番茄种子消毒方法探究
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摘要
番茄种子表面常常带有病原微生物,故在培养番茄无菌苗时,需要进行消毒。传统番茄种子的消毒剂是氯化汞,而其较大的毒性和消毒原理限制其在实验室及实际生产上的应用。因此,本研究开发出一套新的采用无毒或低毒试剂的消毒方法来制备番茄无菌苗,从而消除重金属离子对蛋白质的变性作用,并排除杂菌污染,进而为探究蛋白类物质对番茄的促生作用做前期实验方法的研究铺垫。对氯化汞、次氯酸钠和过氧化氢三种消毒剂采用正交实验,选取各自合适的浓度和时间梯度联合洗衣粉和75%乙醇消毒效果进行最佳消毒条件的摸索。结果表明,20%次氯酸钠消毒5 min以及8%次氯酸钠消毒+1%吐温20消毒1 min的消毒效果较好,但吐温20对幼苗的苗重有一定的抑制作用。运用单因素实验设计对正交实验结果进行验证和进一步摸索消毒条件,结果表明,次氯酸钠消毒对种子萌发率影响不大,次氯酸钠浓度在10%～40%的差异不显著,故最佳的消毒方式为10%次氯酸钠消毒5 min。通过蛋白质溶液进行消毒条件的验证,最终表明,建立的番茄种子消毒方法可以用于研究活性物质对番茄的生理作用,且消毒效果较好。
There are pathogenic microorganisms on the surface of tomato seeds, so it is necessary to sterilize for cultivating the aseptic seedlings of tomato. The traditional disinfectant for tomato seed is mercuric chloride, but its toxicity and sterilization principle limit the application in the laboratory and practical production. Therefore, this study developed a set of new sterilization method by using non-toxic or low-toxic disinfectants in order to eliminate the denaturation effect of heavy metal ions on proteins and exclude the microbial contamination, thus preparing for the exploration of growth-promoting effect of protein on tomato. We used the orthogonal experiment, selected the suitable concentration and time gradient of three kinds of disinfectants, mercuric chloride, sodium hypochlorite and hydrogen peroxide and explored the best sterilization conditions with the combination of washing powder and 75% alcohol. Results show that 20% sodium hypochlorite sterilization 5 minutes or 8% sodium hypochlorite and 1% Tween20 sterilization 1 minutes is better, but Tween20 has certain inhibition to the weight of seedlings. Using the single factor experimental design to verify the results of orthogonal experiment and further explore the sterilization conditions, the results show that the sodium hypochlorite sterilization has little effects on seed germination rate and the concentration of sodium hypochlorite in 10% ～ 40% has no significant difference. So the best way of sterilization is 10% sodium hypochlorite sterilization 5 minutes. The verification of sterilization conditions by using protein solution finally indicates that this established tomato seed sterilization method can be used to study the physiological function of active substances to tomato and the sterilization effect is better.
There are pathogenic microorganisms on the surface of tomato seeds, so it is necessary to sterilize for cultivating the aseptic seedlings of tomato. The traditional disinfectant for tomato seed is mercuric chloride, but its toxicity and sterilization principle limit the application in the laboratory and practical production. Therefore, this study developed a set of new sterilization method by using non-toxic or low-toxic disinfectants in order to eliminate the denaturation effect of heavy metal ions on proteins and exclude the microbial contamination, thus preparing for the exploration of growth-promoting effect of protein on tomato. We used the orthogonal experiment, selected the suitable concentration and time gradient of three kinds of disinfectants, mercuric chloride, sodium hypochlorite and hydrogen peroxide and explored the best sterilization conditions with the combination of washing powder and 75% alcohol. Results show that 20% sodium hypochlorite sterilization 5 minutes or 8% sodium hypochlorite and 1% Tween20 sterilization 1 minutes is better, but Tween20 has certain inhibition to the weight of seedlings. Using the single factor experimental design to verify the results of orthogonal experiment and further explore the sterilization conditions, the results show that the sodium hypochlorite sterilization has little effects on seed germination rate and the concentration of sodium hypochlorite in 10% ～ 40% has no significant difference. So the best way of sterilization is 10% sodium hypochlorite sterilization 5 minutes. The verification of sterilization conditions by using protein solution finally indicates that this established tomato seed sterilization method can be used to study the physiological function of active substances to tomato and the sterilization effect is better.
引文
[1]杨华,崔元玗,孙晓军,等.加工番茄种子带菌检测与种子消毒处理试验研究[J].新疆农业科学,2005,42(5):345-348
[2]葛体达,宋世威,姜武,等.不同甘氨酸浓度对无菌水培番茄幼苗生长和氮代谢的影响[J].生态学报,2009,29(4):1994-2002
[3]魏丽丽,刘国军,王瑞刚.表面消毒剂对番茄无菌苗生长及其内源IAA的影响[C].呼和浩特:培养创新型人才、推进科技创新、推动转变经济发展方式——内蒙古自治区第六届自然科学学术年会优秀论文集,2011
[4]杨岚,唐琳,刘建国,等.番茄种子两种灭菌法效果评价[J].贵州医药,2008,32(1):26-28
[5]刘思言,王丕武,关淑艳,等.番茄种子消毒方法及愈伤组织诱导的初步研究[J].河南农业科学,2012,41(3):113-115
[6]唐琳,刘建国,马欣荣.两种灭菌方法对番茄种子灭菌效果的评价[J].种子,2008,27(9):17-18
[7]吴志刚,宋明,王志敏,等.番茄组织培养中无菌苗培养条件的优化[J].中国农学通报,2006,22(4):335-337
[8]鲁黎明,安影.不同消毒剂对烟草种子消毒效果及萌发的影响[J].种子,2012,31(4):93-95
[9]雷天翔,蔡晓剑,周党卫,等.三种茄科植物种子无菌苗繁殖体系的初建[J].中药材,2015,38(3):447-450
[10] Kasselaki AM, Goumas D, Tamm L, et al. Effect of alternative strategies for the disinfection of tomato seed infected with bacterial canker(Clavibacter michiganensis subsp. michiganensis)[J]. NJAS-Wageningen Journal of Life Sciences, 2011,58(3-4):145-147
[11]吴华,高志鹏,陈高,等.不同消毒处理对豇豆种子无菌培养的效应[J].江汉大学学报:自然科学版,2015,43(5):389-392
[12]胡重怡,郑少清,陈兴江,等.烟草无菌苗培养前的种子消毒技术研究[J].中国烟草科学,2007,28(2):45-46
[13] Lindsey BE, Rivero L, Calhoun CS, et al. Standardized Method for High-throughput Sterilization of Arabidopsis Seeds[J]. Journal of Visualized Experiments, 2017(128):1-7
[14] Mickail KY, Bishay F, Farag NS, et al. Tomato seed treatments and seed storage to control bacterial spot or scab[J].Agricultural Research Review, 1970,48:323-327
[15] Siviero P. Tomato seed disinfection from bacteria and Tom MV[J]. Informatore Agrario, 1991,47(5):61-74
[16]董飞,陈运起,刘世琦,等.种子消毒方法对大葱无菌苗培养的影响[J].中国蔬菜,2011(18):74-76
[2]葛体达,宋世威,姜武,等.不同甘氨酸浓度对无菌水培番茄幼苗生长和氮代谢的影响[J].生态学报,2009,29(4):1994-2002
[3]魏丽丽,刘国军,王瑞刚.表面消毒剂对番茄无菌苗生长及其内源IAA的影响[C].呼和浩特:培养创新型人才、推进科技创新、推动转变经济发展方式——内蒙古自治区第六届自然科学学术年会优秀论文集,2011
[4]杨岚,唐琳,刘建国,等.番茄种子两种灭菌法效果评价[J].贵州医药,2008,32(1):26-28
[5]刘思言,王丕武,关淑艳,等.番茄种子消毒方法及愈伤组织诱导的初步研究[J].河南农业科学,2012,41(3):113-115
[6]唐琳,刘建国,马欣荣.两种灭菌方法对番茄种子灭菌效果的评价[J].种子,2008,27(9):17-18
[7]吴志刚,宋明,王志敏,等.番茄组织培养中无菌苗培养条件的优化[J].中国农学通报,2006,22(4):335-337
[8]鲁黎明,安影.不同消毒剂对烟草种子消毒效果及萌发的影响[J].种子,2012,31(4):93-95
[9]雷天翔,蔡晓剑,周党卫,等.三种茄科植物种子无菌苗繁殖体系的初建[J].中药材,2015,38(3):447-450
[10] Kasselaki AM, Goumas D, Tamm L, et al. Effect of alternative strategies for the disinfection of tomato seed infected with bacterial canker(Clavibacter michiganensis subsp. michiganensis)[J]. NJAS-Wageningen Journal of Life Sciences, 2011,58(3-4):145-147
[11]吴华,高志鹏,陈高,等.不同消毒处理对豇豆种子无菌培养的效应[J].江汉大学学报:自然科学版,2015,43(5):389-392
[12]胡重怡,郑少清,陈兴江,等.烟草无菌苗培养前的种子消毒技术研究[J].中国烟草科学,2007,28(2):45-46
[13] Lindsey BE, Rivero L, Calhoun CS, et al. Standardized Method for High-throughput Sterilization of Arabidopsis Seeds[J]. Journal of Visualized Experiments, 2017(128):1-7
[14] Mickail KY, Bishay F, Farag NS, et al. Tomato seed treatments and seed storage to control bacterial spot or scab[J].Agricultural Research Review, 1970,48:323-327
[15] Siviero P. Tomato seed disinfection from bacteria and Tom MV[J]. Informatore Agrario, 1991,47(5):61-74
[16]董飞,陈运起,刘世琦,等.种子消毒方法对大葱无菌苗培养的影响[J].中国蔬菜,2011(18):74-76