Cryopreservation of sweet potato shoot tips by vitrification
Date
1997
Authors
Pennycooke, Joyce C., author
Hughes, Harrison G., advisor
Ward, Sarah, committee member
Towill, Leigh E., committee member
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Journal ISSN
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Abstract
The conservation of vegetatively propagated germplasm is problematic. In vitro conservation of sweet potato has been achieved using normal and limited growth regimes. However, although the storage of in vitro plants is advantageous, it is not the method of choice for long term conservation because normal growth conditions require frequent subculturing which makes it labor intensive and costly. Also, frequent subculturing over a prolonged period of time may lead to undesirable consequences such as, contamination, selection and chromosomal aberrations. Cryopreservation offers the simplest and most economical way for the conservation of plant germplasm and vitrification is the preferred method to accomplish this. Previously, Towill and Jarret (1992) reported that vitrified sweet potato shoot tips exhibited callus formation and that the vitrification protocol developed included high levels of within and between treatment variability for explant survival. The purpose of this study was therefore to, examine the various steps of the cryopreservation procedure in order to generate a reproducible shoot tip cryopreservation protocol for sweet potato. In vitro grown sweet potato shoot tips were excised at 0, 3 or 10 hr in light after an 8 hr dark period. Excised shoot tips were precultured in 2% sucrose in MS for 24 hr and 0.3, 0.5 or 0.75 M sucrose in MS for an additional 24 hr. Precultured shoot tips were loaded with two different cryoprotective solutions for 20 or 60 min at 22 °C and then dehydrated with a concentrated vitrification solution (PVS2) for various lengths of time at 22 °C. Following dehydration, shoot tips were cooled by placing them along with a small drop of PVS2 on thin strips of aluminum foil, in 0.2 ml PVS2 in polypropylene straws or in 1 ml of PVS2 in cryo-vials. These were then plunged in nitrogen 'slush' (ca. -209 °C). Cryoprotectant-exposed shoot tips as well as vitrified shoot tips were recovered on MS media with various modifications (mineral composition, surfactant/ antioxidant, hormonal content). Another type of vitrification technique, the encapsulation / dehydration vitrification procedure, was also pursued for the cryopreservation of sweet potato shoot tips. The highest survival (67%) of vitrified encapsulated shoot tips was achieved after 4 hr dehydration corresponding to a moisture content of 18.1 %. To our knowledge, this is the first reported survival of cryopreservation of sweet potato shoot tips using the encapsulation / dehydration procedure. For the solution-based vitrification method, shoot tip survival after both the dehydration step and cooling in nitrogen slush was strongly dependent on the preculture condition (sucrose concentration). The best survival was achieved from shoot tips (PI 290657) excised from in vitro plants soon after the 8 hr dark period, with a 24 hr preculture in 0.3 M sucrose prior to loading in 2M glycerol + 0.4 M sucrose for 1 hr and then dehydrated with PVS2 for 16 min at 22 °C respectively. A fast cooling rate was beneficial and this was achieved by placing the shoot tips along with a small drop of PVS2 on thin strips of aluminum foil prior to plunging in nitrogen slush. Survival of cryopreserved shoot tips was promoted significantly (P = 0.0001) in the absence of NH4+ in the recovery medium for 5 days prior to transfer to regular MS medium. In this study, we report an improved vitrification protocol in that survival after dehydration and cooling results in the resumption of shoots initiated directly from treated shoot tips. Most important for any genebank is the regeneration of shoots so as to reduce the risk of somaclonal variation. Using the same recovery medium, for all four lines tested, relatively good survival levels (ranging from 62% - 97 %) were achieved, and most important, all lines regenerated shoots after cooling to cryogenic temperatures.
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Subject
Plant cells and tissues -- Cryopreservation
Sweet potatoes -- Germplasm resources -- Cryopreservation