Joyce Maschinski, Center for Plant Conservation and Valerie Pence, Cincinnati Zoo & Botanical Garden
Crotalaria avonensis is an endangered legume endemic to Florida that produces few seeds. In vitro shoot cultures of multiple genotypes have been grown at CREW to provide genetic diversity for restoration and for tissue cryopreservation. These cultures harbor a bacterium, identified as Paenibacillus sp., which may be a natural endophyte in the species. The bacterium grows slowly and does not appear to inhibit the in vitro propagation of the species, but its effect on the recovery of shoot tips after the stress of cryopreservation was investigated. Samples banked using encapsulation vitrification and representing 63 genotypes were evaluated after 4 - 15 years in liquid nitrogen. The rate of recovery growth of samples with visible bacteria was significantly less than samples without bacteria. Similarly, when newly banked shoot tips of 15 genotypes were cryopreserved using an improved technique, droplet vitrification, and were recovered, the presence of antibiotic in the medium significantly increased the percent of shoot tips showing recovery growth. Whereas C. avonensis shoots can be propagated, rooted, and acclimatized in the presence of this bacterium, recovery after the stress of cryopreservation is reduced when the bacteria are present. An increasing number of plant species are being shown to have endophytes in the wild and removing such endophytes may not be possible or desirable in culture. These results with C. avonensis demonstrate the potential for controlling the negative effects of such microorganisms in vitro. This is one example of the particular challenges that may be presented in working with wild species and conserving endangered exceptional plants. Supported in part by grants from the Institute of Museum and Library Services.