VEGETATIVE PROPAGATION MICROPROPAGATION : AN IMPORTANT TOOL IN THE CON S ERVATION OF ENDANGERED HA W AIIAN PLANT S Nellie Sugii and Charles Lamoureux (808) 988-3177 [email protected] [email protected] Harold L. Lyon Arboretum University of Hawai‘i at Manoa 3860 Manoa Road Honolulu, Hawai‘i 96822 Abstract About half the taxa of native Hawaiian vascular plants are endangered in the biological sense. Some 25% have already been federally listed as endangered or threatened (272 endangered, 10 threatened); 49.5% of federally listed endangered plant species native to the U.S. are Hawaiian species. About 300 more have been significantly depleted and are currently treated as Species of Concern. Many endangered Hawaiian plants are exceedingly rare. Eleven taxa are currently known from only a single specimen of each remaining in the wild; more than 100 species currently have 20 or fewer plants remaining in the wild. Many rare Hawaiian plants have recalcitrant seeds, and standard seed storage techniques will not provide satisfactory long-term storage for maintenance of genetic diversity. In 1991 Lyon Arboretum initiated a project to apply any appropriate micropropaga- tion techniques to the conservation of rare Hawaiian plants. These techniques include tissue culture and cloning, as well as embryo culture, immature and mature seed culture. When material is available, embryo and seed cultures are the techniques of choice, in order to maximize genetic variability. The objectives of the project include (a) prevention of extinction of Hawaiian plant taxa, (b) propagating plants for use in approved restoration and reintroduction projects, and for garden use, and (c) main- taining an in vitro genetic safety net for the most critically endangered taxa. Lyon Arboretum works cooperatively with four other Hawaiian botanical gardens in the Center for Plant Conservation’s network, various state and federal agencies officially concerned with plant conservation and endangered species, private conservation agencies such as The Nature Conservancy, environmental organizations, and major private landowners, in joint conservation efforts. To date more than 80 federally listed endangered taxa have been successfully grown at Lyon Arboretum using micropropagation techniques. For several taxa specimens produced by micropropagation have been used for restoration / reintroduction proj- 43 VEGETATI V E PRO P AGATION ects. Advantages of micropropagation are endemic. The Angiosperm numbers for the production of mass quantities of include relatively low cost long-term represent the highest percentage of clonal material for the commercial ag- storage of large quantities of material, endemism found within a single large ricultural industry. Another area where and maintenance of whatever total island group anywhere in the world. micropropagation has been receiving genetic variability exists in very small Hawaii’s high rate of plant endemism an increasing amount of interest is that populations. For taxa with 20 or fewer can be attributed to its geographical of plant genetic conservation. plants remaining, our goal is to propa- isolation from continents and other gate every individual. Some taxa, when islands, isolation between and among reduced to small population sizes, ex- the individual islands, and its highly Micropropagation at Lyon hibit inbreeding depression manifested diverse microclimates. In the islands Arboretum in various ways. In two species studied elevations range from sea level to 4,205 In 1991, Lyon Arboretum initiated the which each had one remaining wild in- meters (13,796 feet), and average an- rare Hawaiian plant project utilizing dividual, the plants flowered regularly nual rainfall ranges from 178 to over micropropagation as a tool for plant but viable seeds were never produced. 11,430 millimeters (from 7 to over genetic conservation. The objectives of Both species produced embryos, which 450 inches). Following the advent of this project were to (a) prevent further aborted spontaneously before maturity. human discovery and colonization, extinction of Hawaiian plant taxa, (b) In both cases embryo culture resulted significant decline and in many cases propagate plants for use in approved in production of healthy seedlings. extinction of native Hawaiian plant restoration and reintroduction projects, taxa has occurred. and also for garden use, and (c) initiate Introduction Today, over one half of all the Hawai- and maintain an in vitro germplasm ian vascular plants are biologically collection of the critically endangered Hawaii, an isolated archipelago in endangered with approximately 25% plants included within the genetic the middle of the Pacific, is a favorite federally listed as endangered or threat- safety net listing. Lyon Arboretum vacation spot for many around the ened (272 endangered, 10 threatened) works cooperatively in joint conserva- world. Hawaii is known for its won- and another 25% significantly depleted tion efforts with four other Hawaiian drous natural displays such as erupting to be treated as species of concern botanical gardens in the Center for volcanoes, pristine white sand beaches, (approximately 300). Eleven taxa are Plant Conservation’s network, and clear blue seas and also its unique flora so rare that they are currently known with various state and federal agen- and fauna. What is surprising to many, only from a single specimen remaining cies officially concerned with plant is that the plants that have become in the wild. In addition, there are a conservation and endangered species, symbolic of Hawaii, such as the Bird more than a hundred species that are private conservation agencies such as of Paradise, orchids, plumeria and known from 20 or fewer remaining The Nature Conservancy, environmen- even the pineapple are not natives but wild individuals. Many of the remnant tal organizations, and major private recent human introductions. In fact, populations have been reduced so far landowners. the about half of all the plants growing that normal regenerating capabilities Theoretically, there exists a potential to without cultivation in Hawaii today have been impaired or rendered non- produce entire plants from small pieces are of foreign origin, and have been functional. of plant tissue, or explants. When introduced by human activity, mostly Plant micropropagation is a technology explants are placed in vitro, literally within the past 200 years. that has been developing and redefined meaning under glass, in an environ- Currently, there are approximately continuously over the past 30 years. ment free of microorganisms and onto 1000 native Hawaiian species of An- It has become an indispensable tool an appropriate nutrient rich medium, giosperms, of which 90% are endemic, in the areas of biotechnology, genetic they can regenerate into plants. Plant and 150 Pteridophytes of which 70% engineering, and in plant propagation, 44 VEGETATI V E PRO P AGATION explants for micropropagation are remain in a juvenile or juvenile-like but mature adults, suitable material either vegetatively or sexually derived. state. Therefore, it is possible to main- can still be collected from certain areas The most commonly used vegetative tain these plants for periods of several where the tissues mature less rapidly explants include apical, axillary and months to a few years in small vessels than in the rest of the plant. Plant tis- root meristems, stem nodes, stem within controlled environments. sues that tend to be more juvenile are internodes and leaves. They produce generally found on the lower branches In the micropropagation of Hawaiian plantlets called clones, which share of the plant. Suckers arising from the natives, it is of utmost importance to an identical genotype with each other stem, root, or stump also display de- preserve the integrity of the original as well as the original parent. These layed maturation. plant genotype; therefore any variables clones can eventually be multiplied which may jeopardize the genetic Apical and axillary meristems, when to produce more plants in a process stability during the culturing process induced to produce direct shoot re- known as cloning or clonal propaga- must be minimized. The ability to es- generation have been known to possess tion. Sexually derived explants such as tablish in vitro cultures of plants that greater genetic stability. Plants derived seed, embryos, ovules, and pollen pro- can regenerate normally and maintain from unorganized callus cultures origi- duce plantlets with unique genotypes. a high degree of genetic stability is nating from non-meristematic tissue One of the goals for all Hawaiian taxa dependent upon several factors. The such as stem internodes and leaves are that have 20 or fewer plants in the selection of suitable plant material and generally thought to be more geneti- wild is to vegetatively propagate the explants, proper surface disinfestation, cally unstable and their use discour- remaining individuals for the purpose plant medium and culture conditions aged. Sometimes non-meristematic of germplasm collection and storage. should all be considered. At Lyon explants are the only kind available for Seeds are also collected from these Arboretum, the majority of the plant micropropagation and callus culture individuals when available but in many material submitted is wild collected the only way to regenerate plants. In cases, inbreeding depression is exhib- and the establishment of an in vitro this case, organogenic or shooty type ited within these small populations culture