2.1 Plant materials and pretreatment
In this study, approximately 100 flower buds of “sour orange” (a variety in which seeds are formed) were harvested from citrus trees (approximately 15 years old) in the Citrus Research Center greenhouse in April 2020 (Figs. 1A and B). Flower buds with a diameter of 3–5 mm were selected for the study. The harvested sour orange flower buds were stored in the dark at 4 ℃ for 24 h and sterilized by immersion in 70% ethanol for 30–60 s. Thereafter, the flower buds were dried in a sterilized filter for approximately 2–3 h to completely dry the alcohol in the aseptic workbench. Petals of the buds were aseptically removed using a small forceps and scalpel, and the anthers were collected in a 60-mm diameter Petri dish containing solid medium (the medium composition is explained in the next section). Subsequently, based on the efficiency of the anther culture, liquid N6 liquid medium—supplemented with GA3 (1 mg/L) and spermidine (200 µM)—was added or not on two types of medicinal culture media (Dunwell 2010) (N6 and Murashige and Skoog (MS) basic media supplemented with plant growth hormone) (Fig. 2C).
2.2 Anther medium composition and culture condition
Induction of callus and embryo from an anther culture was performed on an N6 medium (Chu 1978) supplemented with Nitsch and Nitsch vitamins (Nitsch and Nitsch 1969) and MS medium (Murashige and Skoog 1962) containing MS vitamin, supplemented with sucrose (50 g/L) and malt extract (500 mg/L). Moreover, the following growth regulators were added to the two media: 0.2 mg/L 2,4-dichlorophenossiacetic acid (2,4-D), 0.2 mg/L a-naphthalene acetic acid (NAA), 1.0 mg/L kinetin (KI), 0.8 mg/L 6-benzyladenine (6-BA), 0.43 mg/L Zeatin (ZI), and 0.44 mg/L thidiazuron (TDZ). The pH was adjusted to 5.8 using 1 N KOH and 0.1 N HCl, and 0.8% (w/v) agar was added. Anthers were cultured in the dark at 4 ℃ for the first 14 d and then transferred to a 16-h photoperiod at 25 ± 2 °C.
2.3 Plant regeneration
The callus cells induced in the anther culture medium were transferred to a somatic cell induction medium (MS medium supplemented with 500 mg/L of malt extract and 164 mM of lactose) according to Jin et al. (2007) and cultured for six weeks. Somatic embryos were transferred to MS medium supplemented with TDZ (0.5 mg/L), GA3 (1 mg/L), malt extract (500 mg/L), sucrose (50 g/L), and gelrite (0.2%) to induce germination. The embryos in which the shoots formed were transferred and cultured in a magenta box containing GA3 (0.5 mg/L), malt extract (500 mg/L), sucrose (50 g/L), and agar (8 g/L) in MS medium. After six weeks of culture, roots were induced. Each of the newly formed shoots was grafted onto the cultivated citron root. Subsequently, these plants were transferred from the greenhouse to the external natural environment for acclimatization.
2.4 Genetic analysis of regenerants
To verify whether the plants obtained using anther culture were haploid-derived, a simple sequence repeat (SSR) marker primer specific to the heterozygote was manufactured by Biomedic (Korea) (Table. 1), and polymerase chain reaction (PCR) was performed. Total genomic DNA was extracted using automatic nuclear extraction (MX 16, Promega, Madison, WI, USA) from approximately 0.2 g of plant material (leaves) and stored at -20 °C until further use. The PCR reaction solution comprised genomic DNA 15- in AccuPower® Multiplex PCR PreMix (Bioneer, Corp., Daejeon, Korea) [250 μM dNTP, 1.5 mM MgCl2, 1.0 unit Taq DNA polymerase, 10 mM Tris-HCl (pH 9.0), and 40 mM KCl]. Next, 20 ng and 0.5 μM of primer were added to adjust the volume to 20 µL. The PCR reaction was amplified 35 times at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s after DNA denaturation at 94 °C for 5 min, followed by elongation at 72 °C for 30 min. The PCR amplification products were confirmed using the QiAxcel Advanced System (Qiagen, Hilden, Germany) electrophoresis apparatus.
2.5 Polyploidy analysis
To verify the ploidy of the plants obtained through anther culture, the leaves of each plant were collected using in vitro grafting and an analysis sample was prepared according to Chiancone et al. (2006). The ploidy of the prepared samples was determined using flow cytometry (CyStain UV Precise PAm Flugplatz 13 02828; Sysmex Partec GmbH, Görlitz, Germany). Approximately 0.1 g of the sample (plantlet leaves obtained from anther culture) and heterozygous donor plant leaves of the sour oranges were chopped using a sharp razor blade and added to a plastic Petri dish with 0.5 mL nuclei ectration buffer 2 ml staining buffer. The sample was filtered through 30-µm Partec CellTrics (Sysmex Partec GmbH Am Flugplatz 13 02828 Görlitz, Germany) directly into the sample tube, and the test tube was loaded into the machine for analysis. In total, 109 regenerant lines were analyzed.
2.6 Phylogenetic analysis
Analysis of the genetic relationship between the plant and donor plant obtained using anther culture was performed according to previously reported methods (Jin et al. 2016; Jin et al. 2018), and the entire internal transcribed spacer (ITS) region (ITS15.8S-ITS2) in the nuclear ribosomal DNA was analyzed. PCR was performed for the entire ITS region of nuclear ribosomal DNA using a primer combination of ITS1F1 primer (5ʹ-GAAGGATCATTGTCGACCTGCCAGCAGACG-3ʹ) and ITS2R2 primer (5ʹ-GACCTGGGGTCGCAATGCGAGCGCCGCTT-3ʹ) (Jin et al. 2016, 2018). The amplified PCR product was identified as a band by electrophoresis on a 1.2% agarose gel at 100 V for 30 min. The amplified product confirmed on agarose gel was obtained using the GeneAll® Gel purification kit (GeneAll Biotechnology, Co., Seoul, Korea), and the purified amplification product was cloned using the pLUG-Prime® TA-Cloning Vector kit (iNtRON, Korea) (Jin et al. 2018). The cloned PCR product was sent to Solgent (Solgent Co., Daejeon, Korea) to determine the nucleotide sequence. The nucleotide sequence of the determined nuclear ribosomal DNA ITS region was analyzed by Jin et al. (2018) and edited with Bioedit (Hall 1999), and phylogenetic relationships were analyzed using MEGA 5.2 software.