A CLEAN START

Propagating rare plants in The Huntington’s Tissue Culture Lab By Traude Gomez Rhine

When it comes to cleaning When it comes to sterilizing plants, culture container, we throw out all the cacti, Ann Miguel has tried everything. cacti cause the most trouble. One prob - plants in it.” She has scrubbed bleach between the lem is that the meristem—the plant tis - Plant tissue culture, also called micro - spines with a toothbrush and stuck sue from which new cells are formed— propagation, hinges on totipotency— cactus pieces into a sonicator, a little lies just beneath the areoles from which the ability of plant cells to regenerate contraption that removes dirt by using the spines grow. Porous spines are a nat - whole plants. A small piece of sterilized ultrasound. She has even tried vacu - ural home for dirt, mold, and spores— plant tissue can ultimately produce thou - uming a furry specimen. On this day the enemies of plant tissue culture. sands of plants. Miguel helps things along Miguel has decided to pull out the dirty spines and areoles from an Echinopsis ‘Reverie.’ “With cleaning a A small piece of sterilized plant cactus, it’s hit-or-miss,” Miguel says matter-of-factly as she plucks away tissue can ultimately produce prickly pieces with tweezers. No, Miguel is not some neat freak who is taking her love for succulents thousands of plants. to extremes. She’s a volunteer in The Huntington’sTissue Culture Lab, a small After discarding the spines, Miguel with doses of nutrients and organic plant room in the Botanical Center managed cuts the cactus specimen into little hormones such as cytokinins and auxins, by Sean Lahmeyer, The Huntington’s pieces, each no bigger than a thumb - which can speed up growth or stimulate plant conservation specialist. Lahmeyer nail, and puts them into a small con - root production. Imagine taking a single has been diligently working with vol - tainer of common household bleach, blueberry and, just by adding a few unteers and interns over the past two the kind you might use to remove dashes of key ingredients, producing an years to integrate plant tissue culture stubborn stains from a white shirt. entire blueberry pie. techniques intoThe Huntington’s con - She wonders whether to go for five Tim Harvey—another volunteer on servation practices. The lab looks like a or 10 minutes this time around—five the project—is quickly increasing the simple kitchen where you might micro- minutes may not eradicate all the germs, numbers of a historic agave from the wave a lunch. Instead of heating lasagna, but a 10-minute soak may kill the tis - Desert Garden. Sitting in the lab’s though, Miguel is busily eradicating sue. One tiny spore can produce fungal “hood,” a sterile work station, Harvey microbes. Her goal is to mass produce contamination. “It’s easy to lose a few is slicing and dicing a sample intermit - plants from the tissue of any one spec - weeks’ work from just a little dirt,” says tently cleaning his sharp instruments imen, but she needs to sterilize the plant Miguel, opting for the longer soak. with a propane torch while sterilized tissue first if she is to have any success. “Once we see contamination in a tissue air blows potential contaminants away

HUNTINGTON FRONTIERS 23 Volunteer Tim Harvey divides tissue samples and places them in a container. Page 22: Sterilized plant tissue in the lab can help ensure the survival of many rare plants, including the Agave mapisaga var. lisa . Photos by Lisa Blackburn and John Sullivan .

from the already sterile material. Harvey is dividing tissue culture propagations “We’re making them immortal,” of Agave guiengola , a plant that came to The Huntington from Mexico in the 1960s. In the following decades, it pro - says Tim Harvey. duced only one pup—the vernacular for an offset, or new plant.Then, as agave Lab in the Botanical Center, built in “I look down the road a hundred are prone to do, the original plant died 2000; work in the lab began in earnest years,” says Folsom. “What if the person after its first and only flowering. With in 2006. Folsom knew the technique in my position then says, ‘These 50 tissue culture taken from that pup about would be critical to furthering the species are extinct in the world and a year ago, The Huntington now has institution’s conservation practices you had them?’ It would almost be about 200 plants.Without tissue culture, within its gardens. As far as Folsom is criminal if we didn’t do all we could it would have none. “We’re making concerned, The Huntington not only to conserve them now.” them immortal,” says Harvey. has the unique capacity to engage in Folsom says that micropropagation Jim Folsom, the Telleen/Jorgensen a robust program of conservation for will one day become as routine as more Director of the Botanical Gardens, had its world-class succulent collection, it traditional forms of propagation are to- the foresight to include aTissue Culture bears the responsibility. day, such as grafting or hand-pollination.

24 Fall /Winter 2009 “If we don’t use every means at our disposal to reproduce the agaves that are dying, then we’re going to lose them,” lab work he says. Sean Lahmeyer likes to say that the Tissue Culture Lab became its own field An ambitious commercial nursery of dreams—once they built it, two volunteers with the right skills and expert - might produce 10 million plants annu - ise seemed to appear out of nowhere. Tim Harvey, a biochemist who has ally through tissue culture. Most poin - worked for Amgen, and Ann Miguel, a biophysicist who has worked at settias and orchids now on the market are Caltech, now spend every Tuesday in the lab, along with Cody Howard, an produced that way. The Huntington, of intern from the University of Arkansas. course, is not in the business of selling plants for profit, but it does propagate Perhaps it’s unusual that The Huntington’s Tissue Culture Lab is a volunteer- hundreds of plants annually for its driven program. The Huntington, however, is a volunteer-driven institution, International Succulent Introductions attracting highly skilled and motivated people to work in real ways throughout (ISI) program. Former Huntington its gardens and galleries. Why are Miguel and Harvey willing to donate an botanical director Myron Kimnach entire day each week, along with providing their scientific expertise pro helped found the program in Berkeley bono? “They believe in rare plant conservation and are fascinated with in 1956; it was formally adopted by fig uring out just what makes a plant grow,” says Lahmeyer. “It’s an intel - The Huntington in 1989 and continues lectual challenge that keeps them coming.” to propagate and distribute new and rare succulents to collectors, scientists, and research institutions. Ex-situ—or off- site—cultivation is a stopgap for plants that otherwise will go extinct as their habitats disappear. Conservationists believe it’s better to safeguard threat - ened plants in “captivity” than not to months, a bounty that would usually the potential once again to fulfill its have them at all, much like building a take at least five years to achieve. European orders. seed bank for ensuring the survival of Even with a program like ISI, The Cleanliness is not always a mere as many species as possible. Huntington’s ambitious reach has had its means to an end. Folsom says botani - Before the recent successes of the limitations.The Convention on Inter- cal gardens and institutions must be Tissue Culture Lab, it might have taken national Trade in Endangered Species able to produce disease-free plants that decades to propagate some of the slow- (CITES) is an international treaty that they can trade or exchange or they face growing plants in the collection. John regulates plant and animal trade between dire consequences. Presently Belgium Trager, curator of The Huntington’s countries. In recent years CITES has is losing, through disease, several cultivars Desert Garden collections and director tightened its restrictions regarding inter- from its National Collection of Camellias. of ISI, brought an agave into the col - national plant import and export. ISI Representatives from the National lection in the 1980s. Over the inter - shipments to Europe stopped in 2003, Collection have asked Lahmeyer ifThe vening 20 years he propagated a few a huge disappointment for the many Huntington can reintroduce some of offsets and now has a dozen plants. By collectors of succulents who’ve long those historic cultivars. propagating some plants from seeds or worked with The Huntington to Over the past three years, Lahmeyer from cuttings, through pollination or enhance their collections. However, has attempted to grow through tissue germination, it might take five or more sterile material in tissue culture flasks is culture Camellia japonica ‘Duc de Brabant,’ years to yield a good crop. Using tissue exempt from CITES regulations because Camellia japonica ‘Willmetta,’ and Camellia culture of one plant, Trager’s agave has there’s no concern that such shipments japonica ‘Comte de Tol.’ He’s drawing yielded 200 new plants in just nine will transmit diseases, pests, or pathogens. from The Huntington’s rich camellia months. In another instance, the vol - Clearly such specimens were not har - collection; Henry E. Huntington’s per - unteers in the Tissue Culture Lab vested illegally from the wild.This year, sona l landscaper William Hertrich turned one aloe hybrid into 300 in six The Huntington’s ISI program has imported significant Belgian camellias

HUNTINGTON FRONTIERS 25 The volunteers in the Tissue Culture Lab turned one aloe hybrid into 300 in six months.

to The Huntington early in the 20th “A lot of tissue culture is trial and have to pay close attention and try a century. So far success has been elusive. error,” says Lahmeyer. “The challenge variety of methods before you find The propagations have suffered fungal is in discovering the right combina - the one that works.” Despite the chal - contamination or just haven’t grown. tions of hormones and nutrients. You lenges, Lahmeyer is heartened by the prospects of such international con - servation partnerships. “This is a great use of our lab,” he says. Back in the lab, Harvey has finished slicing and dicing the sterile agave pieces, and Miguel then drops several pieces into a gooey concoction—sugar and vitamins mixed into water and agar, which is essentially a sterilized soil. The sugar is necessary since the plant tissue can’t yet produce its own; and the agar, a seaweed byproduct, turns the water into a gel that keeps the plants from sloshing around. Miguel (a retired biophysicist) and Harvey (a retired biochemist) are now faced with the challenge that makes most use of their scientific back - grounds—determining the right mix of hormones to make the plants grow. Hormones comprise the critical chem - istry that can be used to encourage plant growth. By applying small doses in the right combinations, Miguel and Harvey can prompt stems and leaves to

begin by washing your hands

1. Sterilize plants: Plants are naturally contaminated with microorganisms. Bleach or alcohol can be used to sterilize the small pieces of plants, known as explants.

2. Place sterilized plant tissue into growth media: Explants are then placed directly into a container of sugar, salt, vita mins, and plant hormones. A gelling agent, usually purified agar, is added to solidify the liquid media.

3. Add hormones: Doses of auxin often result in a proliferation of roots; an excess of cytokinin may yield shoots. As cultures grow, pieces are typically sliced off and transferred—or subcultured—to new media.

4. Transfer shoots to potting soil: As shoots emerge from a culture, they may be sliced off and rooted with more auxin to produce plantlets that, when mature, can be transferred to potting soil.

26 Fall /Winter 2009 grow first; when the plant is big enough, nurtures them until they are ready to about conservation, when he evoked the hormones encourage roots. Often be sent out into the world. the words of environmentalist Aldo an approach doesn’t work and the plant “The more we send out there the Leopold: “The first rule of intelligent won’t grow at all. “We’ll end up try - better,” she says, looking forward to the tinkering is to save all the pieces.” ing a lot of different approaches,” says new plants she andTrager will be offer - The Huntington is fulfilling that Harvey. “When something works easily ing in the 2010 ISI catalog. rule and then some. it gives us hope.” She is credited with creating a num - Finally, the plants are ready for the ber of aloe hybrids, four of which— climate-controlled growing room, ‘Princess Jack,’ ‘Gargoyle,’ ‘Dragon,’ and where they thrive under about 16 hours ‘dz’—will be the first tissue culture of artificial light each day. Successful plants offered to collectors and insti - plants that survive for six months or so tutions through ISI. Any surplus plants Traude Gomez Rhine is a freelance writer without succumbing to fungal contam - from the program become available for based in Pasadena. In the spring/summer ination are finally taken outside intoThe Huntington plant sales. 2006 issue of Huntington Frontiers , she Huntington’s greenhouse, where suc - The lab’s success puts a different spin wrote about the International Succulent culent propagator Karen Zimmerman on a sentiment Folsom had expressed Introductions program.

Left: Volunteer Ann Miguel places sterilized tissue in a climate-controlled growing room. Right: Succulent propagator Karen Zimmerman takes over when the plants become big enough for potting soil. Here she inspects a batch of Aloe ‘dz.’ Photos by Lisa Blackburn . Opposite: Aloe ‘Princess Jack,’ one of four aloe hybrids that have been successfully propagated in the Tissue Culture Lab. Photo by Karen Zimmerman .