Ve g e t a t i v e Pr o p a g a t i o n

Micropropagation : An Im p o r t a n t To o l in t h e Co n s e r v a t i o n o f En d a n g e r e d Ha w a i i a n Pl a n t 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 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 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 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 , 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-

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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 Ve g e t a t i v e Pr o p a g a t i o n

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 is often times hampered by callus is preferred due to its greater and result in plants, which produce no this fact. genetic stability. Viable mature seeds are generally propagated using con- seed, or seed that abort spontaneously The selection of suitable plant mate- ventional greenhouse practices but before they reach maturity. In the case rial not only pertains to the type of are placed into in vitro culture if where no viable mature seed are pro- explant used for culturing, but also to germ­plasm storage is requested. The duced, the immature seed, before they the time of harvest, juvenility of the optimal time to collect seed is just abort, can be collected and embryo or material and the general health of the before fruits dehisce or fall from the ovule culture attempted. Another prob- plant. Harvesting of vegetative cuttings plant. Seeds collected in this manner lem encountered with Hawaiian plants should be timed to coincide with the are not only assured of their parentage, is that many have recalcitrant seed and plant’s active vegetative growth cycle. but also easier to disinfest due to less standard seed storage techniques have Many times this is not possible due to microfloral contamination. proved to be inadequate. In these cases, two main factors, which are inadequate in vitro germination and storage of the information on the plant’s horticultural Initiation of in vitro cultures are usual- seedlings is currently the only way to requirements, and difficulty in obtain- ly more successful if explants are taken preserve the genetic diversity contained ing plant material due to limited access. from plants which are in reasonably within the seeds. Once the clones or Micropropagation of juvenile plants is good health. Any decline caused either seedlings are established in in vitro often much easier than with their by disease or senescence tends to be ir- culture, the plantlets continue to grow mature counterparts. While many of reversible and continue to progress in miniaturized leaves and stems and the plants in the wild are not juvenile the explants during micropropa­gation.

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Many of the critically endangered Ha- throughout the disinfestation process in 95% ethanol and quickly flamed waiian plant populations are very small, must be done to prevent over steriliza- prior to placing onto the germination fractured, older and in decline due to tion. Very dirty material may require medium. a multitude of environmental distur- one or more pretreatments before the The media formulations used at Lyon bances resulting in premature death of actual sterilizing of the final explant. Arboretum for micropropagation are existing plants and the lack of regenera- The pretreatments we use are long based on the Murashige and Skoog tion within the natural population. The water rinses, 3% hydrogen peroxide medium, commonly known as MS. poor condition of these species makes dips, 70% ethanol dips and Physan 20 This medium was originally developed collection of viable material suitable for soaks. The most common disinfectant for the rapid growth of tobacco tissue in vitro culture and successful micro- is sodium hypochlorite, which is found cultures, but has become one of the propagation extremely difficult. as a 5% solution in household bleach. most utilized medium formulations For disinfestation, the soaking solu- in the tissue culture of many kinds tions are prepared fresh by diluting the of plants and plant tissue worldwide. Techniques Employed bleach to a 5 (1 part bleach to 4 parts Different variations of this medium are water) and 10 (1 part bleach to 9 parts When material is collected for micro- made for the purpose of inducing spe- propagation, it is important that it is re- water) percent dilution with 1 drop of cific plant responses at specific stages ceived in the lab as quickly as possible. Tween20 added for every 100 ml of of in vitro plant growth. In Hawaii, Once in the laboratory, the material solution made. Vegetative propagules the plant genetic conservation effort is trimmed, washed and prepared for are usually soaked initially in the10% encompasses a considerably large and disinfestation and any extra material is bleach solution for 15-30 minutes. In diverse number of plant species where sent to the greenhouse for propagation. a petri dish containing 5% bleach solu- very little or no previous propagation Almost all of the vegetative cuttings tion, the explants are trimmed further information is known. In almost all and seed that are sent to Lyon are wild into final explant pieces with the aid cases, there is no time or sufficient or field collected. Primarily due to the of a dissecting microscope. The final plant material to optimize the culture high level of microflora found on and explants are placed into a 5% solution conditions, which makes appropriate within wild collected plant tissue, they for 1-15 minutes, rinsed well with media selection difficult. Media selec- must be subjected to a harsh chemical sterile water then placed onto the ap- tion is usually determined by the plant regimen before clean explant mate- propriate plant medium. itself and the type of culture desired. rial can be obtained for sterile culture. Intact immature fruits are usually There are basically three different types Sometimes the explants are lost during soaked in a 10% bleach solution for of tissue culture performed at the Lyon the disinfestment treatment before the Arboretum micropropagation lab; seed, 30 minutes to 3 hours. The contain- material can be sufficiently cleaned. If embryo and organ culture. ers with the fruit are brought into the wild collected vegetative cuttings can be transfer hood and the seeds excised Whole seeds may be sown in vitro established in the greenhouse, the new under aseptic conditions. The seeds are to produce intact plants. Seeds are growth is used as starting material for either left whole, or ovule or embryo germinated and maintained on a me- micropropagation since they are much culture is performed. Mature seeds are dium containing only one half of the cleaner and easier to disinfest. extracted prior to sterilization if they MS macro and micronutrients (1/2 Treatments and soaking times will vary are still in the fruit. The seeds are MS) with no hormones. Seeds that are according to how dirty the plant mate- washed well in water then soaked in immature, very small or rare benefit rial is and also on the sensitivity of the a 10% bleach solution for 15 minutes from in vitro germination. Embryo plant tissue to the cleaning agent. Care- to overnight. To enhance germination, culture entails the excision of the em- ful monitoring of the plant material the seeds may be scarified or dipped bryo from the seed coat. Embryos are

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placed onto a 1/2 MS medium with Examples of Restoration over 300 plants were produced no hormones but may contain a higher in the micropropagation lab by sugar concentration, coconut water or of Endangered in-vitro ovule culture. Several charcoal. Hawaiian Species Using plants have been returned to the Micropropagated Plants site where they are planted in The regeneration of plants from veg- a fenced area, and many others etative plant material that possess mer- We present four examples of successes are being grown successfully in istematic or non-meristematic tissue is attained: a nursery devoted to endangered accomplished through organ culture. 1. Delissea undulata. This species is plant culture at Volcano on the Organogenesis is encouraged, which a woody lobeliad with a single island of Hawai`i. With continued is the development of plant organs palm-like unbranched stem that protection from cattle chances of directly on the explant surface or upon may reach 10 meters in height restoration appear to be good. an intervening callus phase. A 1/2 MS and 5 centimeters in diameter, 2. pinnatifida. This lobeliad medium supplemented with hormones with a crown of leaves each about is a sparsely branched shrub to 3 such as auxins and cytokinins is used 20 centimeters long. Populations meters tall, with pinnately lobed to induce shoot and root growth. The were known originally from four leaves as much as 60 centimeters ratio of auxin to cytokinin is often islands; three of these disappeared long. It occurred in the southern manipulated to promote one type of during the nineteenth century. Wai`anae Mountains of O`ahu. growth as opposed to the other. In The Hawai`i Island population Although it was last collected in some cases, auxin is omitted during the was known to have persisted in 1965, it was known to survive as initiation of the cultures to promote small numbers until 1971 but oc- a single plant in the Palikea area. shoot growth and an auxin added later. curred only in an area devoted to This plant had been observed for Once whole plants are regenerated, cattle ranching, and was thought several years growing on a nearly they are maintained on a hormone to have disappeared in the early vertical slope that offered some free medium. 70’s. It is listed as “Extinct” in protection from feral goats. It the 1990 Manual of the Flower- To date more than 80 federally listed had never flowered and no natural ing Plants of Hawai`i. In 1992 endangered taxa have been successfully reproduction had been observed. at Pu`uwa`wa`a Jon Giffen, a grown at Lyon Arboretum using mi- Dr. Gregory Koob, then in charge wildlife biologist with the Hawai`i cropropagation techniques. For several of the Lyon micropropagation State Division of Forestry and of these taxa, specimens produced by lab, collected two lateral buds Wildlife found a single plant, 2 micropropagation have been used for from this plant and eventually meters tall, that had been knocked restoration and reintroduction projects. produced clonally more than 500 over by cattle but survived grow- In addition about 80 more species of new plants. The site with the ing horizontally over a deep hole concern have been grown successfully. that was the entrance to a lava remaining wild plant is now a Ware also concerned with conservation tube. He propped up the plant, preserve managed by The Nature of genetic resources of old Hawai- fenced it to keep cattle away, Conservancy of Hawai‘i, and ian crop plants, and are successfully and shortly afterward it began when a management plan is ad- maintaining in vitro about 70 unique flowering. This single individual opted several plants produced by Hawaiian cultivars of kalo (taro), Co- proved highly fertile. Immature micropropagation will be returned locasia esculenta. fruits were collected before birds to the wild. In the meantime two or insects could damage them, and plants flowered in the Lyon green-

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house in 1995. These were hand `Iniki in 1992 only two plants pollinated and several seeds were remained, both badly damaged. produced, although they aborted However, prior to the hurricane before reaching maturity. However the Lyon micropropagation lab a few new seedlings were produced obtained young fruit and pro- from embryos taken from very duced about 500 seedlings from young seeds. Also, several clon- immature seeds. Several have been ally produced plants were sent to returned to the State Division of a private reserve on the island of Forestry and Wildlife and are be- Kaua‘i, where they have thrived, ing planted on the original site. flowered, and produced viable seeds. Thus, to the extent that the original plant was heterozygous it Conclusions now seems possible to restore at These examples demonstrate that spe- least a small, amount of genetic cies can be rescued from extinction, variability to this species. multiplied using various micropropa­ gation techniques, and restored in the 3. Cyanea superba. Plants of this spe- wild, when appropriate management of cies are palm-like trees to 6 meters their habitats can be undertaken. But, tall, with leaves to 1 meter in given the magnitude of the problem, length. Cream-colored flowers to work has just begun. 8 centimeters long are produced on 35-centimeter long pendent racemes. The species is confined Literature Cited to the northern Wai`anae moun- tains of O`ahu. Five plants are Wagner, W.L., D.R. Herbst and S.H. known to remain in the wild, in Sohmer. 1990. Manual of the Flow- . 2 Vols., the Pahole State Natural Area Re- ering Plants of Hawai`i 1854 pp. University of Hawai`i serve. Many plants were produced Press, Bishop Museum Press; in the micropropagation lab using Honolulu. immature seed culture; about 100 have now been returned to the wild, and the first flowers have just been produced on these plants.

4. Cyanea asarifolia. This lobeliad is a shrub less than one meter tall, with small leaves and white flow- ers about 3.5 centimeters long. It was described as a new species from a collection made in 1970 at Anahola Valley on Kaua`i. The only known colony contained about 10 plants. After Hurricane

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