In vitro colchicine treatment and plantlet regeneration
The E. crus-galli was collected from the experimental field of Hubei University in Wuhan City (30°34′N, 114°20′E). Young panicles, 1–2 cm long, of hexaploid E. crus-galli were used as explants. After disinfection, the young panicles were cut into 1- to 2-mm sections and transferred to solid N6 (Chu et al. 1975) induction medium supplemented with 2 mg L− 1 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L− 1 6-benzyladenine (BA), 1 mg L− 1 1-naphthaleneacetic acid (NAA), 5% (w/v) sucrose, and 0.75% (w/v) agar (pH 6.0) to induce callus formation in the dark at 25°C. Approximately four weeks later, vigorous calli were transferred to the liquid N6 induction medium containing 0.01, 0.05, or 0.1% (w/v) colchicine and incubated at 25°C on an orbital shaker (110 rpm) for 24, 48, or 72 h. Medium without colchicine (0.0%) was used as the control. Each treatment consisted of 30 calli. The colchicine-treated calli were rinsed three times with sterile distilled water and cultured onto fresh solid colchicine-free N6 induction medium to re-start growth. After 7–10 days, the calli were transferred to MS (Murashige and Skoog 1962) differentiation medium supplemented with 1 mg L− 1 BA, 2 mg L− 1 kinetin (KT), 0.3 mg L− 1 NAA, 3% (w/v) sucrose, and 0.75% (w/v) agar (pH 6.0) and cultured in the light at 25°C to differentiate. Shoots regenerated from calli were transferred to rooting medium containing half-strength MS supplemented with 0.3 mg L− 1 BA, 0.5 mg L− 1 NAA, 2% (w/v) sucrose, 0.75% (w/v) agar, and 0.03% (w/v) activated carbon (AC) (pH 6.0). Roots were prevalent after approximately 7–10 days, at which time the plantlets were acclimated for three days in the culture room, then washed in tap water, and transplanted to the open field.
Ploidy analysis by flow cytometry
Flow cytometry analysis was performed as follows. Approximately 0.5 cm2 of young leaf tissue from in vitro grown plantlets was chopped with a sharp razor blade in 0.5 mL of Partec HR-A buffer (Partec high-resolution nuclei extraction solution), in a plastic Petri dish. The sample was filtered through a 30-µm Partec CellTrics cell strainers directly into the sample tube, with 2 mL of HR-B buffer (Partec high-resolution DAPI staining solution). The sample was stained for 5 min at room temperature, and the relative fluorescence of total DNA was measured by Sysmex CyFlow Ploidy Analyser II (Sysmex Partec GmbH, Münster, Germany) instrument.
Chromosome counts
After the plantlets had been transferred to the field for about two weeks, root tips were excised from the plants and pretreated with 2 mM 8-hydroxyquinoline for 2 h at room temperature and then fixed in fresh Carnoy’s fixative [methanol/acetic acid, 3:1 (v/v)] overnight. Then, the root tips were rinsed in 75 mM KCl for 30 min at room temperature, digested in an enzyme mixture containing 2% cellulase and 2% pectinase at 28°C for 4 h, washed three times in distilled water, and incubated in distilled water at room temperature for 20 min. These root tips were then placed on precooled slides and squashed in the presence of the fixative. The slides were heated over an alcohol flame to dry the fixative, stained with 5% Giemsa in Sorensen’s buffer at room temperature for 1 h, washed under a stream of tap water, and then dried at room temperature. The chromosomes were observed under an Olympus BX51 light microscope (Olympus Corporation, Tokyo, Japan) and photographed.
Stomatal observation
Stomatal measurements were made on completely developed flag leaves of field-grown plants. The lower epidermis was observed under a light microscope. The length and width of each stoma and the corresponding guard cells were measured with a micrometer. The stomata numbers within the grid squares were then counted to calculate the stomatal density. 30 stomata sizes and densities were measured randomly on three leaves for each population.
Morphological observations
Morphological characteristics of dodecaploid and hexaploid plants, namely plant height, tiller number, flag leaf length and width, leaf thickness, panicle length, anther length, grain length and width, total grain number, filled grain number, and seed set were measured. Total grain number, filled grain number, and seed set were measured on 10 panicles, and the other characteristics were measured on 30 biological replicates for each population.
Determination of plant nutrient concentrations
The nutrient concentrations of the leaves of dodecaploid and hexaploid plants, such as concentrations of water, crude protein (CP), crude fat (ether extract, EE), crude fiber (CF), crude ash (CA), and nitrogen-free extract (NFE), were determined in hexaploid and dodecaploid plants sampled two months after planting. In addition, the nutrient concentrations of total carotene (TCA), calcium, phosphorus, iron, and free amino acids were also determined. The samples were weighed (fresh weight) then dried at 105°C for 30 min, before being dried at 80°C for 8 h. The water contents of the samples were obtained from fresh weight minus dry weight, relative to fresh weight. The nutritional concentrations were determined according to Zhang (2016): CP was determined by the Kjeldahl method, EE was determined by the Soxhlet extraction method, CF was determined by the acid-alkali washing method, and CA was determined by the dry ash method. NFE was calculated using the equation: NFE (%) = Dry matter (%) - [CP (%) + EE (%) + CF (%) + CA (%)]. TCA was determined by extraction with acetone and colorimetric (λ = 450 nm) determination. For elemental analysis, the samples were digested with concentrated nitric acid-perchloric acid at 200°C, and the concentrations of calcium, phosphorus and iron were determined using an inductively coupled plasma-optical emission spectrometer (ICP–OES) (Thermo Fisher Scientific, USA). Free amino acids concentrations were determined by high-performance liquid chromatographic (HPLC) (Agilent, USA). The concentration of each nutrient was determined on three biological replicates for each sample.
Data analysis
The data were analyzed using SPSS Statistics Version 24.0 (IBM, Armonk, NY, USA). Significant differences among the means of dodecaploid and hexaploid material were identified using the two-sample t-test.