消毒方法对红花种子发芽率的影响和种子环境细菌研究

doi:10.16035/j.issn.1001-7283.2018.06.026

摘要/Abstract

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

马梦雪,赵玲玲,唐思,陈贤军,覃瑞. 消毒方法对红花种子发芽率的影响和种子环境细菌研究[J]. 作物杂志, 2018 (6): 162–167

Ma Mengxue,Zhao Lingling,Tang Si,Chen Xianjun,Qin Rui. The Effects of Different Disinfection Methods on Seed Germination and Study on the Environmental Bacteria in Safflower (Carthamus tinctorius L.)[J]. Crops, 2018(6): 162–167

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水平 Level	因素Factor
水平 Level	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	—

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

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

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