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Category — plant research

Just Cloning Around: Getting to the Root of Genetic Diversity

In August, CREW staff traveled down to Daniel Boone National Forest in Kentucky to accompany the US Forest Service in their annual survey of our Cumberland sandwort experimental outplanting. The Cumberland sandwort is a small, delicate plant that clings precariously to the sandy soil of sandstone rock formations in the Cumberland Plateau of southern Kentucky and northern Tennessee. It is endangered, in large part due to trampling by hikers and people scavenging for Native American artifacts. CREW has been working with the US Fish & Wildlife Service to help preserve and protect this little plant.

Cumberland sandwort (Arenaria cumberlandensis) in bloom.

Cumberland sandwort (Arenaria cumberlandensis) in bloom.

Using seeds collected in 1994 from Pickett State Park in Tennessee, CREW established 10 genetic lines of the Cumberland sandwort in our tissue culture lab, and we banked each line in liquid nitrogen in our CryoBioBank. In 2005, to test whether the plants grown by tissue culture could be used to re-establish a population if needed, CREW and the US Forest Service planted 77 plants from our tissue culture lab in a sandstone cave in Daniel Boone National Forest that had a habitat similar to the native habitat of the sandwort. On our August 2013 trip to that experimental outplanting, we counted 160 plants!

collecting

CREW staff collecting leaf samples. Left: Omer Donmez Right: Megan Philpott

It looks like our experimental outplanting is flourishing in its new location, so now we want to look at the genetic diversity of the outplanting. Genetic diversity in a population is important because it allows the population to adapt to changing environments, or to survive and develop resistance to disease. Although we had started with 77 plants, they were all clones of the 10 original genetic lines established in our tissue culture lab. On our trip, we collected leaf samples from 35 individuals to analyze using microsatellite markers. For comparison, we also traveled down to the two locations in Pickett State Park that our original seeds were collected from and collected about 150 total leaf samples from the two large populations. Now we’re working on extracting DNA from the nearly 200 leaf samples collected this August. We should be able to compare the amount of genetic diversity generated in the experimental outplanting with the amount of genetic diversity naturally found in the original populations. This study is part of a larger study where we are evaluating the genetic stability of tissues that have been stored in liquid nitrogen for up to 20 years – a project that is supported in part by a grant from the Institute of Museum and Library Services, as well as CREW’s Eisenberg Fellowship. It should give us insight into how to manage and protect threatened and endangered plant populations more effectively. Keep an eye out for our results!

Sandstone rock habitat of Cumberland sandwort

Sandstone rock habitat of Cumberland sandwort

September 3, 2013   No Comments

Two Decades on Ice

1050612  Kris Lindsey, CREW Research Associate, and Dr. Dani Ballesteros, CREW Post-doc, removing samples from one of the Frozen Garden’s storage tanks.

Kris Lindsey, CREW Research Associate, and Dr. Dani Ballesteros, CREW Post-doc, removing samples from one of the Frozen Garden’s storage tanks.

This year CREW’s Plant Division is starting work on an exciting project:  Removing samples from the Frozen Garden of CREW’s CryoBioBank, some of which have been frozen in liquid nitrogen at -196oC for up to 25 years.  The official title of the project is:  Evaluating Two Decades of Seeds, Spores, and Tissues in CREW’s CryoBioBank: Cryostorage as a Tool for Ex Situ Conservation in Botanical Gardens, and the work is part of a National Leadership Grant from the Institute of Museum and Library Services (IMLS).  CREW has one of the oldest and most diverse frozen collections of wild plant materials in the world.  The Frozen Garden includes seeds, spores, embryos, gametophytes of mosses and ferns, pollen, and shoot tips from a wide variety of species, many of which are endangered.

The sample box is quickly moved to a small box with liquid nitrogen, to keep all the samples in the box frozen, while some are removed.

The sample box is quickly moved to a small box with liquid nitrogen, to keep all the samples in the box frozen, while some are removed.

Over the course of the next three years we will be removing samples from the collection to test their viability and to look at their genetic integrity.  Close to 1000 samples are targeted for the project—about one-third of the collection.  In addition, some samples that have been frozen in liquid nitrogen have had duplicate samples that were stored under other conditions:  -20oC (which is the temperature most seed banks use), 4oC (refrigerator temperature), and at about 22oC (room temperature).  These will provide valuable comparisons with the samples stored in LN.

Removing the targeted sample from the sample box.  The box with the rest of the samples is then quickly moved back into the storage tank.

Removing the targeted sample from the sample box. The box with the rest of the samples is then quickly moved back into the storage tank.

Working on this project will be a team effort.  Dr. Valerie Pence and Kris Lindsey are being joined by CREW’s new post-doc, Dr. Dani Ballesteros, Megan Philpott, a Ph.D. student and her advisor, Dr. Theresa Culley, from the Department of Biological Sciences at the University of Cincinnati.  Dani will be primarily working on studies of survival, while Megan will be looking at the genetics of the samples.  During the course of the project she will also travel to the USDA-ARS National Center for Genetic Resources Preservation (NCGRP) lab in Ft. Collins, CO, to work with Dr. Christina Walters on DNA degradation studies of the samples.

Megan Philpott, UC Ph.D. student, preparing solutions for DNA isolation.

Megan Philpott, UC Ph.D. student, preparing solutions for DNA isolation.

Removing the samples is a look at the past, reviving plant materials that have been in “suspended animation” for up to two decades.  But, it will also provide valuable information and guidance for the future, as botanical gardens work to preserve the rich plant diversity of the world using the best practices available.  Look for updates as this project unfolds in the coming months.

July 2, 2013   3 Comments

Buttercups Return Home

Autumn Buttercup

On June 8, 336 plants of the Autumn buttercup (Ranunculus aestivalis) propagated at the Carl H. Lindner Jr. Family Center for Conservation and Research of Endangered Wildlife (CREW) were planted in a restoration project at a preserve near Panguitch, Utah.  This small region of Utah is the only place in the world these plants are known to exist.  The species was first discovered in 1894, but by 1979, it was thought to be extinct.  A population of 400 plants was found in 1982, but within 6 years the numbers had plummeted to less than 20 plants.  At that point, The Nature Conservancy purchased the land to protect the buttercups, but the buttercup population did not rebound.

RaInVitro2012bIn the early 2000s, CREW’s Plant Division developed a protocol for propagating the plant through tissue culture, thanks to funding from the Institute of Museum and Library Services.  The process started with a few seeds, and each seed produced a genetically unique clone that was then multiplied through tissue culture—a technique in which the tissues are grown on an artificial, sterile medium.  The shoot-producing cultures initiated in this way can be propagated indefinitely, but when plants are needed, the shoots are transferred to a root-inducing medium. Once roots are formed, the plants can be moved to soil.

The Sevier Nature Preserve

The Sevier Nature Preserve

Once these protocols were established, CREW’s Plant Division became part of unique team that formed to restore the buttercup population in its native habitat.  The team includes The Nature Conservancy, U.S. Fish and Wildlife, The Arboretum at Flagstaff, and Weber State University.  June’s outplanting was the third and largest the team has attempted.  A planting in 2007 produced some survival in areas with appropriate moisture, but a planting in 2010 was quickly eaten, it is assumed, by voles.  The site was also being overgrown by other vegetation, so the restriction on grazing, which was done originally to reduce pressure on the plant, came into question.

Autumn Buttercup Plant

Autumn Buttercup Plant

Based on these previous experiences, the new planting has fourteen sites, seven each in the grazed and ungrazed areas, and half of the 24 plants at each site will be protected from herbivores both above and below ground.  Water availability was recorded for each plant, as well as the initial plant size, and number of leaves and flowers.  Traps were set for several nights at the time of the planting in order to provide information on the current small mammal population at the site.  All of this data will be evaluated by faculty and students at Weber State University.

Planting in their new home.

Planting in their new home.

It definitely took a team effort between the collaborating partners’ staff and students, as well as volunteers, to accomplish digging the holes (through very tough sod), organizing the plants, taking the data, and carefully putting each plant in its place.  It was wonderful to see the plants, which had been sent to Flagstaff from CREW in test tubes over the past several years, as robust, healthy plants in soil, ready to take on the dry winds and heat of their new Utah home.  Later in the summer we should get the first reports on how they are doing.

 

June 13, 2013   2 Comments