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Yongjian Chang and Barbara M. Reed

Cold hardiness and cryogenic survival of micropropagated pear (Pyrus cordata Desv.) shoots were evaluated after pretreatments with ABA and sucrose. Shoot cold hardiness increased by 3 °C, and cryopreserved shoot tip growth increased by 17% after a 4-week 150 μm ABA pretreatment. Low temperature (LT) pretreatments improved the recovery of cryopreserved P. cordata shoot tips. Six to 10 weeks of LT were required for reaching high cryopreservation recovery. ABA and LT treatments produced significant synergistic effects on both cold hardiness and cryopreservation recovery. ABA shortened the LT requirement for high cryopreservation growth from 10 to 2 weeks. The optimal treatment for recovery of cryopreserved shoot tips was a 3 week culture on 50 μm ABA followed by 2 weeks of LT, while the maximum cold hardiness (-22.5 °C) was obtained with 150 μm ABA and 2-week LT. A 4 week culture on 150 μm ABA at 25 °C induced dormancy in 74% of shoot tips, but had little effect on cryopreservation growth unless combined with LT. Control and ABA-treated shoot tips, lateral buds, and leaves had similar cold hardiness (-10 to -12 °C), but LT and LT+ABA-treated shoot tips survived the lowest temperatures (-17 to -23 °C), lateral buds next (-15 to -20 °C), and finally leaves (-14 to -18 °C). An increase in the preculture-medium sucrose concentration from 2% to 7% combined with 2-week LT significantly increased cryopreserved shoot tip growth (0% to 75%) and decreased the LT50 from -7.8 to -12.4 °C. The optimal shoot pretreatment for successful recovery of cryopreserved P. cordata shoot tips was a 3 week culture on either 50 μm ABA or 5% to 7% sucrose medium followed by 2 weeks of LT, and increased shoot tip growth from zero to >70%. Chemical name used: abscisic acid (ABA).

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Matthew G. Blanchard, Erik S. Runkle, Arend-Jan Both, and Hiroshi Shimizu

.g., temperature and light) influence plant growth and development. The rate of plant development is controlled by the mean daily temperature (MDT) of the apical meristem (e.g., shoot tip) ( Faust and Heins, 1993 ; Niu et al., 2001 ; Roberts and Summerfield, 1987

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Fatemeh Haddadi, Maheran Abd Aziz, Hossein Kamaladini, and Seyed Ali Ravanfar

formation has increased during the last few years. There are no reports on the effect of zeatin on strawberry shoot-tip proliferation. The second objective of this study was to determine the most efficient regeneration system, along with root formation from

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Jean Carlos Bettoni, Aike Anneliese Kretzschmar, Remi Bonnart, Ashley Shepherd, and Gayle M. Volk

). Compared with the cost of creating duplicated field collections, it is cost-effective to establish cryopreserved back-up collections ( Benson, 2008 ). Although there are many established cryobanks that conserve vegetative propagules such as shoot tips or

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Anne K. Logan, Justin A. France, James M. Meyers, and Justine E. Vanden Heuvel

Excessive vine vigor often leads to issues such as poor canopy ventilation, inconsistent fruit ripening, and a high incidence of cluster rot. Hedging, or shoot tipping, is one common approach to managing vigorous vegetative growth in vertical

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Allison D. Oakes, Tyler Desmarais, William A. Powell, and Charles A. Maynard

–10 d to 3–4 d, and also by placing plantlets in the dark during that time ( Oakes et al., 2013 ). In addition, acclimatization success can generally be improved by increasing the number of adventitious roots produced in culture and keeping the shoot tip

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Leigh E. Towill and Gayle M. Volk

Arabidopsisthaliana shoot tips provide a model to study processes important for cryopreservation. Cryopreservation was accomplished using both vitrification and two-step cooling methods. With vitrification methods, shoot formation after liquid nitrogen (LN) exposure was as high as 100% and 95% for shoot tips exposed to PVS2 at 0 °C and to PVS3 at 23 °C, respectively. A two-step cooling method also gave greater than 90% survival if shoot tips were cooled at 0.3 °C per minute to below –30 °C before immersing the samples into LN. The high levels of shoot formation after LN exposure in Arabidopsis thaliana shoot tips will allow the use of mutants to examine how alterations in biochemical, metabolic, and developmental processes affect survival and growth.

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Joyce C. Pennycooke and Leigh E. Towill

Cryopreservation offers the simplest and most economical way for the long-term conservation of germplasm and vitrification is the preferred method to accomplish this. Undefined endogenous compounds are produced during plant growth and shoot tip preculture conditions. These may influence “cryopreservability” and interact with cryoprotectants that are artificially added during the cryogenic protocol. We are beginning to examine these aspects to improve cryopreservation. Nodal segments of PI 296057 were propagated on a hormone-free modified Murashige and Skoog (MS) solid medium and were grown with 16 hr/8 hr photoperiod. Shoot tips were excised at 0, 3 or 10 hr in light after the dark period. Excised shoot tips were precultured in 0.06 M sucrose in MS for 24 hr and 0.3 M sucrose in MS for 24 hr and then treated with 0.4 M sucrose plus 2 M glycerol for 20 min or 1 hr before being dehydrated in PVS2 [30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide in MS and 0.4 M sucrose[for 10, 16 or 26 min at 22°C. Shoot tips were placed on thin strips of aluminum foil, which were folded to enclose the shoot tips and then immersed in a liquid nitrogen (LN) slush. Rapid warming and dilution were achieved by transferring the foil strips from LN into 3 ml of 1.2 M sucrose at 22°C for 20 min. All cultures were incubated in darkness for 2 days then dim light for 3 days before transfer to the usual light intensity. Elimination of iron and nitrogen from MS medium in post thaw culture for 5 days increased the viability of LN-treated samples. Maximum survival after LN exposure was achieved with excision immediately after the dark photoperiod, cultured for 1 hr in 0.4 M sucrose plus 2 M glycerol and exposed for 16 min in 100% PVS2 at 22°C. Previously, Towill and Jarret (1992, Plant Cell Reports 11: 175–178) reported that surviving shoot tips developed callus and a variable percentage subsequently formed shoots. In this line all surviving shoot tips eventually formed shoots.

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Leigh E. Towill

Papaya shoot tips, obtained either from seedlings or from in vitro plants, survived liquid nitrogen (-196°C) exposure using a vitrification procedure. Vitrification is a technically simple method but requires large concentrations of cryoprotectants. These were added in two steps, first slow addition of dimethylsulfoxide (DMSO) and PEG-8000, and subsequent fast addition of ethylene glycol (PG). The final concentration before cooling was 40% EG, 7.8% DMSO, and 10% PEG-8000. Both rapid cooling and rapid warming rates were required. Differential scanning calorimetry (DSC) was used to determine that the external solution vitrified upon cooling. It could not be demonstrated by DSC that cells within the shoot-tip vitrified, but since both DMSO and EG rapidly permeate plant cells, vitrification within the cells seems a likely explanation for retention of viability.

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Xiuli Shen, William S. Castle, and Frederick G. Gmitter Jr

). However, this in vitro protocol was not successful using shoot tips taken from mature male C . cunninghamiana trees in our preliminary experiment. Therefore, our objectives were: 1) to investigate the effects of plant growth regulators (PGRs) (type