Plant material and seed collection
The mature plants of V. planifolia were maintained in a greenhouse at Taoyuan District Agricultural Research and Extension Station at Taoyuan City, Taiwan. Anthesis generally occurs in late April each year (Fig. 1A). For the pod setting, flowers were hand-pollinated by transferring the pollinia onto the stigma of the same flower (Fig. 1B). Developing pods (Fig. 1C) were collected at regular intervals for morphological measurement, histology, and seed germination experiments. In January the next year, pods began to mature and turned yellow (Fig. 1D). In each experiment, at least three pods were collected at regular intervals after pollination.
Evaluation of asymbiotic germination percentage
The pods of different developmental stages were surface-sterilized with a 1.8% sodium hypochlorite solution with one drop of a wetting agent (Tween 20, Sigma-Aldrich) for 15 min. After surface sterilization, the capsules were cut open, and seeds were scooped out with forceps onto the culture medium. To ensure the seed quality and developmental stages of each capsule, the remaining seeds of each capsule were fixed and examined under a microscope. The culture medium used was 1/2 Murashige and Skoog (MS) (Murashige and Skoog 1962) supplemented with 2 mg l− 1 glycine, 0.5 mg l− 1 niacin, 0.5 mg l− 1 pyridoxine HCl, 0.1 mg l− 1 thiamine, 1 g l− 1tryptone, 20 g l− 1 sucrose, and solidified with 7 g l− 1 agar (plant cell culture, tested, powder; all Sigma-Aldrich). The pH value was adjusted to 5.7 before autoclaving at 121 °C and 1.2 kg cm2 for 20 min. An amount of 10 ml medium was placed into each culture tube (20 × 100 mm). After sowing, the cultures were incubated in a growth room under a 16/8 hours photoperiod with daylight fluorescent lamps (20W, China Electric, Taipei) at light intensity 30 µmol m− 2 s− 1. Germination percentage was recorded 60 days after sowing.
Effect of seed pretreatments on germination
To examine the effectiveness of different concentrations and durations of sodium hypochlorite pretreatments in improving germination, mature seeds at 135 DAP were collected. The pretreatments included soaking the seeds in 0.5%, 1%, 2% or 4% sodium hypochlorite solution with one drop of Tween 20, for 15, 30, 45, 60, 75 or 90 min. In control, seeds were soaked only in water. After pretreatments, seeds were washed three times with sterilized distilled water, then inoculated on 1/2 MS medium as described above.
Histological and histochemical studies
Developing seeds were collected and fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.1M phosphate buffer (pH 6.8) for 48 h at room temperature. Seeds were then dehydrated in an ethanol series, then infiltrated gradually (3:1, 1:1, and 1:3 100% ethanol: Technovit 7100, 24 hr each) by using Technovit 7100 resin (Kulzer & Co., Germany), followed by three changes of pure resin. Seeds were then embedded in resin, as described by Yeung and Chan (2015). Sections of 3-µm thick were cut using a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with periodic acid–Schiff (PAS) procedure for structural carbohydrates and counterstained with 1% (w/v) amido black 10B in 7% acetic acid for protein (Sigma-Aldrich, St. Louis, MO, USA) or 0.05% (w/v) toluidine blue O (TBO, Sigma-Aldrich) for general histological staining (Yeung, 1984). For detecting the deposition of cuticular material in developing seeds, sections were stained with 1 µg ml− 1 Nile red (Sigma-Aldrich) for 1 min, then washed in running tap water for 3 min. The fluorescence pattern of Nile red was viewed under an epifluorescence microscope (Axioskop 2, Carl Zeiss AG, Germany) equipped with the Zeiss filter set 15 (546/12 nm excitation and 590 emission). All images were recorded by using a CCD camera attached to the microscope.
Rooting and acclimatization of in vitro seedlings
After 150 days of culture, developing protocorms with roots were transferred onto seedling growth medium: 1/2 MS medium supplemented with 20 g l− 1 sucrose, 1 g l− 1activated charcoal, 20 g l− 1 potato homogenate, and 7 g l− 1 agar for growing seedlings described by Lee (2011). The potato was boiled for 10 min, then peeled and cut into about 1-cm3 sections, then homogenized with a kitchen blender. The pH of the medium was adjusted to 5.6 before autoclaving at 121 °C for 20 min. An amount of 100 ml medium was dispensed into a 500-mL culture flask. After transferring to the seedling growth medium, flasks were placed in a growth room under a 16/8 hours photoperiod with daylight fluorescent lamps (20W, China Electric, Taipei) at light intensity 30 µmol m− 2 s− 1. After 90 days of culture in the seedling growth medium, seedlings of about 10 cm tall with 4 leaves were taken out of flasks.
Experimental design and statistical analysis
All experiments were performed in a completely randomized design and repeated three times; 12 replicates (culture tubes) were used for each treatment, with one explant planted in each plate. Data were analyzed by using analysis of variance (ANOVA) with Fisher’s protected least significant difference test at P < 0.05. All data were analyzed with SAS v9.0 (Cary, NC, USA).