Eric Menges is conducting demographic and fire ecology studies.

Tissue culture propagation protocols have been developed for this species at CREW (Cincinnati Zoo & Botanical Garden) and used to propagate plants for outplanting in Florida in collaboration with Bok Tower Garden and Archbold Biological Station. Shoot tip cryopreservation protocols have also been developed and used to bank multiple genotypes of this species in CREW's CryoBioBank. Bernadette Plair and Valerie Pence (Cincinnati Zoo and Botanical Garden) are micropropagating this species to increase their numbers for research and possible reintroduction efforts (Plair and Pence 2000)

Tissue culture propagation protocols have been developed for this species at CREW (Cincinnati Zoo & Botanical Garden) and used to propagate plants for outplanting in Florida in collaboration with Bok Tower Garden and Archbold Biological Station. Shoot tip cryopreservation protocols have also been developed and used to bank multiple genotypes of this species in CREW's CryoBioBank.

Tissue culture lines and cryopreserved shoot tips of multiple genotypes are maintained at CREW.

Crotalaria avonensis, a federally endangered legume which produces few seeds, is found in only 3 populations in the South Central Florida scrub. Several years ago, the Cincinnati Zoo and Botanical Garden (CREW) was asked to develop an invitro propagation and shoot tip cryopreservation protocol for this species. CREW has since cultured and cryopreserved over 120 genotypes representing all 3 populations and has contributed plants to a restoration project. For cryopreservation, small shoot tips about 1mm in length are dissected and put through a cryoprotective protocol and banked in liquid nitrogen. In earlier work, the Encapsulation Vitrification method was used but more recently Droplet Vitrification has proved to be more effective as a cryopreservation method. Over the years of maintaining the cultures, it was noted that there was a subtle growth of bacteria that could be detected visibly in most of the cultures, but which did not appear to hinder their growth. This bacteria has been identified by colleagues in Cincinnati Children's Hospital as a species of Paenibacillis using mass spectroscopy with time of flight. Information from this literature indicates that Paenibacillis is a common soil bacterium which can also be a plant endophyte and it has been reported from invitro cultures of other species.As part of an Institute of Museum and Library Services (IMLS) funded study, 136 samples of shoot tips (63 genotypes) were removed from long-term storage of 4 to 15 years in liquid nitrogen. All of these had been cryopreserved using the Encapsulation Vitrification method. While there was survival in most of the samples some bacteria were seen around the pieces in these recovery plates. Even though some of the pieces that were growing also showed bacteria it was most noticeable around the pieces that weren't growing. During the evaluation of the survival of samples that showed some bacteria and those that did not, it was observed that there was significantly more survival at 4 weeks in visibly clean cultures (using antibiotic Rifampicin) than those that showed some Paenibacillis bacteria. Similarly, when newly banked shoot tips of 15 genotypes were cryopreserved using the improved technique of Droplet Vitrification, and were recovered, the presence of Rifampicin in the medium significantly increased the percent of shoot tips showing recovery growth. Based on these results, the focus was then placed on growth of stock cultures. While the cultures do grow without the additional antibiotic, in some cases there is an increase in growth in the presence of the antibiotic. Observations show that when tissues are removed from the antibiotic the visible bacteria will return. Summary and Conclusions: (1) The subtle bacterium found in stock cultures of Crotalaria avonensis has been identified as Paenibacillus, and has been identified as an endophyte in other species. (2) While it does not appear to have a strong effect on the growth of the cultures, it can significantly inhibit the amount of recovery after cryopreservation. (3) Further studies are underway to identify the bacterium from newly collected tissues of Crotalaria avonensis. (4) With the increasing understanding of the roles of endophytes in plant growth, when dealing with rare species, it may not be appropriate to discard valuable lines if such endophytes appear and do not inhibit growth and the production of plants. They should, however, be confirmed as an endophyte in the species. (5) Even as a normal endophyte, they may inhibit recovery of shoots after cryopreservation, a situation that can be improved by the use of an antibiotic in the recovery medium. (Maschinski for Valerie Pence. 2018)

Crotalaria avonensis, a federally endangered legume which produces few seeds, is found in only 3 populations in the South Central Florida scrub.  Several years ago, the Cincinnati Zoo and Botanical Garden (CREW) was asked to develop an invitro propagation and shoot tip cryopreservation protocol for this species.  CREW has since cultured and cryopreserved over 120 genotypes representing all 3 populations and has contributed plants to a restoration project.  For cryopreservation, small shoot tips about 1mm in length are dissected and put through a cryoprotective protocol and banked in liquid nitrogen.  In earlier work, the Encapsulation Vitrification method was used but more recently Droplet Vitrification has proved to be more effective as a cryopreservation method.  Over the years of maintaining the cultures, it was noted that there was a subtle growth of bacteria that could be detected visibly in most of the cultures, but which did not appear to hinder their growth.  This bacteria has been identified by colleagues in Cincinnati Children's Hospital as a species of Paenibacillis using mass spectroscopy with time of flight.  Information from this literature indicates that Paenibacillis is a common soil bacterium which can also be a plant endophyte and it has been reported from invitro cultures of other species.  As part of an Institute of Museum and Library Services (IMLS) funded study, 136 samples of shoot tips (63 genotypes) were removed from long-term storage of 4 to 15 years in liquid nitrogen.  All of these had been cryopreserved using the Encapsulation Vitrification method.  While there was survival in most of the samples some bacteria were seen around the pieces in these recovery plates.   Even though some of the pieces that were growing also showed bacteria it was most noticeable around the pieces that weren't growing.  During the evaluation of the survival of samples that showed some bacteria and those that did not, it was observed that there was significantly more survival at 4 weeks in visibly clean cultures (using antibiotic Rifampicin) than those that showed some Paenibacillis bacteria.  Similarly, when newly banked shoot tips of 15 genotypes were cryopreserved using the improved technique of Droplet Vitrification, and were recovered, the presence of Rifampicin in the medium significantly increased the percent of shoot tips showing recovery growth.  Based on these results, the focus was then placed on growth of stock cultures.  While the cultures do grow without the additional antibiotic, in some cases there is an increase in growth in the presence of the antibiotic.  Observations show that when tissues are removed from the antibiotic the visible bacteria will return.  Summary and Conclusions:  (1) The subtle bacterium found in stock cultures of Crotalaria avonensis has been identified as Paenibacillus, and has been identified as an endophyte in other species.  (2) While it does not appear to have a strong effect on the growth of the cultures, it can significantly inhibit the amount of recovery after cryopreservation.  (3) Further studies are underway to identify the bacterium from newly collected tissues of Crotalaria avonensis.  (4) With the increasing understanding of the roles of endophytes in plant growth, when dealing with rare species, it may not be appropriate to discard valuable lines if such endophytes appear and do not inhibit growth and the production of plants.  They should, however, be confirmed as an endophyte in the species.  (5) Even as a normal endophyte, they may inhibit recovery of shoots after cryopreservation, a situation that can be improved by the use of an antibiotic in the recovery medium.  (Maschinski for Valerie Pence. 2018)

Tissue culture lines and cryopreserved shoot tips of multiple genotypes are maintained at CREW.

Tissue culture propagation protocols have been developed for this species at CREW (Cincinnati Zoo & Botanical Garden) and used to propagate plants for outplanting in Florida in collaboration with Bok Tower Garden and Archbold Biological Station.  Shoot tip cryopreservation protocols have also been developed and used to bank multiple genotypes of this species in CREW's CryoBioBank.

Eric Menges is conducting demographic and fire ecology studies. This plant has been successfully propagated in ex situ collections, and Historic Bok Tower Gardens maintains individuals of this species as a part of the National Collection. Bernadette Plair and Valerie Pence (Cincinnati Zoo and Botanical Garden) are micropropagating this species to increase their numbers for research and possible reintroduction efforts (Plair and Pence 2000)

A narrowly distributed central Florida endemic, known from only three sites in Polk and Highlands counties, Florida. The mass development occurring on the Central Florida Ridge threatens this species. The site with the largest population suffers fragmentation due to a housing development.

The principal cause of decline for this species is the conversion of the high pineland and scrub for development for agricultural purposes (principally citrus groves), and for commercial, residential, and recreational purposes. Other threats include o

Several hundred individuals in 2 populations known from only 3 sites. Only two of the populations are protected and occur in the Lake Wales Ridge Ecosystem Carl Project Site (Saddle Blanket Lakes and Carter Creek) and Avon Park Lakes in Highlands and Polk Counties (FNAI 2001).

Bea Pace is monitoring this species at Saddle Blanket Preserve.

Use prescribed burns to maintain the historical structure of the Lake Wales Ridge. Control non-native invasive plant species. Purchase privately-held lands where this species occurs. Research is needed on demographic processes, pollinators, seed dispersers and seed viability.

Maintain plants in ex situ collections. Research propagation and seed germination methods.