Colchicine Induction of Tetraploid and Octaploid Drosera Strains from D. Rotundifolia and D. Anglica
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© 2021 The Japan Mendel Society Cytologia 86(1): 21–28 Colchicine Induction of Tetraploid and Octaploid Drosera Strains from D. rotundifolia and D. anglica Yoshikazu Hoshi1*, Yuki Homan1 and Takahiro Katogi2 1 Department of Plant Science, School of Agriculture, Tokai University, 9–1–1 Toroku, Higashi-ku, Kumamoto 862–8652, Japan 2 Graduate School of Agriculture, Tokai University, 9–1–1 Toroku, Higashi-ku, Kumamoto 862–8652, Japan Received September 21, 2020; accepted October 15, 2020 Summary Artificial tetraploid and octaploid strains were induced from the wild species of Drosera rotundifolia (2n=2x=20) and D. anglica (2n=4x=40), respectively. The optimal condition of colchicine-treatments for poly- ploid inductions was determined first. A flow cytometry (FCM) analysis showed that the highest mixoploid score of D. rotundifolia was 20% in the treatment of 0.3% for 2 days (d), or 0.5% for 3 d, while the highest mixoploid score of D. anglica was 20% in the treatment of 0.5% for 2 d. Next, to remove chimeric cells, adventitious bud inductions were carried out using the FCM-selected individuals in both species. One strain from a total of 360 colchicine-treated leaf explants in each species had pure chromosome-double numbers of 2n=40 (tetraploid) in D. rotundifolia and 2n=80 (octaploid) in D. anglica. In both species, the guard cell sizes of the chromosome- doubled strains were larger than those of the wild types. The leaves of the chromosome-doubled strains of D. ro- tundifolia were larger than those of the wild diploid D. rotundifolia, while the leaves of the chromosome-doubled strains of D. anglica were smaller than those of the wild tetraploid D. anglica. Keywords Chromosome, Tetraploid, Octaploid, Genome size, Colchicine, Flow cytometry, Drosera rotundifolia, Drosera anglica. The genus Drosera (sundew), which is the second- derivative species with large plant size and could be largest genus of carnivorous plants (Seine and Barthlott easily distinguished from the other species by high 1994), consists of nearly 150 species with high ge- chromosome number and large genome size seeming to netic diversity (Rivadavia et al. 2003, Eschenbrenner arise through natural polyploidization event (Kondo and et al. 2019). Many Drosera species are a natural source Segawa 1988), even though it is quite closely related to of pharmacologically important compounds (Banasiuk D. rotundifolia based on cytological and molecular data et al. 2012), creating 200–300 registered medications (Wood 1955, Hoshi et al. 2008). Thus, we expect that D. for coughs and pulmonary diseases in Europe (Baran- anglica is a candidate species for the breeding improve- yai and Joosten 2016). Nowadays, huge amounts of the ment of sundews, as well or better than D. rotundifolia. plants in these species are required for a continuing Polyploid induction with chromosome doubling is an demand from pharmaceutical companies. Especially, a important protocol for plant breeding because even in representative species D. rotundifolia L., which is the nature the polyploids generally show strong environ- main herb of the medicine “Droserae Herba,” has been mental adaptation and diverse variations (Wang et al. traditionally used in the treatment since the 17th century 2020). The artificially induced polyploid plants also of- (Paper et al. 2005). The other species such as D. anglica ten appear to have superior characteristics to the progen- Huds., D. burmannii Vahl, D. indica L., D. intermedia itor in morphology, metabolite, and yield performance Hayne, D. madagascariensis DC., D. peltata Thunb. and (Xiong et al. 2006, Zhou et al. 2020). Therefore, the D. ramentacea Burch. ex DC. are also officially permit- chromosome doubling technique for inducing polyploids ted for pharmaceutical purposes in European countries has been reported for some plant species (Soltis et al. (Baranyai and Joosten 2016). Except for D. burmannii, 2003, Hannweg et al. 2013). However, the breeding to D. indica, and D. peltata, all the officially permitted spe- create the new polyploid strain has not yet been exten- cies taxonomically fall into section Drosera, according sively carried out in this genus. to the taxonomic system of Seine and Barthlott (1994) Expecting a high yielding effect of Drosera species [formerly classified within series Eurossolis of section as medicinal herbs, the present study aims to establish Rossolis in subgenus Rorella of Diels’ (1906) classifica- artificially chromosome-doubled strains induced from tion]. In this section, D. anglica is a phylogenetically the wild species of D. rotundifolia and D. anglica. The optimal condition of colchicine-treatments for polyploid * Corresponding author, e-mail: [email protected] inductions was determined first, and then screened in- DOI: 10.1508/cytologia.86.21 dividuals in each species were characterized by FCM, 22 Y. Hoshi et al. Cytologia 86(1) chromosome counting, and guard cell measurements. Fluorescent staining with DAPI Moreover, the chromosome-doubled strains were com- For mitotic chromosome observations, the DAPI pared with the wild types of each species to infer the staining technique was performed according to Hoshi speciation of the studied member of section Drosera. and Kondo (1998 a, b) and was simplified the procedure for Drosera chromosome counting. Root tips in vitro Materials and methods were collected and pretreated with 0.05% colchicine for 2 h at 18°C before fixation in 45% acetic acid for 30 min Plant materials on ice. Then, they were hydrolyzed in a mixture of Two wild types of Drosera rotundifolia L. (acces- 1 M HCl and 45% acetic acid (2 : 1) at 60°C for 7 s. The sion No. 010816sera1, 2n=2x=20) and D. anglica Huds. hydrolyzed root-tip meristems were isolated on glass (Accession No. KF-01, 2n=4x=40) were used as the slides, and well-spread meristem cells in 45% acetic original strains for polypoid inductions. These materi- acid were squashed under coverslips. After removing als were obtained from tissue-cultured seedlings from frozen coverslips, the chromosomes were air-dried at aseptic-treated seeds sown on half-strength Murashige– RT. Then, the chromosomes were stained with 1 µg mL-1 Skoog’s (1/2 MS) basal medium (Murashige and Skoog DAPI in McIlvaine’s buffer, containing 50% glycerol. 1962), supplemented with 3.0% sucrose and 0.2% gellan The chromosomes stained with DAPI were observed gum (pH 5.7 before autoclaving), and were subcultured under an epifluorescence microscope (BX51, Olympus) in the same medium. These strains were maintained in with a U-MWU2 filter. Digital images were taken with vitro in the Laboratory of Plant Environment Science, a DP73 digital camera (Olympus) on the fluorescence Department of Plant Science, School of Agriculture, To- microscope. More than 100 metaphase cells were ob- kai University. served in each strain to check for chromosomal aberra- tions and chimeric cells in each artificial strain. Since Polyploid induction the Drosera chromosomes at mitotic metaphase have The procedure of colchicine treatment was employed no primary constriction or localized centromere (Kondo following the protocol of Tungkajiwangkoon et al. and Lavarack 1984, Kondo and Segawa 1988, Sheikh (2016). Adventitious buds were produced from young et al. 1995, Hoshi and Kondo 1998a, b, Shirakawa et al. leaves by tissue culture in 1/2 MS liquid medium supple- 2011), they could not be classified by the conventional mented with 1.0% sucrose after transferring. The buds method using the position of the localized centromere were soaked in 0%, 0.1%, 0.3%, and 0.5% colchicine (Levan et al. 1964). Therefore, the chromosome clas- solutions for 1, 2 and 3 d (30 adventitious buds per treat- sification of Drosera followed Kondo (1976), and the ment). They were then cultured on 1/2 MS solid medium nomenclature for the karyotype symbols followed Shira- supplemented with 3.0% sucrose and 0.2% gellan gum at kawa et al. (2012). Chromosome sizes were defined 25°C under continuous light conditions. as s=small chromosome (shorter than 1.0 µm), and m=middle chromosome (1.0–2.4 µm). Ploidy analysis by FCM Ten young leaves cultured in vitro were chopped in Observation of leaf guard cells 1.0 mL of a nuclei isolation buffer (NE buffer) contain- Guard cell lengths and widths of the wild strains and ing 50 mM Tris–HCl (pH 7.5), 50 mM Na2SO3, 140 mM the colchicine-induced strains were measured on 30 2-mercaptoethanol, 2 mM MgCl2, 2% (w/v) PVP K-30, guard cells per strain. Guard cell sizes were measured 1% (v/v) Triton X-100, and 25 µg mL-1 propidium io- from the lower epidermis in the middle parts of the dide. Then, the chopped samples were filtered through leaves using ImageJ software (ver. 1.45s). a 48-µm nylon mesh and centrifuged with 12,000 rpm for 2 min at room temperature (RT). After centrifug- Results ing, the pellets including isolated nuclei were suspended with 0.2 mL NE buffer. After incubating the samples Effect of colchicine treatment for 5 min at RT, the DNA contents of nuclei were mea- The results of survival rates and polyploid frequencies sured using a flow cytometer (Guava EasyCyte 12HT of D. rotundifolia and D. anglica after colchicine treat- microcapillary flow cytometer, Millipore). Five thousand ments are shown in Table 1. A few treatments caused nuclei acquired at a flow rate of 0.12 µL s-1 were used phytotoxic effects. The survival rates were recorded for each FCM measurement and at least three replicates after 10 weeks. The control groups for 1 d treatments were measured for each strain. Young leaves of Oryza of D. rotundifolia and D. anglica had the highest sur- sativa L. ‘Nipponbare’ (2C value=0.91 pg, Uozu et al. vival rate (Table 1). The other control groups of longer 1997) as a reference standard were used to estimate ge- treatments showed lower survival rates than those of nome size in the absolute unit.