Using Anti-fungal Agents to Reduce Fungal Contamination for Micropropagation in the Classroom
Jason Okazaki
Mentor: Roger Shane Gold Department of Biology, Brigham Young University-Hawaii, 55-220 Kulanui St., Laie Hawaii

Introduction
Micropropagation is a method used for the multiplication of tissue culture in vitro. Fungal contamination is a major problem during explant micropropagation, as fungal growths greatly reduces survival and shoot proliferation. Fungal contamination is especially a problem in undergraduate teaching labs where inexperience and suboptimal culturing conditions tend to amplify the problem. The use of antifungal agents in culture may help alleviate these problems (Brown et al. 1982, Sheilds et al. 1984, Hauptmann et al. 1985, Tynan et al. 1993) . The purpose of this study was to explore the use of antifungal chemicals on Saintpaulia ionantha (African Violet), Daucas carota (Carrot), and Passiflora edulis (Passionfruit) by testing the efficacy of five commonly used antifungal compounds (Miconazole, PPM, Amphotericin B, Benomyl, Nystatin) as gauged by monitoring rates of fungal contamination and explant survival during in vitro micropropagation.

Results
There was a significant difference between the different antifungals used when comparing explant survival (p=0.011). PPM at 1.5 ml/L showed the best result with 75.0% of P. edulis explants surviving.

Conclusions
All explants of D. carota were contaminated throughout the experiment. This may have been due to improper sterilization of the explants. A longer sterilization of explants or more concentrated ethanol and hypochlorite solution may have been needed during the sterilization step. To optimize chances of survival, PPM at 1.5 ml/L should be used with P. edulis explants. Results showed that explants used at these concentrations (p=0.000) were affected least by fungal contamination (Figure 4). This meant that PPM affectively decreased the probability of fungal contamination with minimal effect to the explant survival when compared to other antifungals and concentrations used in this experiment. To optimize chances of callus tissue development, Miconazole at 30 mg/L should be used with P. edulis explants (p=0.001). The growth of callus tissue for P. edulis increased by three to five explants as the antifungal concentration increased (Figure 5). Explants grown on P. edulis at 30 mg/L also developed callus tissue at the fastest rate with ten explants developing callus tissue after three weeks (Figure 6). These results allude to the fact that Miconazole may stimulate callus tissue development, and increase the rate of development of P. edulis when used in media for micropropagation. Perhaps introducing antifungals to growth media allows explants to focus less on survival and more on callus development. Passiflora edulis had the greatest amount of tissue cultures that survived (39.4%) and developed callus tissue (48.3%) compared to S. ionantha (Figure 7). The data showed that P. edulis were least affected by antifungals and specified concentrations, and are better adapted to handle antifungal treatment in future micropropagation studies.

All explants of D. carota were contaminated at all antifungal concentrations throughout the experiment.

Figure 3: Fungal contamination of D. carota explants

Figure 4: Explant contamination ( PPM

), cell death ( ■ ), and survival ( □ ) for

Materials and Methods
Sterilization of Plant Tissue Culture in Clean Box (Figure 1): • Immersed explants in a 70.0% (v/v) ethanol solution for 40 sec. • Immersed explants in a 2.5% (v/v) sodium hypochlorite solution with 0.1% (v/v) Tween-20 for 15 min. (Dornelas and Vieira 1994). • Rinsed explants four times with sterile distilled water • All sterilized explants were trimmed into 1 cm x 1 cm pieces Agar Medium Proceedures: •Explants were deposited on MS agar medium (Murashige and Skoog 1962). ―4.4 g/L MS medium; 3% sucrose; 1.2% agar (Becerra et al. 2004) ―2 mg/L BAP (6-benzyl aminopurine) and antifungals at specified concentrations added after autoclaving (Table 1)
Incubation Proceedures (Figure 2): • Explants were incubated at room temp. (25°C) • Used Sylvania Gro-Lux bulbs (40W)
Table 1: Antifungal Concentrations

There was a significant difference between the antifungals used when comparing callus tissue development (p=0.001). Miconazole at 30 mg/L showed the best result with ten P. edulis explants developing callus tissue.

Miconazole at 30 mg/L showed the fastest callus tissue development rate with ten P. edulis explants developing callus tissue in three weeks.

Literature cited
Figure 5: Callus tissue development for explants of S. ionantha (□ ) and P. edulis (■) grown on Miconazole Figure 6: Callus tissue development rate for explants of P. edulis grown on Miconazole
Becerra, D.C., A.P. Forero, and G.A. Gongaro. 2004. Age and Physiological Condition of Donor Plants Affecting In Vitro Morphogenesis in Leaf Explants of Passiflora edulis f. flavicarpa. Plant Cell, Tissue and Organ Culture 79(1): 87-90. Brown, D.M., Groom, C.I., Cvitanik, M., Brown, B., J. I.Cooper, and J. Arditti. 1982. Effects of Fungicides and Bactericides on Orchid Seed Germination and Shoot Tip Cultures InVitro. Plant Cell, Tissue and Organ Culture 1: 165-180. Dornelas, M.C., and M.L.C. Vieira. 1994. Tissue Culture Studies on Species of Passiflora. Plant Cell, Tissue and Organ Culture 36: 211-217. Hauptmann, R.M., J.M. Widholm, and J.D. Paxton. 1985. Benomyl: A Broad Spectrum Fungicide For Use In Plant Cell and Protoplast Culture. Plant Cell Reports 4: 129-132. Murashige, T, and F. Skoog. 1962. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tobacco Tissue Cultures. Physiol. Plant 1: 474-497. Paul, A.L., Semer, C., T. Kucharek, and R.J. Ferl. 2001. The Fungicidal and Phytotoxic Properties of Benomyl and PPM in Supplemented Agar Media Supporting Transgenic Arabidopsis For Space Shuttle Flight Experiment. Appl. Microbiol. Biotechnol. 55: 480-485. Sheilds, R., S.J. Robinson, and P.A. Anslow. 1984. Use of fungicides on Plant Tissue Culture. Plant Cell Reports 3: 33-36. Tynan, J.L., Conner, A.J., R.C. Macknight, and R.T.M. Poulter. 1993. Miconazole: An Effective Antifungal Agent for Plant Tissue Culture. Plant Cell, Tissue and Organ Culture 32: 293-301. Watts, J.W., and J.M. King. 1973. The Use of Antibiotics in the Culture of Non-Sterile Plant Protoplast. Planta (Berl.) 113: 271-277.

There was a significant difference between the different tissue cultures used in this experiment (p=0.000). Explants of S. ionantha showed a total survival of 16.1% and callus tissue development of 16.7%. Explants of P. edulis showed a total survival of 39.4% and callus tissue development of 48.3%.

Acknowledgments
I would like to thank my mentor Roger S. Gold for his help throughout the whole project. I would also like to thank Randy Day for his help in statistical analysis, and the BYUH biology faculty for their advice and revisions of this paper. The financial assistance of the FAST program is gratefully acknowledged.

Figure 2: Incubation of explants Figure 7: Total survival ( □ ) and callus tissue development ( ■ ) for S. ionantha and P. edulis explants

Figure 1: Clean Box

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