Plant genebanks preserve genetic material in a surprising variety of forms, including
tiny, one-millimeter shoot tips.
Shoot tips are the growing points in plant tissues.
They can be excised from plants and preserved in liquid nitrogen for long-term storage.
Shoot tips contain the exact genetic makeup as the original plant and can be used to preserve
material that can’t be stored as seed.
As an added bonus, viruses or viroids present in plant tissues are sometimes eliminated
during the cryopreservation process.
This is called cryotherapy and can be used to eliminate pathogens from infected plants.
While shoot tip cryopreservation opens more possibilities for preserving plant genetic
resources, it is a labor-intensive process and is not suitable for every situation.
Shoot tips are an appropriate conservation target in the following circumstances:
(1) The original genetic composition must be conserved.
Unlike seeds, shoot tips do not capture the diversity within a population—they capture
the genetic composition of an individual.
This is useful for preserving specific cultivars such as Chardonnay grapes or Golden Delicious apples.
(2) The plant can’t be preserved as dormant buds.
Dormant bud cryopreservation is a much faster means of storing vegetative tissues; however,
it’s only successful for some cold-hardy, woody species.
Shoot tip preservation works on a wide range of plants including those from both tropical
and temperate places as well as trees, shrubs, and small herbaceous plants.
Shoot tip methods have also been used to cryopreserve rare and endangered species that are maintained
in tissue culture.
(3) Tissue culture systems are available.
We typically use tissue culture plants to produce clean and uniform shoot tips.
We also need tissue culture systems to recover the shoot tips after cryopreservation.
They’ll need to be provided with extra nutrients and sugars to grow into fully functional plants.
(4) Cryopreservation methods are available.
Shoot tip cryopreservation methods are highly specialized for each crop.
Even if published methods are available, testing and modifications may still be needed to ensure success.
(5) Infrastructure for long term preservation is available.
Tissue culture facilities, as well as access to liquid nitrogen, cryotanks for long-term
storage, specially trained technical support, and documentation systems are necessary.
At the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado, we
use shoot tip cryopreservation to securely back-up a huge variety of crops from the living
collections in the USDA’s National Plant Germplasm System.
We preserve shoot tips from strawberries, potatoes, mint, coffee, and so much more.
Shoot tips may be easily overtaken by bacteria and fungus.
This is why they are processed in sterile environments and why we typically source material
from aseptic cultures.
However, we can sometimes use surface sterilized plants grown in a greenhouse, screenhouse,
or growth chamber.
Plant materials should be of uniform age and physiological state.
Plants may need pretreatments for successful cryopreservation.
This could require cold temperature treatments, or the addition of hormones, antioxidants,
or salicylic acid to the medium on which they are grown.
After any pretreatments, uniform shoot tips are carefully excised to 1 to 2 mm in size.
Shoot tips may then undergo any necessary preculture steps.
They are then cryopreserved using one of four common methods:
(1) Vitrification.
In vitrification, shoot tips are treated with cryoprotectant solutions that replace free
water with chemicals that protect cells and promote glass formation within cells during
the cooling process.
These solutions may be toxic to the cells, so exposure times must be precise.
Shoot tips must then be quickly cooled to liquid nitrogen temperatures.
Increasingly, this is done by adhering shoot tips to foil strips to increase the cooling
rate of the samples when they are submerged into liquid nitrogen.
Cooled foil strips are then placed into vials and stored in cryotanks.
(2) Encapsulation-vitrification.
In this method, shoot tips are embedded into calcium alginate beads, treated with cryoprotectants,
loaded into vials, plunged into liquid nitrogen, and stored.
(3) Encapsulation-dehydration.
Shoot tips are embedded into calcium alginate beads, treated with high sugar solutions,
then the beads are air dried to an optimal moisture content.
The beads are loaded into vials, frozen, and stored.
(4) Two-step cooling.
Shoot tips are loaded into vials along with a less toxic cryoprotectant solution.
These are slowly cooled to -30 degrees Celsius, then plunged into liquid nitrogen, and stored
in cryotanks.
For all cryopreservation methods, a successful recovery method is required.
Foil strips or vials with shoot tips are warmed, and the cryoprotectants diluted into a sucrose
solution.
Shoot tips are typically plated onto a medium and grown in tissue culture, however, in cases
such as Citrus, we instead micrograft the shoot tips onto tissue cultured rootstock plants.
The tricky part of shoot tip cryopreservation is that every single step of the procedure
must work for the plants to be successfully preserved.
Often, it takes years to develop a shoot tip cryopreservation method for a new crop, especially
for crops with a broad range of cultivars or species.
While the barriers to shoot tip cryopreservation may be large, scientists across the world
have been working on this for decades.
Thanks to this continued work, we are increasing our ability to preserve a diverse range of
crops and securing the future of our clonal collections.