mengxue,Zhao Lingling,Tang Si,Chen Xianjun,Qin Rui   

  1. College of Life Sciences, South-Central University for Nationalities/Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China, Wuhan 430074, Hubei, China
  • Received:2018-05-09 Revised:2018-10-11 Online:2018-12-15 Published:2018-12-06

摘要:

红花(Carthamus tinotorius L.)种子在发芽和组培中染菌情况严重,发芽率较低,针对这一现象,对供试红花种子进行消毒以降低染菌率并提高发芽率。选择种子预浸泡时间、消毒剂种类、消毒时间3个因素,采用L9(3 4)正交试验,以种子的发芽率、发芽势和染菌率为考察指标,综合分析种子消毒效果。结果表明,消毒剂种类是影响红花种子消毒效果的最重要因素。红花种子最佳消毒方法为种子消毒前用无菌水预浸泡12h,再经2% NaClO消毒10min,染菌率仅为8%,发芽率高达90.67%。另外,对分离纯化消毒后种子所带的细菌16S rDNA(GenBank登录号:MH190221)进行了PCR扩增,序列Blast结果显示,该菌为阿耶波多芽孢杆菌(Bacillus aryabhatti),初步判断该菌可能为红花种子内生菌。该研究结果为快速获得生长良好的无菌苗、后续的组织培养以及遗传转化奠定了基础。

关键词: 红花, 种子消毒, 发芽率, 正交试验, 细菌

Abstract:

Contamination was a serious problem in seed germination and tissue culture of safflower (Carthamus tinctorius L.). In our study, seeds were disinfected in order to reduce the contamination rate.Three factors including soaking time, disinfecting agents and disinfecting time were used for L9(3 4) orthogonal experiment. Results were comprehensively evaluated by using the weighted scoring method based on indexes such as germination rate, germination vigor and contamination rate. Orthogonal test showed that the disinfecting agent was the most important factor affecting the seeds germination. Among various treatments, seeds soaked in sterile water for 12h followed by the 2% sodium hypochlorite (NaClO) for 10min was the best disinfecting method in safflower seeds which yield a contamination rate of 8% and a germination rate of 90.67%. In addition, 16S rDNA (GenBank Accession No. MH190221) of a bacterium was isolated from seeds and purified after disinfection which was amplified by PCR. BLAST results showed that it belonged to Bacillus aryabhatti which was most likely to be entophytes in safflower seeds. The results of this study provided an important basis for obtaining well-grown aseptic seedlings, tissue culture as well as genetic transformation.

Key words: Carthamus tinctorius L., Seed disinfection, Seed germination rate, Orthogonal experiment, Bacteria

表1

正交试验因素水平"

水平
Level
因素Factor
A预浸泡时间(h)
Soaking time
B消毒剂
Disinfecting agent
C消毒时间(min)
Disinfecting time
D对照
Control
1 0 0.1% HgCl2 10
2 6 2% NaClO 15
3 12 10% H2O2 20

表2

正交试验设计L9(34)对种子发芽率的影响"

处理
Treatment
A
预浸泡时间(h)
Soaking time
B
消毒剂种类
Disinfecting agent
C
消毒时间(min)
Disinfecting time
D
对照
Control
染菌率(%)
Contamination
rate
发芽势(%)
Germination vigor
发芽指数(%)
Germination index
发芽率(%)
Germination
rate
综合评分
Overall score
T1 0 1(0.1% HgCl2 10 1 9.33 34.67 33.65 64.67 0.27
T2 0 2(2% NaClO) 15 2 19.33 34.00 38.68 71.33 0.15
T3 0 3(10% H2O2 20 3 24.00 24.00 28.95 48.67 -0.06
T4 6 1(0.1% HgCl2 15 3 3.33 50.00 45.37 75.33 0.45
T5 6 2(2% NaClO) 20 1 10.67 55.33 57.22 80.67 0.39
T6 6 3(10% H2O2 10 2 27.33 36.00 40.02 61.33 -0.01
T7 12 1(0.1% HgCl2 20 2 2.00 53.33 51.10 85.33 0.53
T8 12 2(2% NaClO) 10 3 8.00 59.33 63.17 90.67 0.48
T9 12 3(10% H2O2 15 1 18.67 44.67 46.08 67.33 0.17
K1 0.36 1.24 0.74 0.83
K2 0.83 1.02 0.77 0.67
K3 1.18 0.11 0.86 0.88
k1 0.12 0.41 0.25 0.28
k2 0.28 0.34 0.26 0.22
k3 0.39 0.04 0.29 0.29
R 0.28 0.38 0.04 0.07

图1

不同处理对种子发芽率、发芽势和发芽指数的影响"

表3

各因素间方差分析"

因素Factor 偏差平方和
Square of deviance
自由度
Degrees of freedom
F
A预浸泡时间(h)
Soaking time
0.113 2 37.667*
B消毒剂种类
Disinfecting agent
0.247 2 82.333*
C消毒时间(min)
Disinfecting time
0.003 2 1.000
D对照Control 0.000 2

表4

验证试验结果"

编号
Number
发芽率
Germination rate
发芽势
Germination vigor
染菌率
Contamination rate
1 91.33 60.00 7.33
2 90.67 60.67 7.33
3 90.00 58.00 8.67
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