Plant material and preparation
Dry Syntrichia caninervis gametophytes were collected from the Gurbantunggut Desert in Xinjiang-Uyghur Autonomous Region, China (44º32ʹ30ʺN, 88º6ʹ42ʺE) and kept in the dark in a paper sack at room temperature. Dry gametophytes were fully re-hydrated on filter paper saturated with distilled water (9 mL) in glass petri dishes for 24 h at 25ºC, with light at a photosynthetic photon flux density (PPFD) of 50 μmol/m2/s. To remove surface impurities and sand, the fully hydrated S. caninervis was transferred to a glass beaker, stirred lightly using a glass rod for 3 min, ﬁshed out using sieve, then put into another beaker. The washing was repeated ﬁve times. The washed gametophytes were placed on ﬁlter paper in Petri dishes, prior to regeneration.
Cultivation of vegetative fragments
The vegetative fragments (leaf, stem, rhizoid) and entire gametophyte of S. caninervis were separately sown on peat pellet (JiffyCorp., Manitoba, Canada). For reducing contamination, we placed a peat pellet in tissue culture vessels (350 mL, height 108 mm, diameter 75 mm, caliber 69 mm, lid with air hole), then dry autoclaved for 30 min at 121 ºC. 100 mL distilled water was applied to sterile peat pellet, after a few minutes, peat pellet swelled by drinking water, prior to cultivation (Additional file 1). The whole gametophyte regeneration was conducted as follow: we picked 80 fully hydrated gametophytes (as mentioned in Plant material and preparation), and separated 20 repeats, every repeat included 4 individuals. The leaf regeneration was conducted as follow: we randomly selected and sampled 10 gametophytes, the juvenile and green leaves were chosen for propagation and cultivated in peat pellet, isolated juvenile and green leaves using the method of Stark et al. . Each leaf placed on the substrate, adaxial surface up, in the growth chamber. The stem regeneration was conducted as follow: we cut stem apices (with juvenile and green leaves) and rhizoid, just kept middle part, and put them on peat pellet. The rhizoid was detached and sown on peat pellets. Growth chambers were set with a 16 h /8 h photoperiod, light intensity was about 150 μmol/m2/s, and day/night temperatures of 25 ºC /15 ºC. Relative humidity in the chamber was approximately 60% for the duration of the experiment.
The whole plant, stem, leaf and rhizoid were sown and grew on peat pellet, we observed them in situ. Regeneration process were observed through a stereomicroscope (SZX-16, OlympusCorp., Tokyo, Japan) , and photographs were taken using a digital camera (DP74, OlympusCorp., Tokyo, Japan). Adobe Photoshop software (ver. 6.01, Adobe Systems Inc., San Jose, USA) was used to edit the digital images. The regeneration of whole gametophytes noted on 0 d, 3 d, 6 d, 9 d, 12 d, 15 d, 19 d, 23 d, 32 d, 56 d. The regeneration of stems observed at 0 d, 4 d, 6 d, 9 d, 12 d, 16 d, 32 d, 56 d. The regeneration of protonema and shoot from leaf were noted on 0 d, 4 d, 6 d, 9 d, 12 d, 16 d, 32 d, 56 d. The regeneration of rhizoid observed at 0 d, 12 d, 16 d, 32 d, 56 d.
After 56 d cultivation, the extension of protonema filaments from different explants measured by ImageJ (the National Institute of Health, Wisconsin, USA). The linear distance from the edge of original explants (leaf, rhizoid, stem, entire gametophyte) to the furthest extent of protonema extension was determined.
Water content measurement
Room temperature was 25 °C, relative humidity was 30%. 32 d S. caninervis were placed on ﬁlter paper, and redundant water on plant surfaces was absorbed using another piece of ﬁlter paper. We weighted 200 milligram (mg) as Wt, and then were placed in the Petri dish, and weighed (Dwt) after drying in an oven for 15 min at 105 °C, and at 65°C for 48 h. The water content (g H2O /g Dwt) was calculated as (Wt – Dwt)/Dwt. The ratio was measured three biological replicates and six technical replicates and then averaged.
Gametophyte regeneration assay
Every plantlet bottle containing one peat pellet, and 4 individuals were sown on each pellet. Shoot or protonema occurrence, days to shoot or protonema emergence, and shoot number were recorded for entire regeneration stage (days 56). Regeneration rate (%) was determined as the number of gametophytes produced protonema or shoot divided by the total number of sown gametophytes. Shoot growth rate (%) was determined as the number of shoots divided by the number of days from emergence to the end of the experiment (days 56).
Chlorophyll content was measured according to the methods described by Ritchie 2006. We collected 40 mg fresh weight S. caninervis from 32 d old complete plant. Pigments were extracted by incubation of the entire plant in 2 mL of 96% ethanol (room temperature about 25ºC) for 4 h in darkness with constant agitation. The extracts were centrifuged at 10000 rpm for 2 min and the supernatants removed for analysis. Supernatants were analyzed spectrophotometrically at wavelengths of 470 nm, 649 nm, and 665 nm using a UV-visible spectrophotometer (Biomate 3S, Thermo Fisher Scientific, Waltham, USA). The concentration of the chlorophyll a, b and total carotenoids were determined using the following equations: Chl a=13.95*OD665-6.88*OD649, Chl b =24.96*OD649-7.32*OD665, and Car= (1000*OD470-2.05*Chl a-114.8*Chl b)/245. The total pigment content in mg/g =N*C*V/W, “N” represent dilution ratio, “C” represent pigments concentration (mg/mL), “V” represent the volume of extracting solution (mL), “W” represent sample fresh weight (g).
Fluorometric assessment photosynthetic performance
Photosynthetic performance of regenerated S. caninervis assessed by pulse amplitude modulated ﬂassessed using a portable chlorophyll fluorometer (PAM 2500) (Heinz, Walz, Germany). Measurements of chlorophyll ﬂuorescence were recorded in situ. The saturation pulse method was used to calculate the Fv/Fm. Fo and Fm were measured in the dark after dark adaption for greater than 30 min. The parameter settings were based on the recommendations of Zhang et al. . The Y(II) of samples were measured at ambient light, saturating pulses were applied to determine the maximal fluorescence yield during actinic illumination, Fm’ and the steady-state level luorescence during actinic illumination F. The values of Y(II) were calculated by Y(II) = (Fm’− F)/Fm’. All parameters were measured three biological replicates and three technical replicates and then averaged.
All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) 16.0 software (SPSS Inc., Chicago, USA). Data were compared using one-way ANOVA, a post hoc LSD test was used to examine difference in the significance of ANOVA results, and values were considered statistically significantly different at P < 0.05, or distinctly statistically different at P < 0.01. Error bars represent standard deviations.