Plant material and culture initiations
For culture initiations, immature samaras were collected from a variety of surviving green and white ash source trees in areas enduring EAB infestation by multiple cooperators in Michigan, Ohio, and Pennsylvania in three different years (2013, 2015 and 2018). Each year’s culture initiations had different objectives and the tested induction treatments and source tree genotypes used varied among the years, so they are described separately. Table 1 summarizes all of the different seed sources, seed collection dates, explant types, plant growth regulator (PGR) treatments and explant numbers for each of the three years of culture initiations.
2013 culture initiations
Samaras from four lingering female white ash trees located in Indian Springs Metro Park Farmland Trail, Oakland County, Michigan (designated LA101, LA102, LA114, and LA116; Fig. 1) were harvested by Ohio State University collaborators on July 7, August 8, and September 16, 2013, and shipped on cold packs via next day delivery to the University of Georgia, Athens, Georgia. On the final collection date, samaras were harvested from only two trees, 102 and 116, and no cultures were initiated from the harvested fruit due to weevil-infested seeds, aborted seeds, or zygotic embryos that appeared to be fully mature. The mother trees had canopy ratings of average to healthy and none of them showed signs of damage by EAB.
Samaras were surface disinfested by washing them in 70% ethanol for 20 s, 10% Roccal-D Plus (9.2% didecyl dimethyl ammonium chloride, 13.8% alkyl dimethyl benzyl ammonium chloride, 1% bis-n-tributyltin oxide; Pfizer) for 3 min, 70% ethanol for 20 additional seconds, 10% Roccal for 3 additional minutes, 50% Clorox (8.25% sodium hypochlorite) for 5 min, sterile deionized water for 3 min, and sterile 0.01 N hydrochloric acid (HCl) for 3 min, followed by three additional rinses in sterile deionized water of 3 min each. Following surface disinfestation, samaras were dissected to remove the seeds and half of the seeds were dissected to remove the zygotic embryos. Whole seeds or embryos dissected from seeds were cultured in 60 mm plastic Petri plates containing semisolid induction-maintenance medium (IMM; Andrade and Merkle 2005), which was a modified woody plant medium (WPM; Lloyd and McCown 1980), with 30 g/l sucrose, 1 g/l filter-sterilized L-glutamine, and either 2 (low auxin treatment) or 4 (high auxin treatment) mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and gelled with 3 mg/L Phytagel (Sigma). Four explants were cultured per Petri plate. For each collection date/source tree combination, three repetitions (Petri plates) were used per each auxin treatment/explant type (zygotic embryo versus embryo with seed coat) combination. Cultures were incubated in the dark at 25 °C. After 1 month, they were transferred to fresh IMM with the same concentration of 2,4-D. Explants showing evidence of embryogenesis induction after two months in culture (or in some cases, longer) were transferred individually to plates of IMM with 2 mg/L 2,4-D and maintained by transfer to fresh medium every three weeks.
2015 Culture Initiations
This set of initiations was conducted to generate additional culture lines from two of the same lingering ash parents used for the 2013 initiations, plus five additional parents in the same Oakland County, Michigan population. Again, all source trees were female white ash trees (designated LA102, LA111, LA112, LA113, LA115, LA116 and LA132). Based on results from 2013, samaras were only collected on a single date (August 11). Shipping conditions, surface disinfestation, dissection and culturing were all as described above for the 2013 initiations, except all cultures were started from zygotic embryo explants cultured on IMM with 2 mg/L of 2,4-D. A total of 30 zygotic embryo explants was cultured (10 Petri plates with 3 explants per plate) for each of the seven source trees. After one month, explants were transferred to fresh medium with 2 mg/L of 2,4-D. Explants showing evidence of embryogenesis induction after two months in culture (or in some cases, longer) were transferred individually to plates of the medium with 2 mg/L 2,4-D and maintained by transfer to fresh medium every three weeks. Since the only “variable” in this initiation was source tree, no statistical analysis was performed on the embryogenesis induction data. Although different induction treatments were not tested, we are including this information since some of the embryogenic culture lines resulting from this work were used in later germination and conversion experiments.
2018 Culture Initiations
The culture initiation experiment in 2018 had two objectives: (1) to compare an alternative PGR treatment to previously used PGR treatments for embryogenesis induction, and (2) to attempt to initiate embryogenic cultures from seeds resulting from controlled pollinations between lingering ash individuals. For this experiment, open-pollinated samaras were collected by a cooperator from five lingering white ash trees (#1-FA, #2-FA, #3-FA, #4-FA, #6-FA) and one lingering green ash tree (#5-FP) in four Pennsylvania counties (Allegheny, Beaver, Butler and Mercer) on August 9, 2018. In addition, we cultured immature zygotic embryos from full-sibling seeds of two separate families. Each family resulted from controlled pollinations between two separate pairs of lingering green ash parents (DC6 × DC5 and DC4 × DC2) that had been confirmed to have increased EAB defenses using EAB egg bioassays, as previously described in Koch et al. (2015). Pollinations were conducted by scientists at the USDA Forest Service Forestry Sciences Laboratory in Delaware, Ohio. For controlled pollinations, pollen was collected by gently brushing or shaking from dehiscing male flowers directly into a glass beaker. To separate pollen from any debris it was passed through a 53 μm nylon sieve and used fresh or transferred into glass vials that were sealed and placed into 50 ml polypropylene tubes containing packets of silica gel and stored at − 80 °C until use. Viability of all pollen was confirmed at the time of use by looking at pollen tube formation on artificial media as described in Koch and Carey (2004). Pollination bags were placed over dormant branches prior to female flowers becoming receptive. Receptivity was tested using methods described by Galen and Plowright (1987) and once receptive (in late April to early May), the bags were briefly removed, and pollen was applied directly to the stigmas of individual flowers using a paintbrush. Pollination bags were removed when stigmas were no longer receptive or when all other male ash trees in the vicinity were finished dehiscing. Seed was monitored for developing embryos and was harvested for somatic embryogenesis when the embryos were 1 to 3 mm long, which was on July 25, 2018 for DC6 × DC5, and on August 14, 2018 for DC4 × DC2. Harvested seed were kept at 10° C until shipped to the Merkle Lab. Shipping conditions, surface disinfestation and dissection were all as described for the 2015 culture initiations. Immature zygotic embryos were cultured on IMM supplemented with 2 mg/L 2,4-D, 4 mg/L 2,4-D or 0.1 mg/L picloram. For all the 2018 culture initiations, at least 15 zygotic embryos were cultured for each source tree by PGR treatment combination, with 3 embryos cultured per 60 mm Petri plate, except for the DC4 × DC2 embryos. Because that cross had a very low filled seed percentage, only 6–8 zygotic embryos were cultured from it per PGR treatment. Culture maintenance was the same as described for the 2015 cultures, except that cultures from explants initiated on picloram were maintained by monthly transfer to fresh medium with that PGR.
Statistical analysis of initiation experiment data
Three months following culture initiations in 2013, 2015 and 2018, each explant was scored for whether or not it showed evidence of embryogenesis induction (i.e., produced embryogenic callus or somatic embryos). For the 2013 initiation, percentages were calculated for each collection date, plant growth regulator treatment and genotype as well as the different combinations of these variables. Following arcsine transformation of the percentage data, one-way analysis of variance (ANOVA) was performed to test for the effects of initiation date, PGR treatment, explant treatment (embryo versus whole seed) and their interactions on embryogenesis induction using R statistical software (R Core Team 2021). Means comparisons were conducted using Tukey’s HSD test. Because numbers of explants per source tree × PGR treatment were unbalanced in the 2018 initiation experiment, the GLM function of R was used to analyze these data for the effects of PGR treatment and genotype on embryogenesis induction. This analysis did not employ induction percentage data, but individual explant data whereby each successfully induced explant was scored as “1” and lack of induction was scored as “0” for the GLM analysis.
Somatic embryo germination and conversion experiments
Effects of pre-germination treatments (cold, activated charcoal, gibberellic acid) on germination and conversion
Two experiments were conducted to test the effects of pre-germination treatments on germination and conversion of lingering white ash somatic embryos from cultures initiated in 2013 and 2015. While the two experiments tested some of the same treatments, the sources of the somatic embryos were different, so the two experiments are not replicates and are described separately.
In the first experiment, which used two culture lines initiated in 2013 (LA114-17B and LA114-20B), 4–6 mm long cotyledonary-stage somatic embryos were harvested individually from clusters of embryos proliferating via repetitive embryogenesis on Petri plates of semisolid IMM with 2 mg/L 2,4-D. Embryos were transferred to 60 mm plastic plates containing semisolid embryo development medium (EDM; Andrade and Merkle 2005), which was the same as IMM, but lacking PGRs, and given a pre-germination cold treatment at 8º C for 15 weeks in the dark in a refrigerator. Following cold treatment, all embryos were transferred to 125 ml Erlenmeyer flasks containing 30 ml liquid EDM and shaken overnight on a rotary shaker at 90 rpm, to remove residual 2,4-D. Before transfer to germination treatments the next day, embryos received a final wash by pipetting approximately 200 ml of liquid EDM over the embryos under mild vacuum using a Büchner funnel. Embryos were then transferred to 100 mm plastic Petri plates containing one of four treatments for germination: semisolid EDM, semisolid EDM supplemented with 10 mg/L gibberellic acid (GA3), semisolid EDM with 0.5 g/L activated charcoal (AC), or semisolid EDM with both AC and GA3. Ten somatic embryos were placed on each plate and 3 repetitions (plates) were used per treatment for each culture line. Table 2 summarizes the treatments and numbers of somatic embryos tested per treatment x genotype combination in this experiment. Plates with embryos were incubated in a lighted incubator under cool white fluorescent lights (100 µmol·m− 2·sec− 1) with 16 h day lengths at 25° C. Germination and conversion were scored at 12 weeks. Embryos were considered to be germinated when they exhibited taproot elongation from the radical and converted when they had produced both roots and shoots. Somatic seedlings were removed from in vitro conditions and potted in moistened peat:perlite:vermiculite (1:1:1) mix in 4-inch plastic pots, which were placed on top of water-saturated perlite in clear plastic dome-covered trays (to maintain humidity) under cool white fluorescent lights (80 µmol·m− 2·sec− 1) and 16 h day lengths. Somatic seedlings were watered and fertilized with 10 ml of Miracle-Gro fertilizer weekly. For acclimatization, vents on the domed trays were slowly opened over the following two months until dome were removed completely and the somatic seedlings were transferred to the greenhouse to continue growth.
The second germination and conversion experiment used somatic embryos from two lingering white ash embryogenic cultures lines initiated in 2015 (LA112-10 and LA111-2). Embryos for this experiment were produced from suspension cultures. To initiate suspension cultures, approximately 0.5 g of embryogenic culture material of each line was inoculated into 30 ml of liquid IMM in 125 ml Erlenmeyer flasks and grown on a rotary shaker at 90 rpm in the dark at 25° C. Suspension cultures were fed every 3 weeks by decanting off the old medium and adding 30 ml of fresh IMM. At approximately 9 weeks, suspensions were size-fractionated by pouring them through nested stainless steel CELLECTOR® sieves (Bellco Glass) with pore sizes of 860 μm and 38 μm, such that cell clumps with diameters between the two pore sizes were collected on the 38 μm sieve. Material that collected on the 38 μm sieve was backwashed from the sieve with liquid EDM using a pipette, and collected on Nitex nylon mesh (Sefar America, Depew, NY, 30 μm pore size) using a Büchner funnel under mild vacuum. Cells were cultured along with the nylon mesh on semi-solid EDM in 100 mm plastic Petri plates. Plates were incubated in the dark at 25° C to allow somatic embryos to develop. Once populations of somatic embryos had developed on EDM, cotyledonary-stage embryos at least 2 mm long were harvested and transferred to fresh plates of semisolid EDM to enlarge for 2–3 weeks. Then, the mature embryos were given one of four treatments to test their effects on germination: (1) “Planting” (radical inserted into the medium in 100 ml of semisolid EDM in a GA-7 vessels (Magenta Corp.), with incubation in a lighted growth chamber at 22º C under cool white fluorescent light at 100 µmol·m− 2·sec− 1 with 16 h of light per day, (2) Same as 1, except on using EDM supplemented with 10 mg/L GA3, (3) Same as 1, except embryos were first given an 8-week pre-germination cold treatment at 4º C, prior to planting in GA-7 s, and (4) Same as 3, except embryos were transferred to GA-7s of EDM with 10 mg/L GA3 before placing them in the lighted incubator. Each clone by treatment combination was represented by three GA-7 vessels, with nine embryos per GA-7. Table 2 summarizes the treatments and numbers of somatic embryos tested per treatment x genotype combination in this experiment. Germination and conversion were scored after 12 weeks. Germinated somatic embryos from this experiment were removed from culture and acclimatized using the same procedure detailed for the previous experiment.
In vitro versus ex vitro somatic embryo conversion
This experiment compared conversion percentage and early growth measurements of pre-germinated somatic embryos under vitro versus ex vitro conditions. It employed somatic embryos derived from embryogenic suspension cultures using the same procedure described above for the first germination and conversion experiment for producing synchronous populations of embryos via size fractionation and plating. Four embryogenic ash culture lines (LA112-10, LA111-7 A, LA115-5, and LA102-2) were used in the first replication of this experiment. Approximately five weeks following fractionation and plating of the suspension cultures, 3–4 mm-long embryos that developed from the plated material were individually harvested using a dissecting microscope, transferred to 60 mm plastic Petri plates containing semisolid EDM and allowed to enlarge for another two weeks in the dark at 22° C before being given a pre-germination cold treatment at 8º C for eight weeks in the dark. The plates were then removed from the cold and somatic embryos were transferred to 100 mm plastic Petri plates of semisolid EDM with 10 mg/L GA3 and 0.5 g/L activated charcoal. The embryos were stored in a dark growth room at 22° C for 1 week and subsequently moved to a lighted incubator at 22º C under cool white fluorescent light at 100 µmol·m− 2·sec− 1 with 16 h of light per day until radicles (taproots) elongated to at least 1 cm. The germinated embryos from each clone were divided into two groups for the two treatments. For the in vitro conversion treatment, 20 germinated embryos representing the each of the four lines were planted, five per vessel, in GA-7 vessels (Magenta Corp.) with 100 mL semisolid EDM with no GA, but with 0.5 g/L activated charcoal, and returned to the lighted incubator. For the ex vitro conversion treatment, 20 germinated embryos from each of the same lines were transferred to Fafard #3 potting mix in Hilson Rootrainer planting containers (Beaver Plastics). To plant the germinants in potting mix, a 1 cm deep hole was pushed into the potting mix with a forceps tip; then, holding the germinated embryo with forceps, its root tip was positioned into the hole so that the cotyledons remained above the surface. The planting containers with germinants were placed on top of water-saturated perlite in clear plastic dome-covered trays (to maintain humidity) under cool white fluorescent lights (80 µmol·m− 2·sec− 1) and 16 h day lengths and watered and fertilized with 10 ml of Miracle-Gro fertilizer weekly. For both treatments, data for conversion percentage, somatic seedling shoot length and numbers of leaves and first order lateral roots were recorded after 10 weeks. Table 3 summarizes the treatments and numbers of somatic embryos tested per treatment x genotype combination in this experiment. A second replication of this experiment was performed, although due to the failure of one of the culture lines to produce sufficient material, only three culture lines (LA112-10, LA115-5, and LA102-2) were used.
Statistical analysis for germination and conversion experiments
Data from both germination and conversion experiments were analyzed using R statistical software (version 3.12; R Foundation for Statistical Computing) using analysis of variance. Germination and conversion percentage data were subjected to arcsine transformation prior to analysis, although original percentage data are shown in the graphs. Means comparisons for treatments were conducted using Tukey’s HSD test.
Common garden study
Lingering ash somatic seedlings and control trees were planted in April 2018 in a fenced nursery space (~0.9 hectare) at the Whitehall Forest, University of Georgia, Athens, Georgia. EAB was detected in neighboring counties by that time, although not in Athens-Clarke County. We used a randomized complete block design with six blocks, five rows, and four columns. Blocks were sized 12 × 12 m and spaced 12 m from each other. Rows and columns were spaced 3 m from each other. Species/types planted included: (1) Manchurian ash grafted saplings (EAB resistant controls); (2) conventional green ash seedlings (EAB susceptible control 1); (3) conventional white ash seedlings (susceptible control 2) and (4) white ash somatic seedlings (test trees). The white ash somatic seedlings represented nine clones derived from open-pollinated seeds collected from Michigan lingering ash source trees. Sizes of the seedlings at planting varied widely with type/species, ranging from ≤ 1 m in height for most of the somatic seedlings up to ≥ 2 m for the Manchurian ash grafts. We randomly assigned five trees of each type to rows and columns within each block. Hence, 30 trees were planted for each type, except that 26 white ash somatic seedlings were planted in the garden. Plants were fertilized annually in the spring with 10-10-10 (N:P:K), watered by an irrigation system (once a week in spring and fall, twice a week during summer; irrigation was turned off during the winter). The nursery space was mowed as needed to reduce competition from other plants.
We measured the diameter and height of the plants soon after planting (early June 2018), September 2018, April 2019, October 2019, July 2020, and November 2020. Since trees were of different sizes at planting and we took measurements at different time intervals, we calculated the relative growth rates (RGR) during 2018–2020 for the diameter (cm) and height (cm) of each plant type/species (Manchurian, green, white, and lingering white ash). The formula used for RGR was: [loge(Diameter2 or Height2)–loge (Diameter1 or Height1)]/ (Time2− Time1). Time was calculated for each plant per year as 2018 (T1 = date of planting, T2 = fall measurement); 2019 (T1 = spring, T2 = fall); and 2020 (T1 = spring, T2 = fall). RGRs were averaged for all five plants per type within a block, and then across each tree species (N = 6).
At each measurement time, the saplings were assessed for any evidence of EAB activity by checking the foliage for adult feeding and any evidence of larval presence (D-shaped exit holes, bark splitting, epicormic branching, etc.) (Smith et al. 2015). Survivorship of trees was also noted. We placed and monitored 5 purple panel traps in April/May of each year (except 2020 because of shut-down due to COVID-19) at the nursery’s perimeter for assessing EAB adult activity in the general area.