Pollination

Flowers for histochemical examination were selected according to the pollen tube kinetics results, at anthesis, two, and three days after pollination. Two flowers - 10 styles - per day were fixed in 2.5% glutaraldehyde in 0.03M saline phosphate buffer pH7.3 for 4 h [78 (link)]. Then the pistils were washed in 0.03M saline phosphate buffer and sequentially dehydrated in an ethanol series (30%, 50%, 70%, and 96%), leaving them one hour in each ethanol concentration. The gynoecia were left for five days in the embedding solution at 4ºC, and then embedded in JB4 plastic resin (Polysciences Inc., 0226A). Both longitudinal and transversal sections 2μm thick were cut on a LEICA EM UC6 ultramicrotome with a glass knife and then placed onto distilled water on a glass slide previously coated with 1% gelatine. Polysaccharides were stained with periodic acid shift reagent-PAS [79 (link)] counterstained with 0.02% Toluidine Blue for general structure, and proteins with 0.25% Naphtol Blue Black in 1% acetic acid [80 (link)]. Also 0.07% calcofluor white for cellulose [48 (link)] and other polysaccharides [52 (link)], 0.01% auramine in 0.05M phosphate buffer for cutin and lipids [81 (link)], and 0.01% acridine orange in 0.03% phosphate buffer, pH7.4 [82 ] were used to observe the stylar morphology.
Slides were observed under bright field LEICA DM2500 microscope carrying 100W light source, and photographs were obtained with a Leica DFC320 camera linked to the software Leica Application Suite. Fluorescence observations were done with the same microscope provided with an epifluorescence source and connected to a CANON Power Shot S50 camera linked to the CANON Remote Capture software. Filters used were 355/455 nm for calcofluor white, and 450/510 nm for auramine and acridine orange stained sections.

Multivariate and univariate models were used to investigate statistical relationships between the cone-warming treatment and response variables. Effect sizes of cone-warming on responses were then estimated as described below. All analyses were conducted in R (version 3.5.1, R Core Team 2018). Seedlings in the common garden demonstration from each type of cone-warming bag (glassine versus plastic bubble-wrap packaging material, both inside of a polyester pollination bag) were treated the same because there was no statistically significant difference between the effect of the two types of cone-warming bags on seedling traits. Multivariate models were built using both principal component analysis (PCA; via the function prcomp) and permutational multivariate analysis of variance (PERMANOVA; via the function adonis) separately for the following three categories of response variables: (1) plant growth traits including bud phenology, (2) foliar spectra, and (3) mycorrhizal assemblages. The first principal component of the PCA generated from variables, belonging to one response category at a time, was used as the response in linear mixed effect models. Next, PERMANOVAs were executed using Euclidean distance matrices composed of aggregated response variables for each of the three response categories (plant growth, spectra, and mycorrhizae), separately, specifying seed source tree as a random effect. Univariate linear mixed effect models were also fitted for all response variables, specifying seed source tree nested within stand, and raised-bed box (where models would allow), as random effects using functions from the R package lme4. In both multivariate and univariate models, seed mass was tested for inclusion as a covariate via AIC comparisons. Models with the smallest AIC were favored, and when models competed with AIC values within 2 AIC units of the smallest AIC, the simplest model structure with the least predictors was selected for subsequent ANOVAs. Satterthwaite approximation of denominator degrees of freedom was specified for all omnibus F-tests of fixed effects as well as type III sum of square ANOVAs for models that included interactions between seed mass and warming treatment. Type II sum of square ANOVAs were specified for models that included seed mass as an added covariate. The magnitude of the variance explained by the cone-warming fixed effect was estimated by Cohen’s Local f², which is suitable for use with mixed models for which denominator degrees of freedom must be approximated, and is suitable for use with unbalanced experimental designs [34 (link), 35 (link)]. Input for the calculation of Cohen’s Local f² includes marginal R² goodness-of-fit values both from models with and without the factor of interest, as follows: $$f^2=\frac{R_{\mathit{with}}^2-R_{\mathit{without}}^2}{1-R_{\mathit{with}}^2}$$
$R_{\mathit{with}}^2$ refers to the marginal coefficient of determination from a model containing a fixed factor of interest, and $R_{\mathit{without}}^2$ refers to the marginal coefficient of determination from the same model with the fixed factor of interest removed. For instance, in this study the warming treatment was present in the $R_{\mathit{with}}^2$ model and omitted from the $R_{\mathit{without}}^2$ model. Cohen’s Local f² effect sizes ≥ 0.02, ≥ 0.15, and ≥ 0.35 are respectively considered small, medium, and large [35 (link), 36 (link)]. Code and data related to this work are accessible through the Knowledge Network for Biocomplexity.

Plants of B. formosana were collected and molecularly identified by Industrial Technology Research Institute, Hsinchu, Taiwan. Mature capsules were collected 3–4 months after pollination, sterilized in 70% (v/v) ethanol for one minute, followed immersed in 1.5% NaOCl for 20 min, and then rinsed twice with sterile distilled water. Sterile seeds were collected from capsules and cultured on callus-inducing medium according to Gamborg et al. [20 (link)] with some modifications. Briefly, seeds were inoculated on B5 medium supplemented with 2 mg/L 6-Benzyladenine (6-BA), 1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 25 g/L sucrose, and 0.7% agar at 25 °C in the dark. After 2 months, calli were transferred to a new medium for subsequent subcultures and proliferation. Subcultures were carried out at 30-day intervals. The materials used in this study were harvested from 45 days cultured calli of 10–15 generations and extracted by SFE-CO₂ after lyophilization. The extract was dissolved in DMSO and used in the following experiments.

Based on HPLC data from the T0, two transgenic lines (E and F) with the highest artemisinin content were chosen for the development of T1 progeny. To prevent cross-pollination, the T0 plants were covered with paper bags. The seeds from the T0 lines were harvested and stored in the warehouse of the school of agriculture and biology, Shanghai Jiao Tong University. During the spring of 2019, the seeds were cultivated in a 50:50 peat-lite/sand medium and kept in the germination room at 25°C with 16/8 hours of light/dark to obtain T1 transgenic progeny plants. Each line produced a total of 24 independent T1 plants, which were then subjected to DNA extraction and PCR analysis to confirm the presence of the inserted genes. Positives were transferred to the field and grown for further research.