|Joung, Hyang young|
|Kamo, Kathryn - Kathy|
Submitted to: CryoLetters
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/22/2007
Publication Date: 10/31/2007
Citation: Joung, H.J., Cantor, M., Ellis, D.D., Kamo, K.K. 2007. Cryopreservation of Gladiolus shoot tips derived from cormels. CryoLetters. 48:251-255. Interpretive Summary: The preservation of gladiolus plants, especially genetically engineered plant lines, is very labor intensive. The plants must be grown each year following dormancy, harvested, and then stored for the next year. Growth of genetically engineered plants requires special conditions to prevent possible release into the environment. We have developed a method for preserving shoot tips of gladiolus in the freezer. Shoot meristems, 1-2 mm, were excised from the corm, placed at 4ºC on medium in culture, and then incubated in plant vitrification medium. Meristems treated with plant vitrification medium were then placed in liquid nitrogen for deep freezing. Following deep freezing for various periods, the meristems were thawed out and cultured. Plants have been recovered from the meristems, and the regenerated plants look phenotypically normal.
Technical Abstract: Gladiolus shoot tips, 1-2 mm, were excised from in vitro and greenhouse-grown cormels of cultivars ‘Peter Pears,’ in vitro-grown cormels of ‘Jenny Lee,’ field-grown cormels of the breeding lines 02-943A, 02-900, 02-926, and field-grown cormels of the cultivar ‘Double Delight.’ The highest frequency of excised shoot tip recovery and growth (85-100%) of in vitro-grown ‘Peter Pears’ and ‘Jenny Lee’ occurred when shoot tips were cultured on Murashige and Skoog’s (MS) medium lacking plant growth regulators. Growth of excised shoot tips from the three field-grown lines was similar (70-92%) on either MS medium lacking hormones or supplemented with 2 mgl-1 kinetin. The factors that affected growth of shoot tips following vitrification were size and dormancy status of the excised shoot tip, whether the source plants were grown in vitro or ex vitro, cultivar, and time in Plant Vitrification Solution 2 (PVS2). The conditions for vitrification examined include preculture time for the excised shoot tips, sucrose concentration of the preculture medium, incubation time in the PVS2 vitrification medium, PVS3 vitrification medium, addition of Supercool X1000 to PVS2 medium, and slow cooling using a programmable freezer. The highest survival of shoot tips was achieved using greenhouse-grown ‘Peter Pears’ (80%), followed by in vitro-grown ‘Peter Pears’ (54%), and field-grown 02-943A (55%). Incubation times of 120 and 60 min in PVS2 were optimal for ‘Peter Pears’ and 02-943A, respectively. All shoots that have grown following vitrification were phenotypically normal both in vitro and in the greenhouse.