洋葱种子消毒和无菌苗培养新方法

doi:10.13560/j.cnki.biotech.bull.1985.2022-0983

摘要/Abstract

摘要：

为了获得简便、高效、低污染的洋葱种子消毒及无菌苗培养方法，通过控制植物组培抗菌剂（PPM）的pH值、消毒浸泡时间及培养浓度，对洋葱种子消毒及无菌苗培养进行研究。结果表明，PPM（20 mL/L，pH 2）浸种24-48 h消毒效果可以达到10% NaClO消毒10 min水平，且不影响种子发芽，8 d发芽势超过90%。污染微生物分析鉴定了4种真菌（Alternaria alternata、Fusarium proliferatum、Fusarium oxysporum、Stemphylium vesicarium）和2种细菌（Phytobacter diazotrophicus、Atlantibacter hermannii）可以通过洋葱种子进行传播，抑菌试验明确了PPM可有效的抑制污染微生物的生长。培养基中添加PPM可以有效的降低污染率，高浓度PPM（1-9 mL/L）可以抑制洋葱幼苗的生长，而低浓度PPM（0-0.5 mL/L）对洋葱幼苗生长无明显抑制作用。探索开发了一套操作简便无需繁琐冲洗的洋葱种子消毒以及高发芽率、低污染率无菌苗培养方法。

关键词: 洋葱, 种子, 消毒, 无菌苗, 植物组培抗菌剂（PPM）

Abstract:

This work aims to obtain a simple, efficient and low-contamination method for onion seed disinfection and aseptic seedling culture. The sterilization of onion seeds and the culture of sterile seedlings were studied by controlling the pH value, disinfection soaking time and culture concentration of plant preservative mixture（PPM）. The results showed that the disinfection effect of soaking seeds in PPM（20 mL/L, pH=2）for 24-48 h reached the level of 10% NaClO disinfection for 10 min, which did not affect seed germination, and the germination potential exceeded 90% in 8 d. The analysis of contaminated microorganisms found that four species of fungi（Alternaria alternata, Fusarium proliferatum, Fusarium oxysporum, and Stemphylium vesicarium）and two species of bacteria（Phytobacter diazotrophicus and Atlantibacter hermannii）were transmitted through onion seeds, and the bacteriostasis test confirmed that PPM was able to effectively inhibit the growth of contaminated microorganisms. The medium added with PPM effectively reduced the contamination rate using PPM for seed soaking disinfection; high concentration PPM（1-9 mL/L）inhibited the growth of onion seedlings, while low concentration PPM（0-0.5 mL/L）had no significant inhibitory effect on the growth of onion seedlings. In sum, a set of simple and efficent methods for onion seed disinfection and aseptic seedling culture are explored and developed.

Key words: onion（Allium cepa）, seed, disinfection, aseptic seedling, plant preservative mixture（PPM）

孙亚玲, 李瑞平, 王振宝, 张庶, 刘冰江, 霍雨猛. 洋葱种子消毒和无菌苗培养新方法[J]. 生物技术通报, 2023, 39(4): 212-220.

SUN Ya-ling, LI Rui-ping, WANG Zhen-bao, ZHANG Shu, LIU Bing-jiang, HUO Yu-meng. A New Method for Onion Seed Disinfection and Aseptic Seedling Culture[J]. Biotechnology Bulletin, 2023, 39(4): 212-220.

图/表 9

图1 PPM消毒液pH值对洋葱种子发芽（A）及消毒效果（B）的影响柱形图上方不同字母表示处理间差异显著（P<0.05）。横线上方为卡方值和调整P值。下同

Fig. 1 Effects of pH value of PPM disinfectant on onion seed germination（A）and disinfection（B） Different letters at the top of the column chart indicate significant differences among different treatments（P<0.05）. The Chi square value and the adjusted P value are marked above the horizontal line. The same below

图2 PPM消毒时间对洋葱种子发芽的影响 A1：干种子直播1/2 MS（1 mL/L PPM）；A2：20 mL/L PPM表面消毒后播种至1/2 MS（1 mL/L PPM）；A3-A7：20 mL/L PPM分别浸种消毒3、6、12、24、48 h后播种至1/2 MS（1 mL/L PPM）；A8：10% NaClO消毒10 min播种至1/2 MS；下同

Fig. 2 Effects of PPM disinfection time on onion seed germination A1：Directly sowed dry seeds to 1/2 MS（1 mL/L PPM）. A2：Sowed seeds to 1/2 MS（1 mL/L PPM）after surface disinfection by 20 mL/L PPM. A3-A7：Sowed seeds to 1/2 MS（1 mL/L PPM）after soaked and disinfected for 3, 6, 12, 24 and 48 h respectively by 20 mL/L PPM. A8：Sowed seeds to 1/2 MS after disinfection by 10% NaClO for 10 min. The same below

图3 PPM浸种时间对洋葱种子消毒效果的影响

Fig. 3 Disinfection effect of PPM soaking time on onion seeds

图4 真菌和细菌菌株PCR扩增结果

Fig. 4 PCR amplification of fungal and bacterial strains

表1 不同浓度PPM对4种真菌抑菌率的影响

Table 1 Inhibition rates of different concentrations of PPM in four fungi

浓度 Concentration/（mL·L^-1）	S. vesicarium	A. alternata	F. proliferatum	F. oxysporum
0.25	65.71±5.86a	40.13±4.06a	49.05±3.33a	51.87±3.14a
0.50	72.14±6.84ab	53.62±2.63b	57.87±2.58ab	59.7±7.48ab
1.00	78.08±3.63abc	58.52±3.87b	60.36±2.49b	61.09±6.44ab
2.00	81.6±3.52bcd	62.39±3.07bc	63.25±5.18b	67.46±6.76ab
4.00	91.07±2.52cd	71.16±1.78c	73.74±3.17c	71.67±6.5bc
8.00	92.01±3.43cd	85.14±1.97d	79.96±1.31c	86.44±1.99c
16.00	94.97±2.6d	89.45±2.31d	81.65±2.13c	87.19±3.02c

图5 不同浓度PPM对细菌Atlantibacter hermannii的抑菌效果 A：抑菌效果图；B：抑菌圈直径及显著性，不同小写字母表示差异显著性（P<0.05），抑菌圈 ≤5 mm表示无抑菌效果

Fig. 5 Inhibitory effects of different concentrations of PPM on Atlantibacter hermannii A：Bacteriostatic effect photograph. B：Diameter and significance of bacteriostatic circle, different lower-case letters indicate significant differences（P<0.05）and bacteriostatic circle with diameter ≤5 mm indicates no bacteriostatic effect

图6 不同PPM浓度（mL/L）对洋葱子叶和根生长的影响

Fig. 6 Effects of PPM concentrations（mL/L）on the growth of onion cotyledons and roots

图7 最佳方案在洋葱种子消毒和无菌苗培养上的应用 A-B：4次重复的发芽率和污染率；C-E：6、10、20 d无菌苗

Fig. 7 Application of the optimal scheme in onion seed disinfection and sterile seedling culture A-B：Germination rate and contamination rate of 4 replicates. C-E：Aseptic seedling in 6, 10 and 20 d

图8 最优方案在3种类型洋葱种子消毒及无菌苗培养上的应用 A-F：‘天正201’（6 d）、‘YY18’（6 d）、‘GCH’（6 d）、‘优越’（12 d）、‘佳美’（12 d）、‘卡木依-153’（12 d）

Fig. 8 Application of optimal scheme in disinfection of three types of onion seeds and culture of aseptic seedling A-F：‘TIANZHENG201’（6 d）, ‘YY18’（6 d）, ‘GCH’（6 d）, ‘YOUYUE’（12 d）, ‘JIAMEI’（12 d）, ‘KAMUYI-153’（12 d）

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