Inflorescence

- Grow healthy Arabidopsis plants until they are flowering (see Figure 1A).
Optional: Clip first bolts to encourage proliferation of many secondary bolts. Plants will be ready roughly 4–6 days after clipping. Optimal plants have many immature flower clusters and only few fertilized siliques, although a range of plant stages can be successfully transformed.
- Transform the DNA construct of interest into the appropriate Agrobacterium strain. Grow the transformed Agrobacterium on YEB (or LB) plates containing the appropriate antibiotics in a 28°C incubator.
- Select a colony and resuspend bacteria in 10 μl H₂O. Plate half of the volume immediately as a lawn onto a YEB plate with the suitable antibiotics and incubate at 28°C for (2–3 days), and use the other half to verify the presence of your DNA construct by PCR analysis.
- Collect the densely grown bacteria from the plate by scraping, and resuspend them in 30 ml YEB in a sterile Falcon tube. The OD₆₀₀ should be about 2.0.
- Per transformation prepare 120 ml of 5% sucrose solution containing 0.03% of Silwet L-77 (surfactant; Lehle Seeds), pour solution into a disposable plastic bag and add the bacteria.
- Dip the inflorescences of the plants into the Agrobacterium solution for 10 seconds, under gentle agitation. You should observe a film of liquid coating the plants. The bacteria are distributed to all plant parts including very young flower shoots by gently pressing the outside of the bag with your hands.
- Place dipped plants under a lid or cover for 16 to 24 hours to maintain high humidity (plants can be laid on their sides if necessary). Do not expose to excessive sunlight (the temperature under the lid can get high).
- Water and grow the plants as normal, tying up loose bolts with wax paper, tape, stakes, twist-ties, or by other means. Stop watering as seeds become mature.
- Harvest dry seeds.
- Select for transformants using appropriate antibiotics or herbicides.

A B. napus diversity set population with ninety-six genotypes was grown as in Siles et al. (2021) (link). The seeds were germinated in P24 trays with John Innes Cereal Mix and once they presented four true leaves, they were transferred to a vernalization room with an 8 h photoperiod at 4°C day/night for 8 weeks. Each plant was re-potted in a 2 L pot in John Innes Cereal Mix. Each genotype had five biological replicates and once out of vernalization, all plants were grown in two glasshouse compartments in long-day conditions (16 h photoperiod) at 18°C day/15° night (600w SON-T, high pressure sodium lighting) at a density of 12 pots per m². Once the plants were fully dry and mature, the first five dry pods on the main inflorescence were ignored, and the next three developed pods were collected for scanning. To avoid pod shattering the pods were sprayed with Prism Clear Glaze (Loxley Arts, Sheffield, UK).
For each genotype, three fully dried pods were placed in plastic holders (34mm x 110mm) and packing peanuts were used to keep the samples in place while scanning. The pedicel was cut with a scalpel before placing the pods into the plastic holders. If the pods were too tall to fit in the holders, they were cut into two pieces and were separately scanned. Twelve holders were loaded into the sample changing carousel of a μCT100 scanner (Scanco Medical, Switzerland). This scanner has a cone beam X-ray source with power ranging from 20 to 100 kVp (pre-set and calibrated for 45, 55, 70, 90 kVp) and a detector consisting of 3072 × 400 elements (48 µm pitch) and a maximum resolution of 1.25 µm. Pods were scanned with the X-ray power set at 45 kVp, 200 µA, 9W, with an integration time of 200 ms.

Nine-year-old blueberry (V. corymbosum “Duke”) plants collected from the small berry garden of the Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences were used as experimental material. Experiments were conducted from March 2019 to February 2022 at the Ministry of Agriculture Key Laboratory of horticultural crop germplasm resources utilization, Institute of Fruit Tree Research, Chinese Academy of Agricultural Sciences (Xingcheng, Liaoning, China) and College of Horticulture Sciences, Shandong Agricultural University (Tai’an, Shandong, China). To ensure the consistency of the material, fruits were collected from the top of the inflorescence (first to mature). Samples were taken from plants under the same growth conditions for organ (root, stem, leaf, flower, and fruit)-specific expression. All samples were rapidly frozen in liquid nitrogen and stored at −80°C.

In 2016, initial crosses were carried out by bagging inflorescences of switchgrass ramets in a greenhouse. The majority of crosses occurred between a diverse group of southern genotypes (n = 57) from lowland populations and two lowland genotypes that showed strong winter survivorship as multiple clonal replicates over 6 winters near Arlington, WI. There were also a limited number of crosses between southern genotypes which had not been evaluated for winter survivorship. The winter tolerant genotypes are referred to as Tolerant 1 and Tolerant 2, and they originated from an unknown population originally collected in North Carolina, South Carolina or northern Florida (Timothy DH, personal communication). Collectively, the individuals used for initial crosses will be referred to as Founders. Crosses resulted in 2,058 individuals unevenly distributed across 29 unique F₁ families. The number of individuals per family was the result of variable seed quantity and viability. During the following year, a set of pseudo-F₂ families were generated by crossing randomly selected siblings within F₁ families. This resulted in 1,039 pseudo-F₂ individuals unevenly distributed among 20 full-sib pseudo-F₂ families. Some pseudo-F₂ families were generated from pairs of siblings within an F₁ family, so only 10 F₁ families were represented in the pseudo-F₂ families.
Among controlled greenhouse crosses, the success rate for initial crosses among Founder individuals was 71%, with success defined as resulting in at least one progeny seedling from a parent (mean 36 seedlings per successful cross). Within F₁ sibling matings, used to generate pseudo-F₂ families, the success rate was 20%, but with a mean of 74 seedlings generated per successful cross parent.
All Founder individuals and F₁ parents of pseudo-F₂ families were maintained in a greenhouse and divided into vegetative replicates by dividing crowns. In July 2018, a completely randomized spaced plant nursery was planted with 195 genotypes. The spaced plants were genotypes maintained in 12-plant rows with 0.7 m between and 0.7 m within rows. Weeds were controlled between individuals genotype crowns using roto-tilling and occasional hand weeding. The nursery contained a minimum of two vegetative replicates per individual. In 2019, vegetative replicates reserved from Founder individuals and F₁ parents of pseudo-F₂ families were used to replace individuals that were lost to winterkill in the spaced plant nursery during the first winter.
In addition, an unreplicated, stratified by genotype spaced row nursery (unique genotypes planted with 0.7 m between rows and 0.3 m within rows) was established of the F₁ families and pseudo-F₂ families in the spring of 2018. Each row contained 10 unique genotypes. In the summer of 2018, heavy rain and standing water in sections of the nursery and resulted in uneven and poor plant vigor. To account for establishment damage that was unrelated to winter survival, fall vigor ratings were made on a scale of 0 to 5 during September in 2018 and 2019. Fall vigor was then used as a covariate for the subsequent spring vigor scores. A fall vigor score of 5 indicated a healthy switchgrass plant and zero indicated a deceased plant.
Winter survivorship scores and heading date was measured for each individual in both nurseries during 2019, 2020, and 2021 (spring vigor only). Spring vigor was recorded using a scale from 0–20, with 20 indicating no visible damage and 0 indicating mortality. Heading date was recorded as the date in which panicles were observed on at least 50% of an individual's tillers.