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Category — CREW

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

Sumatran Rhino Harapan is Happy to be Home

Harapan happily  wallowing

Harapan happily wallowing

In the middle of August at the Cincinnati Zoo it is hot, uncomfortable, and difficult to stay cool. Unless, of course, you’re a rhino. Sumatran rhinos, Suci and Harapan, are built to tolerate this muggy,  hot weather. At least, their ancestors have adapted to it. In the wild, Sumatran rhinos find relief from the heat in wallows that have filled with rain water. These pools also keep biting flies and insects from bothering them. The rhinos frequent these wallows and put their own personal touch or signature on them by rolling, digging the sides, and likely spraying urine on the trees and vegetation nearby. These behaviors tell other rhinos and animals in the area that this wallow already belongs to someone and to please leave it the way they found it.  Here in Cincinnati, Suci and Harapan also have their wallows and they put them to good (and frequent) use. It’s our jobs as keepers to keep the wallows, and all exhibit areas (inside and out), fresh and clean. Yes, clean mud is difficult to attain but necessary. We do this by adding fresh water daily and periodically adding fresh soil. In the wild fresh rain water is added almost daily and during the dry season a rhino will move on to another wallow near or closely adjacent to a stream.

Handsome Harapan

Handsome Harapan

Harapan, the handsome young stud born here in April of 2007, and who has spent some time in Florida and San Diego California over the years, returned here on July 3, 2013.  His behavior indicates he is happy to be home. Most days he eats his first meal of ficus browse ( tree cuttings) and fruits and vegetables and then he saunters over to the wallow to get himself good and muddy. The unique part of his behavior is that he then goes into his pool for a quick rinse. Next he wanders inside and checks up on the keepers to see how our day is going. Then, repeats all the steps over again. People often ask zoo keepers if the animals “ know you “ or know they are back home. After this experience, I can honestly say Yes.   Harapan is without a doubt happy to be here and we are happy to have him home.

‘ Till next time- Paul Reinhart, Cincinnati Zoo Ungulate dept.

August 30, 2013   4 Comments