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Stepwise Disinfestation Reduces Contamination of Huperzia Lucidula

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Stepwise Disinfestation Reduces Contamination of Huperzia Lucidula PROPAGATION & TISSUE CULTURE HORTSCIENCE 38(4):565–567. 2003. dium contained (per liter): 50 mg MgSO4·7 H2O; 26.5 mg CaCl2·2H2O; 50 mg K2PO4; 50 mg NH4Cl; 2 g dextrose; and half-strength Stepwise Disinfestation Reduces Murashige and Skoog (1962) basal salts micronutrients. Semi-solid medium con- Contamination of Huperzia lucidula tained 0.8% Phytagar (Gibco , Invitrogen, Carlsbad, Calif.) as a gelling agent. The pH Shoot-tips and Gemmae was adjusted to 5.1–5.3 before autoclaving (Whittier, 1986, 1998). Alice Spurlin Waegel1 Standard disinfestation. Three lots of 48 Division of Arts and Sciences, Neumann College, Aston, PA 19014 shoot-tip cuttings were washed in 250 mL tap water with one drop of detergent (Lemon Joy, Additional index words. shining club moss, in vitro culture, contamination, surface Proctor and Gamble, Cincinatti) for 1 min; then sterilization explants were immersed in 70% ethanol for 30 s with agitation. Individual explants in 25-mm Abstract. Shining club moss, Huperzia lucidula (Michaux) Trevisan, can be propagated in ×100-mm tubes were then treated with 1% w/v tissue culture, but it is difficult to obtain sterile explants. When standard disinfestation sodium hypochlorite/0.15% Tween 20 solution methods were used, 100% of H. lucidula apical shoot-tips disinfested for 10, 20, or 30 for either 10 min (lot A), 20 min (lot B), or 30 minutes with 1% sodium hypochlorite/0.15% Tween 20 became contaminated or browned min (lot C). Cuttings were then washed once within 3 weeks. Since this club moss is highly contaminated, a stepwise disinfestation pro- with 10 mL sterile distilled water, trimmed to cedure was developed to eradicate spore forming microbes. Based on the principle that remove bleach damaged areas on stem bases vegetative microbial cells are more easily destroyed than spores, stepwise disinfestation and cultured on semisolid medium. induced spore germination before explant treatment with sodium hypochlorite. A 48-hour Repeat disinfestation: All cuttings (144) disinfestation treatment resulted in 50% explant survival without visible contamination. undergoing standard disinfestation eventu- Fewer explants (22%) were successfully disinfested when a 24-hour stepwise disinfestation ally became contaminated and received a was used. Applying the stepwise disinfestation method to contaminated cultures after a repeat disinfestation with 1% w/v sodium standard disinfestation protocol produced an additional 15.6% contamination-free club hypochorite/0.15% Tween 20 for 20 min, moss shoot-tips. After successful surface disinfestation, H. lucidula shoot-tips and gemmae rinsed as above. When contamination persisted established in vitro grew normally. after several additional treatments, affected explants were either stepwise disinfested or Shining club moss, Huperzia lucidula (Mi- are relatively ineffective against spores, discarded as the explant size diminished fol- chaux) Trevisan, is an attractive northeastern surface sterilizing club moss explants can lowing trimming of damaged stems. United States native that deserves better be difficult. Stepwise disinfestation. Explants were representation in today·s gardens. Efforts Toovercome the spore problem, a stepwise washed in tap water with one drop of detergent have been made to propagate this plant since disinfestation procedure was developed for for 1 min, immersed in 70% ethanol for 30 s the 1700s (Barrows, 1935), but these efforts decontaminating Huperzia lucidula shoot- tip with agitation, then treated with 1% sodium have been fraught with difficulties. Growing cuttings and gemmae prior to culture. This hypochlorite /0.15% Tween 20 for 20 min. H. lucidula from spores is not practical since new decontamination process was based on a Single explants were removed from the bleach the germination rates are low and growth method to sterilize heat labile culture media, and rinsed in individual 25- × 100-mm tubes is slow, regardless of the growing medium, described as fractional sterilization (tyndal- containing 10 mL sterile distilled water for 10 light, temperature, and alcohol treatments lization) by the 19th century British scientist min on a shaker (160 rpm). After rinsing, each (Barrows, 1935). Only 0.3% of Huperzia John Tyndall. Stepwise disinfestation, like explant was placed in a culture tube containing lucidula spores germinated in vitro after 9 tyndallization, operates on the principle that 10 mL of liquid medium and incubated on the months, with none producing mature game- vegetative microbial cells are more easily shaker (under cool-white fluorescent lights, tophytes in culture (Whittier, 1986, 1998). killed than spores. However, stepwise disin- 50 µmol·m–2·s–1, 16 h-photoperiod) at room Based on this information, mass production festation uses sodium hypochlorite solutions to temperature for 24 h. of shining club mosses from spores appears kill vegetative microbes, whereas tyndalliza- At the end of the 24-h incubation period, extremely unlikely. tion uses heat. In both processes, a bactericidal 10 mL of 2% sodium hypochlorite/0.3% Tween Although cultivating shining club moss treatment (heat or bleach) is followed by an 20 (double strength) was added to each culture from spores has met with little success, incubation period under conditions favorable tube, mixed with the equal volume of medium growing sporophyte cuttings and gemmae for the germination of microbial spores. Af- in the tube and shaken for 20 min. Individual has been more productive (Barrows, 1935). ter incubation, the bactericidal treatment is explants were removed and placed in tubes Cuttings root readily in a mixture of peat and repeated to kill newly produced vegetative with 10 mL sterile distilled water for a 10- sand under greenhouse conditions so they may cells, followed by another incubation and a min rinse with agitation. In the 24-h stepwise also grow in tissue culture. One difficulty with third bactericidal treatment—the intent being disinfestation procedure, club moss explants culturing H. lucidula is obtaining sterile ex- that all fungal spores and bacterial endospores were trimmed after rinsing to remove bleach plants. Shining club moss grows close to the will have germinated and the resulting vegeta- damaged basal ends, and cultured on club ground and has subterranean gametophytes tive cells killed. moss agar. In a 48-h stepwise disinfestation associated with mycorrhizal fungi, so explants procedure, club moss explants were incubated tend to be highly contaminated with soil mi- Materials and Methods in club moss broth (after rinsing) for a sec- crobes, including spore forming bacteria and ond 24-h period and treated again with double fungi. Since standard chemical disinfestation Explants. Huperzia lucidula shoot-tip strength sodium hypochlorite solution for 20 methods using sodium hypochlorite (bleach) cuttings and gemmae were collected from min before rinsing, trimming and culturing woodlands in southeastern Pennsylvania and on semi-solid medium. northwestern Delaware in Sept. 2000 (144 After culture, explants were inspected Received for publication 11 Mar. 2002. Accepted for shoot-tips), Oct. 2000 (28 shoot-tips and daily for evidence of microbial growth, using publication 17 Sept. 2002. This study was made pos- sible with the assistance of Sherry Kitto, who provided 8 gemmae), and Aug. 2001 (30 gemmae). a stereoscopic microscope. Contaminated cul- facilities, instruction, and encouragement during my Explants were transported and maintained at tures were subjected to additional stepwise sabbatical leave at the Plant and Soil Sciences Dept., 4 °C for 1–2 d until disinfested and cultured. disinfestation or discarded. College of Agriculture, Univ. of Delaware. A typical specimen is shown in Fig. 1A. Statistical analysis. Club moss explants (n 1Professor. Culture medium. Club moss liquid me- = 18 for each group) were treated with either HORTSCIENCE, VOL. 38(4), JULY 2003 565 16-7256, p565-567 565 6/29/03, 3:05:12 PM PROPAGATION & TISSUE CULTURE nal explants were discarded due to refractory contamination and diminished size. Besides reducing explant size, repeated exposure to sodium hypochlorite caused pale stripes to appear on the microphylls of H. lu- cidulacuttings. This “zebra phenomenon” did not persist; in the absence of further exposure to sodium hypochlorite, green color returned to the explant after a few weeks in culture. GreenH. lucidula shoot-tips free of visible contamination (Fig. 1B) began to grow in vitro after 5 weeks. First the apex would broaden, increasing in width from §2 mm to 4 mm in Fig.1. (A) Huperzia lucidula in its native habitat. Shoots 14–20 cm. (B) Shoot-tip in culture 5 weeks after diameter. Next, shoot-tips split and branched initiation, height 32 mm. (C) Gemma producing shoot in vitro 5 weeks after initiation, height 6 mm. dichotomously in the typical Lycopodium pattern. Cultured H. lucidula gemmae grew differently than apices, but within the same a 20-min standard disinfestation (control), a better than the 24-h treatment (22%) through time frame. After 5 weeks in axenic culture, 24-h stepwise disinfestation or a 48-h stepwise 38 d (Fig. 3). After a 48-h stepwise disinfes- gemmae produced small shoots with more disinfestation. All explants were monitored for tation of 30 gemmae collected in Aug. 2001, rounded microphylls than a mature sporo- visible contamination for 38 d. The percent- 50% remained free of visible contamination phyte (Fig 1C). After the shoot emerged and age of contaminated cultures over time was for 6 months. obtained a height of several millimeters, one calculated for each group and the Student·s t Both 24- and 48-h stepwise disinfestation fi to two roots developed from the proximal root test (1 tailed) was used to determine the mean results were signi cantly different from 20 primordium of the gemmae and penetrated 3-4 test difference between the control group and min standard disinfestation results with the mm into the culture medium. Unlike Gillen t P each stepwise disinfestation group. Student's test ( 0.01for 24 h vs. 20 min and Basile (1986), who reported abnormal P standard disinfestation; 0.001 for 48 h growth of gemmae in culture, the gemmae Results vs.
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