Overcoming challenges in seed production of Bonamia menziesii, a critically endangered and Hawai’i-endemic vine Rhian K Campbell and Brenna Fowler 1: Introduction to B. menziesii B. menziesii inflorescence in the nursery; image: Manser L. Bonamia menziesii is a critically endangered perennial vine in the Convolvulaceae family. It is the only species of its genus endemic to Hawaii. It has been listed as endangered since 1994 by IUCN and USFWS but little research has been conducted or reported (Bruegmann & Caraway, 2003). On Kaua'i, suitable habitat is varied and includes lowland dry, mesic or wet forest from steep slopes to level ground (elevations between 566 and 1,127 m (1,858 and 3,695 ft) (Critical habitat; plants on the Hawaiian Islands, 2003). Ovate, glabrous leaves- image: Manser, L. The notable morphology of this plant includes twining branches that are pubescent in early growth stages. It has dark green foliage and small tubular flowers that are monoecious. Petals are white and covered in beige pubescence, no more than 1 inch in length. Leaves are ovate, glabrous and opposite on vines. When established in the wild, plants grow woody at the base, climbing up neighboring trees (USFWS, 2011). The primary threats to B. menziesii are habitat degradation, competition with non-native vegetation, insect and/or animal predation, and reproductive failure from lack of pollinators (Endangered and Threatened Wildlife and Plants; Designation of Critical Habitat for 60 Plant Species from the Islands of Maui and Kaho'olawe, HI, 2003). Examples of extreme weather witnessed on Kaua'i in the last year include flooding events, slope erosion, landslides, and wildfires. The damage to native habitat caused by these events such as these have increased opportunities for non-native plants to establish. The observed reduction of fruit set over the last 10 years has resulted in decreased genetic diversity within the Kaua’i populations and in the further decline of the species. 3: Methods- Observations and Pollination Aborted seed pod; image: R. Campbell Clonal propagation is the last resort for conservation collections. However without viable seed being produced by our wild populations of B. menziesii for many years it was our only option. After improving our success rates with clonal propagation of B. menziesii in 2018, our next goal was to produce viable seed for storage and propagation. Our plants flowered abundantly with no fruit set for over 6 months. We started to hand pollinate, both selfing and outcrossing, within our collections. These pollinations set fruit, however, all of the fruit aborted prematurely. Healthy vs. aborted seed pod; images: R. Campbell Observations of the fruit and stem under a microscope revealed exit holes, likely from the larvae of a small predatory insect that had oviposited eggs into the fruit or stem. We also observed multiple species of fruit fly and flying insects present on the stems, flowers, and fruit. As a protective measure, we began placing mesh bags on all fruit once the flowers showed signs of successful pollination. Observed insects on B. menziesii; images: Campbell, R. During the course of controlled pollinations, it became clear that spontaneous pollinations were also occurring. There were many observations of small ants entering and exiting fertile flowers (with pollen present). After two months of hand pollinations, the practice was discontinued because the plants were setting fruit successfully without intervention. We continued to bag developing fruit and collected all fruit as it matured. Ants observed; images: Campbell, R. 4: Methods- Fruit Development and Germination Fruit development of B. menziesii; images: Campbell, R. After three months of pollination and bagging fruit on the B. menziesii vines, we had 60 fruit set that contained 1 - 4 seeds per pod. The first seed harvested were small and potentially immature as none of the seed we planted germinated successfully. B. menziesii have successfully germinated after being dried and stored in our seed bank so the logical first step was changing our substrate/germination methods. Harvest and immediate seed dissection yielded green plant tissue; image: Campbell, R. Since there had been no successful germinations, we dissected several freshly harvested seed to determine if the embryos were viable. Inside we observed green, healthy seed leaves and embryonic tissue. Seed measurement, B. menziesii; Images: R. Campbell Because the freshly harvested seed were viable, we began to tweak the conditions for germination and designed a small germination study. Half of seeds were sowed on moist filter paper in petri dishes under 24 hour lighting in our temperature controlled lab and half were sowed subsurface in a mixture of perlite and vermiculite on a shaded table in the greenhouse. 1: Seed soaked for 10 minutes; image: Trask, A. 2: Half placed on moist filter paper in petri dish, inside of a cool, non-sterile lab environment; image: Trask, A. 3: Half sowed subsurface in mixture of perlite and vermiculite, placed on shaded table in greenhouse; image: Campbell, R. The test was too small in scale to produce highly meaningful results; however, it resulted in successful germinations in both treatment groups. Our initial sowings had all been done on the surface of black cinder. We now hypothesize that moisture and depth of sowing are the two most important factors for germination. Germination success!; image: Campbell, R. 5: Conclusions and Next Steps B. menziesii infloresence; image: Campbell, R. We have now established protocols for successful clonal propagation, pollination, fruit production, and seed germination of B. menziesii on Kaua’i. Moving forward we can apply these techniques to establish comprehensive ex-situ conservation collections. These collections can then be used to shore up wild populations. In addition further research can be done in monitoring and identifying the predatory insects which are causing premature fruit drop. The hope is that we can prevent this species from becoming extirpated on Kaua’i. Conservation in Hawaii tends to focus on those species which are closest to extinction. By starting earlier, and working with plants that have reduced but still viable populations, we increase our chances of success. Dried up seed in dissected fruit; image: Campbell, R. 2: Current Collections at NTBG B. menziesii flower buds; image: Manser, L. Maintaining nursery stock at NTBG allows horticulturalists and scientists access to populations for research and restoration. In establishing our long term nursery collections we have been able to develop techniques for vegetative propagation of B. menziesii. For the last year we have been able to observe and collect data on flowering cycles, pollination mechanisms, and insect predation. Our most recent challenge has been determining appropriate methods for seed propagation. Accession tags used in the horticulture center; image: Manser, L. As of June 2019, there are 34 seeds and 32 plants in propagation in the conservation nursery and 168 seeds in storage in the seed bank and laboratory. Seeds are stored in a temperature controlled environment of 5, -18, and -80°C. Cuttings from 2018 study; image: Magers, R. (2018) In June of 2018, wild collected and F1 propagated cuttings were taken and given comparative trials to research efficiency of root growth. Techniques included callusing, utilization of 5 types of rooting hormones and then placement in constant watering under misters (Magers, 2018). These collections were successful propagules that bore flowers and fruit used in cross pollination and germination trials for phase 2. 6: Acknowledgements and References Discussion for future / References Presented by Rhian Campbell, Conservation Nursery Technician, and Brenna Fowler, Horticulture and Living Collections KUPU member.