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

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

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.

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.