Flaming Fabaceae —Using an Alcohol Flame to Break Seed Dormancy Nellie Sugii | n 1991, the Lyon Arboretum on O‘ahu initiated the Rare Hawaiian Plant Pro- Igram utilizing micropropagation as a ABSTRACT tool for plant genetic conservation. The mission of this project was to prevent fur- Soaking Fabaceae seeds in alcohol and then passing them through a flame for ther extinction of Hawaiian plant species, 2 to 3 s is a quick and easy way of promoting germination of at least 8 species propagate plants for use in approved of Hawaiian legumes. In addition to stimulating germination, the technique restoration and reintroduction projects, effectively sterilizes seeds for in vitro germination for mircropropagation or for and initiate and maintain an in vitro germination in the greenhouse. germplasm collection of the “critically endangered” plants included within the KEY WORDS Genetic Safety Net List (GSNL). The Micropropagation, greenhouse, threatened and endangered, GSNL generated by the Hawai‘i Rare Plant recalcitrant, legume Restoration Group is a collection of plant taxa identified as “critically endangered” NOMENCLATURE and given top management priority. ITIS (2002) Plant micropropagation is a technol- ogy that has been developed and rede- fined continuously over the past 30 years and has received an increasing amount of interest as a propagative method for plant genetic conservation (Dodds 1991). In Hawai‘i, micropropagation is commonly used to propagate many Hawaiian plant taxa, including our most “critically endangered” for germplasm storage, ex situ use, and restoration and reintroduc- tion projects. Micropropagation is espe- cially suitable when: 1) the plant species are difficult to propagate using conven- tional propagative methods; 2) viable plant propagules are rare due to inbreed- ing depression or difficulty in accessing plants; 3) plants have very small, recalci- Photos by Nellie Sugii trant, or immature seeds or spores; or 4) only poor quality propagules are available Figure 1. Flaming the alcohol off a Canavalia Figure 2. A Canavalia napaliensis seedling about due to unhealthy parent stock. seed enhances germination. 2 wk after treatment. 46 NATIVEPLANTS | SPRING 2003 In Hawai‘i, a total of 13 native genera bench top if you plan to germinate the Within 24 h seeds swell up to 33% with 16 endemic species occur within seeds in a greenhouse). Keep a petri greater than their original size and within the family Fabaceae (Wagner and others dish or some other cover close at hand 3 to 4 d germination is visible. With 1999). The fruit is usually a legume and in case you set the alcohol on fire. Canavalia napaliensis,we had a 7.5-cm- typically possess hard, smooth seeds. Smothering the flame by covering the long (3-in) seedling in just 2 wk (Figure Physical dormancy is present, caused by beaker will extinguish the fire. 2). One main advantage of this technique an impervious seed coat that requires 3. Place the seeds into the beaker and let is that it saves us time—without flaming, scarification to initiate the germination them soak for approximately 1 min these seeds normally take at least 6 mo to process (Janick 1979). (do not rinse off bleach solution). germinate in our shadehouse. At the Lyon Arboretum we work with 4. With forceps, extract seeds one or two very small amounts of Fabaceae seeds and at a time and pass them briefly (2 to 3 have found that germination can be expe- s) through a flame (Figure 1). REFERENCES dited as well as enhanced by quickly pass- 5. Extinguish seeds by dropping them ing alcohol-coated seeds through a flame into sterile water (if you plan to ger- Dodds JH. 1991. Introduction: conservation of and allowing the alcohol to burn. Flaming minate seeds in the greenhouse, you plant genetic resources—the need for tissue is apparently effective in weakening the need only clean water). You may also culture. In Dodds JH, editor. In vitro meth- hilum, which then allows water imbibi- extinguish the seeds in vitro by plac- ods for conservation of plant genetic tion to occur. The heat also efficiently ster- ing them directly onto the agar sur- resources. United Kingdom: Chapman and ilizes seeds for in vitro seed sowing or even face in your culture vessel. Hall. p 1–9. for propagation in a clean greenhouse [ITIS] Integrated Taxonomic Information System. seed tray. So far, we have successfully Seed germination in vitro does not 2002. Biological names. Version 4.0 (on-line propagated 8 endemic Hawaiian species require any special media formulation. In database). URL: http://www.itis.usda.gov (ac- (Table 1) with the following procedure. the laboratory, we use a half-strength cessed 1 Nov 2002). Murashige and Skoog (MS) medium Janick J. 1979. Horticultural science, 3rd edition. (Murashige and Skoog 1962) without New York (NY): WH Freeman and Company. FLAMING PROCEDURE hormones, solidified with Phytogel ®, and Murashige T, Skoog F. 1962. A revised medium pH adjusted to 5.7 to 5.8.After extinguish- for rapid growth and bioassays with tobacco 1. Soak seeds in a 10% Clorox™ bleach ing the seeds on the agar, the culture ves- tissue cultures. Physiologia Plantarum solution for 15 min, providing gentle sels with seeds are kept at approximately 15:473–497. agitation. 22 °C (72 °F) under a 14-h light and 10-h Wagner WL, Herbst DR, Sohmer SH. 1999. Man- 2. In a transfer hood, pour 95% ethyl alco- dark cycle. In the greenhouse, we sow ual of the flowering plants of Hawai‘i, revised hol (you may substitute with 70% iso- seeds into trays containing a 3:1 (v:v) per- edition. Honolulu (HI): University of Hawai‘i propynol) into a small beaker or lite:vermiculite seed mix and place them Press. Bishop Museum Special Publication 97. fireproof container (you may also do under 50% shade with prevailing Hawai- this as a “non-sterile” technique on the ian temperature conditions. AUTHOR INFORMATION TABLE 1 Nellie Sugii Species in the Fabaceae successfully germinated using an alcohol flame treatment. Assistant Researcher Harold L Lyon Arboretum Scientific name Common name University of Hawai‘i at Manoa Acacia koa Gray koa 3860 Manoa Road Honolulu, HI 96822 Caesalpinia kavaiensis Mann. uhiuhi [email protected] Canavalia kauaiensis Sauer. Kaua‘i jackbean Canavalia napaliensis St. John Makaha Valley jackbean Erythrina sandwicensis O.Deg wili wili Sesbania tomentosa Hook. & Arn. ‘ohai Sophora chrysophylla Seem mamane Vicia menziesii Spreng. Hawai‘i vetch Vogel O‘ahu cowpea Vigna o-wahuensis 47 NATIVEPLANTS | SPRING 2003.