利用流式细胞术鉴定桑树染色体倍性的方法

Establishment of Morus L. Chromosome Ploidy Identification Method Using Flow Cytometry

利用流式细胞术可以快速、准确地鉴定植物染色体倍性,但需要针对不同植物样本的制备及具体操作对应用方法进行选择和优化。为了建立适合桑树染色体倍性快速鉴定的流式细胞术应用方法,以桑树幼叶为材料,比较以不同解离液及机械解离方法和不同离心漂洗次数制作样本用流式细胞术检测的效果,并用不同染色体倍性桑品种的幼叶为材料,以效果最佳的方法进行验证试验。优化的样本制备方案为:采集0.2 g桑树幼叶,置于预冷的平面玻璃上,加入Mg SO4解离液后,用双面刀片快速切碎,转移至培养皿中静置3~5 min,用300目细胞筛网过滤至1.5 m L离心管中,得到500μL单细胞核悬浮液,再加入PI溶液(最终质量浓度为50μg/m L)和RNase A溶液(最终质量浓度为30μg/m L),4℃避光环境下染色30 min,经300目细胞筛网过滤后用流式细胞仪检测。如果采集叶片后不能立即进行检测,可在-80℃下保存待测。试验结果还表明,用在-80℃保存10 d和40 d的冷冻幼叶与用新鲜幼叶制备的样本所得到的检测效果相似。初步建立的适合桑树染色体倍性鉴定的流式细胞术应用方法效果最佳,具有细胞碎片少、主峰清晰、杂峰少等优点。

Flow cytometry is a rapid and accurate technology for identifying plant ploidy. However, for certain plants, specific operation methods should be employed and optimized for sample preparation. This paper is aimed at establishing a rapid method suitable for Morus L. ploidy identification using flow cytometry. By using Morus L. young leaves as experimental material, we compared the effects of different dissociation buffers, mechanical dissociations and centrifugation times on identification of chromosome ploidy by flow cytometry. Furthermore, we verified the optimal method using mulberry young leaves of different chromosome ploidy. The results indicated that the following steps constitute a suitable sample preparation method for mulberry ploidy identification： taking 0.2 g young mulberry leaves as material, adding MgSO4 dissociation buffer, chopping the leaves quickly on chilled plain glass with double-edged razor blade, transferring the sample into a petri dish and keeping static for 3 to 5 minutes, and sieving the sample into a 1.5 mL centrifugation tube using 300 mesh cell strainer. The resultant single nucleus suspension （500 μL） was added with PI solution （50 μg/mL） and RNase A solution （30 μg/mL）, stained for 30 minutes under dark condition at 4 ℃, sieved with 300 mesh cell strainer, and finally sent to testing on flow cytometer. If no instant testing can be carried out after leaves are collected, they can be stored under -80 ℃ for later use： In addition, we found that the samples prepared using frozen leaves preserved for 10 d and 40 d at -80 ℃ and using fresh leaves had little difference on the test effect. We preliminary established a suitable method for identifying Morus L. chromosome ploidy using flow cytometry which could achieve optimal identification result that includes less cellular debris, clear main peak and less miscellaneous peaks, etc.