There’s a rescue helicopter, but it doesn’t actually land on the roof of the hospital for the world’s most endangered plants. Rather, the tissue culture lab at the National Tropical Botanical Garden (NTBG) on Kauai sits in a trim red bungalow with a peaked roof primarily suitable for butterfly landings. There’s still life-or-death drama in the business of botanists working to save the rarest of the rare plants. For example, only one individual plant of Cyanea kuhihewa, a gray stem topped by a tuft of straplike leaves, remains in the wild. In May, a select crew traveled by helicopter to the north side of the island of Kauai to visit the plant for a few days.
Waiting in the lab for the helicopter’s return was Susan Murch, a biologist who commutes from Toronto, Ontario. She’d come to the lab at 7 a.m. to make sure she was ready when the crew arrived from the airport 15 minutes away. She was awaiting precious cargo: She had asked that the crew members, right before they started home, (gasp) pick a Cyanea leaf.
Murch works as a fertility doctor for plants, and she does much the same thing that other fertility specialists do. She brings the latest in hormone chemistry and cell physiology to the aid of faltering reproduction. She deals with extreme cases and has tended to endangered plants in Egypt and Costa Rica, as well as in North America.
This Cyanea needs her badly. The botanic garden grows a few of the plants, but they’re all offspring of the same parent. That’s hardly an ample genetic foundation on which to rebuild a whole species. Even the genetic variation of one more plant, that loner in the wilderness, would help.
Last year, the wild Cyanea bloomed, and the botanic garden spent $1,500 to send a helicopter with pollen from a garden plant for the flower. Like so many desperate fertilization attempts, though, this one failed, and the wild plant didn’t set seeds.
This year, Murch has been shuttling between Toronto and Kauai as she sets up the lab to help the island’s rarest plants have seedlings of their own. When the Cyanea rescue team arrived with a zip-sealed plastic bag holding a wet paper towel and one leaf, Murch started disinfecting the leaf and snipping it into bits that she may someday be able to coax into whole new plants.
In the world of mammals, high-tech reproductive successes have made front-page news. Yet when Murch succeeds at creating offspring for species hundreds of times more rare than the recently cloned gaur or mouflon, it’s months or maybe a year before even readers of In Vitro Cellular and Developmental Biology see how it all turned out.
The projects may be unsung and chronically underfunded, but Murch argues that for the most depressing cases of impending plant extinction, these technologies offer real hope.
Small beginnings
Murch explains that, yes, she really is trying to make new plants from a leaf instead of a seed. This approach can work because plant cells are totipotent, meaning that if they are given the right cues, they can grow into an entire new plant.
The effort to find those cues and regenerate whole plants from bits of tissue dates back to the first years of the 20th century. Scientists found that some snippets of leaves and other plant parts maintained in the laboratory could change into unspecialized plant cells. Then researchers demonstrated they could make such blobs of tissue grow into either roots or shoots depending on the ratio of two critical plant hormones, auxins and cytokinins. Later, a pair of research teams demonstrated that providing the right regulatory chemicals to undifferentiated carrot cells could make them start forming an embryo. Once scientists have an embryo, they can coax it into an adult plant.
Today’s tissue culture specialists continue to search for the right ratio of the right hormones to trigger development of more and more species. Much effort also goes into maintaining sterile conditions and coming up with the best blend of nutrients to feed particular plants. For example, Murch often adds regular grocery-store sugar, since the first nubbins of tissue that she nurtures may not be up to photosynthesizing efficiently for themselves. B vitamins often go into the mix, too, because many plants normally rely on soil bacteria to supply them.
Since the early days with blobs of carrot tissue, commercial growers of food and ornamental plants have come a long way with these techniques. Lab researchers found tissue culture expands the possibility of studying corn, soybeans, and other crops. Growers have also explored the techniques. Commercial strawberries and potatoes are propagated this way, as are geraniums and African violets.
On the edge
“When I first learned about tissue culture, my very first thought was, ‘Why do we still have endangered plant species?'” Murch says. It seemed to her that tissue culture labs should be able to take even the rarest species and create dozens of new plants.
Even though it’s turned out to be more complicated than that, Murch remains upbeat, even when she goes to Hawaii. There, the fragile flora evolved without many mammalian or insect predators but has recently been attacked by a multitude of invaders. The state has 292 plants on the federal endangered species list, including 150 species comprising fewer than 50 individuals. And of these, 11 species have fewer than five representatives left on Earth.
Soon after Murch got her lab set up in February, she started with the direst cases, such as the hard-luck shrub called Kanaloa kahoolawensis. The last part of its name comes from the Hawaiian island Kaho’olawe, which is 13 miles long and, at its widest, 8 miles across. Goats and other animals introduced by Europeans flourished there, destroying native vegetation, and the U.S. Army and Navy used the island for target practice between 1941 and 1990.
Botanists Ken Wood and Steve Perlman of the National Tropical Botanical Garden were exploring the island in the early 1990s when they discovered two unusual sprawling shrubs on a tall rock just offshore. The shrubs’ flowers, in tufts like a mimosa’s but the color of cream, bloomed over blunt oval leaves.
Wood and his colleague taxonomist David Lorence had never seen the plant before, but they consulted a specialist who knew the pollen well. The relatively smooth grains with grooves, typical for a legume, had been turning up in samples of ancient soils all over the islands.
Paleontologists had concluded that the mysterious plant releasing this pollen must once have dominated the lowland landscape.
The researchers published the official description of the plant in 1994, declaring it unusual enough not just to be designated as a new species but to have its own new genus. Lorence assigned a generic name that honors the Hawaiian god Kanaloa.
The plants seem to be “mostly male,” as Lorence puts it: most of the blooms grow only male parts. But one year, biologists found three seeds from one of the plants. That happy fluke has yielded two shrubs that now sit in a place of honor at the entrance to the botanic garden’s rare-plant nursery.
Unfortunately, that burst of luck faded. Horticulturists at the facility have repeatedly failed to propagate the plant by cuttings or grafts. “The greenhouse staff is very excellent,” says Murch. Their record glows with innovations in coaxing little-studied species into reproducing, so if they didn’t manage, Murch deems the prospects grim.
Waiting for more seeds began to seem unpromising, too. Drought hit Kaho’olawe so hard that in 2001, island managers sent helicopter expeditions out to water the plants. “The helicopter puts one skid down on a boulder about half the size of your desk and you get out–carefully,” recalls island restoration manager Paul Higashino. Then the helicopter went back to pick up 400 pounds of 5-gallon water containers. Keeping an eye out for unexploded ordnance on the rocky slope, the emergency-watering crew directed the container drop.
Even with three waterings, one of the plants withered to what a casual observer, or less of a diehard optimist than Higashino, might call dead. “I don’t want to say that on my watch one died,” he says.
When Murch arrived in Kauai, she approached the problem by observing the two Kanaloa plants growing near her lab’s front door. From these–by then, two-thirds of the world’s K. kahoolawensis population–Murch noted details of the leaf-bud structure and other clues to what sort of hormones and nutrients might work. Unfortunately, there were parts of the plants that she couldn’t study, such as the root system.
“One of the challenges of working on the last few [plants] is that you can’t destroy anything,” she says.
She quickly decided that the petioles, the little stems that connect a leaf blade to a twig, looked most promising as a source of unspecialized tissue. However, Murch counted four fungal diseases and three bacterial ailments afflicting the plants, not to mention infestation by an abundance of insects. The last remnants of a species often are sickly, she says.
Murch gave some plucked leaf-stem samples a big wallop of antibiotics and then spent 3 weeks weaning the plant bits from the drugs by reducing the dosage a little every 48 hours. Only then did she expose the tissue to cocktails of hormones and nutrients. A round of tests generally takes 7 to 10 days, after which Murch works out another set of cocktails and tries again. “You just have to work through the possibilities,” she says.
The Kanaloa bits didn’t do much for several rounds of testing, but finally, embryo tissue started to form. What did the trick was a mild auxin mixed with a strong cytokinin, one used commercially for defoliating cotton plants to ease harvesting.
When she saw her success, did she whoop and hug people? “No!” she says. “You don’t get excited until you can do it again.”
The youngsters aren’t out of the climate-controlled growth chamber yet, but the planet now has a new generation of 20 Kanaloa seedlings about an inch tall. This summer, Murch is working on repeating the experiment.
Global colleagues
Among tissue culture specialists, scientists who specialize in discovering the requirements of rare species don’t reach anywhere near the numbers of what Murch calls “the corn-and-soybean crowd.” Yet the rare-plant specialists have figured out how to grow dozens of rare plants.
The Lyon Arboretum in Honolulu has had at least limited success in figuring out how to culture some 300 rare species, says Nellie Sugii. One of her more dramatic projects began with a batch of little green fruits–they looked a bit like grapes, she says–on a stem delivered by Ken Wood in 1998. He’d visited one of the last half-dozen known members of Tetraplasandra flynii, a tropical tree species, and found half-ripe fruit.
Sugii extracted the embryos and nurtured the botanical equivalents of premature babies. After 6 months, with the embryos turning brown, “I was worried,” she says. She couldn’t bear to throw them away though, and suddenly they began to grow. “Now, in the lab, you can’t throw anything away,” she says.
The Royal Botanic Gardens, Kew in England also hosts a sweeping effort to propagate rare, fragile, or cantankerous species from around the world. They’ve been particularly successful with sterile-tissue-culture propagation of arid-zone succulent plants, such as cacti, which tend to rot when disturbed.
Some 30 rare North American plants are now under study by tissue-culture specialists at the Cincinnati Zoo. Endangered species of pawpaw trees in Florida, for example, grow what botanists call recalcitrant seeds, which don’t survive drying and freezing in seed banks. “We started with the four-petaled pawpaw–the shoots looked really good, but we couldn’t get roots for a long time,” says Valerie Pence. She and her colleagues there have now succeeded in culturing three of these pawpaws, she reported in June at the Portland, Ore., meeting of the Society for In Vitro Biology. Her colleague Bernadette Plair also reported a way around the seed-bank problem. For the dwarf pawpaw, the researchers can now pack shoot buds inside gelatin beads, freeze them, and then thaw the plant tissue for culture.
Praveen Saxena, who presides over a test-tube garden of plant tissue from around the world, points out another plant dilemma that culturing techniques might solve. Saxena, a plant physiologist at the University of Guelph in Ontario, is working with Costa Rican scientists to find a trick for culturing their native Guaiacum sanctum. The tree’s slow-growing wood is so hard that people have used it to make bowling balls and propeller-shaft bearings for ships.
Traditional healers have relied on the plant, and modern medical researchers are now testing its potential against asthma. With so many uses and such slow growth, the population has shrunk sadly. Saxena says that, so far, tissue culture for this tree has proved “very difficult,” but he’s betting the process will produce new plants more quickly than seeds do. In this species, a tree can take more than 200 years to flower.
In other cases, culturing could save a species from overardent collecting and offer health benefits, too.
Saxena and Murch are finishing a study on the endangered goldenseal, a popular North American medicinal herb. The researchers bought bottles of the herbal preparation and sent them for chemical testing. Some samples turned out to carry dangerously high concentrations of lead, accumulated by the plant’s root.
Saxena and Murch have now fine-tuned a procedure for tissue culture of the plant that offers herb suppliers an alternative to ravaging the last of the wild supplies and, incidentally, keeps the lead out of the product. Fortunately, the researchers’ tests show that an ingredient that’s purported to be medicinally active remains high in the cultured version.
Murch can list many more uses for the tissue-culture approach. Her main message, she says, is a bracing counterbalance to a lot of reports of the gloomy state of the planet’s botanical resources. “It’s not a hopeless situation,” Murch says.
And although she isn’t ready to celebrate yet, the little bits of Cyanea leaf have shoots and are “looking good.”
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