Sigma xi presentation 2017

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Creating a Model to Restore Endangered Plant Species through Saving Cypripedium reginae

Creating a Model to Restore Endangered Plant Species through Saving Cypripedium reginaeKatherine DuanPhillips Academy, Andover, MA

Mentor: Dr. Peter FaletraNew Hampshire Academy of Science Copyright Elaine Faletra

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The World is Facing Sixth Mass Extinction, the Holocene ExtinctionWe are entering what many scientist believe is the sixth mass extinction, the Holocene Extinction. The Holocene Extinction is anthropogenic or caused by humans activities, including deforestation, strip mining, air and water pollution, and climate change.For about the past 50 years, conservation through habitat protection has been the most common approach in the fight against plant extinction. Unfortunately, this approach has not worked (Swarts and Dixon, 2009). Restoring endangered species to the wild might be another approach, but worldwide plant restoration efforts are rare. For restoration efforts to be most effective, they should be based on a model system to combat biodiversity loss. Orchids are a Red Flag Organism for biodiversity loss because of their sensitivity to changes in their environment. This makes them a prime subject for modeling a restoration effort.

Figure 1: Species Extinction and Human Population

The Washington Post: Earth is on brink of a sixth mass extinction, scientists say, and its humans faulthttps://www.washingtonpost.com/news/morning-mix/wp/2015/06/22/the-earth-is-on-the-brink-of-a-sixth-mass-extinction-scientists-say-and-its-humans-fault/?utm_term=.9d85c0fe21ee

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Cypripedium reginaeThe Showy Ladys SlipperAn endangered terrestrial orchid native to the mid-northern and northeastern U.S. as well as bordering regions of CanadaTakes 8-10 years to reach maturity in the wildReproduces mostly through rhizomes and less so through seeds Less than 1% of its seeds will germinate and less than 1% of those will reach maturityTypically flowers in June and produces a mature seedpod or fruit by late AugustNatural habitat is a fen in which neutral or slightly basic water is constantly percolatingIs endangered because of its low germination and survival rates, habitat loss, and habitat degradation. In NH, there are less than 5 locations where this plant is thriving in the wild

The Eshqua Bog, a natural fen in Hartland, Vermont, in late fallCyp. reginae blooming in June

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HypothesisBy understanding Cypripedium reginaes life cycle in the wild and everything about its tissues, anatomy, and in vitro growth requirements, a complete model for restoring it to the wild can be created. My ultimate goal is for this model to be used by conservationists around the world for other species.

Methods & Results Creating a Restoration Model through a Full Plant Lifecycle StudyEstablishing foundation through basic researchFlower anatomyPollinator varietyFruit ProductionHistologyPropagating in controlled environmentSeed viability over 4 yearsThree methods of vernalizationHydroponics to improve seedling survivalEstablishing protected habits and involving communityProtected habits constructionPublic involvement

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A Class II Laminar Flow HoodMany of the experiments in this five-year study required a laminar flow hood (clean bench) to maintain sterile cultures. A class II laminar flow hood was designed and built. Sterility tests were conducted at three fan speeds: medium, medium-high, and high. Six sterile culture vessels were opened for 1 minute at the 3 different speeds. Two culture vessels were unopened and served as a control group. Three tests of the hood showed that it maintained sterility at all speeds, with no contamination. Over 2,500 seedlings were produced per year using this hood. These seedlings were used as raw materials in other experiments.

Final hood assembly The hood in use

Installing the hoods fan

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OvaryPouchStaminodeStigmaPollen FlowerStructureOvaryPollen on the antherStigma, the female reproductive structureStaminode, shields the stigmaLeaves, petals, sepals, and pouch are covered by numerous hairs

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PossiblePollinatorsIn mid June of 2015, videos and photos of possible pollinators were recorded in Warren, NH using an Olympus SH-1 digital camera.

Toxomerus geminatus (Syrphid fly)

Butterfly or moth

Immature stilt bug

Bee or wasp

Swallowtail butterfly

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Fruit ProductionOver a four-year period starting in 2013, Cyp. reginae flowers were counted in June on individual plants in three different location in New Hampshire. In the fall of each year, the number of fruit (seedpods) was counted per plant.As shown in the table below, fruit production ranged from 13% to 44% over the years 2013 to 2016 in fen 1 and from 11% to 44% over the years 2013 to 2016 in fen 2. No fruit were produced in 2016 at the Lyme NH location.

Cyp. reginaeYearFen 1- Warren, NHFen 2- Warren, NHLyme, NHflowerfruit% fruitflowerfruit% fruitflowerfruit20139444%6233%00201414536%9444%00201515213%19211%00201617318%23939%30

Cyp. reginae Seedpod in Late SummerTable: Cypripedium reginae Fruit Production

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Histological Analysis of Ovules Per SeedpodCyp. reginae tissue was processed using a Leica ASP300S tissue processor according to manufacturer's instructions. All tissues were embedded into paraffin using a TissueTek embedding console (Tec 5 EMA1). The paraffin embedded tissues were sectioned at 4 microns thick using a Leica microtome (MODEL RM2255) and floated onto a 57 degree Celsius water bath, captured on glass slides, dried, and stained with a Leica Autostainer XL according to the manufacturer's instructions. Images were captured using a Leitz Dialux 20 with a Fujifilm HC-300Z digital camera.

Specimen embedded in paraffin wax

Embedding specimen in hot paraffin wax

Capturing images of specimens

Cyp. reginae tissue was processed using a Leica ASP300S tissue processor according to manufacturer's instructions. The tissues were treated using the following steps:

1. Formalin (10% neutral buffered)1 hr 2. Formalin (10% neutral buffered) 1 hr 3. 70% Alcohol 1 hr 4. 90% Alcohol 1 hr 5. Absolute alcohol 1 hr 6. Absolute alcohol 1 hr 7. Absolute alcohol 1 hr 8. Xylene 1 hr 9. Xylene 1 hr 10. Xylene 1 hr 11. Paraffin wax 1 hr 12. Paraffin wax 1 hr 13. Paraffin wax 1 hr

All tissues were embedded into paraffin using a TissueTek embedding console (Tec 5 EMA1). The paraffin embedded tissues were sectioned at to 4 microns thick using a Leica microtome (MODEL RM2255) and floated onto a 57 degree Celsius water bath, captured on glass slides, dried, and stained with a Leica Autostainer XL according to the manufacturer's instructions. The slides were stained using the following steps:

HEAT FIXED 15 min. 1. Xylene 90 sec. 2. Xylene 90 sec. 3. Xylene 90 sec. 4. Alcohol 200 proof 30 sec. 5. Alcohol 190 proof 30 sec. 6. Water wash 30 sec. 7. Gill's Hematoxalyn (nuclear stain)1 min. 8. Water wash 10 sec. 9. Mild acid rinse solution 60 sec. 10. Water wash 30 sec. 11. Bluing reagent 30 sec. 12. Water wash 30 sec. 13. Alcohol 190 proof 30 sec. 14. Orange G sol'n (cytoplasmic counterstain) 2 min. 15. Alcohol 190 15 sec. 16. Alcohol 190 15 sec. 17. EA solution (cytoplasmic counterstain) 4 min. 18. Alcohol 190 60 sec. 19. Alcohol 190 60 sec. 20. Alcohol 200 30 sec. 21. Alcohol 200 30 sec. 22. Xylene 60 sec. 23. Xylene 60 sec. XYLENE EXIT 10 min.

Images were captured using a Leitz Dialux 20 with a Fujifilm HC-300Z digital camera.

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Histological Analysis of Ovules Per Seedpod

Figure 1: Cross section of the ovary

Figure 2: Enlarged view of 2 ovules

The number of ovules bounded by a specific area in was counted and the number of ovules per cubic mm was found to be ~624 ovules;The total number of ovules per seedpod was calculated to be around 1.2 million which far outnumbers the amount of seeds each seedpod produces by at least a factor of 4. This suggests that 1 in 4 ovules is fertilized. Figure 1: Cross section through the ovary while the plant is flowering. The circular arrangements of nucleated cells (nuclei stain dark purple) are unfertilized ovules. The image was taken at 40 x magnification.Figure 2: Enlarged view of two ovules with the upper left ovule revealing a central mother cell that will later complete meiosis and form the mature megagametophyte. The image was taken at 400x magnification.

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Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old SeedsSeeds from mature, un-dehisced (unopened) seedpods of Cypripedium reginae collected in late September to early October of 2011, 2012, 2013, and 2014 were used in this experiment. After the seedpods were harvested, they were allowed to dry for about a week. The harvested seeds were stored at 5 C.

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Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old SeedsSeeds were cultured and monitored for both germination and development according to the procedure published by Sokolski et al, 1997 and Faletra et. al, 1997.

Stage-1: untreated seed with dark brown embryo

Stage-2: seeds treated with bleach

Stage-3: early germination with enlarged embryo

Stage-4: embryo has breached the outer seed-coat

Stage-5: seedling that has developed roots and shootsSterilizing seeds, weakening their seed-coats using bleach solutionInoculating seeds

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Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old SeedsAs shown by the four colored lines representing the 4 di