Nestled in Manoa Valley is a 200-acre arboretum and botanical garden and what looks like small cottages that are actually laboratories storing hundreds of threatened and endangered plant species.

For years, Hawaii’s native plants have been vulnerable to extinction due to invasive species and climate change. To help prevent that, researchers store samples of the rarest plant species in test tubes and seed banks kept at low temperatures at the Lyon Arboretum.

But keeping propagated plants in glass tubes is time-consuming and expensive. Instead, researchers in the coming months at the Micropropagation Laboratory and Seed Conservation Laboratory will use cryopreservation to protect at-risk plant species for the future.  

Devon Gordon, who works at Lyon Arboretum’s Micropropagation Laboratory, holds an empty rack pulled from an unfilled cryogenic storage tank, a specialized vacuum flask for storing liquid nitrogen. Eventually the plant samples will be prepared for cold storage. About 30,000 samples can fit in a single storage unit. Cory Lum/Civil Beat/2022

Cryopreservation is a storage technique that exposes plants to ultralow temperatures without killing them. The plant samples will be introduced to liquid nitrogen, which will flash freeze them at minus 196 degrees Celsius. Then they will be stored in two, 2-by-2-feet tanks. 

The concept is new to Hawaii, which started experimenting with cryopreservation in 2020, but the practice already has been used at the Cincinnati Zoo and Botanical Garden, and in other countries including Australia and India. 

“If we have a backup of them, they’re just extinct in the wild.” — lab technician Nate Kingsley

Devon Gordon, who leads the grant-funded cryopreservation project at the Micropropagation Laboratory, said he’s still working on a protocol that safely freezes plants into icy glass while keeping their cells alive and preventing lethal freezer burn.

Since each plant species reacts differently to the cryopreservation treatment, according to Gordon, he has to develop a protocol so that each plant species is safely frozen.

“If you just take a plant and freeze it, it produces ice crystals in the cells, and it can kill a lot of those cells,” Gordon said. “If you take spinach and put it in the freezer then take it out, it’s going to be mushy after.”

There are 33,000 plant samples stored at the Lyon Arboretum’s Micropropagation Laboratory. Cory Lum/Civil Beat/2022

Hawaii has been dubbed the “Endangered Species Capital of the World,” with about half of its 1,400 native plants in need of conservation, according to the Department of Land and Natural Resources.

Conservationists are trying to preserve samples of threatened and endangered plants in these labs with the ultimate goal of reintroducing them into the wild. There’s no concrete timeline for reintroduction because each species varies. Some species only exist in laboratories because they’re already extinct in the wild.  

But pests, diseases and climate change continue to threaten their existence.

“There’s no way for them to go back currently because there’s a lot of invasive species that have taken over their habitats,” Gordon said. “So (for) the people that work in the field, it’s a lot for them to clear out all of the weeds and put up a fence to keep all the invasive species out.”

Why Cryopreservation?

Currently the seed lab has 75% of Hawaii’s plant species in seed form, and the remaining 25% is in the micropropagation lab, which keeps live plant cuttings in test tubes filled with clear gel and nutrients. 

The plants can’t be left in the test tubes for more than 10 years because of concerns they would mutate and their survival rate would be affected, according to Gordon. 

The 570-square-foot Micropropagation Lab holds 33,000 plants from 275 species growing in test tubes. But the lab could become crowded as the plants outgrow their test tubes and need to be recut.

“The nice thing about cryopreservation is that it condenses that,” Gordon said.

“Theoretically, you can fit about 30,000 potential plants in one of those tanks,” he added. “That would save us a lot of space and time.”

Nate Kingsley, lab technician at the Seed Conservation Lab, shows equipment that dries the seeds thoroughly before the seeds can be frozen. Cory Lum/Civil Beat/2022

For now, the cryopreservation methods will be used on plant species in the Micropropagation Lab.

Nate Kingsley, lab technician at the Seed Conservation Lab, said he hopes the seeds will get a chance to be a part of the cryopreservation project. His lab holds 550 plant species in conventional refrigerators and freezers kept at minus 18 degrees Celsius. It holds up to 29 million seeds, the largest of six seed labs in the state.

Depending on the plant species, the shelf life of the seeds can last decades. Kingsley said some plant species that he checks on are from the 1990s and early 2000s.

“If we can have them in long-term storage and know that they can last without decreasing in viability substantially in 20 years, then the species are never really fully considered extinct,” Kingsley said. “If we have a backup of them, they’re just extinct in the wild.”

Continued Experiments

In order to develop a proper protocol for other experts to follow, Gordon said he’s continuing to experiment with different plant species using cryopreservation methods.

In his experiments, he cuts the tip-top of a plant to about 1 millimeter, about the same thickness of a fingernail. Then he takes the small plant cuttings, dabs a solution on them, then uses liquid nitrogen to quickly freeze them to prevent ice crystals from forming.

Gordon is conducting experiments with liquid nitrogen to preserve endangered and and threatened plant species. Cory Lum/Civil Beat/2022

But he noted that a challenge to conducting these experiments is that he can’t do the same protocol with every plant because species respond differently to liquid nitrogen and the type of solution he puts on them.

He said he’s trying to determine the best survival rate, noting a recent petri dish experiment in which most of the plants died but a few are still alive.

“It is a lot of work,” Gordon said. “But once we have the protocol established, I’m able to use that protocol for that plant species.”

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