Endopolyploidy screening of explants
Natural populations of Epidendrum fulgens Brongn (Orchidaceae) were sampled in the Restinga vegetation of the Atlantic rainforest in Florianópolis, (27°46'50.74"S and 48°29'11.98"W; 27°39'2.15"S and 48°28'6.25"W; 27°37'25.02"S and 48°27'18.05"W). Vegetative offshoots, produced in the floral stalks, were carefully removed from healthy plants of each population and transplanted to 2 L pots containing a mix of autoclaved sand and commercial substrate (Tropstrato HT®) in a 1:1 ratio. These plants were used as the source of materials for the present study. For obtaining in vitro plantlets, E. fulgens seeds were harvested from mature pods, sterilized, and sown as described by Voges et al. (2014).
Flow cytometry (FC) was used to analyze the endopolyploidy level of the following organs/tissues: Pollinia, petals, labella, root tips, leaf basal region, leaf tips, protocorm bases, and protocorm apex. Pisum sativum cv. Ctirad (Fabaceae) (2C = 9.09 pg DNA) was used as an internal reference standard for the flow cytometry analysis. Seeds of P. sativum were kindly provided by Dr. Jaroslav Doležel from the Institute of Experimental Botany of the Czech Academy of Sciences. Nuclei from the samples and leaves of the reference standard (~50 mg) were simultaneously extracted by chopping with a razor blade (Galbraith et al. 1983) on 2 mL ice-cold Otto-I buffer (Otto 1990). The nuclei suspension was filtered through a 40 µm nylon mesh (BD Falcon) and centrifuged at 150 g for 5 min. The supernatant was removed with a pipette and the pellet was resuspended after the addition of 100 µL of fresh ice-cold Otto-I buffer. The nuclei suspension was stained in the dark for 30 min in 500 µL of Otto-II buffer (Otto 1990) supplemented with 50 µg mL-1 of propidium iodide (PI; Biotium) and RNase-A (Sigma-Aldrich).
PI fluorescence was measured with a BD FACSCantoTM II flow cytometer, equipped with an Argon Laser (488 nm), at the Laboratório Multiusuário de Estudos em Biologia, Federal University of Santa Catarina (LAMEB/UFSC). The position of the peaks from the samples and the reference standard was settled by analyzing the first run with each sample separately. The G1 peaks were assigned to a specific channel and the equipment voltage and gain were kept constant throughout the analyses.
Flowing software 2.5.1 was used to process the data. First, we analyzed dot-plots of fluorescence intensity on a linear scale vs. forward scatter light on a logarithmic scale. A polygonal region including all PI-stained nuclei was created on the dot-plots, from which gated histograms of fluorescence intensity in linear scale were created. Linear gated regions were created on histograms to obtain statistics of intact nuclei only.
The numbers of endopolyploid nuclei were used to calculate the cycle value (Barow and Meister 2003), with the formula:
Where, n is the number of nuclei with C-values of 2C, 4C, 8C, and 16C, respectively.
The genome size was calculated based on the ratio between the 2C fluorescence intensity peaks from the samples and the internal reference standard. The value was multiplied by the DNA C-value of the reference standard (Doležel and Bartoš 2005). To convert DNA content in picograms (pg) to base pairs (bp), we considered 1 pg = 0.978 x 109 bp (Doležel et al. 2003).
Protocols for PLB induction and plantlet regeneration
Experiments for PLB induction were performed using all the organs/tissues used on the endopolyploidy analysis. They are described separately below.
PLB induction using flower parts as explants
Flower buds were collected before anthesis and superficially sterilized with ethanol 70% for 1 min and sodium hypochlorite 0.5% for 5 min, followed by 3 rinses in sterile distilled water. Buds were opened under sterile conditions and the petals, labella, and pollinia were excised. Explants were inoculated in Petri dishes containing 25 mL of half-strength MS medium (Murashige and Skoog 1962) supplemented with 20 g L-1 sucrose, 250 mg L-1 polyvinylpyrrolidone (PVP), and different concentrations of plant growth regulators (PGR). The PGR compositions were the four different treatments of the experiment: (i) 20 µM Thidiazuron (TDZ), (ii) 30 µM TDZ, (iii) 9.3 µM 2-isopentenyladenine (2iP) + 36 µM 2,4-Dichlorophenoxyacetic acid (2,4-D), and (iv) control medium without PGR.
Petals and pollinia were inoculated intact. Labella were cut in half, and ovaries were sliced in 1 mm thin cell layers (TCL). The numbers of explants inoculated per treatment were 32 for pollinia and labella and 64 for petals. Each explant was considered as a repetition. After 60 days of the inoculation, data of PLB induction and oxidation rates were collected. For pollinia, the length of the pollen tube was also measured.
Defining the best TDZ concentration for PLB induction from leaf explants
Leaves from in vitro plantlets were used to define the optimal concentration of PGR for PLB induction. Then, roots and protocorm bases were inoculated on this optimal concentration to compare the induction rate between the three different explants, as described below.
For obtaining in vitro plantlets, E. fulgens seeds were harvested from mature pods, sterilized, and sown as previously described. After four months of sown, plantlets with 4-5 leaves and roots were selected to extract leaf explants. Leaves (≈ 1 cm) were inoculated with the abaxial face down in Petri dishes containing half-strength MS medium (Murashige and Skoog 1962), supplemented with 20 g L-1 sucrose and different concentrations of TDZ (0 µM, 3 µM, 6 µM, 9 µM, 12 µM, and 15 µM). Eight leaves were inoculated per Petri dish and a minimum of 40 leaves per treatment. The Petri dishes were sealed and kept in the dark at 25 ± 2 °C.
PLB induction using different explants
The best TDZ concentration for leaves was used to induce PLB from protocorm base and root tips. The explants were obtained from the asymbiotic seed germination as previously described. After one month of sown, homogenous protocorms, with shoot apex and before the first leaf emission, were selected and used as explants. A 1 mm width TCL from the protocorm base was excised from each protocorm under sterile conditions and inoculated in test tubes containing 5 mL of MS medium supplemented with 20 g L-1 sucrose and 10 µM TDZ. The apexes were discarded. Root tips and leaves (≈ 1 cm) were obtained from four-month-old in vitro plantlets, as previously described, and inoculated in test tubes with the same medium used for protocorm bases. A total of 109 root tips, 49 protocorm bases, and 32 leaves were inoculated.
Eight weeks after inoculation, data from oxidation, PLB induction rates, and number of PLB per explant were collected. Statistical analysis was performed with generalized linear models (GLM) using binomial distribution and logit link function, considering explants as categorical, and growth regulator concentrations as explanatory variables. All GLM analyses and Figures were produced on the R environment (R Core Team, 2019) using the car (Fox and Weisberg 2011), MASS (Venables and Ripley 2002), and ggplot2 (Wickham 2016) packages.
Microscopic features of primary and secondary PLB
Leaves with PLB and PLB clusters were fixed in glutaraldehyde (2.5%) in sodium phosphate buffer (0.1 M, pH 7.2) for 24 h and then dehydrated in ethanol series (20%, 40%, 60%, 70%, 80%, 90% and 100%) for 30 min each. For light microscopy, samples were embedded with histo-resin (Leica Historesin, Germany) and block-polymerized. Transversal slices (5 µm) were performed in a microtome, stained with toluidine blue (O’Brien et al. 1964) and analyzed in an BX-40 microscope (Olympus, Japan). For scanning electron microscopy, dehydrated samples were submitted to critical point drying (EM CPD 030/Leica, Germany), fixed in aluminum stubs with carbon tape, coated with gold/palladium (EM SCD 500/Leica, Germany), and examined on a scanning electron microscope (JEOL, JSM-6390LV) at LCME/UFSC.
Cytogenetic stability of plants regenerated endopolyploid explants
Induced PLBs were transferred to test tubes containing 10 mL of PGR-free MS medium supplemented with 1.5 g L-1 activated charcoal and maintained in a 16 h photoperiod at 25 °C ± 2 °C. Subcultures were performed every 8 weeks, and plants were obtained after ca. 24 weeks.
The ploidy of plantlets regenerated from leaves, protocorm bases and root tips were analyzed with FC using the same methodology previously described. Their ploidy level was compared with P. sativum as an internal reference standard, and asymbiotically seed-derived plantlets were used as a control for ploidy level. PLB-regenerated plantlets obtained from leaves (n = 27), protocorm bases (n = 20) and root tips (n= 1) were analyzed. Histograms presenting peaks with poor quality and high coefficients of variation (≥ 5%) were discarded.