ENDOGENOUS CONTAMINATION IN MICROPROPAGATION OF CASTANEA SATIVA MILL.

In in vitro tissue culture, contamination of explants can be caused by endogenous microorganisms. In this study, bacteria were isolated from seven clones of chestnut. Material was collected in three different stages: vegetative rest, bud formation and fruit maturation. The buds were submitted to different treatments of disinfection: hot-water, H₂O₂ and Ca-hypochlorite. 109, 72 and 61 bacteria isolates were obtained, respectively, as well as a total of 25 yeasts isolates. The treatment with Ca-hypochlorite demonstrated more effectiveness than H₂O₂ treatment. The hot-water treatment was more effective on Gram-negative organisms, and the growth of Gram-positives was only delayed. Contamination depends on the sample’s vegetative stage, localization and explant type. The “Colutad” clone was selected for micropropagation because it presented high resistance to the ink-disease. Thus, buds were collected from adult and juvenile trees in the field and juvenile trees in a greenhouse. The buds were surface-sterilized and cultivated in GD media supplemented with BAP. In the establishment phase, the explants from the field presented higher levels of contamination than those that were grown in the greenhouse. Also, explants from the greenhouse presented the highest rate of multiplication. Explants that had not developed in 13 weeks and had no contamination were smashed and cultured in NB. After 5 days of incubation, isolation was made on NA and some of them developed bacteria and yeasts. This result confirms the endogenous origin of these microorganisms. Bacillus and Pseudomonas were the most abundant isolated genera. In rooting phase, 10 isolates were also obtained.