tissue culture

Valerie Pence and Megan Philpott, Cincinnati Zoo & Botanical Garden

Crotalaria avonensis is a Florida endemic found in three populations and characterized by low seed production. In the late 1990s, CREW developed protocols for tissue culture propagation from field collected shoot cuttings as well as cryopreservation methods. In order to develop a genetically representative collection for conservation, in vitro lines were established from shoots collected at all three populations from 2008-2012. Plants were produced and sent to Bok Tower Garden for further growth and for use in an outplanting by Archbold Biological Station. The resulting collection of genotypes in culture at CREW provides an example of the challenges of a genetically diverse collection of an exceptional species. C. avonensis cultures require maintenance subculturing every 2-3 months. Only a low number of replicates could be maintained for each genotype, resulting in some loss of genotypes over time. Cryopreservation offered a solution to this challenge and over the course of 16 years, a number of lines were cryopreserved. In a study of lines stored for 5.5 Ð 16 years in liquid nitrogen, there was no change in average viability of the collection in storage, although specific survival differed by genotype. A cost estimation indicated that cryopreservation could decrease the cost of maintaining the collection over 20 years by at least 1/3. Genetic analysis of the collection and the wild populations is also underway in order to determine the genetic representation of the collection.

Contributing Author(s): 
Date Recorded: 
Friday, May 3, 2019

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.

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Date Recorded: 
Friday, May 4, 2018

Although many rare plants have seeds that can be stored by conventional methods, not all species have seeds that can live after drying or freezing. Sometimes called "Exceptional plants, " these species have a wide range of variation. Some produce few or no seeds, thus they cannot be seed banked, others have seeds or spores that will die if dried or frozen, while others have seeds that can tolerate drying, but not freezing, and another group have seeds that live less than 10 years at freezing temperatures. Recent studies suggest that there are many rare plant species that need alternative storage for conservation. This video describes a procedure for  testing whether rare plants can be stored in liquid nitrogen successfully.

Date Recorded: 
Friday, March 1, 2019

Peter Zale and Matt Taylor, Longwood Gardens

Several species of Spiranthes native to the Eastern U.S. are considered rare, threatened or endangered by federal and state agencies. Using the Pennsylvania endangered Spiranthes casei as a model species, experiments were designed to determine optimal conditions for in vitro seed germination and seedling development. Seeds were collected in November 2015 from 10 individual plants found in three subpopulations in Elk and McKean counties, Pennsylvania, and air-dried for six weeks. Seeds were surface sterilized for 10 or three minutes in a 10% bleach solution, then plated onto a commercially available terrestrial orchid seed germination media: P723, M551 or K400 (Phytotechnology Labs, Shawnee Mission, KS) with 5 replicate plates. Seed germination ranged from 24 to 60 % and occurred on all three media only with the 3-minute treatment. None of the seeds treated with bleach for 10-minutes germinated and visual inspection revealed badly damaged embryos. After shoot initiation, 150 seedlings were transferred to individual test tubes on one of two media (P723 or P658) and each was given one of the three 24-hour light/dark photoperiod treatments for 10 months: 24/0, 18/6, or 0/24. Seedling survival and growth occurred in all treatments, but seedlings on P723 with the 24/0 or 16/8 photoperiod treatments had a significantly greater fresh weight, leaf length, number of roots and root length than light treatments on P658 and dark treatments. Results indicate Spiranthes seeds can be damaged by extended chemical scarification times and the light is essential for optimal seedling growth.

Contributing Author(s): 
Date Recorded: 
Friday, May 4, 2018