Zea mays ssp. parviglumis (teosinte) seeds Ames 21785 and Ames 21789 were obtained from the USDA Agricultural Research Service seed repository.¹ Bulk amount of Zea parviglumis seeds can be purchased from a private retailer Restoration Seeds.² Culture media used in this work are listed in Supplementary Table S1.

Teosinte seeds (Figure 1A) were sterilized twice following the protocol described by Martinez and Wang (2009) before being placed on germination media (Martinez and Wang, 2009). About 10–50 seeds were submerged in 50ml of seed sterilization solution (Supplementary Table S1) in an Erlenmeyer flask with a stir bar. The flask was placed on a stir plate set to 220rpm. The sterilization of the seeds was carried out at room temperature (RT, 22–25°C) for 20min. The seeds were then rinsed three times with autoclaved deionized water (dH₂O) to remove any residual bleach.

The sterilized seeds remained in sterile water for 5–18h at RT to soften its hard casing. Once softened, the casing was mechanically removed using a pair of clean nail clippers. The “naked” seeds (Figure 1B) were then sterilized using the same sterilization and rinse procedure as described above.

The “naked” teosinte seeds were germinated in a 25×100mm Petri dish containing germination media ½ MS or MSVS34-P2.2 (Supplementary Table S1) with seven seeds per plate (Figure 1C). The Petri dishes were left unwrapped and placed in a clean transparent plastic container [10cm (H)×30cm (L)×18cm (W)] with lid and incubated in a biological incubator at 28°C with 16/8h (day/night) photoperiod and 100–200μmol/m²/s light intensity measured using a LI-250A Light Meter (LI-COR Biosciences, Lincoln, Nebraska, United States). The teosinte plants were allowed to grow for 1–3weeks until they reached a height of ~5–9cm. If needed, the lids of Petri dishes were removed to allow plantlets to grow upright in the covered plastic container.

To prepare whorl segments (WSs) for callus culture, teosinte plantlets of appropriate sizes (Figures 1D,E) were harvested. Inside a laminar flow hood the germinated seedlings were cut to collect the WSs (Figure 1G) using a Number 10 Royal Tek surgical blade. The first cut was a lateral incision along the length of the plant creating two long segments (Figure 1F). Holding the two pieces together at the top of the plant using a pair of forceps, 1mm cuts were made perpendicular to the first cut. The 1mm pieces of leaf tissue were then evenly spaced onto callus induction media MSW57 or 605B (Supplementary Table S1, Figure 1F). The plates were left unwrapped, placed in a clean plastic box with lid as described above, and incubated at 28°C in the dark.

Callus formation and subsequent growth occurred over the next 6–8weeks. WS pieces that began to form embryogenic callus were subcultured onto fresh callus induction media every 2–3weeks. Four to six weeks after callus induction, embryogenic teosinte calli were moved to maturation growth medium (13329iaa, Supplementary Table S1) containing BAP and indole-3-acetic acid (IAA) to promote shoot formation. Callus tissues on maturation media were cultured in the dark at 28°C. After 14days in the dark, the calli were moved into the light [16/8h (day/night), 100–200μmol/m²/s] at 28°C for 7–21days.

For rooting step, callus pieces with developing shoots were placed on rooting medium (272iba, Supplementary Table S1) with indole-3-butyric acid (IBA) to induce root growth. To prevent overgrowth, the calli were placed evenly on the rooting plates with no more than eight pieces per plate. The rooting process was carried out under light [16/8h (day/night), 100–200μmol/m²/s] at 28°C for 14–28days (subculture every 2weeks when needed). During the rooting step, callus with healthy and vigorously growing shoots were closely monitored. To encourage root formation, any excessive callus materials associated with these growing shoots were removed.

Once roots were established, healthy plants were transferred to soil. Excess growth medium associated with the roots was removed and roots were rinsed and cleaned with water. Plantlets were carefully separated using forceps and surgical blades. Rooted plants were transferred into 3 sq. in (19cm²) plastic insert (1801 deep inserts, T.O. Plastic, Clearwater, MN, United States) containing pre-wetted soil mix (Sungro^® Professional Growing Mix Sunshine^® Mix #1). The inserts were placed in a tray (27cm×54cm) and covered with a clear plastic humidity dome. The plants were grown in a growth chamber at 28°C with 12/12h (day/night) photoperiod and 250–320μmol/m²/s light intensity (~1.7m below the lights) for 7days. Developing plants were transplanted into one gallon (3.8L) plastic pots until maturation. Tassels began to form 8–12weeks after being placed in soil. To prevent cross-pollination, tassels were removed and stored separately in a beaker of water away from the silks of the teosinte. Plants were fertilized with Peters Excel 15-5-15 fertilizer as needed.

Plasmid pKL2155 (Figure 2A; Supplementary Figure S1) and pAHC25 (Christensen and Quail, 1996; Figure 2B) were used for bombardment experiments. The plasmid pKL2155 (9,492bp) was designed in this work. The construct carries a mutant acetolactate synthase (Als) gene (Okuzaki et al., 2007) driven by sorghum Als gene (SbAls) promoter as selectable marker gene and a recombinant red fluorescent protein tdTomato gene (Shaner et al., 2004) driven by a double 35S promoter (Omirulleh et al., 1993) as a scorable marker gene (Figure 2A; Supplementary Figure S1). pKL2155 was constructed using Gibson assembly (Gibson et al., 2009). First, four different fragments were PCR amplified using primers, plasmid DNA templates (Supplementary Table S2a), and 2× Phusion High-fidelity DNA polymerase master mix (Thermo Fisher Scientific, MA, United States). For each fragment, 0.5μM primers and 5–10ng of plasmid DNA template were used for a 50μl reaction volume. Detailed thermocycling conditions are as follows: initial denaturation at 98°C for 30s, 35cycles of 10s at 98°C, 30s at 52.1–62.8°C, 30–75s at 72°C, and followed by final extension at 72°C for 5min. Annealing temperature and extension time varied depending on the fragment to be amplified (Supplementary Table S2a). PCR products were resolved on 1% agarose gel and corresponding DNA fragments were purified using the QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instruction. Amplified DNA fragments and the linear pJET1.2 DNA (Thermo Fisher Scientific, Waltham, MA, United States) were assembled using the HiFi DNA assembly master mix (New England Biolabs, Ipswich, MA, United States) according to the manufacturer’s protocol. Two microliters of the assembly mix were used for Escherichia coli (DH5α) transformation as described by Fronger and Hall (2007).

The plasmid pAHC25 is a 9,618bp in size and contains a selectable marker gene, bar, and a scorable marker gene, gus. Both genes are driven by separate maize ubiquitin promoters (Christensen and Quail, 1996).

Plasmid DNA was isolated and purified using QIAprep Spin Miniprep Kit (Qiagen GmbH, Hilden, Germany) per manufacture instruction.

Two different methods were used for coating of gold particles with DNA for biolistic transformation. The spermidine/CaCl₂ method was based on the procedure for biolistic transformation of maize immature embryo as described previously (Wang et al., 2020). Briefly, in an Eppendorf tube, 1.5mg surface-sterilized 0.6μm gold particles were mixed with 2–3μg of plasmid DNA, CaCl₂ and spermidine. After coating, the gold/DNA complex was pelleted, washed by 100% ethanol (Decon 200 Proof Pure Ethanol Alcohol, Decon Labs Inc., King of Prussia, PA) and resuspended in 110μl of 100% ice-cold ethanol. For each shot, 10μl of gold/DNA suspension was aliquoted onto the center of each macrocarrier and allowed to air-dry prior to the bombardment. Typically, each bombarded plate received ~150μg of gold particles and 200–300ng of plasmid DNA in this work.

The majority of the bombardment experiments described in this work used a transfection reagent TransIT^®-2020 (Mirus Bio LLC, Madison, WI, United States) for coating DNA onto the gold particles following the protocol described in Miller et al. (2021) with modifications (Miller et al., 2021). Specifically, 1.5mg of surface-sterilized 0.6μm gold particles were homogenously suspended in 90μl of dH₂O using an Ultrasonic Cleaner (Lab Safety Supply HB-3818B sonicator, Grainger, Lake Forest, IL, United States). Three μg of plasmid DNA (300ng/μl in dH₂O) was added to the gold suspension and mixed by vortexing for 10s on high setting. Next, 1μl of TransIT-2020 (ratio 0.33μl TransIT-2020 per μg DNA) was added and the tube was vortexed on high for 15s. The Eppendorf tube was then vortexed in the upright position for 10min on low setting to allow contents to settle to the bottom of the tube. The Eppendorf tube was centrifuged at 9,391×g for 1min to pellet the gold. Using a pipette, the supernatant was removed without disrupting the gold/DNA pellet. Finally, 110μl of 100% ice-cold ethanol were added and the tube was then sonicated for 5s to ensure the homogenous suspension of gold/DNA. Ten microliter of gold/DNA were dispensed onto each macrocarrier and allowed to air-dry prior to the bombardment. Similar to spermidine/CaCl₂ coating method, each bombarded plate using the TransIT-2020 coating received ~150μg of gold particles and 300ng of plasmid DNA in this work.

For pre-bombardment osmotic treatment, approximately 1g of embryogenic callus derived from multiple leaf pieces of a single plant was placed onto the center of a MSW57 osmotic plate (Supplementary Table S1) in a circle ~2.5cm² in diameter. The plates were incubated in the dark at 28°C for 4 to 24h prior to the bombardment. Bombardment of teosinte callus pieces was carried out directly on the MSW57 osmotic plates using the Biolistic PDS-1000/HE particle delivery system (Bio-Rad) inside a sterile flow hood as described (Wang et al., 2020). The specific parameters were 6cm target distance, 650psi rupture discs, and 26.5mmHg vacuum pressure.

Bombarded calli were immediately moved from MSW57 osmotic medium onto fresh callus induction medium without any selective reagent (Supplementary Table S1) and incubated in the dark at 28°C. Seven to eleven days after the bombardment, the calli were moved onto callus induction medium with appropriate selection agent.

Teosinte callus bombarded with pKL2155 were cultured on callus induction medium containing 0.2mg/L of ethametsulfuron (EMS) after the resting stage. After the first round of selection, dead calli were discarded and the healthy ones were subcultured onto fresh selection media. Selection on callus induction media with 0.2mg/L EMS continued for 4–6weeks. Healthy EMS resistant calli were transferred onto maturation medium 11329iaa (Supplementary Table S1) containing the selective reagent 0.2mg/L EMS. Callus tissues on maturation media were cultured at 28°C in the dark. After 14days in the dark the maturation plates culturing EMS resistant calli were placed under the light [16/8h (day/night), 100–200μmole/m²/s] at 28°C for another 7–21days.

Teosinte tissues bombarded with pAHC25 were moved to MSW57 containing 2mg/L of bialaphos. Two weeks after the selection, healthy proliferating calli were moved onto callus induction medium containing 5mg/L of bialaphos. An optional third subculture to callus induction medium containing 5mg/L of bialaphos was used to increase the size of the transgenic calli if needed.

Developing shoots were placed on rooting medium 272iba (Supplementary Table S1) with either 0.2mg/L of EMS (for pKL2155) or 2mg/L bialaphos (for pAHC25) for root development. Rooted putative transgenic plants were acclimated and brought to maturity in a growth chamber as described in the section “Callus Induction, Maintenance and Regeneration” above.

Genomic DNA was extracted from ~2cm² clippings of teosinte leaf tissue using the protocol described in Edwards et al. (1991). PCR analysis of the genomic DNA was used to detect the presence or absence the transgenes in regenerated T0 teosinte plants and the progenies using the primers and conditions listed in Supplementary Tables S3a,b.

Genomic DNAs were extracted from teosinte leaf materials as described by Richards et al. (1994). A teosinte single copy gene, ZvMEK1, an ortholog of maize ZmMEK1 (Hufford et al., 2021), was selected as a reference gene for the transgene copy number estimation. To generate standard curves, a 3,797bp plasmid (pKL2347) containing fragments of the reference gene ZvMEK1 (261bp), gus gene (263bp) and SbAls promoter (299bp) was constructed using Gibson assembly (Gibson et al., 2009). These fragments were amplified by PCR using the primers listed in Supplementary Table S2b and the Q5 master mix (New England Biolabs, Ipswich, MA, United States). PCR products were purified using the QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany) and assembled with the linearized pJET1.2 DNA (Thermo Fisher Scientific, Waltham, MA, United States) using the HiFi DNA assembly master mix (New England Biolabs, Ipswich, MA, United States) according to the manufacturer’s instruction.

Transgene quantification was carried out using TaqMan PCR. Quantitative PCR (qPCR) primers and probes were designed using the PrimerQuest™ Tool (IDT, Coralville, IA, United States), and the probes with compatible dyes (HEX for ZvMEK1; FAM for gus and SbAls promoter) and double quenchers (ZEN/Iowa Black™ FQ) were synthesized by IDT (Coralville, IA, United States). Each qPCR was performed in a 20μl reaction volume with the following ingredients (final concentration): 0.5μM ZvMEK1 primer pair, 0.5μM gus or SbAls promoter primer pairs, 0.25μM probe for ZvMEK1, 0.25μM probe for gus or SbAls promoter, 0.01pg–1ng pKL2347 plasmid DNA (2.57×10³–2.57×10⁸ copies) or 10–20ng of teosinte genomic DNA, and 1×PrimeTime™ Gene Expression Master Mix (IDT, Coralville, IA, United States). Real-time PCR was carried in an Mx3005p qPCR system (Agilent, Santa Clara, CA) using the fast-cycling condition as instructed by the manufacturer: 3min at 95°C for polymerase activation followed by 40cycles of amplification with 5s at 95°C for denaturation and 30s at 60°C for annealing/extension. All reactions were performed with four technical replicates. Transgene copy numbers were estimated using the standard curves generated by the pKL2347 DNA copy numbers and the qPCR cycle threshold values (C_t). The amplification efficiencies were 98% for the SbAls primers (r²=0.999), 107% for the gus primers (r²=0.999), and 98–107% for the ZvMEK1 primers (r²=0.999). Because the single copy reference gene has two alleles in the genome, a single copy transgene would have a 1:2 ratio of gus or SbAls promoter to ZvMEK1.

Genomic DNA was extracted from leaf tissue of T0 and T1 plants using cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). Fifteen micrograms of gDNAs were digested with SacI enzyme and separated on a 0.9% (w/v) agarose gel by electrophoresis. The DNAs were transferred to and cross-linked to a membrane (BrightStar Plus; Thermo Fisher Scientific, Waltham, MA, United States). The membrane was prehybridized using Church’s buffer (Church and Gilbert, 1984) for 4h at 65°C. The probe was designed to detect the gus gene. It was amplified by PCR from the plasmid pACH25 using primers Gus-F (TTG GGC AGG CCA GCG TAT CGT) and Gus-R (ATC ACG CAG TTC AAC GCT GAC). The 421bp product was purified and subsequently labeled with ³²P-dCTP using the Prime-it II kit (Agilent Technologies, Santa Clara, CA, United States). The labeled probe was purified (Illustra Probequant columns, VWR, Radnor, PA, United States) and incubated with the DNA membrane overnight at 65°C. The membrane washing and film development were done following standard protocols (Green and Sambrook, 2012).

Teosinte seeds do not readily germinate after harvest (Mondrus-Engle, 1981). To germinate naturally aged seeds (minimum 3months after harvest in this work) for progeny analysis, seeds were sterilized and the hard casing were removed as described above in “Seed sterilization and explant preparation.” A sterile 100mm×25mm Petri dish was lined with one piece of 7cm or 12cm diameter Whatman filter paper in the flow hood. Autoclaved distilled water was used to wet the filter paper, with a 1ml pipette. The sterile “naked” seeds were placed on the pre-wetted filter paper. The petri dish was wrapped with a porous tape and incubated at 26°C in the dark for 4–7days until sprouting. The germination is monitored daily. To prevent drying of the germinating seedlings, additional sterile water was added, as needed, to the filter paper to keep it moistened constantly. The sprouts with established roots and shoots were planted into soil as described above in “Callus induction, maintenance, and regeneration.”

To accelerate germination of freshly harvested and delicate teosinte seeds produced from tissue culture plants grown in a confined growth chamber, germination was carried out in vitro without surface sterilization using bleach or ethanol. The seeds were soaked in a filter-sterilized gibberellic acid (GA3) solution (1mg/L), at 26°C for 24h on the lab bench under ambient lighting. The hard casing of seed was aseptically removed. The “naked” seeds were placed, embryo axis up, in a ½ MS germination medium (Supplementary Table S1) supplemented with 1mg/L GA3. The plates were incubated in a biological incubator at 28°C with 16/8h (day/night) photoperiod and 100–200μmol/m²/s light intensity. The germination could be observed as early as 10days after the GA3 treatment.

Callus tissue and the T1 seeds of pKL2155 were screened for tdTomato gene expression using fluorescent microscope (Olympus SZH10 stereo microscope with Texas red filter, EX 535–585nm, EM 605–690nm) or using a NIGHTSEA dual fluorescent protein flashlight and filter glasses (NIGHTSEA LLC, Lexington, MA, United States).

Putative transgenic T0, T1, and T2 plants generated from the bombardment of pAHC25 were phenotyped using GUS stain as described (Jefferson et al., 1987).

pAHC25 transgenic T2 progeny was tested for herbicide resistance using the protocol described previously (Frame et al., 2006). For screening of glufosinate tolerance, fully expanded leaves from 2-week-old teosinte seedlings were used. Glufosinate solution (150mg/L glufosinate plus 0.1% Tween 20; freshly prepared from the herbicide Liberty®, BASF, Ludwigshafer, Germany) was applied onto one-third of the leaf surface (from tip of the leaf). Q-tips soaked with the glufosinate solution were used to gently rub both sides of the leaves to ensure the herbicide contact and penetration. Plants were assessed for damage 2days after the leaf painting.

Statistical analysis for plant regeneration using different germination and callus induction media was conducted using a z-score test for two independent proportions.³ Pairwise comparisons were made for all six combinations among the four different treatments.

An efficient regeneration protocol is a pre-requisite for establishing a genetic transformation protocol using a tissue culture system. To overcome the limited production of immature embryos in teosinte plants, we used seedlings germinated from mature seeds as an alternative starting material. Unlike maize, teosinte is resistant to inbreeding. As a result, the seed accessions exist as populations (Fukunaga et al., 2005). Therefore, it is expected that the seed germination and callus initiation rates may differ between plants. In this study, we chose to use seed accessions Ames 21785 and Ames 21789 acquired from USDA Agricultural Research Service seed repository and Zea parviglumis seeds from Restoration Seeds (Talent, OR, United States). We chose the Restoration Seeds (RS) for its bulk quantity needed in the experiments.

To ensure uniform seed germination and to minimize contamination in vitro, the hard casing covering teosinte seed was removed. Twice disinfected (before and after removal of the casing) “naked” seeds were then placed on germination media (Figures 1A–C). Two types of germination media were evaluated (Supplementary Table S1). The ½ MS medium was a simple MS/sucrose-based medium, and MSVS34-P2.2 was MS-based medium supplemented with maltose and growth hormones cytokinin (BAP) and auxin (picloram). MSVS34-P2.2 was modified based on MSVS34 medium used by Sidorov et al. (2006) for maize seedling-derived callus (Sidorov et al., 2006).

The “naked” teosinte seeds could be germinated from both media. Table 1 shows typical germination rates for RS seeds, with 75.7% on ½ MS and 65.7% on MSVS34-P2.2. Because of the presence of picloram, plants germinated from the MSVS34-P2.2 medium grew abnormally with curled and stunted shoots (Figure 1E) when compared with the plants from the ½ MS medium (Figure 1D).

Teosinte seedlings were first dissected to 2–3cm-long whorl segments (Figures 1F,G). Each WS was considered as an explant. Then the segments were cut into 1mm pieces and placed on callus induction media (Supplementary Table S1). To test and track the degrees of callus initiation ability, these pieces were arranged on the media according to their anatomical positions on the segment (Figures 1F,H). Two to three weeks after the initiation, calli were readily formed from pieces that contain shoot apical meristematic region of the first node of the teosinte seedling (Figure 1H and Supplementary Figure S2A). While not all pieces derived from the same plants formed callus, most of RS seedlings tested on both media produced various callus cultures as embryogenic callus (Figure 1I, Supplementary Figure S2B) and non-embryogenic callus (Figure 1J, Supplementary Figure S2B). The callus induction frequency was calculated as percentage of callus-producing WSs in total dissected WSs used in the experiment (Table 1).

To improve regeneration ability of callus culture, we compared two different callus induction media MSW57 (Sidorov et al., 2006) and 605B (modified from Masters et al., 2020). Both were MS/sucrose-based media supplemented with silver nitrate and auxin (picloram in MSW57 and dicamba in 605B, Supplementary Table S1). Leaf nodal pieces from seedlings germinated in ½ MS or MSVS34-P2.2 were divided equally and placed on either MSW57 or 605B for callus induction (Figure 3). As shown in Table 1, greater than 90% of callus induction could be achieved for all four treatments. The callus generated on each growth medium was morphologically consistent, displaying a range of phenotypes including type I (Figure 1I, Supplementary Figure S2B), pre-type II embryogenic callus and watery, loose-structured non-embryogenic callus (Figure 1J, Supplementary Figure S2B). Both MSW57 and 605B callus induction media performed well regardless of germination media, generating callus in >90% of the WSs placed on each medium. However, the regeneration rates were different between seedlings germinated on MSVS34-P2.2 (50 and 70%) vs. ½ MS (0 and 18%). Statistical analysis using two-tailed z-test of difference in proportion indicated that regeneration rates of treatments 3 and 4 were significantly higher than that of treatment 1 (p<0.01, Table 1 and Supplementary Table S4). This suggests that using MSVS34-P2.2 as a germination medium can result in higher plant regeneration rates with either MSW57 (treatment 3) or 605B (treatment 4) as callus induction media (Table 1 and Supplementary Table S4). The regeneration rates of treatments 2 and 4 were significantly different at the level of p<0.05, but not at p<0.01. The regeneration rate differences between treatments 2 and 3 as well as treatments 3 and 4 were not significant (Supplementary Table S4). Here, regeneration rate was measured as the number of rooted shoots divided by the total number of WSs used for callus initiation.

Plants regenerated from the experiments were moved to soil and brought to maturity. Fertile seeds were successfully recovered from regenerated plants. A portion of the regenerated teosinte plants displayed a tassel-ear phenotype, but seeds could be recovered from these plants. These results showed that the inclusion of auxin in germination medium plays critical role in plant regeneration in teosinte.

The embryogenic callus (Figure 1I) forming from WS pieces was often lighter in color and more dense than non-embryogenic callus (Figure 1J), which was often watery and darker. Callus cultures of similar appearance and derived from the same plant did not always maintain the same ability to regenerate into fertile plants. Supplementary Figure S3A shows regeneration ability from two callus lines that were derived from two pieces originated from one single WS. Initially, both pieces produced vigorously growing calli consisting of both embryogenic and non-embryogenic types. Interestingly, these two morphologically similar callus lines displayed markedly different regeneration ability.

Teosinte callus could retain its regeneration competency for several months, but the rates diminished with age of the cultures. For example, in one experiment we observed that callus of Ames 21785 retained 100% regenerability after 8months but less than 50% of the callus could be regenerated after 15months (Supplementary Figure S3B).

Our methods included a 3-week treatment on maturation medium (13329iaa, Supplementary Table S1) when the callus cultures were transitioned from dark to light for shoot induction. The inclusion of this step was essential to ensure the formation of shoots, whereas in the absence of this step no plants were recovered from the callus culture (Supplementary Figure S3B).

Before starting our transformation experiments, we conducted experiments to create kill curves using the selective reagents on non-transformed callus tissues. We tested two selective reagents (bialaphos for the bar gene, and EMS for the Als gene) that are commonly used in maize transformation and determined the efficacy of each reagent in teosinte. Our results showed that 5mg/L of bialaphos and 0.2mg/L of EMS were sufficient to arrest callus growth in teosinte (Supplementary Figure S4).

Two plasmid DNA constructs, pKL2155 (Figure 2A, Supplementary Figure S1) or pAHC25 (Christensen and Quail, 1996; Figure 2B) were used for bombarding teosinte embryogenic callus cultures. Callus chosen for the bombardment experiments were healthy and proliferate actively with some embryogenic sectors (Figure 1I). The callus cultures were treated in osmotic containing medium prior to the bombardment (Supplementary Table S1; Wang et al., 2020). Majority of the experiments used a transfection reagent TransIT®-2020 for DNA/gold particle coating (Miller et al., 2021). Transient transformation comparison has shown that DNA/gold microprojectiles coated using TransIT®-2020 performed equally well compared to that coated using the traditional spermidine/CaCl₂ reagents (Supplementary Figure S5). Because TransIT®-2020 coated DNA/gold suspension formed less aggregates, it was easier to handle during the microprojectile loading process.

Table 2 summarizes 10 independent bombardment experiments carried out within a 5-month period. All the experiments were performed using embryogenic callus culture generated from teosinte RS seedlings. Each experiment used bulked callus pieces derived from several WSs (ranged from 1 WS in Exp #3 and #7, and 15 WSs in Exp #6). The age of callus cultures ranged from 34days (Exp #6) to 104days (Exp #1). The bombardment used pKL2155 plasmid, which contains the tdTomato gene for visual screening and the herbicide-resistant Als gene for transgenic plant selection. Transient tdTomato expression could be visualized 4days after the bombardment on most callus tissues (Figures 4A,B). Four to six weeks after incubating the callus on selection (callus induction media containing 0.2mg/L EMS), proliferating calli could be identified. Some resistant callus pieces displayed strong red fluorescence (Figures 4D–I). The expression of the tdTomato gene on callus tissue was strong; the red fluorescence could be visualized readily by using a hand-held NIGHTSEA BlueStar flashlight and filter glasses (NIGHTSEA LLC, Lexington, MA, United States).

A total of five EMS resistant callus pieces were generated and regenerated (Table 2). They were derived from four independent bombardment experiments. A total of 49 rooted plants were produced from these five events (Figures 5A,B and Supplementary Table S3c). PCR analyses were performed on 27 T0 plants that survived in soil using three different pairs of primers (Supplementary Tables S3a,b). All 27 plants were tested positive for at least one transgene from pKL2155 (Figure 6A, Supplementary Figure S6 and Supplementary Table S3c). Transformation frequency (TF) was calculated as the percentage of PCR confirmed T0 event of the total callus-producing WSs bombarded. TF of the four successful bombardment experiments ranged from 6.67% (Exp #6) to 50% (Exp #1). Note that in Exp #1, each plate was bombarded twice using the same parameters. Excluding Exp #1, average TF from nine independent experiments was 4.17%.

Quantitative PCR analysis was used to estimate the transgene copy numbers in the five T0 events. We selected a single copy ZvMEK1 gene, an ortholog of maize ZmMEK1 (Hufford et al., 2021), as the reference gene for the qPCR analysis. The qPCR analysis for pKL2155 events examined the transgene region containing the SbAls promoter. We chose to analyze the SbAls promoter region to avoid detecting endogenous Als gene in teosinte. Estimated transgene copy numbers in the five pKL2155 T0 events were low (Table 3). Two events (2155-B and 2155-E) have approximately one copy Als transgene; one event (2155-A) has two copies; two remaining events (2155-C and 2155-D) have less than one copy. The less than one copy number could be attributed to the possible mosaic nature of the transgene in these T0 plants.

qPCR analysis was also performed on four seedlings germinated from the 2,155-A event (Table 3), showing a similar copy number (1.49±0.73) in the T1 plants compared to its T0 parent (2155-A, 1.61±0.28). Transgene tdTomato expression in the progeny was monitored in the T1 seeds. Red fluorescence could be readily detected and visualized under a fluorescent microscope on the “naked” seed but not on the intact seed (Figure 7A).

Bombardment experiments using pAHC25 were carried out using callus derived from Ames 21789 seedlings. Out of three bombardments, one GUS-positive event (Figure 4C) was produced. This event was self- or sib- pollinated in a growth chamber (Figures 5C,D). Southern blot hybridization analysis was performed on six T0 plants and two T1 plants (Figure 6B). Five of the six T0 plants showed two dominant bands hybridized with the ³²P-labled gus probe, suggesting at least two transgene insertions in the genome (Figure 6B). The different patterned T0-1 plant did not survive the soil transfer; therefore, no further investigation was conducted.

qPCR analysis of the gus transgene was performed on four T2 plants of the pAHC25-1 event: two GUS-positive and two GUS-negative plants. It revealed that the two GUS-negative plants indeed did not have the transgene, whereas the two GUS-positive plants have ~4 transgene copies integrated in the teosinte genome (Table 3). This discrepancy likely reflects the zygosity of T0 vs. T2 transgenic plants. If the T2 plant was a homozygous for the transgene, its copy number is likely doubled compared to the T0 plant, which was a hemizygous for the transgene.

Phenotyping of the pAHC25-1 T1 progeny showed that 27 (77.1%) were GUS-positive and eight (22.9%) were GUS-negative. T2 progeny analysis of 19 seedlings showed 14 (73.7%) GUS-positive and five (26.3%) GUS-negative plants (Figures 7B–E). All GUS-positive plants showed resistant to herbicide glufosinate while the GUS-negative plants showed necrosis on the portion of the leaf surface where the herbicide was applied (Figure 7F).

Figure 8 presents a flowchart of our current transformation process used to produce embryogenic callus and transgenic T0 events. The process involves typical steps required for most biolistic-mediated transformation. Bombardment experiments can be conducted 6–8weeks after the initiation of the callus from whorl segments. Four to six weeks after the selection, herbicide-resistant embryogenic callus culture can be placed onto maturation media with selection agent for shoot induction. Once the cultures are moved to light, rooted plantlets can be produced within 3–4weeks. The entire process takes approximately 4–7months for obtaining transgenic plantlets from the day of seed germination.