Preservation Methods for Jerusalem Artichoke Cultivars

Preservation methods were evaluated forjerusalem artichokes (Helianthus tuberosus L.) to facilitate the back-up of field collections. In vitro plant cultures were established from field-harvested tubers for nine jerusalem artichoke cultivars obtained from the Plant Gene Resources of Canada, Saskatoon Research Center, Saskatoon, Canada. Most cultivars could be maintained at 5 °C or under 25°C growth room conditions for at least 6 months. Excised shoot tips from in vitro cultures were cryopreserved using plant vitrification solution 2 (PVS2; 15% w/v ethylene glycol, 15% w/v dimeth yl sulfoxide, 30% w/v glycerol, and 13.7% w/v sucrose in half strength media salts) as a cr y oprotectant. PVS2 exposures of 15 or 30 mm at 0 °C resulted in an average regrowth of 34% across five jerusalem artichoke cultivars after liquid nitrogen exposure. Jerusalem artichokes can be successfully maintained using both reduced temperature and cryopreservation approaches. Jerusalem artichoke (Helianthu.s tube rosu.s L.) is a cold-hardy North American wild relative of the sunflower, Helianthus annuus L. Jerusalem artichoke plants were spread from Saskatchewan east to Ontario and south to Arkansas and Georgia by Native Americans who cultivated them for edible tubers that can be baked, steamed, or boiled. Raw tubers can be sliced or shredded for salads. The tubers contain high levels of inulin, which has been pursued as a bulking agent for artificial sweeteners (McLaurin etal., 1999). Sincejerusalem artichoke plants produce large quantities of biomass with few inputs, this crop has also been considered for silage production (Seiler, 1993). Furthermore, Helianthus breeders have interest injerusalem artichokegermplasm since Scierotinia scierotiorum (Lib.) resistance has been identified through tissue culture screening (Cassells and Walsh, 1995). The USDA National Plant Germplasni System maintains seven clonal accessions of H. tuberosus; however, 165 H. tuherosus (L.) accessions are propagated using tubers at Plant Gene Resources of Canada. These accessions are not currently distributed or listed on the Germplasm Resources Information NetworkCanadian version (GRIN-CA) database. Since the tubers must be dug and replanted every 3 years,jerusalem artichoke plants are relatively high maintenance compared to many seedReceived for publication 30 Sept. 2005. Accepted forpublication 3 Nov. 2005. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Technical assistance was provided by Kate Rotindo, Jackie Harris, and Dallas Kessler. 'To whom reprint requests should be addressed; e-mail gvolk lamar.colostate.edu . propagated species. Labor costs and field space also make maintaining duplicate cultivars at separate locations prohibitively expensive. This research was performed to identify desirable methods for maintaining back-up collections of clonally propagated H. tuberosus in genebanks. Methods for in vitro culture of H. tuberosus have been published (Cassel Is and Walsh, 1995; Devi and Rani, 2002; Roche and Cassells, 1996; Gamburg et al., 1999). Once established as in vitro plants, many temperate crops can be stored for extended lengths of time at low (5 to 15 °C) temperatures (Reed, 1992, 1993). Jerusalem artichoke tubers easily over-winter in Canadian field conditions, but a test for reduced-temperature medium-term storage of H. tuberosus in vitro cultures has not been published. Successful cryopreservation protocols allow for long-term storage of clonally propagated species. While H. tubemsus suspension cell cultures have been successfully cryopreserved (Harris etal., 2004; Swan etal., 1999), there are no reports of the cryopreservation of H. tubero.su.s shoot tips. The use of shoot tips limits the risk ofsomaclonal variation that can occurwhen plants are maintained as suspension cell cultures (D'Amato, 1985; Harding, 2004; Scowcroft 1984). Shoot tip cryop reservation protocols have been developed for many temperate plant species, but they have not been shown to be generally applicable. Vitrification solutions such as plant vitrification solution 2 (PVS2) (Sakai et al., 1991) and plant vitrification solution 3 (PVS3) (Nishizawa et al., 1993) reduce ice crystal formation by dehydrating or by altering water properties (Volk and Walters, 2006); however, constituents of the solutions can be cytotoxic (Steponkus et al., 1992). Cultivars within a species differ in their responsiveness to PVS2 and PVS3, though the basis for different responses is not known. Medium-term cold storage and long-term cryopreservation approaches are complementary. Cultivars introduced into tissue culture can be maintained in vitro until resources for ciyopreservtng shoot tips for long-term storage are available. This research was performed to identify a method by which clonally propagated H. tuherosus accessions could be backed up within genebanks. Materials and Methods Plant materials. In mid-August, tubers were harvested from nine H. tuberosus cultivars grown in the fields at the Plant Gene Resources of Canada, Saskatoon Research Centre, Saskatchewan, Canada (Table I). Oneyear-old field plants had not begun flowering and had small (<I-cm-diameter) tubers. Tubers were sent to the National Center for Genetic Resources Preservation, Ft. Collins, Cob, and stored at 4 °C for up to 30 d until introduction into tissue culture. Tubers and rhizomes have many buds that provide shoot tips for introduction into tissue culture. Segments (5 mm) of field-grown tubers or rhizomes were surface-sterilized with either 70% isopropanol for 2 mm, 70% isopropanol for 5 mm, 95% ethanol for 2 mm, or 70% ethanol for 5 min followed by a 20% commercial bleach (1.8% sodium hypochlorite) + 0.0001% Tween 20 (Sigma, St. Louis, Mo.) for 20 mm. After three 10-min sterile water rinses, 20 shoot tips were excised under sterile conditions and plated onto one of two media. Growth medium I contained MS salts and vitamins, 0.1 mgL ' gibberellic acid and 15 gL ' sucrose (Cassells and Walsh, 1995) and growth medium 2 contained MS salts (Murashige and Skoog, 1962), MS-G, I mg-L-' thiamine HCI, 1 mgL ' indole acetic acid, 20 gL-' sucrose, and 9 gL agar. Table I. Jerusalem artichoke accession information. All varieties are maintained at the Plant Gene Resources of Canada, Saskatoon Research Centre, Saskatoon, Canada. Alternative Date Accession Canadian of no. no. Name Origin acquisition JA 14 NC 10-16 HM-3 Manitoba, Canada 1970 JA 37 NC 10-40 Comber Manitoba, Canada 1974 JA 38 NC 10-41 B.C. #1 Native American collection, Canada 1976 JA 40 NC 10-43 USDA-PI Unknown 1976 JA 41 NCIO-44 Sunchoke-Fiesda's California 1976 JA 64 NCIO-79 W-3X Branching 7611 Canada 1979 JA 92 NC 10-114 Industrie Russia 1984 JA 103 NCIO-130 #1 Ontario, Canada 1988 JA 172 SR SR Unknown Unknown 80 HORTSCIENCE VOL. 4 1(1) FEBRUARY 2006 j Storage temperature (°C)