Creating a Model to Restore Endangered Plant Species through Saving Cypripedium reginae
Katherine DuanPhillips Academy, Andover, MA
Mentor: Dr. Peter FaletraNew Hampshire Academy of
Science
© Copyright Elaine Faletra
The World is Facing Sixth Mass Extinction, the “Holocene Extinction”
We 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
Cypripedium reginaeThe Showy Lady’s Slipper An endangered terrestrial orchid native to the mid-northern
and northeastern U.S. as well as bordering regions of Canada Takes 8-10 years to reach maturity in the wild Reproduces mostly through rhizomes and less so through
seeds Less than 1% of its seeds will germinate and less than 1% of
those will reach maturity Typically flowers in June and produces a mature seedpod or
fruit by late August Natural habitat is a fen in which neutral or slightly basic
water is constantly percolating Is 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 fall
Cyp. reginae blooming in June
Hypothesis
By understanding Cypripedium reginae’s 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 research
Flower anatomy Pollinator variety Fruit Production Histology
Propagating in controlled environment Seed viability over 4 years Three methods of vernalization Hydroponics to improve seedling survival
Establishing protected habits and involving community
Protected habits construction Public involvement
FullLifecycle
Study
Flower Anatomy
& Pollinator
Variety Fruit Productio
n
Histology
Seed Viability
Vernaliza-tion
Methods
Hydroponics
Protected Habits
&Public
Involvement
A Class II Laminar Flow Hood Many 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 useInstalling the hood’s fan
Ovary
Pouch
Staminode
Stigma
Pollen
FlowerStructure Ovary
Pollen on the anther
Stigma, the female reproductive structure
Staminode, shields the stigma
Leaves, petals, sepals, and pouch are covered by numerous hairs
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 bugBee or wasp
Swallowtail butterfly
Fruit Production Over 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. reginae
Year Fen 1- Warren, NH Fen 2- Warren, NH Lyme, NH
flower fruit % fruit flower fruit % fruit flower fruit
2013 9 4 44% 6 2 33% 0 0
2014 14 5 36% 9 4 44% 0 0
2015 15 2 13% 19 2 11% 0 0
2016 17 3 18% 23 9 39% 3 0Cyp. reginae Seedpod in Late Summer
Table: Cypripedium reginae Fruit Production
Histological Analysis of Ovules Per Seedpod Cyp. 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
Histological Analysis of Ovules Per Seedpod
Figure 1: Cross section of the ovary
Figure 2: Enlarged view of 2 ovules
Using the volume equation for a prolate ellipsoid V, it was estimated that the average Cyp. reginae seedpod volume is about , where a;
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.
Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old Seeds
Seeds 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 ºC.
Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old Seeds Seeds 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 shoots
Sterilizing seeds, weakening their seed-coats using bleach solution
Inoculating seeds
Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old Seeds As shown by the four colored lines representing the 4 different years during which seedpods
were harvested, there appears to be little correlation between the age of seeds and percent germination rates over a 7 month period, since seeds from 2011 had nearly the same germination rate as those of seeds from 2014. Seeds collected in 2011, 2012, 2013, and 2014 had a germination percentage of 77.6%, 7.0%, 36.0%, and 98.6% respectively after about six months.
Percentage of Germination in Seeds Collected in 2011 - 2014
Comparing Seed Viability Among 1, 2, 3, and 4 Year-Old Seeds The chart represents 3 stages of seedling development in sterile culture from seeds collected
over 4 years. Seeds collected in 2013 and 2014 reached stage 5 faster than seeds collected in 2011 and
2012, with 31.00% of seeds reaching stage 5 in the 2013 sample and 92.6% in 2014 sample while 27.0% of seeds reached stage 5 in the 2011 sample and no seeds reached stage 5 in the 2012 sample.
This suggests seeds have a certain viability upon harvesting and storage has little or no effect. To more carefully elucidate the cause, one might conduct an experiment where the same seedpod is used to inoculate seeds in 4 successive years.
Percentage of Germination with Stages in Seeds Collected in 2011 - 2014
Comparing 3 Methods of Vernalization Three methods of vernalization were tested:
1) Removing sterile seedling from their medium, washing them free of agar, and stratifying seedlings in organic matter – a common method of vernalization
2) Removing sterile seedling from their medium, washing them free of agar, storing bare-root seedlings in containers with small amounts of tap water
3) Storing seedlings at 3 - 5 ºC in a refrigerator in their original culture vessels without transfer to new vessels
Of the three methods of vernalization, the first method of stratifying in organic matter was the least effective with over 77.8% loss. Storage as bare roots was the second most effective but remained with a high level of loss at 62.5%. The storage of seedlings in their original culture vessels was by far the most effective with ~0% loss (>99% survival).
Washing medium off of seedlings
Method-1: storing in organic matter (77.8% loss)
Method-3: store in their original culture vessels (~0% loss)
Method-2: storing bare-boot (62.5% loss)
Refridgeration at 5º C for all methods
Maximizing Survival of Transplanted Seedlings with Hydroponics After vernalization, seedlings transplanted to soil had a 33% survival rate. To
increase the survival of vernalized seedlings, a novel hydroponics system was designed. A two-chamber automatic pumping system was built to propagate un-vernalized and vernalized seedlings in a soil-less medium.
A propagation tray measuring 18 x 103 cm with a depth of 10 cm held the seedlings in their soil-less medium. This tray rested in a larger reservoir tray measuring 30.5 x 122 cm with a depth of 10 cm. Holes were drilled into the propagation tray to allow the solution to circulate with an automated pump system.
The propagation tray was filled halfway with Growstone® Hydro stones with a half inch layer of coarse coconut fiber (coir) on top. A one inch layer of fine coir was placed on top of the coarse coir.
Initial design
Drilling holes in propagation tray
Setting the propagation tray into reservoir
Growstone® Hydro stones
Cloth lining
Coarse coir
Fine coir
Maximizing Survival of Transplanted Seedlings with Hydroponics The reservoir tray was filled with 10 liters of a 1/10 strength dilution of
Murashige and Skoog basal salt solution to provide inorganic nutrients. The weights of seedlings which were un-vernalized, vernalized for 8 months, and
vernalized for 15 months were recorded. The seedlings vernalized for 8 months were divided into two sub-groups: one with healthy root-tips, the other with black and unhealthy root-tips.
The seedlings were laid on top of the fine coir. The seedlings were covered with half an inch of fine coir and were illuminated with a 120 cm T5HO LED Sun Blaster® grow light for approximately 14 hours per day to mimic springtime day length.
Preparing coir for seedlings Planting seedlings Finished hydroponic system
15-Month
8-MonthHealthy/Blacktip
Mixing Murashige and Skoog salt solution
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7
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04 5 6 7 9
6 7 8 8 7 8 7 8 8 9 9 9 111112 913 14 17 16
93
7
15
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 04 4 4 4 6 8 91112141517 181819181920 222222 23 24
2934
4346 48 47 45
Comparing Growth of Seedlings 15 Month Unvernalized 8 Month
Date
Aver
age
Plan
t Hei
ght i
n m
m
The first seedlings emerged after 8 days only in the vernalized groups. After 82 days, the average height for the un-vernalized seedlings was 0 mm, for seedlings
vernalized for 8 months it was 45mm, and for seedlings vernalized for 15 months it was 15mm.
The plant to shoot ratio was 0% for un-vernalized, 79% for seedlings vernalized for 8 months, and 36% for seedlings vernalized for 15 months.
The data indicates that seedlings do not grow unless they go through a cold dormancy; however, extended cold dormancy is detrimental to seedling growth.
Since seedlings grown in organic media, including soil, emerge after 2-3 weeks and grow slowly thereafter, this hydroponics system substantially accelerates seedling emergence and growth.
Maximizing Survival of Transplanted Seedlings with Hydroponics
Plant to ShootRatio = 36%
Plant to ShootRatio = 79%
Plant to ShootRatio = 0%
Measuring plants
Young plants
The shoot emergence rate is the ratio of emerged shoots to total number of shoots on the seedling planted per test group.
One common belief is that the larger and heavier the seedling is, the stronger it is and more likely to grow. This graph compares the weight of the seedling to the plant-to-shoot ratio.
Vernalization is the most important factor in seedling growth with which weight becomes inconsequential.
Comparing 15-month and 8-month groups, the data demonstrated that proper duration of vernalization has a much higher impact on seedling growth than weight differences.
Comparing the black root-tip group with the healthy root-tip group, the data showed that heavier seedlings had higher plant-to-shoot ratio.
The number of seedlings in each test group was small (about 18 seedlings per group), and further experimentation is needed to support these observations.
Maximizing Survival of Transplanted Seedlings with Hydroponics
Unvernalized
8 Month Healthy Root
8 Month Blacktip
8 Month
15 Month
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90
0.18
0.07
0.22
0.11
0.14
0%
77%
83%
79%
36%
Shoot Emergence Rate vs. Average Seedling Weight
Plant to Shoot Ratio Average Weight (g)
Maximizing Survival of Transplanted Seedlings with Hydroponics
During the hydroponics experiment, beginning on January 16 mold growth caused some browning and seedling shoot die-back. This graph shows that seedlings with a faster initial growth rate were more likely to survive the mold.
12/25
/16
12/29
/161/2
/171/6
/17
1/10/1
7
1/14/1
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1/30/1
72/3
/172/7
/17
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7
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73/3
/173/7
/17
3/11/1
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Seedling Growth for 8-Month Vernalization Group
Date
Plan
t Hei
ght i
n m
m
Mold begins to grow on 1/16/2017
Establishing Protected Habitats & Involving Community
From the years 2013 to 2015, five controlled habitats were created in locations that received approximately eight hours of direct sunlight. The top 12 inches of the soil were removed and half of the original soil was mixed with an equal amount of Vermont Compost® and peat moss. Approximately a cup of pelletized lime was added to reduce the acidity of the soil. The hole was lined with plastic and punctured to allow drainage. The hole was filled with the planting mixture and seedlings were planted about an inch below the surface to ensure roots and shoots were not exposed. In the fall, the sanctuaries were covered with straw. Seedlings were monitored in the spring for new shoots.
Digging a 12inch deep hole
Mixing top soil with organic compost
Lining with plastic and puncture for drainage Filling with soil mixture
Planting seedlings an inch below surface
Covering with straws before the first frost
Keep the soil moist and make a shade if too sunny in summer
Establishing Protected Habitats & Involving Community
In 2013 through 2015 five controlled habitats were established as sanctuaries on both private and public land. The oldest sanctuary in Lyme, NH, created in 2013, was seeded with about 45 six- to twelve-month-old seedlings. In the summer of 2015, it had about 18 healthy ~3-year-old plants, with the largest about 25 centimeters tall. In the summer of 2016, it had 16 healthy plants all exceeding 25 centimeters tall, three of which flowered. This brings the time to maturity to within a 4-5 year range.
In the spring 2014, two controlled habitats were created in Warren, NH. One was seeded with about 15 six to nine month-old seedlings. In the summer of 2015, it had 11 healthy plants with the largest about 5 centimeters tall. In the summer of 2016, it had 10 healthy plants. The other sanctuary was seeded with about 12 six to nine month-old seedlings. In the summer of 2015, it had about 7 healthy plants with the largest about 5 centimeters tall. In the summer of 2016, it had 7 healthy plants all exceeding 4 centimeters tall. Weeding with scissors
The first sanctuary in Lyme NH built in 2013, 3 plants flowered in 2016
Educating a community member while building a sanctuary on her property
Establishing Protected Habitats & Involving Community
In the fall of 2014, a controlled habitat was created in Unity, NH. It was seeded with about 36 three-month-old seedlings. In the spring of 2015, it had about 16 healthy plants. In the spring of 2015, a sanctuary was established in Hanover, NH. It was seeded with about 35 approximately 9-month-old seedlings. In the early summer of 2015, it had about 12 healthy plants. In the spring of 2016, only 2 seedlings emerged after an animal dug up the habitat.
Through the New Hampshire Orchid Society and the Native Orchid Conference community awareness was raised and volunteers to host protected habits were gathered.
Educating community members at 2015 New Hampshire Orchid Show
Giving presentation at The 2015 Native Orchid Conference
Findings or conclusionThis model reduced the life cycle of Cyp. reginae from 10 years to 5 years, and efficiently generated thousands of seedling annually. The results also show in considerable detail: the critical stages of Cyp. reginae’s life cycle both in vitro and in situ. The pollination recordings supported the previous observations of the trap flower directing
pollinators to ensure crosspollination. Some of these pollinators appear to be syrphid flies while other insects such as bees, wasps, moths, and butterflies visited the flowers.
It is encouraging to know that seeds can be stored for at least four years without loss of viability.
The vernalization study and hydroponics study support the ability to efficiently propagate large numbers of these orchids for restoration.
The histology revealed about a 4 to 1 ratio of ovules to seeds. There is a long-held theory that a plant must have a certain number of fertilized ovules before it decides to invest energy in producing a fruit.
A possible future experiment would look more thoroughly into the histological relationship among ovaries, ovules, and seeds.
Lastly, it seems that my hypothesis was supported and a model for species restoration can be made based on a thorough understanding of that species’ in vitro and in situ characteristics.
Acknowledgement and ReferencesI would like to thank the New Hampshire Academy of Science STEM Center and Crossroads Academy for letting me use their lab and facilities to conduct my research. I am grateful for the support of Elaine Faletra, her help with histology photos, and use of her flower pictures in this presentation. I would like to thank Dr. Ezequiel Rivera for his help with analyzing histology photos. I am grateful for the Crossroads Academy lady’s slipper club for giving me a foundation for my research. I would also like to thank my parents for their help and support. I am especially grateful for my mentor and advisor Dr. Peter Faletra for all his unwavering support, guidance, and for always challenging, inspiring, and believing in me.
References:1) Extraterrestrial Cause for the Cretaceous-Tertiary Extinction.
Author(s): Luis W. Alvarez, Walter Alvarez, Frank Asaro, Helen V. Michel Source: Science, New Series, Vol. 208, No. 4448 (Jun. 6, 1980), pp. 1095-1108 Published by: American Association for the Advancement of Science. Stable URL: http://www.jstor.org/stable/1683699
2) Progress and challenges in geochronology. Renne, P.R., Ludwig, K.R., Karner, D.B., 2000. Science Progress, 83: 107-121.
3) Terrestrial Orchid conservation in the age of extinction. Swarts and Dixon, Annals of Botany 104: 543-556, 2009.
4) Habitat loss and extinction in the hotspots of biodiversity. Brooks TM, Mittermeier RA, Mittermeier CG, et al. 2002. Conservation Biology 16: 909-923.
5) Axenic seed culture and micropropagation of Cypripedium reginae. Sokolski K., Dovholuk A., Dovholuk L., Faletra P., 1997. Selbyana 18, 172-82.
6) IUCN Annual Report. International Union for Conservation of Nature, 1997-98,
7) Saving Cypripedium reginae. Faletra, P., Dovholuk, A., King, T., Sokolski, K., 1997. Orchids, February 1997, pp.: I3 8-143.
Dr. Peter Faletra & Elaine Faletra
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