When collecting, preserving, and storing plant genetic material, seeds may not always be the
best option. This could be because the seeds are not available, the seeds are not tolerant
of storage conditions, or the seeds do not contain the desired genetic information.
In some of these cases, such as apple, we can instead preserve their dormant buds.
Dormant buds refer to the buds that form on a woody tree or shrub during a growth period
and will expand only after they overwinter and experience warm spring weather.
These buds can be either vegetative or floral. Vegetative buds make shoots with
leaves while floral buds will make new flowers. For preservation purposes, it’s the vegetative
bud we want so that we can grow a new shoot after the buds are recovered from storage.
Dormant bud preservation methods are primarily used within agricultural genebanks,
however there are opportunities to use this technology for other conservation
purposes. These techniques are best suited for cold-hardy plants. That means that our
current technology is not suitable for tropical or subtropical species, and even some temperate
crops can’t be preserved as dormant buds if their growing conditions aren’t cold enough.
For successful preservation, twigs must be harvested from winter-dormant plants
and stored before they lose dormancy. This means they must be kept cold and processed
quickly, either on-site or shipped overnight to the processing facility. Once in storage,
these dormant buds can last for decades until they are needed to replenish dwindling field material,
at which point they can be recovered and grown into healthy plants
using a recovery system appropriate to each crop. Long-term preservation projects require a lot of
planning. Some considerations for dormant bud cryopreservation are as follows.
(1) Are dormant buds the appropriate conservation target?
Often it is much more appropriate to store seeds of wild species
since they capture much more genetic diversity. Dormant buds should be considered if a specific
individual must be conserved, or if seeds cannot be successfully obtained and stored.
(2) Is the source plant growing in a winter hardy location and available for sampling?
To ensure successful cryopreservation, budwood should not only come from a cold hardy species—it
must also come from plants grown in a hardy location.
Those plants must also have enough budwood so the parent tree is not adversely affected,
and so the conservation team has sufficient material to preserve the individual. In addition,
the appropriate permits and permissions are needed to sample the plant for long-term preservation.
(3) Are facilities and technical expertise available for processing and long-term storage?
Dormant bud cryopreservation requires specialized equipment, such as a method
to cut the bud sections, cold rooms with low humidity for bud drying, controlled rate freezers,
and a long-term commitment to maintain liquid nitrogen storage tanks. This may require that
personnel be trained or that collaborations be established to perform the necessary tasks.
(4) What research is needed to test the available methods?
Literature reviews must be performed to determine if the species has been successfully preserved as
dormant buds. It is equally important to have a method for preserved buds
to be grown into complete plants. Prior publication
doesn’t necessarily ensure success for the desired individuals and specific locations,
so additional feasibility experiments will be needed. Initial experiments may
reveal that the collecting, processing, or regrowth stages still require modification.
While these techniques may not be appropriate for every situation,
an established program for dormant bud cryopreservation can become a quick and efficient
way to preserve large quantities of material that might otherwise be lost due to pests, diseases,
and other environmental conditions. Apples are the largest clonally
propagated crop preserved at the USDA National Laboratory for Genetic Resources Preservation.
Here at the NLGRP in Fort Collins, Colorado, we have successfully preserved apple cultivars
and wild apple species, as well as collections of sour cherry, willow,
currents, and more. This method could also be used to conserve rare plant species. A preliminary
trial in 2021 showed that Salix arizonica could be successfully cryopreserved. Here is an overview of
the dormant bud cryopreservation procedure used in the USDA national genebank system.
Twigs with dormant buds are collected from winter-hardy trees. In this case, sour cherry
twigs are collected in upstate New York in early to mid-January and shipped overnight to the NLGRP
in Colorado. Upon arrival, the twigs are cut into uniform segments, each containing a healthy
dormant bud. A small subset of segments is dried and weighed to establish the moisture content.
Meanwhile, the remaining dormant buds are placed into a cold room.
There they will be dried to reduce water and ice damage. The segments are weighed
periodically to determine the change in moisture content. When the desired moisture is obtained,
the bud segments are sealed into plastic tubes. The tubes are placed into a controlled rate
cooler that drops the temperature one degree per hour until it reaches -30 degrees Celsius.
These are then plunged into ultra-cold liquid nitrogen vapors for long-term storage.
To ensure that preservation was successful, one to two tubes must be recovered from liquid nitrogen
and used for regrowth assessments. Traditionally, the buds are recovered and rehydrated in moist
peat moss. After one to two weeks, the buds can then be grafted onto the appropriate rootstocks.
The grafted trees are grown in greenhouse conditions
and bud viability is assessed once the shoots are well-established.
In an alternative regrowth assessment, segments can be warmed and planted in
high-humidity conditions, using antimicrobial treatments to promote healthy growth.
Initial viability can be assessed by observing shoot growth.
These shoots can then be introduced into tissue culture to regenerate complete plants.
Dormant bud cryopreservation has been successfully used to conserve thousands
of plants in our national genebank system and has the potential to be applied to many more species.
While research will likely be necessary to apply these methods to new plant collections,
this process allows us to preserve plant materials that can’t be stored as seeds.
Thanks to this approach, genebanks now have greater options to conserve the
genetic resources from some of our most precious and hard-to-preserve plants.