基于流式细胞仪鉴定树莓倍性方法的建立及应用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0475

摘要/Abstract

摘要：

目的建立一套高效易行检测树莓细胞倍性的方法，鉴定不同树莓种质的细胞倍性，为树莓倍性育种和杂交育种奠定理论基础。方法以树莓叶片为材料，对不同的解离液、叶片来源、叶龄、叶片的保存和运输方式等条件进行优化，建立流式细胞术检测树莓细胞倍性的方法，并使用此方法检测黑树莓、红树莓、黄树莓等不同树莓种质叶片、根以及愈伤组织的细胞倍性。结果流式细胞仪法检测树莓叶片细胞倍性时，解离液Ⅵ（WPB）制备的细胞核悬浮液上机检测效果最好，收集到的细胞核数最多，峰图最好；田间苗和组培苗嫩叶制备的细胞核悬浮液检测效果最好，成龄叶次之；把叶片夹在2层润湿滤纸中间并放于塑料平皿后，置于低温泡沫箱中，直接带着样品上机检测效果更好，将叶片从植株剪下后直接浸泡至装有无菌水的50 mL离心管中，再邮寄样品上机检测的效果次之；流式细胞仪检测叶片的直方图主峰位于荧光强度值10 000附近，用此检测方法鉴定了15个树莓种质叶片细胞的倍性均为二倍体，鉴定了‘龙园双丰’‘波尔卡’的根和愈伤组织的细胞倍性也均为二倍体。结论建立了一套能够检测不同发育时期和不同生长环境下树莓叶片和根以及愈伤组织细胞倍性的方法，并用此方法检测了黑树莓、红树莓和黄树莓共15个不同种质树莓倍性均为二倍体。

关键词: 树莓, 细胞, 倍性检测, 流式细胞术, 叶片, 根, 愈伤组织, 快速鉴定

Abstract:

Objective To establish a set of efficient and easy-to-operate methods for detecting the ploidy of raspberry cells, and then use this method to identify the ploidy of different raspberry germplasms, laying a theoretical foundation for ploidy breeding and hybrid breeding of raspberry. Method Taking raspberry leaves as the material, different dissociating solutions, leaf sources, leaf ages, leaf preservation and transportation methods and other conditions were optimized to establish a method for detecting the ploidy of raspberry cells by flow cytometry. This method was used to detect the cell ploidy of leaves, roots and callus of different raspberry germplasms including black raspberry, red raspberry and yellow raspberry. Result The nuclear suspension prepared with dissociating solution VI (WPB) had the best detection effect when using flow cytometry to detect the ploidy of raspberry leaf cells, with the most collected nucleus and the best peak graph. The nuclear suspension prepared from the young leaves of field-seedlings and tissue-culturing seedlings had the best detection effect, followed by that from adult leaves. The best detection effect was achieved by placing the leaves between two layers of moist filter paper and then putting them in a plastic petri dish, and then placing the plastic petri dish in a low-temperature foam box for direct detection with the sample. The second-best effect was achieved by cutting the leaves from the plant and directly soaking them in a 50 mL sterile water-filled centrifuge tube before mailing the sample for detection. The main peak of the flow cytometry histogram of the leaves was located near the fluorescence intensity value of 10 000. Using this detection method, the ploidy of 15 raspberry germplasm leaf cells was all determined to be diploid, and the ploidy of the roots and callus of ‘Longyuan shuangfeng’ and ‘Polka’ was also determined to be diploid. Conclusion This study established a method capable of detecting the cell ploidy of raspberry leaves, roots, and calli at different developmental stages and under different growth environments. Using this method, a total of 15 raspberry germplasms, including black raspberries, red raspberries, and yellow raspberries, were tested, and all were found to be diploid.

Key words: raspberry, cell, ploidy identification, flow cytometry, leaves, roots, callus tissue, rapid identification

青梦瑶, 田林, 李迎超, 史瑞基, 张瑞杰, 郑奕宸, 孙权, 李寒, 顾玉红. 基于流式细胞仪鉴定树莓倍性方法的建立及应用[J]. 生物技术通报, 2025, 41(11): 228-235.

QING Meng-yao, TIAN Lin, LI Ying-chao, SHI Rui-ji, ZHANG Rui-jie, ZHENG Yi-chen, SUN Quan, LI Han, GU Yu-hong. Establishment and Application of a Method for Identifying Raspberry Ploidy Based on Flow Cytometry[J]. Biotechnology Bulletin, 2025, 41(11): 228-235.

图/表 7

图1 不同来源不同叶龄的树莓叶片

Fig. 1 Raspberry leaves of diverse origins and leaf ages

图2 不同树莓品种鉴定细胞倍性的嫩叶

Fig. 2 Young leaves of different raspberry germplasm

图3 不同解离液解离红树莓叶片的相对核DNA含量直方图图中X轴为荧光强度代表的相对DNA含量，Y轴为细胞核数量，下同

Fig. 3 Histogram of relative nuclear DNA contents in red raspberry leaves dissociated with different dissociating solutionsIn the figure, X-axis is the relative DNA content represented by fluorescence intensity, and Y-axis is the number of nuclei. The same below

图5 不同运输和保存方式的树莓叶片的相对核DNA含量直方图

Fig. 5 Histogram of relative nuclear DNA contents in the raspberry leaves with different transport and preservation modes

图6 不同树莓种质的相对核DNA含量直方图

Fig. 6 Histogram of relative nuclear DNA contents of different raspberry germplasms

图7 红树莓的根和愈伤组织

Fig. 7 The roots and the calluses of red raspberry

图8 流式细胞仪法检测红树莓根和愈伤组织的细胞倍性

Fig. 8 The cell ploidy of red raspberry root and calluses determined by flow cytometry

参考文献 32

[1]	Kshatriya D, Li XY, Giunta GM, et al. Phenolic-enriched raspberry fruit extract (Rubus idaeus) resulted in lower weight gain, increased ambulatory activity, and elevated hepatic lipoprotein lipase and heme oxygenase-1 expression in male mice fed a high-fat diet [J]. Nutr Res, 2019, 68: 19-33.
[2]	Chen L, Xin XL, Zhang HC, et al. Phytochemical properties and antioxidant capacities of commercial raspberry varieties [J]. J Funct Foods, 2013, 5(1): 508-515.
[3]	Wu HZ, Oliveira G, Lila MA. Protein-binding approaches for improving bioaccessibility and bioavailability of anthocyanins [J]. Compr Rev Food Sci Food Saf, 2023, 22(1): 333-354.
[4]	Eng WH, Ho WS, Ling KH. In vitro induction and identification of polyploid Neolamarckia cadamba plants by colchicine treatment [J]. PeerJ, 2021, 9: e12399.
[5]	李秀兰, 宋文芹, 陈瑞阳. 北方几种小浆果植物的核型研究 [J]. 武汉植物学研究, 1993, 11(4): 289-292, 393-394.
	Li XL, Song WQ, Chen RY. Studies on karyotype of some berry plants in North China [J]. J Wuhan Bot Res, 1993, 11(4): 289-292, 393-394.
[6]	林盛华, 张冰冰, 方成泉, 等. 中国树莓属8个种染色体数目与核型 [J]. 园艺学报, 1994, 21(4): 313-319.
	Lin SH, Zhang BB, Fang CQ, et al. Chromosome numbers and karyotypes of 8 species Rubus in China[J]. Acta Hortic Sin, 1994, 21(4): 313-319.
[7]	王小蓉, 汤浩茹, 付华清, 等. 15个引进树莓品种的核型 [J]. 林业科学, 2008, 44(12): 147-150, 196.
	Wang XR, Tang HR, Fu HQ, et al. Karyotypes of 15 introduced bramble cultivars (Rubus) [J]. Sci Silvae Sin, 2008, 44(12): 147-150, 196.
[8]	刘倩. 6种苜蓿属植物多倍体诱导及倍性鉴定研究 [D]. 扬州: 扬州大学, 2014.
	Liu Q. Polyploidy induction and ploidy identification of 6 Medicago spp. germplasms [D]. Yangzhou: Yangzhou University, 2014.
[9]	夏春皎, 李运广, 夏姝, 等. 植物基因组学中的流式细胞分析及分选技术 [J]. 植物学报, 2024, 59(5): 774-782.
	Xia CJ, Li YG, Xia S, et al. Flow cytometric analysis and sorting in plant genomics [J]. Chin Bull Bot, 2024, 59(5): 774-782.
[10]	Faisal M, Abdel-Salam EM, Alatar AA, et al. Induction of somatic embryogenesis in Brassica juncea L. and analysis of regenerants using ISSR-PCR and flow cytometer [J]. Saudi J Biol Sci, 2021, 28(1): 1147-1153.
[11]	Chowrasia S, Nishad J, Pandey R, et al. Oryza coarctata is a triploid plant with initial events of C4 photosynthesis evolution [J]. Plant Sci, 2021, 308: 110878.
[12]	Abdolinejad R, Shekafandeh A, Jowkar A. In vitro tetraploidy induction creates enhancements in morphological, physiological and phytochemical characteristics in the fig tree (Ficus Carica L.) [J]. Plant Physiol Biochem, 2021, 166: 191-202.
[13]	Wu J, Cheng XT, Kong B, et al. In vitro octaploid induction of Populus hopeiensis with colchicine [J]. BMC Plant Biol, 2022, 22(1): 176.
[14]	Parzymies M, Pogorzelec M, Głębocka K, et al. Genetic stability of the endangered species Salix lapponum L. regenerated in vitro during the reintroduction process [J]. Biology, 2020, 9(11): 378.
[15]	Nsabimana A, van Staden J. Ploidy investigation of bananas (Musa spp.) from the National Banana Germplasm Collection at Rubona-Rwanda by flow cytometry [J]. S Afr N J Bot, 2006, 72(2): 302-305.
[16]	Niu YY, Zhou WQ, Chen XY, et al. Genome size and chromosome ploidy identification in pear germplasm represented by Asian pears - Local pear varieties [J]. Sci Hortic, 2020, 265: 109202.
[17]	弓娜, 刘建全, 田新民, 周香艳. 流式细胞术在植物学研究中的应用——检测植物核DNA含量和倍性水平 [J]. 中国农学通报, 2011, 27(9): 21-27.
	Gong N, Liu JQ, Tian XM, Zhou XY. Applications of flow cytometry in plant research—analysis of nuclear DNA content and ploidy level in plant cells [J]. Chin Agric Sci Bull, 2011, 27(9): 21-27.
[18]	Galbraith DW, Harkins KR, Maddox JM, et al. Rapid flow cytometric analysis of the cell cycle in intact plant tissues [J]. Science, 1983, 220(4601): 1049-1051.
[19]	Nosrati H. Relationship between ploidy level and genome size in strawberries [J]. Plant Biosyst Int J Deal Aspects Plant Biol, 2015, 149(6): 1036-1041.
[20]	Rampáčková E, Mrázová M, Čížková J, et al. Pomological traits and genome size of Prunus armeniaca L. considering to geographical origin [J]. Horticulturae, 2022, 8(3): 199.
[21]	邵雪花, 李桂兰, 肖维强, 等. 基于流式细胞仪鉴定番石榴倍性方法的建立及应用 [J]. 生物技术通报, 2024, 40(2): 48-54.
	Shao XH, Li GL, Xiao WQ, et al. Establishment and application of ploidy method for the identification of Psidium guajava by flow cytometry [J]. Biotechnol Bull, 2024, 40(2): 48-54.
[22]	叶天文, 袁德义, 李艳民, 等. 海南油茶的倍性鉴定 [J]. 林业科学, 2021, 57(7): 61-69.
	Ye TW, Yuan DY, Li YM, et al. Ploidy identification of Camelliahainanica [J]. Sci Silvae Sin, 2021, 57(7): 61-69.
[23]	Peng RH, Liu F, Song GL, et al. An efficient method of nuclei extraction in cotton [J]. Agric Sci Technol, 2009, 10(3): 19-21.
[24]	Yang S, Zeng K, Luo L, et al. A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp. [J]. Sci Rep, 2020, 10: 5016.
[25]	Loureiro J, Rodriguez E, Dolezel J, et al. Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species [J]. Ann Bot, 2007, 100(4): 875-888.
[26]	黄雄. 树蕨桫椤的基因组破译、木质部发育及次生代谢物质生物合成途径研究 [D]. 北京: 中国林业科学研究院, 2022.
	Huang X. Genome decoding of alsophila spinulosa, and studies on xylem development and secondary metabolite biosynthetic pathway in tree fern [D]. Beijing: Chinese Academy of Forestry, 2022.
[27]	杨慧娴.雪兔子亚属与雪莲亚属的染色体进化及核DNA含量研究 [D]. 昆明: 云南大学, 2020.
	Yang HX. Studies on the chromosome evolution and nuclear DNA content of Saussurea Subg. Eriocoryne and Subg. Amphilaena [D].Kunming: Yunnan University, 2020.
[28]	Loureiro J, Čertner M, Lučanová M, et al. The use of flow cytometry for estimating genome sizes and DNA ploidy levels in plants [J]. Methods Mol Biol, 2023, 2672: 25-64.
[29]	徐晓丹, 邵维助, 郑伟. 滇山茶多倍体栽培品种的细胞倍性 [J]. 林业科学, 2018, 54(9): 44-48.
	Xu XD, Shao WZ, Zheng W. Ploidy study on polyploidy cultivars of Camellia reticulata [J]. Sci Silvae Sin, 2018, 54(9): 44-48.
[30]	王娅丽, 周利利, 王娜, 等. 利用流式细胞仪快速鉴定棉花倍性的方法比较 [J]. 生物技术通报, 2022, 38(12): 144-148.
	Wang YL, Zhou LL, Wang N, et al. Comparison of methods for rapid determination of cotton ploidy by flow cytometry [J]. Biotechnol Bull, 2022, 38(12): 144-148.
[31]	黎月娟. 桑树种质资源倍性测定及诱导研究 [D]. 南宁: 广西大学, 2019.
	Li YJ. Ploidy determination and induction study of mullberry germplasm resources [D]. Nanning: Guangxi University, 2019.
[32]	裴翡斐.萱草属植物倍性育种研究 [D]. 大连: 大连海洋大学, 2024.
	Pei FW. A study on ploidy breeding of Hemerocallis plants [D]. Dalian: Dalian Ocean University, 2024.