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Title: CONTROL OF BACTERIAL CONTAMINATION IN LARGE SCALE PAPAYA MICROPROPAGATION

Author
item Fitch, Maureen
item LEONG, T - HI AG RESEARCH CENTER
item SAITO, N - HI AG RESEARCH CENTER
item YAMAMOTO, G - HI AG RESEARCH CENTER
item DELA CRUZ, A - HI AG RESEARCH CENTER
item YEH, A - HI AG RESEARCH CENTER
item WHITE, S - HI AG RESEARCH CENTER
item Maeda, Sharyn
item FERREIRA, S - UNIVERSITY OF HAWAII
item Moore, Paul

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Abstract Only
Publication Acceptance Date: 3/20/2003
Publication Date: 5/20/2003
Citation: Fitch, M. M. M., Leong, T., Saito, N., Yamamoto, G., Dela Cruz, A., Yeh, A., White, S., Maeda, S. H.., Ferreira, S., Moore, P. H. Control of bacterial contamination in large scale papaya micro propagation. In Vitro Cell Develop Biology - Plants 39 (Abs) : 19A. 2003

Interpretive Summary:

Technical Abstract: Papayas were micropropagated for research projects for many years, and in 2002 scale up for commercial scale production was initiated. Problems associated with increased propagation were anticipated, but the discovery of slow-growing, cryptic bacteria in the culture population and stocks was, by far, the most devastating. The most serious bacterial pests were those that were barely visible without the aid of a dissecting microscope. Contaminated papayas in culture plates of proliferation medium, modified Murashige and Skoog (MS) medium containing 0.2 mg/L each of benzylaminopurine and naphthalene acetic acid, appeared green and healthy, but when shoots were placed in rooting medium, vermiculite moistened with ½ MS salts and 3% sucrose, the leaves senesced, root formation was slow and poor, and at temperatures above 28oC, the shoots sometimes bleached and died. Assays to screen stocks for contamination status were difficult because the bacteria grew very slowly on standard bacterial culture media, e.g., Luria broth, malt extract, or potato dextrose agar. The most dependable assay for contamination was visual scoring of each plate prior to subculture coupled with tests of stock lines to observe the amount of root development and shoot growth that occurred in rooting medium. If large, dense root masses and deep green, leafy canopies formed in 7-21 days, the stock cultures were presumed to be free of bacteria. Infected stock cultures were decontaminated either by treatment with the addition of carbenicillin at 500 mg/L and/or cefotaxime at 200 mg/L to the proliferation medium for one month or by agitating shoots in 20% commercial bleach for 10 min. Following decontamination, shoots with preformed roots were placed in the rooting medium and root mass and shoot development monitored. Contaminated shoots were placed in photoautotrophic rooting media devoid of sucrose. In contrast to senescent contaminated shoots in sucrose-containing media, the shoots in the photoautotrophic medium remained green and healthy in appearance. Contaminated shoots rooted about 1-2 months later than uncontaminated plants in the sucrose-containing medium. To control future problems with bacterial contamination, each culture plate was examined under a dissecting microscope prior to subculture and stocks were continually tested for rapid root and canopy formation in rooting medium containing sucrose. Although labor intensive, monitoring of all cultures helped minimize the incidence of contamination.