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Category — plant research

Ultra-Frozen Seeds Come to Life after 15 Years

As we described in a previous post, several samples stored in the Frozen Garden of CREW’s CryoBioBank are going to be removed during the next months in order to examine their viability. And, here are results from the first ones: Coming to life after being stored for 15 and 20 years in the ultra-low temperatures of liquid nitrogen (-321 F!!)! And the most interesting result…they germinated as fast and as well as they did the day they were frozen!

Fig.1: Poplar seeds germinating after 15 years of storage in liquid nitrogen

Fig.1: Poplar seeds germinating after 15 years of storage in liquid nitrogen

These are seeds from poplar trees. Seeds from poplars, and also from most willows, germinate very fast because in their natural habitats they have enough water available when they are shed, but also because they must germinate in a short window of time. These trees grow very close to water courses and floodplains where there are frequent disturbances of the substrate in which they grow. The most common disturbance is flooding, which in most cases removes the soil and washes away the seeds and seedlings that are not established. Moreover, during winter, water in the soil can freeze, damaging any ungerminated seeds that remain completely wet along the river banks. For these reasons seeds are shed at the end of the spring and beginning of the summer, when temperatures are warm enough and water availability optimal. Thanks to their fast germination (within 24 hours!) poplars and willows can grow quickly in the summer days, establishing small trees that will be big and strong enough to survive flash flooding and the cold and dry winter. However these seeds are very short lived, and die at ambient conditions in just 2 or 3 weeks if they do not germinate. In the Frozen Garden of CREW at the Cincinnati Zoo and Botanical Garden we have preserved their initial viability and rate of germination for several years; including some samples stored up to 2 decades!! Thanks to these experiments we have demonstrated that seeds from endangered poplars and willows can be preserved for long periods of time, in order to keep them alive for our future generations!

Fig. 2: Seeds germinating in the growth chambers

Fig. 2: Seeds germinating in the growth chambers

Along with poplars and willows, orthodox seeds of several species were removed from liquid nitrogen in June. Orthodox seeds are those seeds that naturally dry during their maturation in the fruit, and thus they can be frozen in seed banks, keeping their initial viability and vigor. So, we could say that they have a “natural” predisposition to be stored at the low temperatures of liquid nitrogen. We have chosen these samples since they do not have special requirements for their recovery and germination, as other samples stored in the frozen garden have (as shoot tips, etc). Germination is the easiest way to analyze seed viability, and, at the same time, it produces seedlings that can be grown to produce plants for future use.

In order to recover seeds from the Frozen Garden, the cryovials with seeds were carefully removed, since dry seeds at very low temperatures are very brittle and we didn’t want to damage them. Then the cryovials were thawed at room temperature for 1 hour. Seeds were re-hydrated in a moist, saturated environment over night to avoid any imbibitional damage, which is damage that can be produced by rapid water uptake when seeds are very dry.

Cryovials

Fig.3: Cryovials containing seeds removed from the Frozen Garden.

Some of the seeds have special requirements for germination because they are dormant. One of these special requirements is a “moist/cold stratification” for several weeks in order to break the dormancy and stimulate germination. It is as if the seeds were resting in the soil of winter! Seeds that did not require stratification were sown in petri dishes on blue filter paper particularly designed for seed germination. Then they were put in a controlled temperature and light growth chamber, and germination was monitored daily.

Fig. 4: Seeds are placed in moist paper towels and placed in the fridge for moist/cold stratification

Fig. 4: Seeds are placed in moist paper towels and placed in the fridge for moist/cold stratification

Seeds from several species have germinated very well after storage in ultra-low temperatures for several years, including the short lived seeds of the endangered plant, native to eastern North America, Plantago cordata (heartleaf plantain). The heartleaf plantain is threatened or endangered in 11 states including Ohio, Illinois and Kentucky because populations have declined or disappeared almost everywhere. The heartleaf plantain is a wetland plant with highly localized distribution which makes it very sensitive to habitat destruction, particularly that occurring for urbanization. We have stored their seeds for 14 years without any decline in their initial germination.

Fig. 5: Seedlings of Plantago cordata after seeds were stored for 14 years in liquid nitrogen.

Fig. 5: Seedlings of Plantago cordata after seeds were stored for 14 years in liquid nitrogen.

More samples will be removed soon! We will keep you updated with the most interesting findings!

September 9, 2013   2 Comments

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