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!
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!
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.
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.
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.
More samples will be removed soon! We will keep you updated with the most interesting findings!
September 9, 2013 1 Comment
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.
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!
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!
September 3, 2013 No Comments
On September 1, 1913—the passenger pigeon was one year away from extinction. Martha, the last of her species, lived here at the Cincinnati Zoo and was an aged bird. Efforts had been made for years to find a mate for Martha that would provide a chance for the species to survive. In truth, the fate of the passenger pigeon had been sealed several decades before by modern communications (telegraph), transportation (rail), rampant commercial-scale harvest of the birds and the felling of large expanses of hardwood forest habitat. For Martha and her species, it was a waiting game. The eyes of the nation watched for the inevitable to happen.
The inconceivable loss of the most common bird species on the planet shook society out of its torpor. There had been billions of passenger pigeons only 50 years before—racing up, down and across the continent like a biological storm, consuming the fruits of the forest in its quest to fulfill their mission to feed, nest and make more pigeons. Few would have believed that there would soon be none. Those that were concerned were not influential enough to prevent it. By the time it was clear to the majority what was going to happen, it was just too late to do anything about it. It was the first time we could be certain that humans had caused a species’ extinction. It was, and is, a heavy burden, yet it was also a catalyst for change.
There is good that came from this extinction. Many species considered common today were on the brink of the same fate at the end of the 1800s. American bison, wild turkey, white-tailed deer and pronghorn antelope were all on the same path to extinction as the passenger pigeon. After the loss of the passenger pigeon, people got to work to save these species from the same outcome. President Roosevelt began the National Parks program and wildlife conservation efforts sprang up all over the country. The wildlife conservation effort we know today was born out of the loss of the passenger pigeon. In a very real way, modern zoos as well as countless other conservation organizations around the globe owe their existence to this one event. It is an impressive legacy and one to be celebrated in the coming year.
Over the next year, the Zoo will celebrate what works in the world of wildlife conservation as a commemoration to Martha. To start, we will renovate the current Passenger Pigeon Memorial at the Zoo to include a hopeful message that celebrates the success of wildlife conservation rather than mourning the loss of a single species. We will highlight the work of our Zoo in species conservation and celebrate the work of others in our community and beyond. We will be blogging each month with updates on the renovation of the Passenger Pigeon Memorial and more, and hope you will join our story and celebration in the coming year.
September 1, 2013 3 Comments