Plant materials
Vines of kiwiberry (A. arguta) were selected from the plant germplasm collection at Warsaw University of Life Sciences (SGGW), located in Warsaw, Poland. Four female cultivars were used in the current study – ‘Geneva,’ of American origin, ‘Weiki,’ of German origin, and two Polish cultivars, ‘Scarlet September Kiwi’® and ‘Bingo’PBR. They all are tetraploids with a chromosome number of 2n = 4x = 116 (Melo et al. 2017). The plants were exposed to open pollination and mature (soft, ready-to-eat) fruits were collected at the end of September in 2018 and 2019. The harvested fruits was divided into two lots. One lot was placed in small, breathable plastic boxes immediately after collecting and was stored at 10 °C in a refrigerator, to be used as the endosperm source for the fresh material (fresh seeds). From the second lot, the pulp and seeds were taken out, washed first in tap water and then in distilled water, dried at room temperature for a few hours, and then stored for one year at room temperature in a glass jar; they were used as the endosperm source for the dry material (dried seeds). The fruits was sterilized, the seeds dissected from the fruit, the seed coats excised, the embryos isolated, and the endosperm tissue dissected (Fig. 1a) according to a protocol previously developed for A. chinensis var. deliciosa (Góralski et al. 2005). The dried seeds after the sterilization were soaked in distilled, sterile water for 24 h at room temperature.
Callus induction
The endosperms isolated from fresh seeds and the one-year-old dry seeds were inoculated on 60-mm Petri dishes with a culture medium based on the Murashige and Skoog (1962) (MS) macro-, microelements, and vitamins (Duchefa), supplemented with 3% sucrose and 0.6% agar (Duchefa) in addition to the PGRs. For the callus induction medium (CIM), the medium described above was supplemented with 0.25 (CIM-0.25), 0.5 (CIM-0.5), or 1.0 mg/l (CIM-1.0) of thidiazuron (TDZ) and Actinidia Endosperm Medium (AEM) with 2 mg/l of 2,4‑dichlorophenoxy-acetic acid (2,4-D) and 5 mg/l of kinetin (KIN). CIM without any PGRs (CIM-0) was used as a control medium. Sealed with Parafilm®, the Petri dishes with endosperm explants were incubated at 25 °C in the dark. The proliferating explants were transferred to fresh medium every three weeks and incubated under the same conditions.
Observations and images were performed using a dissecting binocular microscope (Zeiss Stemi SV 11, Germany) that was equipped with a digital camera (Canon Power Shot G6). The images were processed with CorelDRAW Graphics Suite 2020 software.
Shoot bud development
Calli with visible (with a height of approx. 5 mm) adventitious shoot primordia were transferred to Petri dishes 90 mm in diameter and 25 mm in height (Phoenix Biomedical) containing six types of Shoot Development Medium (SDM). All SDMs contained full-strength MS salts and vitamins (Duchefa), 3% sucrose, and 0.6% agar. SDM0 did not contain any PGRs, while SDM1 was supplemented with 0.5 mg/l of TDZ, SDM2 with 2 mg/l of 6-Benzlyaminopurine (BAP), SDM3 with 2 mg/l of kinetin (KIN), SDM4 with 2 mg/l of zeatin (ZEA), and SDM5 with 2 mg/l of meta-topolin (mT). All cultures were incubated at 25 °C with a 16-h photoperiod under cool-white fluorescent tubes (60–90 µmol m-2s-1) for 60 days.
Rooting and acclimatization
Regenerated shoots with a height of approx. 2–3 cm were excised from the callus clumps. Micro-cuttings were sub-cultured in MagentaTM vessels (Sigma) with four types of Root Induction Media (RIM). All RIMs were provided with half-strength MS salts and vitamins (Duchefa), 2% sucrose, and 0.6% agar (Plant Agar, Duchefa). RIM0 did not contain any PGRs, while RIM1 was supplemented with 0.2 mg/l of indole-3-acetic acid (IAA), RIM2 with 0.2 mg/l of indole-3-butyric acid (IBA), and RIM3 with 0.2 mg/l of 1-napthaleneacetic acid (NAA). The cultures were kept under the same temperature and photoperiod conditions as described above. Shoots with a well-developed root system were removed from the culture vessels and gently washed in a beaker with sterilized distilled water to wash out the RIM. Afterwards, the plants were placed in small pots with commercial substrate for seeding (Substral). The potted plants were watered and covered with a plastic bag to maintain high relative humidity. After two weeks in humid conditions, holes were made in the plastic bags in order to gradually reduce the humidity. Two months later the shoots were transferred from pots to garden soil conditions in the middle of August 2019. The plants were grown under green netting to protect them from direct sunlight.
Light and scanning electron microscopy
Six-week-old intact calli with shoot bud domains were collected from CIM-0.5TDZ and fixed overnight at 4°C in a solution of 5% (v/v) glutaraldehyde in 0.1 M phosphate buffered saline (pH: 7.2). Next, the samples were washed four times in phosphate buffer and dehydrated gradually in an ethanol series, from 10% to 100% (v/v). The basal stem segments from in vitro rooting shoots with 10-day-old root primordia were excised and fixed as described above. The fixed tissues were embedded in Technovit® 7100 synthetic resin and 5-μm-thick sections were cut, stained with 0.1% (w/v) toluidine blue, and finally mounted in Entellan (Merck, Darmstadt, Germany) according to the procedure described by Popielarska et al. (2006). Observations and documentation were performed using the visible light system of a Nikon Eclipse E400 microscope equipped with a Zeiss AxioCam MRe digital camera and Zeiss AxioVision 3.0 software and a Nikon DS-Fi2 with NIS-Elements 4.0 software.
For scanning electron microscope (SEM) analysis, the fixed samples, after dehydration through a graded ethanol series, were dried with CO2 critical-point drying and coated with gold (Jeol JFC-1100 E ion-sputtering system). Observations were made with an SEM (HITACHI S-4700).
Flow cytometry
Flow cytometric (FCM) analysis was performed on leaf samples from 35 rooted regenerated plants obtained from the endosperm-derived callus of ‘Bingo’ seeds from both sources of explants – fresh seeds (15 regenerants) and year-old dry seeds (20 regenerants). Leaves from five seedlings of the tetraploid cultivar ‘Bingo’ grown in vitro were used as a ploidy control. The leaf samples were prepared as previously described (Sliwinska and Thiem 2007) using nuclei isolation buffer (200 mM TRIS, 4 mM MgCl2•6H2O, 0.5% [v/v] Triton X-100; pH=7.5) supplemented with propidium iodide (50 μg/mL) and ribonuclease A (50 μg/mL). Solanum lycopersicum (1.96 pg/2C; Doležel et al. 1992) served as an internal standard. The nuclear DNA content was estimated using a CyFlow SL Green (Partec GmbH, Münster, Germany) flow cytometer equipped with a high-grade solid-state laser with green light emission at 532 nm, a long-pass filter RG 590 E, DM 560 A, as well as side and forward scatters. The nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions of Actinidia/Solanum on a histogram of fluorescence intensities. For each sample, the nuclear DNA content in 5000–8000 nuclei was measured by applying linear amplification. Histograms were evaluated using the FlowMax (Partec GmbH, Münster, Germany) program. The coefficient of variation of the G0/G1 peak of the Actinidia species ranged from 2.43% to 4.91%.
The Cx value was calculated based on the genome size of the control tetraploid plants and was applied to establish a DNA ploidy of regenerants. Cx refers to the DNA content of a monoploid genome with the chromosome base number x (Greilhuber et al. 2005). Thus, the abbreviation 4Cx is used for the 2C DNA content of a tetraploid plant and 6Cx for that of a hexaploid. However, it should be noted that hexaploid plants originated from endosperm possessing 3C DNA content, as compared to the source tetraploid plants.
Data recording and statistical analysis
Statistical analysis was performed with the use of R software v. 3.6 (R Core Team 2020). Confidence intervals (confidence level: 0.95) for proportions were calculated with proportion test (prop.test() function) and the proportions were compared with a use of Fisher’s Exact Test for Count Data (fisher.test()) to examine the effects of the cultivar, the medium, and the source of explant on callus proliferation and shoot bud regeneration; differences were regarded as significant when p was less than 0.05.
Almost 3000 explants were used for this study of endosperm response (Table 1). For each kind of medium, cultivar, and source of explant, observations were made to record the proportion of calli and shoot bud induction (for establishing the efficiency of callus and shoot bud induction). The explants’ response was recorded at four-week (for callus induction) and eight-week (for establishing shoot buds induction) intervals after inoculation.
The experiments were carried out in 1–3 trials and with different numbers of explants.