683: Overcoming Seed Dormancy in the Propagation of Endangered Abutilon menziesii (Malvaceae)
Samantha Faul, Lyon Arboretum Hawaiian Rare Plant Program Seed Conservation Lab & Kupu Conservation Leadership Development Program
Background
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Abutilon menziesii (Malvaceae), also known as ko‘oloa‘ula, is an endangered, endemic Hawaiian plant species found within dry shrublands and forests on O‘ahu, Lana‘i, Maui and Hawai‘i Island at elevations between 200 and 520 meters. A shrub that grows to a height of up to 2 meters, A. menziesii has silver-green, heart shaped leaves and attractive flowers ranging from deep maroon (East Maui and Hawai‘i Island) to light red (O‘ahu and Lana‘i) (Gustafson et al. 2014). Not much is known about traditional uses of ko‘oloa‘ula, though some authors have remarked that its small red flowers would make beautiful lei (Lilleeng-Rosenberger 2016). Threatened by agricultural and urban development and invasive plant and animal species, no more than 10 wild populations of A. menziesii remain throughout all islands (Gustafson et al. 2014).
On O‘ahu, there are only two naturally occurring wild populations of A. menziesii. As part of a Habitat Conservation Plan to mitigate the effects of a Department of Transportation construction project, the State of Hawai‘i Department of Land and Natural Resources, Division of Forestry and Wildlife, began to plant A. menziesii at five additional wild sites in 2004 in an attempt to establish 3 additional self-sustaining populations. By 2013, only one mitigation site met their long-term success criteria (State of Hawaii Department of Land and Natural Resources – Division of Forestry and Wildlife, 2013). In a 2013 draft report, species managers noted that their mitigation efforts were hampered by a lack of seed germination and survivorship. The Lyon Arboretum Seed Conservation Lab (SCL) initiated a research project on A. menziesii to better understand germination behavior. Given anecdotal evidence of low seedling recruitment in natural settings, determining how to break seed dormancy at ex situ facilities where seeds are less threatened by insect and fungal pests will give population managers greater resilience as they work to conserve this endangered native species.
Statement of Problem
Abutilon menziesii seeds are thought to exhibit physical dormancy, which can be broken by scarifying the seed’s water-impermeable seed coat (Baskin & Baskin 2014). However, several Hawaiian seed banks have observed that fresh seeds scarified immediately before sowing often rot. Thus, we hypothesize that this species may instead exhibit combinational dormancy – both physical and physiological dormancy – which may be broken by allowing seeds to sit on growth media for before scarifying.
Introduction
Background
Abutilon menziesii (Malvaceae), also known as ko‘oloa‘ula, is an endangered, endemic Hawaiian plant species found within dry shrublands and forests on O‘ahu, Lana‘i, Maui and Hawai‘i Island at elevations between 200 and 520 meters. A shrub that grows to a height of up to 2 meters, A. menziesii has silver-green, heart shaped leaves and attractive flowers ranging from deep maroon (East Maui and Hawai‘i Island) to light red (O‘ahu and Lana‘i) (Gustafson et al. 2014). Not much is known about traditional uses of ko‘oloa‘ula, though some authors have remarked that its small red flowers would make beautiful lei (Lilleeng-Rosenberger 2016). Threatened by agricultural and urban development and invasive plant and animal species, no more than 10 wild populations of A. menziesii remain throughout all islands (Gustafson et al. 2014).
On O‘ahu, there are only two naturally occurring wild populations of A. menziesii. As part of a Habitat Conservation Plan to mitigate the effects of a Department of Transportation construction project, the State of Hawai‘i Department of Land and Natural Resources, Division of Forestry and Wildlife, began to plant A. menziesii at five additional wild sites in 2004 in an attempt to establish 3 additional self-sustaining populations. By 2013, only one mitigation site met their long-term success criteria (State of Hawaii Department of Land and Natural Resources – Division of Forestry and Wildlife, 2013). In a 2013 draft report, species managers noted that their mitigation efforts were hampered by a lack of seed germination and survivorship. The Lyon Arboretum Seed Conservation Lab (SCL) initiated a research project on A. menziesii to better understand germination behavior. Given anecdotal evidence of low seedling recruitment in natural settings, determining how to break seed dormancy at ex situ facilities where seeds are less threatened by insect and fungal pests will give population managers greater resilience as they work to conserve this endangered native species.
Statement of Problem
Abutilon menziesii seeds are thought to exhibit physical dormancy, which can be broken by scarifying the seed’s water-impermeable seed coat (Baskin & Baskin 2014). However, several Hawaiian seed banks have observed that fresh seeds scarified immediately before sowing often rot. Thus, we hypothesize that this species may instead exhibit combinational dormancy – both physical and physiological dormancy – which may be broken by allowing seeds to sit on growth media for before scarifying.
This experiment provides some support for the hypothesis that A. menziesii has combinational dormancy, given the large difference in normal germination and total germination rates between the initial scarification and one-month delayed scarification treatments (p-adj. values between those groups as calculated using post-hoc Tukey tests are 0.0264418 and 0.0405327 for percent normal and percent total germination respectively). However, the optimal amount of time to delay scarification was much lower than originally hypothesized, with delaying scarification by one month emerging as the best treatment option to maximize the number of normal seedlings produced while minimizing the time spent waiting for seeds to germinate.
To test our hypothesis, we designed a delayed scarification experiment with eight treatments: initial scarification (labeled Init.Scar in tables and figures); scarification after one (Scar.1mo), two (Scar.2mo), three (Scar.3mo), four (Scar.4mo), five (Scar.5mo) or six months (Scar.6mo), and an untreated control (Untr). For this experiment, we used seeds stored in dry, frozen conditions for approximately 17 years.
Experimental Methods
We sowed 4 replicates of 25 seeds for each treatment onto growth paper in two compartment petri dishes in mid-December. The growth paper did not contain any nutrients; it merely retains moisture. The petri dishes were kept within the SCL’s Percival Growth Chamber, which is programmed to a diurnal schedule with average daily high and low temperatures calculated for an average elevation on the island of O‘ahu. Daily high and low temperatures, and the times at which the light in the growth chamber turns on and off are programmed manually each month. When it came time for each treatment to be scarified (in mid-January, February, March, April, May and June), we replaced the growth paper in the dish, rinsed the seeds in tap water, and then lightly nicked the seed coats with a scalpel under a microscope. Every week, we counted the number of normal and abnormal seedlings, and then removed all germinated seedlings and all moldy seeds that gave way when lightly squeezed using tweezers. We counted seedlings as abnormal if they rotted on the dish, failed to green, or did not have both a radicle and shoot. If the growth paper was dry, we also watered the seeds at this time. Occasionally, dishes did dry out between weekly checks. Toward the end of the experiment, the growth chamber reset to a constant light program after a power outage at the Lyon Arboretum. This occurred after the six-month scarification.
Analytical Methods
We performed all data visualization and analysis in R version 3.4.3 (R Core Team 2017). We created box and barplots in the ggplot2 package (Wickham 2016). We focused our analyses on the following statistics:
- Percent normal germination out of 25 seeds sown per replicate
- Percent normal germination out of total germination (normal germination + abnormal germination)
- Percent total germination
After confirming that these three statistics were normally distributed and homoscedastic (had similar variances), we performed three separate one-way analysis of variance (ANOVA) calculations in the base R program. When the p-values were lower than 0.05, we followed the one-way ANOVA calculations with post-hoc Tukey tests.
Methods
To test our hypothesis, we designed a delayed scarification experiment with eight treatments: initial scarification (labeled Init.Scar in tables and figures); scarification after one (Scar.1mo), two (Scar.2mo), three (Scar.3mo), four (Scar.4mo), five (Scar.5mo) or six months (Scar.6mo), and an untreated control (Untr). For this experiment, we used seeds stored in dry, frozen conditions for approximately 17 years.
Experimental Methods
We sowed 4 replicates of 25 seeds for each treatment onto growth paper in two compartment petri dishes in mid-December. The growth paper did not contain any nutrients; it merely retains moisture. The petri dishes were kept within the SCL’s Percival Growth Chamber, which is programmed to a diurnal schedule with average daily high and low temperatures calculated for an average elevation on the island of O‘ahu. Daily high and low temperatures, and the times at which the light in the growth chamber turns on and off are programmed manually each month. When it came time for each treatment to be scarified (in mid-January, February, March, April, May and June), we replaced the growth paper in the dish, rinsed the seeds in tap water, and then lightly nicked the seed coats with a scalpel under a microscope. Every week, we counted the number of normal and abnormal seedlings, and then removed all germinated seedlings and all moldy seeds that gave way when lightly squeezed using tweezers. We counted seedlings as abnormal if they rotted on the dish, failed to green, or did not have both a radicle and shoot. If the growth paper was dry, we also watered the seeds at this time. Occasionally, dishes did dry out between weekly checks. Toward the end of the experiment, the growth chamber reset to a constant light program after a power outage at the Lyon Arboretum. This occurred after the six-month scarification.
Analytical Methods
We performed all data visualization and analysis in R version 3.4.3 (R Core Team 2017). We created box and barplots in the ggplot2 package (Wickham 2016). We focused our analyses on the following statistics:
- Percent normal germination out of 25 seeds sown per replicate
- Percent normal germination out of total germination (normal germination + abnormal germination)
- Percent total germination
After confirming that these three statistics were normally distributed and homoscedastic (had similar variances), we performed three separate one-way analysis of variance (ANOVA) calculations in the base R program. When the p-values were lower than 0.05, we followed the one-way ANOVA calculations with post-hoc Tukey tests.
| Treatment | Percent Normal Germination | Percent Abnormal Germination | Percent Total Germination | Percent Normal Germination/Total Germination | Number of Days Elapsed |
| Init.Scar | 23 | 31 | 54 | 43 | 13 |
| Scar.1mo | 50 | 28 | 78 | 64 | 43 |
| Scar.2mo | 49 | 20 | 69 | 71 | 70 |
| Scar.3mo | 43 | 28 | 71 | 61 | 105 |
| Scar.4mo | 38 | 29 | 67 | 57 | 127 |
| Scar.5mo | 46 | 36 | 82 | 56 | 155 |
| Scar.6mo | 19 | 40 | 59 | 32 | 189 |
| Untreated | 5 | 2 | 7 | 71 | 189 |
Table 1: Key statistics calculated from germination data
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Data Visualization
| Treatment | Percent Normal Germination | Percent Abnormal Germination | Percent Total Germination | Percent Normal Germination/Total Germination | Number of Days Elapsed |
| Init.Scar | 23 | 31 | 54 | 43 | 13 |
| Scar.1mo | 50 | 28 | 78 | 64 | 43 |
| Scar.2mo | 49 | 20 | 69 | 71 | 70 |
| Scar.3mo | 43 | 28 | 71 | 61 | 105 |
| Scar.4mo | 38 | 29 | 67 | 57 | 127 |
| Scar.5mo | 46 | 36 | 82 | 56 | 155 |
| Scar.6mo | 19 | 40 | 59 | 32 | 189 |
| Untreated | 5 | 2 | 7 | 71 | 189 |
Table 1: Key statistics calculated from germination data
Over the course of the experiment, 487 seeds germinated to produce both normal and abnormal seedlings. Table 1 reveals that the highest percent normal germination occurred after delaying scarification by just one month, with similar percent normal germination figures after delaying scarification by two months and five months. The highest percent total germination occurred after delaying scarification by five months, though delaying scarification by just one month resulted in nearly as high a percent total germination rate. Normal germination represented the biggest proportion of total germination after waiting two months to scarify.
In terms of general germination trends, Figures 1-3 reveal a two-fold increase in normal seedlings driving a large increase in total germination if seeds are allowed to sit for one month before scarification. Between the one-month and two-month delayed scarification treatments, normal germination remained approximately the same while abnormal germination decreased, driving an increase in normal germination as a proportion of total germination. Percent normal germination decreased slightly and percent abnormal germination increased slightly as scarification was delayed by three and four months, before both normal and abnormal germination spiked after delaying scarification by five months. Normal germination fell after delaying scarification by six months.
One-way ANOVA calculations on percent normal germination, percent normal germination divided by total germination and percent total germination as grouped by scarification treatment produced p-values of 1.067 x 10-5, 0.1815 and 4.408 x 10-9 respectively. Because there were so many treatments, it was difficult to assign each delayed scarification treatment to distinct groups based on post-hoc Tukey test p-values.
References
Baskin, C. C., & Baskin, J. M. (2014). Seeds: ecology, biogeography, and evolution of dormancy and germination (2nd ed.). San Diego, CA: Elsevier.
Gustafson, R., Herbst, D. R., & Rundel, P. W. (2014). Hawaiian plant life vegetation and flora. Honolulu, HI: University of Hawaiʻi Press.
Lilleeng-Rosenberger, K. E. (2005). Growing Hawai'i's native plants: a simple step-by-step approach for every species. Honolulu, HI: Mutual Publishing.
R Core Team, 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria
State of Hawaii Department of Land and Natural Resources – Division of Forestry and Wildlife. (2013) . Draft DOFAW Abutilon menziesii stabilization plan 2017 [PDF file]. Retrieved from https://dlnr.hawaii.gov/wildlife/files/2013/09/DRAFT-AbuMen-StabilizationPlan_DOFAW_082917-003.pdf.
Acknowledgements
I would like to thank Dr. Marian Chau, Lyon Arboretum Seed Conservation Lab (SCL) Manager, Tim Kroessig, M.S., former SCL technician, and Nellie Sugii, Lyon Arboretum Hawaiian Rare Plant Program Manager, for their mentorship, support, and contributions to this project.
