Flow cytometric analysis of nuclear DNA content in cloudberry (Rubus chamaemorus L.) in vitro cultures Barbara Thiem a,1 , Elwira S ´ liwin ´ska b,  a Department of Pharmaceutical Botany, K. Marcinkowski University of Medical Sciences, Maria Magdalena St. 14, 61-861 Poznan ´, Poland b Laboratory of Molecular Biology and Cytometry, Department of Genetics and Plant Breeding, University of Technology and Agriculture, Kaliskiego St. 7, 85-789 Bydgoszcz, Poland Received 19 July 2002; received in revised form 7 October 2002; accepted 7 October 2002 Abstract Flow cytometry was used to analyze the DNA content of nuclei isolated from four different shoot cultures growing in vitro, and from callus of Rubus chamaemorus . The protocol for isolation of the nuclei of this species was optimized. Flow cytometry proved to be a fast and accurate method for estimation of the genome size of R. chamaemorus , a species containing phenolic compounds in the cytosol, which affect fluorochrome accessibility to DNA. Analyses showed that there is no variation in the ploidy level among different types of tissue cultures: axillary shoots from the 3rd and 20th passage, shoots after cold storage, and shoots developed from alginate-encapsulated buds (artificial seeds). All of these shoots showed the same ploidy level as seedlings obtained from the seeds (2C /8x /56), which served as a control. The results showed that shoots of R. chamaemorus cultured in vitro can be used for propagation and germplasm storage. Callus obtained from wounded generative embryos was mixoploid, consisting of cells with 2C, 4C and 8C DNA content. The estimated genome size of R. chamaemorus is 2C /2.469 /0.05 pg. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Axillary shoots; Callus; Flow cytometry; Genome size; Ploidy; Rubus chamaemorus 1. Introduction Cloudberry */Rubus chamaemorus L. (Rosaceae) */is a herbaceous plant with boreal circumpolar distribution. The edible fruits of economic value in Scandinavia contain a high amount of ellagic acid, a phenolic which has multiple biological properties beneficial to human health, e.g. it is antioxidative and anticarcinogenic [1]. This species, being a glacial relict, is found in Poland only in a few protected reserves and it is listed in the Polish Plant Red Data Book [2]. Micropropagation of R. chamaemorus by initiation of axillary shoots was achieved previously [3]. Two meth- ods of conservation of cloudberry shoot cultures by medium-term cold storage and by encapsulation of shoot buds in calcium alginate hydrogel [4] were developed also. These in vitro culture techniques can be used as an important additional option for preserving this endangered species. However, the use of in vitro cultures for these purposes must be customized to avoid changes in the plant genome. Generally, multiplication via buds is suggested to be the preferred strategy to maintain genetic stability [5]. Bajaj et al. [6] found that propagation from existing meristems reduces or elim- inates the possibility of abnormalities that occurs when other methods are applied. Recently, extensive studies to ascertain plant cytoge- netic stability following micropropagation have been performed on nuclear DNA content and polymorphism, as well as on protein polymorphism [7]. Plants propa- gated in vitro may show somaclonal variation caused by structural chromosome rearrangements, changes in chromosome number or in ploidy level [8,9]. The risk of genetic instability seems to increase with in vitro Abbreviations: BA, 6-benzyladenine; DAPI, 4?,6-diamidino-2- phenylindole; IBA, indole-3-butyric acid; MS, Murashige and Skoog medium; NAA, a-naphthaleneacetic acid; PVP, polyvinylpyrrolidine.  Corresponding author. Tel./fax:  /48-52-340-8722 E-mail addresses: bthiem@mail.am.poznan.pl (B. Thiem), elwira@atr.bydgoszcz.pl (E. S ´ liwin ´ ska). 1 Tel.:  /48-61-852-9057x51; fax:  /48-61-852-9057x61. Plant Science 164 (2003) 129  /134 www.elsevier.com/locate/plantsci 0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(02)00344-8