2 dW 2nd World ld Scientific S i tifi Congress C Challenges in Botanical Research and Climate Change

June 29 - July 4 -- Delft, Delft The Netherlands

Supplementing Seed Banks: In Vitro Methods for Preserving and Propagating Species at Risk
Valerie C. Pence Center for Conservation and Research of Endangered Wildlife (CREW) Ci i Cincinnati ti Z Zoo & Botanical B t i l Garden G d Cincinnati, Ohio, U.S.A.

Cincinnati, Ohio, USA

Population: City: 365,000 Area: 1.9 million, including g areas in the states of Ohio, Kentucky, and Indiana

Cincinnati Zoo & Botanical Garden

Creating Adventure, Conveying Knowledge, Conserving Nature, Serving Community

Second oldest Zoo in the U.S. (1875) Now also a Botanical Garden (1987)

Research Center - CREW

Center for Conservation and Research of Endangered Wildlife

Mission: To use science and technology to understand, preserve and propagate endangered flora and fauna and facilitate the conservation of global biodiversity

Pl Plant Research R h Division Di i i CREW

Endangered Plant Propagation Program

Goal: To use in vitro methods for propagating and preserving endangered plants when traditional methods are not adequate

In vitro methods for propagating and preserving endangered plants

In Vitro Collecting g

In vitro methods for propagating and preserving endangered plants

Tissue culture propagation

In vitro methods for propagating and preserving endangered plants

Cryopreservation

Frozen GardenEPPP Tissue Bank Regional Seed Bank Pteridophyte Bank Bryophyte Bank

Center for Plant Conservation -Collaborators

Over 40 targeted species

15 collaborating gardens, and others . . .

Climate change and endangered plants

Rising sea levels Drought Loss of habitat Increase in temperature Small populations vulnerable to catastrophic events

Ex situ conservation can be used to back up and support wild populations

Tools of Ex Situ Conservation

Living Collections

Cryo tissue Banks Cryo-tissue

Seed/Spore Banks

Seed/Spore Banking

S d/S Seed/Spore B Banking ki Efficient storage of multiple genotypes in a small space Applicable to many species (orthodox seeds and most spores)

But. . .

Species with few or no seeds

Species with recalcitrant seeds

Supplementing Traditional Seed Banking

Embryo cryopreservation

Shoot tip cryopreservation

Species with recalcitrant seeds Species with no seeds or recalcitrant seeds

Using In Vitro Methods

Th Three examples l ...

Hedeoma todsenii Todsens pennyroyal

Lamiaceae South central New Mexico, 6,000-7,000 ft Collaborator: The Arboretum at Flagstaff M Mountain i habitat h bi P d Produces little li l or no seed d

Hedeoma todsenii Tissue culture propagation protocols

Hedeoma todsenii Cryopreservation and Banking

45 40

Pe ercent survi ival

35 30 25 20 15 10 5 0 % Survival Medium D V

Average survival of shoot tips, two weeks after exposure to qu d nitrogen, t oge , us using gt two o methods: et ods D = e encapsulation capsu at o liquid dehydration (n=10); and V=encapsulation vitrification (n=24).

Shoot tips banked (seeds not available)

Hedeoma todsenii - Genetic Analysis

Interfering compounds inhibit DNA analysis, l i except t with in vitro material

A (left): Most parsimonious tree made from RAPD analyses of 12 lines of H H. todsenii todsenii. B (right): population sources for the genetic lines. Matching colors in A and B identify the population source.

Ranunculus aestivalis Autumn buttercup

Ranunculaceae S th Central South C t l Utah Ut h Collaborator: The Arboretum at Flagstaff One population declining to about 20 plants Wet meadow habitat; ; hydrology y gy changing?

Ranunculus aestivalis Outplanting, June, 2007

Acclimation: The Arboretum at Flagstaff Outplanting: Propagation: p g CREW TNC Sevier Valley Preserve Utah Valley State College USFWS funding

Ranunculus aestivalis - outplanting

138 plants augmenting approx 20 plants remaining in the wild a p planted a ted with t wild dp plants; a ts; half a in Half wetter area 92% survival through the summer g better Wetter site doing

Asimina tetramera Four-petal pawpaw

Annonaceae Southeast Florida - scrub habitat Collaborator: Historic Bok Sanctuary Endemic to two counties Occurs only 15 - 35 ft above sea level Recalcitrant seeds

Asimina tetramera Protocols otoco s Cryopreservation Propagation

Asimina tetramera - Germplasm banking

In vitro collecting

Shoot propagation

Cryostorage

Genetic analysis

Conclusions Cryopreservation of in vitro propagated material can supplement seed banking for species with little or no seed or with recalcitrant seeds In vitro propagated plants can be used to test habitat for reintroduction reintroduction, as habitats change In vitro propagated plants may allow genetic analysis when wild tissues are not suitable

Plant Division Staff

(past and present)

Susi Charls Peter Ackerman Kristine Lindsey

and. . . Volunteers and and Students

Bernadette Plair Dr. Doug Winget

Thank you for your attention!