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  • Author or Editor: Joyce C. Pennycooke x
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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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Salinity, drought and temperature frequently limit crop productivity. Transgenic Petunia ×hybrida cv. Mitchell with altered endogenous raffinose family oligosaccharides (RFO) due to over-expression (sense) or under-expression (antisense) of the tomato α-galactosidase gene show that antisense increases in RFO are associated with greater tolerance to freezing stress (Pennycooke et al., 2003). Because vegetative propagules of these antisense lines rooted and established more quickly than their sense counterparts, we hypothesized that antisense lines would also respond to salinity and wilting stress. Salinity treatment plants were exposed to 50-200 mm NaCl graduated 25 mm every 3 days and held at 200 mm for 13 days. Dry-down treatments were watered to pot capacity, then not watered until the onset of wilting. This was repeated in cycles for 26 days. Data were collected on plant growth, root/shoot ratios, and leaf water potential. Fresh and dry weights in four of the six antisense lines exceeded the wild type and sense lines. Osmotic potential for salinity and dry-down plants was 160% to 220% higher than control plants. Pearson correlations revealed that higher osmotic potential was partially associated with higher fresh weight (r = 0.7214, P = 0.02) and root/shoot ratios (r = -0.7414, P = 0.02) in salinity stressed plants. In the dry-down drought stressed plants, osmotic potential was not associated with fresh weight (r = 0.3364, ns) nor root/shoot ratio (r = -0.0431, ns). Salinity stress reduced root mass compared to control and dry down plants. Sense plants grew slowly and were highly variable.

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Previous studies of plant tolerance to low temperature have focused primarily on the cold acclimation response, the process by which plants increase their tolerance to freezing in response to low nonfreezing temperatures, while studies on the deacclimation process have been largely neglected. In some plants, cold acclimation is accompanied by an increase in raffinose family oligosaccharides (RFO). The enzyme α-galactosidase (EC 3.2.1.22) breaks down RFO during deacclimation by hydrolyzing the terminal galactose moieties. Here we describe the isolation of PhGAL, an α-galactosidase cDNA clone from Petunia (Petunia ×hybrida `Mitchell'). The putative α-galactosidase cDNA has high nucleotide sequence homology (>80%) to other known plant α-galactosidases. PhGAL expression increased in response to increased temperature and there was no evidence of developmental regulation or tissue specific expression. Increases in α-galactosidase transcript 1 hour into deacclimation corresponded with increases in α-galactosidase activity and a concomitant decrease in raffinose content, suggesting that warm temperature may regulate RFO catabolism by increasing the transcription of the α-galactosidase gene. This information has potential practical applications whereby α-galactosidase may be targeted to modify endogenous raffinose accumulation in tissues needed for freezing stress tolerance.

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