Overcoming the challenges of endogenous contamination in micropropagation of fruit and nut trees^©

Micropropagation has become a successful technique for fruit and nut tree rootstocks. However, success of a laboratory to produce a high rate of healthy shoots during multiplication stage depends on its ability to maintain cultures free from contamination. Although, all labs can easily start a clean culture from an explant, many labs report a flare-up of bacterial contamination after a few cycles of multiplication despite using their best laboratory practices. This flare-up is often blamed on endogenous bacteria within the micro shoots. This assumes that such bacteria were always present in the shoots, but were in quiescent stage and/or were non-culturable and suddenly they became active and grew on culture media. Such theory is believable since presence of endogenous bacteria in plants is well-known in the literature and only 1% of bacteria are culturable. To overcome this challenge of flare-up of contamination (endogenous or introduced), a laboratory should have a protocol in place to index their stock materials on a regular basis. For culturable bacteria, contamination can be detected by culturing samples of tissue in a nutrient broth for bacteria. For non-culturable bacteria, sections of stems can be eluted in water and the eluate observed under the microscope for bacteria. Bacteria-specific PCR tests are now available and are helpful. These procedures along with shoot tip cultures, occasional use of antibiotic and close visual observation have proved successful at Micro Paradox in maintaining our nuclear stock of walnuts, pistachio, and peach × almond hybrids free from contamination.