Investigations into Selected Pollinator-Friendly Plant Species: Seed Lot Germination, Breaking Dormancy with Plant Hormone Priming and the Influence of Water Stress on Germination

Abstract

The lack of protocols for breaking seed dormancy, inconsistent seed quality, and abiotic stress factors such as drought impede large-scale restoration efforts of pollinator-friendly native plant species. This research explores the germination response, dormancy-breaking techniques, and water stress tolerance in selected pollinator-friendly plant species with characteristics facilitating mechanized rehabilitation protocols and biodiversity enhancement. Forty-two commercial seed lots representing seven plant families with 28 species were evaluated under two alternating temperature regimes (15/25 °C and 20/30 °C) with and without gibberellic acid (GA₃) priming treatments. Six of the twenty-eight species were selected based on pollinator requirements for the monarch butterfly (Danaus plexippus L.) and further examined by priming seeds for 24 h in solutions containing GA₃, kinetin (KIN), and hydrogen peroxide (H₂O₂), or their combinations, to evaluate their dormancy-breaking responses. The effect of water stress on seed germination was assessed in controlled chambers at soil water potentials of −1.08, −0.75, −0.13, and 0 MPa. Initial seed quality of the 42 seed lots revealed that only 62% had greater than 50% germination, while of the same 42 lots, 98% had greater than 50% viability based on the commercial seed label. The difference was largely attributed to seed dormancy. In laboratory studies of the 42 seed lots, GA₃ significantly enhanced germination percentage, and reduced T₅₀ (time to 50% germination) across most seed lots. Overall, germination was higher and faster at 20/30 °C than 15/25 °C. Priming the six selected species with 1.0 mM GA₃ in 0.3% H₂O₂ consistently improved germination compared to the non-primed control after 14 days. Asclepias species (A. incarnata, A. syriaca, and A. tuberosa) exhibited consistently high germination across a broad moisture range of −0.75 to 0 MPa. In contrast, Echinacea purpurea required high moisture levels (−0.13 to 0 MPa) for optimal germination. Monarda fistulosa and Rudbeckia hirta showed their best performance under moderate moisture conditions (−0.13 MPa). Collectively, the use of GA₃ priming to break physiological seed dormancy offers a promising approach to enhance germination and improving the establishment potential of native pollinator species in restoration programs.

1. Introduction

Pollination is a fundamental ecological process essential for maintaining biodiversity, ecosystem functioning, and reproductive success of both wild flora and agricultural crops [1]. Most angiosperms require animal pollinators to complete fertilization and produce seeds, making the plant–pollinator relationship a cornerstone of natural ecosystem stability [2]. It is estimated that 75% of cultivated plants and 90% of wild flowering species depend on insect pollination. Pollinators support the reproductive success of more than 180,000 plant species and over three-quarters of global food crops, playing a significant role in enhancing the yield and quality of agricultural products [3,4,5]. Beyond their ecological role, pollinators provide substantial economic value, with global pollinator-dependent crop production estimated at over $577 billion annually [6].

Many native wildflower species used in pollinator restoration are broadly classified as zoophilous plants, meaning species that rely on animals to transfer pollen. Within this broader category, the species examined in this study fall more specifically under entomophilous plants, which depend on insects as their primary pollination agents [5]. Because both categories accurately describe the ecological role of these species but differ in taxonomic precision, we adopt the more inclusive and restoration-focused term “pollinator-friendly plant species” throughout this manuscript for clarity and consistency.

The rapid decline of pollinator populations worldwide has raised significant concerns [7,8]. Approximately 16% of vertebrate pollinators (birds and bats) and nearly 40% of invertebrate pollinators, including bees and butterflies, are now at risk of extinction [9]. In North America, the eastern population of the monarch butterfly (Danaus plexippus L.) has declined by nearly 80% [10] and several wild bee species have experienced a dramatic decline of 96% since the early 2000s [11]. Despite increasing global recognition of the essential role pollinators play in ecosystem services, they continue to be threatened by habitat loss, climate change, pesticide exposure, and widespread declines in floral resources. Habitat loss is particularly severe in agricultural regions; between 2008 and 2012, for example, 5.7 million acres of U.S. pastureland were converted into cropland, accounting for 77% of total agricultural land conversion during that period [12,13]. Additionally, the shift towards glyphosate-tolerant (Roundup Ready) and other GMO herbicide resistance traits since the late 1990s has accelerated the decline of pollinator food and other nectar plants, contributing substantially to monarch butterfly population losses [14]. These combined pressures underscore the urgent need to restore pollinator-supporting plant communities, an effort that depends heavily on reliable germination and dormancy-breaking protocols for pollinator-friendly plant species.

Restoration initiatives are often limited by the seed issues of pollinator-friendly plant species. In addition to complex and extensive germination regimens, numerous species and seed lots of these species exhibit inconsistent seed quality. The recommended minimum germination of flower species was reported in the Recommended Uniform State Seed Law (RUSSL) [15]. For example, the minimum germination for Echinacea purpurea and Rudbeckia hirta was 60%, Helianthus spp. was 70%, and Gaillardia pulchella was 45%. For those species not listed within the RUSSL guidelines, the minimum recommended germination of commercial seed lots was 50%.

Species may have physiological dormancy, or other types of dormancies [16,17]. Physiological dormancy is particularly prominent in genera of Asclepias, Echinacea, Monarda, and Rudbeckia where the absence of standardized dormancy-breaking protocols contributes to inconsistent seedling establishment in restoration projects. Dormancy release of species with physiological dormancy is governed by tightly regulated hormonal and oxidative pathways: gibberellins promote embryo growth and endosperm weakening, while cytokinins stimulate early cell division and metabolic activation [18,19]. Reactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂), also play a central signaling role during the transition from dormancy to germination by modulating redox balance, promoting cell wall loosening, and enhancing metabolic activation [20,21]. Recent evidence demonstrates that optimized concentrations of H₂O₂ can increase germination speed, improve seedling vigor, and enhance early tolerance to abiotic stress, including drought, as demonstrated in Corethrodendron multijugum [22]. Collectively, these findings highlight the importance of investigating hormone–ROS interactions as a foundation for developing reliable dormancy-breaking strategies in native wildflower species.

Seed imbibition and solution uptake may also be modified by non-ionic surfactants such as Tween 20, Tween 80, and Silwet L-77, which reduce solution surface tension and facilitate faster hydration during priming. Both classical and contemporary studies indicates that appropriate surfactant concentrations can improve water uptake, hydration uniformity, and germination rate, whereas excessive levels may induce membrane leakage or phytotoxicity [23,24,25]. Despite these findings, surfactants remain understudied in native pollinator-friendly plant seed species, and species-specific responses have not been systematically evaluated. Investigating surfactant hormone–oxidative interactions is therefore essential for developing effective germination-enhancement treatments for restoration applications.

Beyond dormancy-breaking treatments, soil moisture availability represents another major factor influencing early seedling establishment in restoration environments. Species vary widely in their germination responses to water stress: Asclepias syriaca and related milkweed species often germinate across a broad moisture range, while others such as Echinacea purpurea require higher water availability for radicle emergence [19,26]. Large-scale meta-analyses further show that germination declines sharply as soil water potential decreases below −0.5 to −0.8 MPa, underscoring the ecological importance of investigating drought sensitivity during early establishment [27]. Such moisture-dependent variation must be considered when designing restoration protocols for heterogeneous field conditions.

Given these challenges, there is a critical need to systematically evaluate dormancy-breaking treatments, oxidative cues, surfactants, and drought responses across multiple pollinator-relevant species. This study addresses three major knowledge gaps limiting the success of pollinator habitat restoration:

• The dormancy-breaking potential of GA₃ and KIN across multiple pollinator-friendly plant seed species.
• The role of oxidative signals (via H₂O₂) and surfactants in combination with GA₃ in enhancing germination.
• The influence of water availability on germination behavior to guide restoration strategies under variable field conditions.

By combining hormonal treatments, oxidative signaling, and surfactants, this study introduces an improved seed enhancement strategy for the establishment of native pollinator-friendly plant species. Quantifying drought stress responses of these species establishes limiting factors on successful stand establishment.

To clarify the connection among the different experimental stages and to guide the reader through the research logic, the workflow is illustrated in Figure 1.

2. Materials and Methods

2.1. Selection of Pollinator-Friendly Plant Species and Acquisition of Seed Lots

The species selected for this study were chosen from pollinator wildflowers identified by the Natural Resources Conservation Service (NRCS) of the United States Department of Agriculture [28]. Seed samples were acquired from Shooting Star Native Seeds, Spring Grove, MN, USA and seed lots were donated by Ernst Conservation Seeds, Meadville, PA, USA. Forty-two commercial seed lots were obtained from the two seed companies, representing 28 different species and 7 plant families, each with its inherent germination and dormancy. Information provided on the seed label of each seed lot was presented on germination percentage, dormancy percentage, total viable seeds, PLS (Pure Live Seed), and 1000 seed weight (TSW), (Appendix A 

Table A1. Taxonomic description of 42 seed lots, including seed source, USDA species code, seed lot number, and seed test results from the label: % germination, dormant and total viable, PLS (Pure Live Seed), and TSW (thousand seed weight). The six selected seed lots (•) were designated for further investigation in subsequent experiments. N/A means Not Available.

Latin Name	Plant Family	Common Name	Company Name	USDA Species	Seed Lot	% Germ	% Dorm	Total Viable	PLS	TSW (g)
Asclepias incarnata L.	Apocynaceae	Swamp milkweed	Ernst Seed	ASIN	•JG040418	85	10	95	92.48	5.38
Asclepias incarnata L.	Apocynaceae	Swamp milkweed	Shooting Star	ASIN	ASCINC463B	77	22	99	93.94	4.70
Asclepias incarnata L.	Apocynaceae	Swamp milkweed	Shooting Star	ASIN	ASCINC263B	14	85	99	94.95	5.04
Asclepias syriaca L.	Apocynaceae	Common milkweed	Shooting Star	ASSY	ASCSYR553A	6	92	98	97.58	5.49
Asclepias syriaca L.	Apocynaceae	Common milkweed	Shooting Star	ASSY	•ASCSYR602A	11	88	99	98.66	5.32
Asclepias syriaca L.	Apocynaceae	Common milkweed	Ernst Seed	ASSY	ASCSYR01-22PA	30	66	96	93.31	4.28
Asclepias syriaca L.	Apocynaceae	Common milkweed	Ernst Seed	ASSY	ASCSYR01-23PA	9	73	82	74.56	3.93
Asclepias tuberosa L.	Apocynaceae	Butterfly milkweed	Shooting Star	ASTU	•ASCTUB670B	90	6	96	93.75	4.36
Asclepias tuberosa L.	Apocynaceae	Butterfly milkweed	Shooting Star	ASTU	ASCTUB463A	18	75	93	92.96	5.31
Asclepias tuberosa L.	Apocynaceae	Butterfly milkweed	Ernst Seed	ASTU	ASCTUB01-21PA	8	71	79	78.72	4.94
Bidens aristosa (Michx.) Britt.	Asteraceae	Burr marigold	Shooting Star	BIAR	BIDARI-2223A	82	8	90	88.67	2.43
Chamaecrista fasciculata (Michx.) Greene	Fabaceae	Partridge pea	Shooting Star	CHFA2	CHAFAS653A	82	4	86	85.87	8.31
Dalea candida Willd.	Fabaceae	White prairie clover	Shooting Star	DACA7	DALCAN053B	93	4	97	94.15	1.20
Doellingeria umbellata (Mill.) Nees	Asteraceae	Flat-topped aster	Shooting Star	DOUM2	DOEUMB103A	N/A	N/A	99	97.60	0.51
Echinacea purpurea (L.) Moench	Asteraceae	Purple coneflower	Ernst Seed	ECPU	PCF311210	78	15	93	90.53	3.87
Echinacea purpurea (L.) Moench	Asteraceae	Purple coneflower	Shooting Star	ECPU	•ECHPUR503A	98	0	98	95.69	3.75
Echinacea purpurea (L.) Moench	Asteraceae	Purple coneflower	Ernst Seed	ECPU	PCF221547	61	30	91	88.52	3.87
Echinacea purpurea (L.) Moench	Asteraceae	Purple coneflower	Shooting Star	ECPU	ECHPUR504A	92	0	92	87.42	3.67
Eryngium yuccifolium Michx.	Apiaceae	Rattlesnake master	Ernst Seed	ERYU	ERYYUC01-190H	95	0	95	92.60	3.79
Eutrochium maculatum (L.) E.E. Lamont	Asteraceae	Spotted Joe-pye weed	Ernst Seed	EUMA9	EUPMAC01-21PA	19	52	71	56.75	0.27
Eutrochium purpureum (L.) E.E. Lamont	Asteraceae	Sweetscented Joe-pye weed	Ernst Seed	EUPU21	EUPPUR462A	35	60	95	89.91	0.45
Eutrochium purpureum (L.) E.E. Lamont	Asteraceae	Sweetscented Joe-pye weed	Shooting Star	EUPU21	EUTPUR463C	51	45	96	60.77	0.44
Gaillardia pulchella Foug.	Asteraceae	Indian blanket	Shooting Star	GAPU	KB011723-14	68	22	90	88.35	2.23
Helianthus petiolaris Nutt.	Asteraceae	Prairie sunflower	Shooting Star	HEPE	QQ034123-31	19	71	90	88.84	4.09
Heliopsis helianthoides (L.) Sweet	Asteraceae	Oxeye sunflower	Shooting Star	HEHE5	HELHEL463A	54	42	96	95.37	3.63
Liatris ligulistylis (A.Nelson) K.Schum.	Asteraceae	Meadow blazing star	Shooting Star	LILI	LIALIG032A	88	10	98	78.29	1.33
Monarda fistulosa L.	Lamiaceaee	Wild bergamot	Ernst Seed	MOFI	MONFIS03-19-2	57	22	79	78.58	0.49
Monarda fistulosa L.	Lamiaceaee	Wild bergamot	Shooting Star	MOFI	•MONFIS463A	99	0	99	98.24	0.42
Monarda punctata L.	Lamiaceaee	Spotted beebalm	Shooting Star	MOPU	MONPUN253B	88	2	90	89.13	0.31
Oligoneuron rigidum (L.) Small	Asteraceae	Stiff goldenrod	Shooting Star	OLRIR	SOLRIG463B	N/A	N/A	99	98.70	0.72
Rudbeckia fulgida Aiton	Asteraceae	Orange coneflower	Ernst Seed	RUFUF	RUDFUL01-22VA	1	96	97	95.00	0.90
Rudbeckia hirta L.	Asteraceae	Black-eyed Susan	Ernst Seed	RUHI2	RUDHIR05-20VT	74	21	95	94.76	0.29
Rudbeckia hirta L.	Asteraceae	Black-eyed Susan	Shooting Star	RUHI2	•RUDHIR463B	99	0	99	98.93	0.29
Solidago nemoralis Aiton	Asteraceae	Gray goldenrod	Ernst Seed	SONE	SOLNEM01-20PA	56	32	88	84.66	0.12
Solidago rugosa Mill.	Asteraceae	Wrinkleleaf goldenrod	Ernst Seed	SORU2	SOLRUG01-20PA	N/A	N/A	41	30.90	0.08
Symphyotrichum novae-angliae (L.) G.L.Nesom	Asteraceae	New England aster	Ernst Seed	SYNO2	ASTNOV01-22PA	63	21	84	67.15	0.27
Symphyotrichum pilosum (Willd.) G.L.Nesom	Asteraceae	Hairy white oldfield aster	Ernst Seed	SYPI2	ASTPIL01-20EK2	54	8	62	61.88	0.34
Symphyotrichum pilosum (Willd.) G.L.Nesom	Asteraceae	Hairy white oldfield aster	Shooting Star	SYPI2	SYMPIL463A	77	13	90	89.98	0.13
Verbena stricta Vent.	Verbenaceae	Hoary vervain	Ernst Seed	VEST	QQ4722-60	31	34	95	94.33	0.98
Verbesina alternifolia (L.) Britton ex Kearney	Asteraceae	Wingstem	Shooting Star	VEAL	ACTALT403A	21	78	99	93.37	3.18
Veronicastrum virginicum (L.) Farw.	Plantaginaceae	Culver’s root	Ernst Seed	VEVI4	VERVIR02-21PA	13	71	84	79.56	0.05
Zizia aurea (L.) W.D.J. Koch	Apiaceae	Golden alexanders	Shooting Star	ZIAU	ZIZIAUR463A	2	96	98	97.29	2.53
					Mean	54	37	91	86.73	2.47

). Seeds of all lots were stored at 4 °C and 30% relative humidity until used for experiments.

2.2. Effect of GA₃ Application and Two Temperature Regimes on Germination and Dormancy of 42 Seed Lots

The effect of GA₃ on germination was conducted on all 42 seed lots to determine their baseline germination response under two temperature regimes (15/25 °C and 20/30 °C). This initial test stage allowed identification of species showing improved germination with GA₃ priming. The two temperature regimes (15/25 °C and 20/30 °C) were selected, and the 15/25 °C was adopted to simulate soil temperatures in spring sowing in the temperate region of Northeastern United States [29], while the alternating 20/30 °C is commonly used in seed testing [30]. Germination chambers (Percival Scientific Inc., model I-36LL, Perry, IA, USA) were set to 20/30 °C (16 h cool/dark, 8 h warm/light), and 15/25 °C (10 h cool/dark, 14 h warm/light), light provided at 3600 lux. Tests were conducted for 14 days and counts were taken daily. The 20/30 °C photoperiod and duration followed International Rules for Seed Testing (ISTA) [30]; however, test duration was limited to 14 days as our criterion for germination was radicle emergence of 2 mm rather than normal seedlings, as required in official germination tests.

The concentration of GA₃ was adopted from the Rules of Seed Testing, Association of Official Seed Analyst (AOSA) [31], and is typically 200–500 ppm (0.5–1.3 mM). Seed vigor and seedling performance are maximized at these concentrations without risk of abnormal seedling growth that may occur at higher doses [31]. GA_3, CAS # 77-06-05 was purchased from Gold Biotechnology Inc., St. Louis, MO, USA. This GA₃ formulation was termed Quick-Dissolve^TM and mixed readily in deionized water. A 1.3 mM (500 ppm) solution was prepared, and the pH of the gibberellic acid solution was adjusted to pH 6 using 50 mM KOH (Mallinckrodt Inc., Paris, KY, USA). The pH adjustment of the GA₃ solution to 6.0 using 50 mM KOH ensured chemical stability and treatment consistency, thereby facilitating a standardized assessment of GA₃ efficacy across different temperature regimes. To apply GA₃, seed samples from each lot were placed on blue blotters (Anchor Paper Co., St. Paul, MN, USA) saturated with solution, placed inside a germination chamber (Percival Scientific Inc., model I-30BL, Perry, IA, USA) set at 15 °C constant and with light at 4500 lux for 24 h, then thoroughly rinsed with deionized water, and dried overnight in ambient environment. For each lot, a minimum of two replicates of 25 seeds were planted with nontreated seeds and GA₃ primed seeds.

The effect of GA₃ was analyzed for paired comparisons at 4, 7, and 14 days for germination studies at 20/30 °C and 15/25 °C for the 42 lots. Using daily counts, the T₅₀ (time required for 50% of the seed lots to germinate) was determined using the following formula [32].

$${\mathrm{T}}_{50}=t_i+{\displaystyle \frac{\left(\frac{N}{2}-n_i\right)\left(t_j-t_i\right)}{(n_j-n_i)}}$$

T₅₀: Time required for 50% germination (days);

t_i: Observation Day before 50% germination;

N: Total number of germinated seeds;

n_i and n_j: Total number of germinated seeds at times t_i and t_j, respectively.

T₅₀ values were only calculated for non-GA₃ treated seed lots with positive and increasing counts at least on days 7 and 14. T₅₀ values were shown as N/A (nonapplicable) for both −GA₃ and +GA₃ comparison for those seed lots not satisfying the positive and increasing count criteria.

2.3. Selection Criteria for 6 Pollinator-Friendly Plant Species for Remaining Studies and Seed Priming Protocol

Six species were selected from the twenty-eight species in Section 2.1 for the remaining experiments based on NRCS/USDA E420B monarch habitat restoration guidelines. Three Asclepias species, A. incarnata, A. syriaca, and A. tuberosa, were included to satisfy program recommendations regarding the required density of milkweed stems per land area (acre), while also providing extended nectar availability and reducing pest pressure associated with reliance on a single species. The remaining three species, Echinacea purpurea, Monarda fistulosa, and Rudbeckia hirta, were chosen to ensure representation of at least two species per bloom period, as required for sustaining pollinator foraging continuity.

The application of compounds to seeds was performed by seed priming, a hydration-based seed soaking treatment with combinations of GA₃, KIN, H₂O₂, and non-ionic surfactants. These treatments involved soaking seeds in solutions for 24 h and seeds subsequently re-dried overnight prior to germination testing. Seeds were primed in a 20:1 priming solution to seeds under temperature and light conditions described in Section 2.3. Seed priming was conducted using a 20:1 solution-to-seed ratio, which ensured that seeds were fully submerged during treatment. Under these submerged conditions, oxygen availability becomes rapidly limiting because diffusion of O₂ in water is substantially slower than in air. One rationale for including 0.3% H₂O₂ in the priming solution was to supply molecular oxygen generated through enzymatic decomposition of hydrogen peroxide by catalase present in seeds during imbibition. This mechanism was documented as an important biochemical source of oxygen for metabolically reactivating seeds under hypoxic hydration conditions [33,34]. It is important to note for all priming treatments that no osmotic agent was added, so the water potential of the solutions was not modified. Therefore, the procedure represents a hydration-based priming rather than classical osmotic priming [35,36,37]. For clarity and consistency with seed technology terminology, the term “priming” is used throughout the manuscript instead of soak or pretreatment.

2.4. The Effect of GA₃ and KIN Priming Treatments with H₂O₂ on Breaking Dormancy

The effects of GA₃ and KIN applied singly and in combination with H₂O₂ were evaluated on five pollinator-friendly species: A. incarnata, A. tuberosa, E. purpurea, M. fistulosa, and R. hirta. Selected seed lots of each species are described in Section 3.3. The concentration of GA₃ and KIN was 1 mM and 0.05 mM, respectively. The GA₃ material and method described in 2.2. KIN, CAS # 525-70-1, was obtained from Gold Biotechnology Inc., St. Louis, MO, USA. KIN was dissolved in 50 mM KOH to prepare an aqueous solution, and this same solution was adjusted to pH 6.0. Similarly, the pH of the GA₃ solution was adjusted to 6.0 using 50 mM KOH to ensure chemical consistency across treatments. Each solution contained 0.3% hydrogen peroxide (H₂O₂). A water priming check and non-priming control was included. Treated seeds were placed on moistened 10 × 10 cm blue blotter paper and subjected to germination tests at alternating temperatures of 20/30 °C as described in Section 2.2. Each treatment had four replicates, with 25 seeds per replicate. Daily counts were recorded, and results were reported for days 4, 7, and 14. At the end of day 14, moldy seeds in each seed lot were also counted. All results were reported as percentages. The study was conducted with a randomized complete block design. Percent values were subjected to arc-sine transformation prior to statistical analysis described in Section 2.7.

2.5. Effect of Non-Ionic Surfactants Priming Applied with Two GA₃ Concentrations on Breaking Dormancy of Asclepias syriaca

Treatments were applied to Asclepias syriaca lot ASCSYR602A (described in Section 3.3) seeds to determine the interaction of gibberellic acid concentration with non-ionic surfactants and to evaluate their potential effect on seed germination/dormancy. The concentrations of components in the priming solutions: 0.3 mM and 1 mM GA₃, and 0.1% Tween 20, Tween 80, and K-wet 20, and 0.01% for Silwet 408. Tween 20 and 80 were purchased from Sigma-Aldrich, St. Louis, MO, USA, K-wet 20 and Silwet 408 were donated by Kannar Agriscience, Lawrenceville, GA, USA, and Momentive Performance Materials Inc., Niskayuna, NY, USA, respectively. Each solution contained 0.3% hydrogen peroxide (H₂O₂). A water priming check and non-priming control was included. To each solution, 50 mM potassium hydroxide (KOH) was used to adjust the pH of all solutions to 6. Seed priming protocol was described in Section 2.3.

Germination tests were conducted using four replicates of 25 seeds. The seeds were sown on 10 × 10 cm blue blotter paper moistened with each solution, placed in a germination chamber set at 20/30 °C with 3600 lux lighting and an 8 h photoperiod. Counts were taken daily, and blotters were re-moistened with distilled water as needed. Results were evaluated on days 4, 7, and 14, and the effect of non-ionic surfactants on preventing mold formation on seeds was also examined at the end of the experiment. Data obtained were adjusted using arc-sine transformation before statistical analysis described in Section 2.7.

2.6. The Effect of Water Stress on Germination of 6 Pollinator-Friendly Plant Seed Species

This study investigated the effects of water stress on seed germination of the 6 seed species described in Section 3.3 and all seed lots were primed with 1.0 mM GA₃ in 0.3% H₂O₂, as described in the same section. To achieve a known and constant soil media moisture content, a particulate sized, proprietary montmorillonite clay was donated by Oil-Dri Corporation of America, Chicago, IL, USA, and used as the germination media in enclosed plastic containers with lids (2.5 × 15 × 24 cm). The media moisture contents were adjusted to 32%, 35%, 42%, 90%, and 92% with resulting soil water potential of −1.08, −0.75, −0.13, 0, and 0 MPa, respectively, as measured by a WP4 Dewpoint, PotentiaMeter (MeterGroup, Pullman, WA, USA). The germination containers with media and seeds were maintained at alternating 20/30 °C with 4 replicates of 25 seeds for each species, with germination counts recorded daily for 14 days (Appendix D Figure A2). From this data set, statistical comparisons were conducted with data at 32, 42, and 92% media moisture water contents on the average of three Asclepias species, E. purpurea, M. fistulosa, and R. hirta. Data was analyzed as described in Section 2.7. This experimental design allowed for the assessment of a controlled water potential on percent germination across multiple pollinator-friendly plant species, providing valuable data for conservation.

2.7. Statistical Analysis

Germination percentage data from all experiments, including GA₃ priming tests on 42 commercial seed lots, GA₃/KIN/H₂O₂ priming, surfactant treatments, and water-stress assays were analyzed using appropriate statistical tests. For the 42-lot GA₃ comparison, paired t-tests were used to evaluate differences between GA₃-treated and nontreated samples. All other experiments were analyzed using one-way ANOVA. Mean comparisons were conducted using Tukey’s HSD or Duncan’s multiple range test, depending on experimental design. Pearson correlation coefficients among germination percentages, T₅₀, labeled germination, and laboratory germination were calculated using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Statistical significance was evaluated at p < 0.05.

3. Results

3.1. Selection of Pollinator-Friendly Plant Species and Acquisition of Seed Lots

Commercial seed lots were sought from seed companies with complete labeled germination test results reporting both % germination and % dormancy, while single seed lots of three species only reported percent viable seeds (Appendix A Table A1). Of the 39 lots that reported % germination and % dormancy, only 24 of the seed lots (62%) had >50% germination, while 98% had >50% viability. Among the eight seed lots for species with stated minimum recommended germination: Echinacea purpurea, Rudbeckia hirta, Helianthus spp., and Gaillardia pulchella [15], only the Helianthus petiolaris seed lot had <60% germination (Appendix A Table A1). The percent PLS (Pure Live Seed) of these 39 lots was generally acceptable and ranged from 98.9 to 56.7. The TSW (thousand seed weight) ranged from 5.49 to 0.05, over a 100-fold difference in seed weight.

3.2. Effect of GA₃ Application and Two Temperature Regimes on Germination and Dormancy of 42 Seed Lots

A wide range of germination values was recorded from the two temperature regimes at 20/30 °C with and without GA₃, with data reported at 4, 7, and 14 days from the 42 lots (Appendix B 

Table A2. Forty-two seed lots described in Table A1. Laboratory germination test results conducted at alternating 20/30 °C with 8 h photoperiod or 15/25 °C with 14 h photoperiod. Samples of each lot were treated with 1.3 mM (500 ppm) GA₃ solution for 24 h at 15 °C with light, followed by drying overnight before the germination test. Germination data shown for 4, 7, and 14 days, and T₅₀ (days) calculated from daily counts. T₅₀ values were only calculated for seed lots with positive and increasing counts on days 7 and 14. T₅₀ values were shown as N/A (nonapplicable) for both −GA₃ and +GA₃ comparison for those seed lots not satisfying the positive and increasing count criteria. Those GA₃ treatments with * had significantly higher percentage germination than non-GA₃ treatment of each comparison. The six selected seed lots (•) were designated for further investigation in subsequent experiments. N/A means Not Available.

		20/30 C								15/25 C
		4 d		7 d		14 d		T50		4 d		7 d		14 d		T50
Latin name	Seed Lot	−GA3	+GA3	−GA3	+GA3	−GA3	+GA3	−GA3	+GA3	−GA3	+GA3	−GA3	+GA3	−GA3	+GA3	−GA3	+GA3
Asclepias incarnata	•JG040418	10	42*	66	88*	78	90*	5.0	4.1	14	26*	58	56	64	72*	5.0	4.6
Asclepias incarnata	ASCINC463B	6	36*	26	66*	26	68*	4.9	3.9	0	0	2	8*	4	12*	7.0	6.5
Asclepias incarnata	ASCINC263B	20	34*	54	54	60	54	4.4	3.4	0	0	20	32*	24	38*	5.0	5.9
Asclepias syriaca	ASCSYR553A	2	32*	10	42*	18	48*	6.8	3.3	2	10*	26	54*	38	74*	5.7	5.8
Asclepias syriaca	•ASCSYR602A	18	54*	42	66*	46	72*	4.6	2.8	28	56*	78	86*	84	90*	4.6	3.8
Asclepias syriaca	ASCSYR01-22PA	12	34*	20	66*	26	74*	4.5	4.2	6	30*	24	56*	34	78*	6.1	4.7
Asclepias syriaca	ASCSYR01-23PA	0	6	10	12	12	14	6.3	5.5	0	0	0	10*	6	30*	N/A	N/A
Asclepias tuberosa	•ASCTUB670B	84	90*	92	90	92	94	2.5	1.9	78	90*	80	94*	84	98*	2.6	2.4
Asclepias tuberosa	ASCTUB463A	22	34*	42	40	44	46	4.0	2.5	14	36*	32	46*	32	50*	5.2	2.9
Asclepias tuberosa	ASCTUB01-21PA	2	2	2	8*	2	8*	N/A	N/A	0	0	0	2	0	12	N/A	N/A
Bidens aristosa	BIDARI-2223A	0	2	6	10	16	30*	8.0	7.8	0	0	0	0	10	14	N/A	N/A
Chamaecrista fasciculata	CHAFAS653A	18	30*	22	38*	26	38*	1.9	2.0	26	14	30	20	32	24	3.4	2.0
Dalea candida	DALCAN053B	96	94	98	98	100	98	1.9	1.6	94	84	94	88	96	90	1.7	1.9
Doellingeria umbellata	DOEUMB103A	0	6	4	12*	18	20	7.8	6.0	0	2	2	10*	10	16*	7.8	6.5
Echinacea purpurea	PCF311210	52	80*	80	94*	82	98*	3.7	2.8	30	72*	70	88*	86	90	4.6	3.2
Echinacea purpurea	•ECHPUR503A	56	78*	92	94	100	96	3.9	2.7	48	74*	92	96	100	100	4.1	3.4
Echinacea purpurea	PCF221547	86	92*	86	94*	88	94*	2.3	1.7	74	86*	96	96	96	98	2.9	2.7
Echinacea purpurea	ECHPUR504A	64	76*	92	100*	98	100	3.5	2.9	36	58*	78	94*	90	96*	4.8	3.4
Eryngium yuccafolium	ERYYUC01-190H	0	0	0	0	0	8	N/A	N/A	0	0	0	0	0	0	N/A	N/A
Eutrochium maculatum	EUPMAC01-21PA	0	4	4	12	10	22*	7.5	6.8	0	0	6	6	14	28*	9.3	8.0
Eutrochium purpureum	EUPPUR462A	12	20*	20	36*	32	48*	5.0	4.5	0	8	8	48*	16	48*	8.0	4.7
Eutrochium purpureum	EUTPUR463C	2	16*	20	38*	30	50*	6.0	4.9	0	8	8	26*	16	30*	7.0	4.4
Gaillardia pulchella	KB011723-14	84	76	90	76*	90	80	3.3	2.8	46	64*	82	80	88	86	3.9	3.6
Helianthus petiolaris	QQ034123-31	10	30*	12	38*	12	38*	3.0	2.6	14	20*	18	20	18	28*	3.5	3.3
Heliopsis helianthoides	HELHEL463A	36	48*	48	66*	62	72	3.7	3.3	22	14	52	46*	68	62	5.0	3.8
Liatris ligulistylis	LIALIG032A	28	54*	52	62*	58	62	4.3	2.6	26	52*	46	74*	60	82*	4.5	3.1
Monarda fistulosa	MONFIS03-19-2	56	54	66	60	74	64*	3.3	3.0	44	56*	60	74*	70	82*	3.6	3.1
Monarda fistulosa	•MONFIS463A	54	68*	56	80*	56	80*	2.6	2.6	46	72*	64	82*	64	86*	3.4	3.0
Monarda punctata	MONPUN253B	92	94	92	96	92	98*	2.6	2.2	86	100*	88	100*	88	100*	2.5	2.3
Oligoneuron rigidum	SOLRIG463B	18	4	40	22	42	40	5.5	6.8	8	6	26	14	46	40	6.0	8.4
Rudbeckia fulgida	RUDFUL01-22VA	0	0	0	0	0	0	N/A	N/A	0	2	2	2	2	2	N/A	N/A
Rudbeckia hirta	RUDHIR05-20VT	62	74*	74	80	86	88	3.3	2.7	78	80	86	90	86	94*	2.9	2.8
Rudbeckia hirta	•RUDHIR463B	92	96	96	96	96	98	2.4	1.6	88	94*	88	94*	88	94*	2.5	1.9
Solidago nemoralis	SOLNEM01-20PA	38	52*	64	88*	80	90*	4.1	3.8	26	42*	80	66*	82	72*	4.5	3.8
Solidago rugosa	SOLRUG01-20PA	16	24*	56	44	60	64	5.2	4.7	20	12	52	44	60	72*	4.6	5.3
Symphyotrichum novae-angliae	ASTNOV01-22PA	32	38	50	56	60	62	3.9	3.7	40	40	50	64*	56	70*	3.5	3.7
Symphyotrichum pilosum	ASTPIL01-20EK2	2	18*	38	64*	64	70*	5.9	4.7	0	6*	44	44	58	66*	5.9	5.7
Symphyotrichum pilosum	SYMPIL463A	0	6	4	18*	22	26	9.2	5.5	0	2	10	10	26	18	7.3	6.8
Verbena stricta	QQ4722-60	4	0	16	18	24	40*	7.0	7.5	0	0	4	8	24	28	10.0	8.5
Verbesina alternifolia	ACTALT403A	0	14*	6	50*	36	86*	9.5	6.4	0	0	2	42*	22	66*	10.2	6.1
Veronicastrum virginicum	VERVIR02-21PA	0	2	14	28*	26	32*	5.9	5.0	0	0	4	20*	18	34*	6.0	5.8
Zizia aurea	ZIZIAUR463A	0	0	0	2	6	14*	N/A	N/A	0	0	0	0	0	6	N/A	N/A

). Focusing on seed lot differences within a species, and to illustrate seed lot differences, three lots of A. incarnata were tested and the 14-day germination at 20/30 °C for JG040418 with GA₃ was 90%. In contrast, for the same comparison of ASINC463B and ASINC263B, the germination was 68%, and 54%_, respectively. Results of these three A. incarnata lots illustrated differences among seed lots with respect to germination and/or dormancy. A. incarnata was not unique and large differences among seed lots in % germination with GA₃ tested at 20/30 °C was observed from A. syriaca, A. tuberosa, M. fistulosa, and R. hirta.

The germination time course was illustrated for six selected pollinator-friendly plant species over a 14-day period under two temperature regimes (20/30 °C and 15/25 °C), with and without GA₃ pretreatment (Appendix C Figure A1). A. incarnata started germination four days after sowing when treated with GA₃ at 20/30 °C and reached 90% germination by day 14, while the nontreated seeds germinated more slowly and only reached 78% by day 14. Similar trends were measured in A. tuberosa, M. fistulosa, E. purpurea, and R. hirta, and in each case, GA₃ enhanced germination percent and rate. Even under the cooler 15/25 °C temperature, GA₃ still had a positive effect, although the germination was generally slower compared to 20/30 °C. These results showed that GA₃ helped break dormancy and speed up germination across different species.

Utilizing the cumulative germination data from 42 seed lots revealed that GA₃ significantly increased the rates and totals of germination. For most species, seeds treated with GA₃ had the highest germination rate at the 20/30 °C temperature regime, resulting in 59% on the 14th day, versus the control at 49% (Figure 2a), while the cooler 15/25 °C temperatures yielded germination of 57% for seeds treated with GA₃ and 47% for those without GA₃ (Figure 2a). Evidence from the T₅₀ values supports that GA₃ pretreatment accelerated germination rate (Figure 2b). Under the 20/30 °C temperature regime, the T₅₀ was reduced from 4.7 to 3.9 days with GA₃-treated seeds. Similarly, under the 15/25 °C regime, GA₃ reduced the T₅₀ from 5.1 days to 4.4 days. These findings clearly indicate that GA₃ pretreatment increases germination percentage and enables seeds to germinate in less time.

The associations between seed lot parameters at selected test conditions were determined by performing a correlation analysis among the obtained values.

Table 1. Correlation coefficients of germination percentage at 4 and 14 days with germination rate (T₅₀), and Label and Lab germination at two temperatures (20/30 °C and 15/25 °C), without and with GA₃.

Correlation Coefficients	20/30 °C −GA3	20/30 °C +GA3	15/25 °C −GA3	15/25 °C +GA3
4-day vs. 14-day	0.86 ***	0.87 ***	0.83 ***	0.85 ***
4-day vs. T50	−0.52 ***	−0.68 ***	−0.53 ***	−0.63 ***
14-day vs. T50	−0.35 *	−0.54 ***	−0.38 *	−0.50 ***
Label germ vs. Lab germ (14-day)	0.61 ***	0.57 ***	0.58 ***	0.44 **

Significance: * 0.05, ** 0.01, *** 0.001.

shows the correlation coefficients of 4 versus 14-day germination varied from 0.83 to 0.87 with significance at a probability level of p < 0.001. This shows a significant positive relationship between the germination percentages from the early count at 4-day with the final count at 14-day. The negative coefficients of correlation at 4-day germination percentage versus T₅₀ ranged from −0.52 to −0.68 all at the same level of probability, p < 0.001, verified that higher percentage germination was associated with faster germination rate. Similar trends were measured with 14-day germination percentages and T₅₀. The positive values of the correlation coefficient between labeled germination and laboratory germination ranged from 0.44 to 0.61 at p < 0.01 or p < 0.001, validating that the labeled germination provides an accurate ranking of laboratory germination test results.

3.3. Seed Lot Selection of the Six Species

Among the commercially available seed lots for each species, a single lot was selected to represent each of the six pollinator-friendly plant species. Selection was made from four lots available for A. syriaca and E. purpurea, three lots for A. incarnata and A. tuberosa, and two lots for M. fistulosa and R. hirta (Appendix A Table A1). Each seed lot was chosen based on two criteria: high germination percentage following GA₃ pretreatment at 20/30 °C (Appendix B Table A2), and sufficient seed quantity for subsequent laboratory studies. The selected seed lot for each species was Asclepias incarnata (lot JG040418), Asclepias syriaca (lot ASCSYR602A), Asclepias tuberosa (lot ASCTUB670B), Echinacea purpurea (lot ECHPUR503A), Monarda fistulosa (lot MONFIS463A), and Rudbeckia hirta (lot RUDHIR463B). The selected lots are denoted with a ● symbol in Appendix A Table A1 and Appendix B Table A2 to clearly distinguish them from the remaining commercial lots.

3.4. Effect of GA₃ and KIN Priming on Breaking Dormancy of Five Pollinator Species

The effects of GA₃ and KIN applications on germination and mold formation during the study were evaluated on days 4, 7, and 14 for the five selected species (A. incarnata, A. tuberosa, E. purpurea, R. hirta, and M. fistulosa), with results presented in

Table 2. (a) Effects of GA₃ and KIN treatments on germination percentage and mold growth at 4, 7, and 14 days for five pollinator species. (b) Species (5 levels) and seed treatment (6 levels) main effects on 4, 7, and 14 DAP germination %.

(a)
Treatments	4			7	14		Mold
	Asclepias incarnata (Swamp Milkweed)
Control	42 ± 3.5 b			59 ± 3.4 c	73 ± 4.7 c		12 ± 2.8 c
H2O	58 ± 12.1 ab			74 ± 5.3 b	90 ± 1.2 b		4 ± 2.8 ab
H2O2	61 ± 3.0 ab			77 ± 1.0 ab	88 ± 3.7 b		1 ± 1.0 a
GA3 + H2O2	67 ± 7.0 a			85 ± 3.4 a	96 ± 1.6 a		9 ± 3.4 bc
KIN + H2O2	66 ± 2.6 a			80 ± 1.6 ab	91 ± 2.5 ab		10 ± 3.5 bc
GA3 + KIN + H2O2	69 ± 2.5 a			81 ± 3.0 ab	88 ± 1.6 b		7 ± 1.9 bc
	Asclepias tuberosa (Butterfly Milkweed)
Control	53 ± 3.4 b			79 ± 2.5 a	80 ± 1.6 b		14 ± 3.8 bc
H2O	78 ± 3.5 a			80 ± 4.3 a	81 ± 3.4 b		6 ± 2.6 a
H2O2	78 ± 3.5 a			80 ± 4.3 a	87 ± 1.9 ab		9 ± 1.0 abc
GA3 + H2O2	86 ± 3.5 a			89 ± 3.0 a	93 ± 1.9 a		12 ± 1.6 bc
KIN + H2O2	83 ± 5.3 a			84 ± 5.6 a	89 ± 1.9 ab		6 ± 1.2 a
GA3 + KIN + H2O2	86 ± 5.0 a			88 ± 4.3 a	88 ± 4.3 ab		15 ± 1.0 c
	Echinacea purpurea (Purple Coneflower)
Control	61 ± 1.9 a			84 ± 1.6 b	88 ± 1.6 c		0
H2O	75 ± 4.4 a			86 ± 4.8 ab	89 ± 4.4 bc		0
H2O2	67 ± 6.4 a			94 ± 1.2 ab	96 ± 2.3 ab		0
GA3 + H2O2	78 ± 5.3 a			95 ± 1.9 a	98 ± 2.0 a		0
KIN + H2O2	78 ± 5.3 a			92 ± 4.3 ab	99 ± 1.0 a		0
GA3 + KIN + H2O2	77 ± 8.0 a			95 ± 2.5 a	95 ± 2.5 abc		0
	Rudbeckia hirta (Black-eyed Susan)
Control	86 ± 2.0 ab			91 ± 1.9 a	92 ± 1.6 b		7 ± 3.0 a
H2O	90 ± 2.0 ab			93 ± 1.0 a	93 ± 1.0 ab		6 ± 1.2 a
H2O2	90 ± 2.6 ab			93 ± 3.4 a	95 ± 1.9 ab		10 ± 1.2 a
GA3 + H2O2	94 ± 2.6 a			95 ± 1.9 a	98 ± 1.2 a		7 ± 1.9 a
KIN + H2O2	94 ± 2.6 a			94 ± 3.8 a	96 ± 2.3 ab		5 ± 3.0 a
GA3 + KIN + H2O2	82 ± 3.8 b			86 ± 2.6 a	94 ± 1.2 ab		10 ± 1.2 a
	Monarda fistulosa (Wild Bergamot)
Control	68 ± 2.8 a			69 ± 1.9 b	70 ± 1.2 c		12 ± 1.6 a
H2O	69 ± 4.4 a			75 ± 1.9 ab	78 ± 2.0 bc		10 ± 1.2 a
H2O2	80 ± 5.7 a			82 ± 4.8 a	84 ± 4.3 ab		7 ± 3.0 a
GA3 + H2O2	69 ± 5.3 a			71 ± 5.0 ab	88 ± 2.8 a		13 ± 3.8 a
KIN + H2O2	70 ± 5.3 a			76 ± 4.9 ab	80 ± 4.6 abc		12 ± 1.6 a
GA3 + KIN + H2O2	78 ± 3.8 a			83 ± 1.0 a	85 ± 1.9 ab		12 ± 1.6 a
(b)
Species	% Germination			Seed treatment	% Germination
	4	7	14		4	7	14
Asclepias incarnata	61 c	76 c	88 b	Control	62 b	76 b	81 c
Asclepias tuberosa	77 b	83 b	86 bc	H2O	74 ab	81 ab	86 bc
Echinacea purpurea	73 b	91 a	94 a	H2O2	75 a	85 ab	90 ab
Rudbeckia hirta	89 a	92 a	95 a	GA3 + H2O2	79 a	87 a	95 a
Monarda fistulosa	72 b	76 c	81 c	KIN + H2O2	78 a	85 ab	91 ab
				GA3 + KIN + H2O2	78 a	87 a	90 ab
p-value	<0.01	<0.01	<0.01		<0.01	<0.01	<0.01

Different lowercase letters within columns indicate significant differences among treatments. Moldy seeds are interpreted as non-viable at the time of imbibition or as seeds that lost viability during priming.

a. In A. incarnata, A. tuberosa, E. purpurea, and M. fistulosa, the combination of GA₃ and H₂O₂ resulted in a significantly greater percent germination compared to the non-primed and primed in water at day 14. For the same comparison for R. hirta, GA₃ + H₂O₂ had significantly higher germination than the non-primed control. Mold was present in germination tests in all species, except that mold was not observed in E. purpurea in any treatment. Table 2b presents the main effects of species and treatment type on germination percentages at days 4, 7, and 14. Across seed treatments, R. hirta and E. purpurea both had high percentage germination (>90%) at days 7 and 14. In contrast, the lowest percentage germination was measured for A. incarnata at day 4 and M. fistulosa at day 14. Among the treatment group means, GA₃ + H₂O₂ consistently resulted in the highest germination percentages (79% on day 4, 87% on day 7, and 95% on day 14), significantly outperforming the control and primed in water groups.

3.5. Effect of Non-Ionic Surfactants Priming Applied with Two GA₃ Concentrations on Breaking Dormancy of Asclepias syriaca

The effects of two GA₃ concentrations with H₂O₂ in combination non-ionic surfactants on the germination percentage and mold formation of A. syriaca seeds were evaluated on days 4, 7, and 14 (

Table 3. (a) Effects of GA₃ treatments with non-ionic surfactants on percent germination at 4, 7, and 14 days (d) and mold growth for Asclepias syriaca (lot ASCSYR602A). (b) GA₃ main effect on percent germination at 4, 7, and 14 days (d).

(a)
PGR Treatment	4 d	7 d	14 d	Mold
Control (nonsoaked)	14 ± 1.2 e	40 ± 3.6 e	42 ± 2.6 e	5 ± 1.9 ab
Water	46 ± 2.8 d	66 ± 4.4 d	68 ± 3.5 d	8 ± 2.6 ab
H2O2 + Water	56 ± 10.1 cd	73 ± 5.3 cd	74 ± 4.9 cd	6 ± 1.6 ab
0.3 mM GA3 + H2O2 + Tween 20	61 ± 6.4 bc	84 ± 2.8 abc	88 ± 1.6 ab	6 ± 1.16 ab
0.3 mM GA3 + H2O2 + Tween 80	65 ± 4.1 abc	84 ± 2.8 abc	87 ± 3.4 ab	3 ± 1.0 a
0.3 mM GA3 + H2O2 + Silwet 408	80 ± 1.6 a	85 ± 2.5 ab	88 ± 0.6 ab	5 ± 1.9 ab
0.3 mM GA3 + H2O2 + Kwet 20	65 ± 5.3 abc	78 ± 2.5 bc	81 ± 5.7 bc	2 ± 2.0 a
0.3 mM GA3 + H2O2 + Water	58 ± 3.4 bcd	87 ± 5.0 ab	89 ± 5.0 ab	9 ± 3.0 ab
1 mM GA3 + H2O2 + Tween 20	79 ± 4.4 a	94 ± 2.6 a	95 ± 1.9 a	5 ± 1.0 ab
1 mM GA3 + H2O2 + Tween 80	72 ± 4.0 ab	89 ± 3.4 ab	93 ± 3.0 a	14 ± 3.8 b
1 mM GA3 + H2O2 + Silwet 408	78 ± 4.7 a	86 ± 2.0 ab	87 ± 1.0 ab	10 ± 6.0 ab
1 mM GA3 + H2O2 + Kwet 20	80 ± 2.8 a	90 ± 3.8 a	92 ± 2.8 a	14 ± 5.3 b
1 mM GA3 + H2O2 + Water	73 ± 4.1 ab	91 ± 1.9 a	93 ± 1.0 a	10 ± 4.2 ab
(b)
Factor II: GA3	4 d	7 d	14 d
0.3 mM GA3	66 B	84 B	87 B
1 mM GA3	76 A	90 A	92 A

Different lowercase (Table 3a) and uppercase (Table 3b) letters within columns indicate significant differences among treatments.

a). In the nontreated control group, final germination was only 42% by day 14. Treatments combining GA₃ and H₂O₂, with or without surfactants, significantly enhanced germination compared to the control (p < 0.01). Only 0.3 mM GA₃ + H₂O₂ with Silwet 408 had greater germination than 0.3 mM GA₃ + H₂O₂ or 0.3 mM GA₃ + H₂O₂ + Tween 20, but only at 4 days. On day 14, most surfactant treatments with 1.0 mM GA₃ resulted in >90% germination.

Overall, non-ionic surfactants showed no additional benefit in germination compared to all 1.0 mM GA₃ + H₂O₂ without surfactant. All surfactants visibly reduced surface tension during seed immersion, which resulted in faster and more uniform wetting of the GA₃ priming solutions. This observation was based on the reduced formation of hydrophobic air pockets on the seed surface when surfactants were present.

The mold appearance scores indicated that certain combinations of GA₃ and surfactants slightly, though non-significantly, increased the incidence of mold (Table 3a). While the control group exhibited 5% mold, this value rose to 14% in treatments combining 1 mM GA₃ with either Tween 80 or K-wet 20, though not significantly different. Therefore, caution should be exercised regarding surfactant role in promoting microbial growth. This elevated mold may be attributed to interactions between surfactant and priming incubation, and further detailed studies are warranted to elucidate the underlying mechanisms. The main effect of GA₃ concentration, regardless of surfactant type, is presented in Table 3b. Seeds treated with 1 mM GA₃ + H₂O₂ consistently exhibited higher germination percentages at all measurement times compared to those treated with 0.3 mM GA₃. On day 4, germination was significantly higher with 1 mM GA₃ (76%) than with 0.3 mM GA₃ (66%). This difference continued over time, with 1 mM treatments reaching 90% on day 7 and 92% by day 14, while 0.3 mM treatments reached 84% and 87%, respectively.

3.6. The Effect of Water Stress on Germination of Six Pollinator-Friendly Plant Seed Species

Germination study was conducted on six pollinator plant species under five distinct media moisture contents (32%, 35%, 42%, 90%, and 92%) with resulting soil water potential of −1.08, −0.75, −0.13, 0, and 0 MPa, respectively. All seeds were primed with GA₃ + H₂O₂, as described in Section 2.3, to mitigate physiological dormancy, allowing the study to isolate the impact of soil moisture levels on germination performance. Although species differed in their quantitative responses to GA₃ concentration, a standardized dose of 1 mM GA₃ was selected for the water-stress experiment because this concentration produced consistent dormancy-breaking effect across all six species in the priming experiment (Table 2a,b). Standardizing GA₃ ensured that differences in germination under water stress reflected moisture-response behavior rather than species-specific hormonal sensitivity. The impact of water stress on germination was better illustrated with a log transformation of time (x-axis) in this time-course germination investigation (Appendix D Figure A2). The highest and fastest germination was recorded at 42% media moisture content, especially for M. fistulosa and R. hirta. The drought stress imposed with 32% media moisture resulted in the lowest germination compared to other moisture contents for each species. Species differences in response to water stress were further validated by the germination data summarized and analyzed in Figure 3. Final germination percentages on day 14 were compared under three distinct moisture regimes: drought (32%), experimentally optimal (42%), and supra-optimal (over-saturation) (92%). Statistical comparisons revealed significant differences (p < 0.05) among treatments within each species, as denoted by different letters atop the standard error bars. The three Asclepias species (A. incarnata, A. syriaca, and A. tuberosa) exhibited similar trends at each moisture level, and data was pooled for analysis. Across all six pollinator-friendly native species, seeds exposed to the optimal moisture level (42%) exhibited the highest germination performance. An acceptable level of germination was arbitrarily set at >30%, but germination was < 30% under drought conditions for E. purpurea, R. hirta, and M. fistulosa. M. fistulosa was shown to be the most sensitive to over-saturation conditions compared to other pollinator-friendly species. These findings reinforce the critical role of adequate moisture availability in supporting robust germination across diverse pollinator-friendly native species and underscore the species-specific sensitivity to soil water potential during early germination stages.

4. Discussion

Seed lot variability emerged as a central constraint for restoration-scale deployment of native pollinator-friendly species. The prerequisite for a successful field restoration of pollinator-friendly plant species is to start with high-quality seeds. Initial seed quality of the 42 seed lots revealed that only 62% had greater than 50% germination based on the commercial seed label (Appendix A Table A1). However, the percent viable seeds for the 42 seed lots showed that 98% had greater than 50 percent viability. The difference was attributed largely to the dormant fraction in each lot. Further, there were large seed lot variations in percent germination within the same species. For the selected six species, the greatest difference among seed lots in percentage points was 71 for A. incarnata, 24 for A. syriaca, 82 for A. tuberosa, 37 for E. purpurea, 42 for M. fistulosa., and 25 for R. hirta (Appendix A Table A1). There was a significant correlation with the labeled germination and 14-day germination test with (r = 0.57 ***) or without (r = 0.51 ***) GA₃ at 20/30 °C (Table 1). Therefore, seed lot selection can be made with the seed label provided by the seed vendor to rank laboratory seed lot performance. Collectively, selection of single lots of each species provided the highest seed quality for further studies.

Our results showed that GA₃-based priming significantly increased both final germination and germination speed across the 42 seed lots tested (Figure 2a,b, confirming its broad dormancy-breaking potential in pollinator-friendly species). Seed dormancy is a key barrier to the successful propagation of many pollinator-friendly native species and the strong GA₃ response observed in our study highlights its effectiveness in overcoming this constraint across diverse taxa. The consistent increase in both final germination and germination speed (lower T₅₀) aligns with the core physiological role of GA₃ in promoting embryo elongation, weakening mechanical resistance, and accelerating metabolic activation. Classical studies identified gibberellins as having a primary role dormancy release through enzyme activation and endosperm weakening [38,39]. More recently, GA₃ was shown to enhance germination in dormant pollinator-friendly or ecologically important species by stimulating reserve mobilization and early metabolic activation [40,41]. Parallel evidence reinforces these mechanisms: GA₃ increases α-amylase activity and soluble sugar mobilization [42], reduces mean germination time [19], and accelerates radicle protrusion via activation of hydrolytic enzymes [33]. GA₃ also shortens after-ripening through enhanced GA biosynthesis and ABA suppression [43], improves embryo growth and enzymatic activation [44], and interacts with light-responsive pathways to promote dormancy release [45]. Collectively, these well-documented physiological responses align directly with the strong negative correlation we observed between T₅₀ and final germination percentage, confirming GA₃’s dual impact on both germination speed and magnitude, an essential requirement for achieving uniform and predictable emergence in direct-seeded restoration systems.

Standardizing GA₃ solution pH (6.0) ensured consistency across treatments, a practice supported by previous studies demonstrating that pH influences GA₃ solubility and uptake efficiency [25]. Although GA₃ is most active in its undissociated acidic form [46,47], our study with moderate pH adjustments can still produce strong, multi-species responses suitable for restoration applications. Hydrogen peroxide (H₂O₂) is well established as a signaling molecule that accumulates during early imbibition, where appropriate concentrations contribute to dormancy release within the “oxidative window” described in previous studies [20,21,48]. In our experiments, treatments containing H₂O₂ performed strongly across several species: GA₃ + H₂O₂ resulted in the highest germination in Asclepias incarnata (96%) and Asclepias tuberosa (93%), while H₂O₂ + KIN further improved germination in Echinacea purpurea, reaching 99%. Although our experimental design does not allow mechanistic inference regarding interaction effects among GA₃, H₂O₂, and KIN, these outcomes demonstrate that H₂O₂-containing priming solutions have practical value when applied at appropriate concentrations. This pattern agrees with recent findings that H₂O₂-mediated redox adjustments can enhance germination capacity and support early stress tolerance [22], underscoring the relevance of controlled oxidative signaling in seed enhancement protocols. Although treatments containing H₂O₂ performed strongly in several species, the experimental design does not allow determination of interaction effects among GA₃, KIN, and H₂O₂, since hormone-only treatments were not included. Therefore, no mechanistic or synergistic conclusions can be drawn from these results.

The integration of non-ionic surfactants into GA₃-based seed enhancement protocols was examined using A. syriaca as a model species. Treatments combining 1.0 mM GA₃ with Tween 20, Tween 80, or K-wet 20 achieved final germination rates exceeding 90%, significantly outperforming non-primed control, and priming in water or 0.3% H₂O₂. However, germination was not improved with surfactants in combination with GA₃ + H₂O₂, compared to GA₃ + H₂O₂ (Table 3a). In contrast, 0.3 mM GA₃, GA₃ + H₂O₂ + Silwet 408 had 80% germination at 4 days, which was significantly greater than GA₃ + H₂O₂ or GA₃ + H₂O₂ + other surfactants (Table 3a). Improvements may be attributed to the surfactant facilitating greater uptake of growth regulators, especially at low concentrations by improving seed coat permeability, thereby enhancing hormonal penetration and action. This is consistent with earlier studies indicating that surfactants reduce surface tension, increasing contact and absorption efficiency [49]. Low doses of non-ionic surfactants were shown to increase germination in wheat [50,51], onion, and lettuce seeds [52]. The results suggest that pretreatment of seeds with non-ionic surfactants may have increased the absorption of GA₃, likely by enhancing water uptake and growth regulator translocation across the seed coat.

However, while surfactants enhanced germination, certain combinations also elevated mold formation, especially with Tween 80 and K-wet 20 at the higher 1.0 mM GA₃ concentration. This outcome implies that non-ionic surfactants, while biologically beneficial in promoting germination, may also modify the microenvironment around the seed, potentially fostering microbial proliferation under the priming condition. As a result, the application of surfactants at a commercial scale should consider both the physiological benefits and the potential phytopathological risks. Further research is needed to optimize concentrations and combinations that balance enhanced germination with minimal microbial risks. Because germinated and moldy seeds did not sum to 100% in several treatments (Table 2a), increased mold incidence likely reflects both shifts in the immediate microenvironment and treatment-induced mortality in a fraction of physiologically weak seeds.

Water availability is a major environmental factor influencing seed germination, particularly in direct-seeded habitat restoration projects. The time-course data (Appendix D Figure A2) and final germination comparisons (Figure 3) demonstrated that seed responses to moisture conditions are species-specific and strongly dependent on the physiological adaptability of each species. For example, the three Asclepias species exhibited broad tolerance across a wide range of soil moisture conditions in our study, suggesting an evolutionary adaptation to variable field environments. This observation aligns with ecological descriptions of these species, where A. tuberosa is known to thrive in dry, sunny habitats, and A. incarnata is typically found in moist areas such as marsh edges but demonstrates adaptability to sunnier and drier conditions if adequate moisture is available [53]. In contrast, Echinacea purpurea demonstrated significantly higher germination only under elevated moisture levels (≥42%), consistent with its ecological preference for more humid environments. This observation aligns with previous studies showing that Echinacea species germinates well with adequate water availability, which supports successful seedling establishment [26]. This niche specific germination response should be considered when selecting species and sowing times for conservation and restoration efforts. In contrast, Monarda fistulosa and Rudbeckia hirta exhibited peak germination at medium moisture conditions (42%), suggesting that they may be more suited to regions with moderate, but stable soil moisture. This pattern underscores the importance of species-specific germination ecology when designing seed enhancement protocols and restoration strategies. The differential responses to water availability not only reflect inherent ecological adaptations but also highlight the necessity of tailoring germination treatments and site preparation methods according to target species.

These findings are consistent with earlier studies that emphasize the role of water potential and water uptake kinetics in regulating dormancy release and radicle emergence [54,55]. The significant decrease in germination under drought conditions (32% moisture) across all species underscores the physiological limitations imposed by low water potential (−1.08 MPa), which likely delays metabolic reactivation and cellular expansion. This highlights the importance of identifying species-specific soil moisture thresholds when planning restoration in drought-prone regions.

From an agronomic perspective, our GA₃ priming protocol may be integrated with an emerging seed-coating technology, MSZP (Multi-seed Zea pellet) [56,57], to facilitate mechanized sowing in restoration programs. Commercial scaleup of MSZP technology, now termed MSP (Multiple Seed Pellets, https://kannargroup.com/products/msp/ (accessed on 15 June 2025), was demonstrated as an effective technology in sowing multiple seeds of milkweed and other species with conventional field planting equipment. However, the performance of such combined approaches under field conditions requires further validation beyond the scope of the present study. In addition, field trials under fluctuating soil moisture regimes will be essential to validate these laboratory-based moisture thresholds and refine recommendations for direct-seeded restoration projects.

5. Conclusions

Selection of high-quality seed lots is essential to the success of any pollinator-friendly plant species initiative. Our recommendation is to request from the seed vendor high-quality seed lots of any desired seed species, and that, when possible, the seed vendor provides complete germination test results including the % germination and % dormancy. This study demonstrated that hydration-based priming using GA₃, particularly the GA₃ + 0.3%H₂0₂ combination, provided the most consistent and effective enhancement of germination performance across multiple pollinator-friendly native plant species. GA₃ priming consistently increased both final germination and germination speed across 42 commercial seed lots, providing a practical and scalable tool for overcoming physiological dormancy. Water-stress assays revealed distinct species-specific moisture thresholds (generally declining sharply below −0.5 MPa), providing operational guidance for seedbed preparation and early-establishment planting. Based on these findings, we recommend GA₃ + H₂O₂ priming for pollinator-friendly seed species with moderate dormancy, along with sowing into soils maintained between 0 and −0.3 MPa to support rapid and uniform emergence. Together, these results provide a standardized, restoration-oriented seed enhancement framework that can be readily adopted by seed companies and conservation practitioners.