Manfredo J. Seufferheld and Cecil Stushnoff
Strawberry plantlets, regenerated from leaf disks, were used as a model system to study the effect of high concentrations of sugars and dehydration on survival during cryopreservation. After cold acclimation, plantlets imbibed for 3 days (one day each) in 0.5, 0.7 and 1.2 M sucrose and (1.0M sucrose + 0.2M raffinose) and desiccated to 25 % moisture (fwb) in alginate capsules consistently survived cryopreservation. Differential scanning calorimetry revealed only a very small exotherm between -20C and -28C during freezing; a glass transition at -50C and a small melting event at -10C during warming. Conversely, samples with the lowest survival rate, had a large nucleation exotherm at -30C and a devitrification exotherm between -70 and -40C. We conclude that imbibition with sugars, coupled with desiccation treatments, may be used to manipulate freeze tender tissues of strawberry to permit successful cryopreservation.
Paul H. Jennings and Cecil Stushnoff
Various carbohydrates have been shown to be associated with stress tolerance in some plant species. Specifically, the content of soluble sugars have been correlated with desiccation tolerance and winter hardiness. We have previously demonstrated that radicles of cucumber seed become progressively more sensitive to chilling injury during the early stages of germination and that cultivar differences exist. Sucrose, raffinose, and stachyose contents of `Poinsett 76' and `Ashley' seed were determined in dry seed during imbibition and at three stages of radicle emergence. The more chilling-tolerant cultivar (Ashley) contained lower raffinose and higher stachyose contents than the less chilling-tolerant `Poinsett 76'. In both cultivars, the contents of raffinose and stachyose declined dramatically between the 1-mm and 5- to 7-mm stage of radicle emergence. At the 1-mm stage, when cultivar chilling-tolerance differences are most pronounced, `Ashley' appears to have a higher content of stachyose and lower raffinose content.
Izulme R. Santos and Cecil Stushnoff
Economically, citrus is the second most-important fruit crop grown worldwide; thus germplasm conservation of commercial cultivars, as well as of wild relatives, is essential. Presently, citrus germplasm has been conserved mainly in field genebanks. This approach is helpful; however, it is costly, exposes germplasm to climatic and biological hazards, and is not a long-term conservation system. Cryopreservation (conservation in liquid nitrogen, at –150°C to –196°C) is a technique that can ensure long-term storage of plant material. Attempts to cryopreserve citrus are restricted to a few reports, but the results obtained are encouraging. The basic purpose of this study is to define cryopreservation protocols for embryo axes and axillary buds of `Pineapple' sweet orange using the encapsulation-dehydration method. Embryo axes encapsualted in Na-alginate beads, precultured with high levels of sucrose and dehydrated over silica gel before freezing in liquid nitrogen had 60% survival. No survival was obtained for buds treated the same way, however buds isolated from plants acclimated at 0°C over a 30-day period survived exposure to –20°C when slow cooled at 2°C/hour. Additional experiments will combine cold acclimation, slow cooling and pre-treatment with sugars and other chemical compounds as an attempt to enhance cold hardiness of axillary buds and obtain survival after freezing in liquid nitrogen. Different approaches will be used to increase embryo axes survival rates.
Thomas E. Marler and Cecil Stushnoff
The influence of plant size on recovery following defoliation of `Tainung 1' papaya was used to study the role of respiratory sink size relative to photosynthetic surface area and the carbohydrate pool size available for remobilization. Defoliated (D) plants at three different ages: oldest, 24 weeks posttransplant (PT), supporting ≈8 weeks of fruit set; intermediate, 10 weeks PT, ≈2 weeks from initial flowering; and youngest, 4 weeks PT, were compared to an equal number of control plants. The oldest plants abscised all fruit <5.5 cm in diameter as a result of defoliation. Increase in stem height and basal circumference ceased on all plants and increase in fruit circumference ceased on the oldest plants following defoliation. Increase in stem height of D plants began again 3 weeks postdefoliation (PD) and returned to that of control plants by 6 weeks PD. Increase in basal circumference of D plants began again 6 weeks PD. Root density was observed on observation windows, and fine roots completely disappeared within 1 week PD. Root density returned to that of control plants by 6 weeks for the youngest and intermediate plants and by 8 weeks for the oldest plants. Increase in fruit circumference of pre-existing fruit for the oldest D plants never returned to that for control plants. These plants began setting fruit again ≈8 weeks PD. Defoliation delayed initial flowering of the intermediate plants 6.5 weeks and of the youngest plants ≈2 weeks. Thus, the greatest impact of defoliation on reproductive growth occurred with the two oldest age groups.
Manfredo J. Seufferheld, Cecil Stushnoff and Philip Forsline
Cryopreservation of mature dormant vegetative buds is a useful method to preserve germplasm of a large number of cold hardy apple cultivars. However, cold tender cultivars have proven to be much more difficult to cryopreserve. Eight cultivars were harvested in September 1993 at Geneva, NY before developing cold hardiness naturally. The twigs were encapsulated with 5% alginate and treated with stepwise imbibition of 0.5 to 1.0 M sucrose. The samples were desiccated over glycerol at 0C. Half of the samples were plunged directly into liquid nitrogen (IN) and the other half were first cooled slowly to -30C. The twigs that had been exposed to prefreezing conditions showed the highest survival (20 to 100%). The samples that were plunged directly in LN survived poorly (0 to 20 %). Samples without encapsulation and no sucrose imbibition had 0% survival. We conclude that this protocol opens up the possibility to expand cryopreservation of cold tender apple cultivars, presently grown only in field gene banks, at great expense and inconvenience.
Virgil Esensee, Cecil Stushnoff and Philip L. Forsline
There is need for backup storage of clonally propagated plant cultivars of numerous taxa. Initial tests, using a protocol developed for dormant apple buds that includes desiccation and slow freezing prior to immersion in liquid nitrogen (-196 C), was not effective with `Valiant' grape. Accordingly, replicates of V. vinifera `Riesling', V. riparia, `Valiant' and a V. amurensis × riparia cross were also tested for survival at –196 C, following desiccation to 25% & 18% water (fwb) and direct immersion into liquid nitrogan. Visual and electrolyte leakage ratings following nine days of dehydration in moist peat were used to assess viability. Direct immersion of desiccated samples resulted in survival for some buds of `Valiant' and a V. amurensis × riparia cross. V. riparia showed some survival when field hydrated and at 25% water, while all buds desiccated to 18% survived. `Riesling' did not survive desiccation, and was killed by all -196 C treatments. The apple protocol was partially effective, in combination with desiccation to 18% in `Valiant' and V. riparia. This is the first report of grape bud survival in liquid nitrogen and more detailed studies are planned.
Imed Dami, Cecil Stushnoff and Richard Hamman
The response of grapevines to methanol was investigated at the Orchard Mesa Research Center in Grand Junction, CO. Optimum sublethal methanol dose levels, based on visual assessments, were 90% for leaves and 100% for trunks for 10 cultivars. Total soluble sugars (TSS) of the berries, monitored every week until harvest, showed significant differences with Muscat Blanc during veraison. Berries from the methanol-treated vines had higher TSS (16.4 °Brix) than controls (15 °Brix). However, no significant differences were observed later in the season when approaching fruit maturity. At harvest, data on yields as estimated by cluster weight, berry weight and berry size showed no differences between the two treatments. Methanol did not enhance cold hardiness of bud tissues. measured by differential thermal analysis. It was concluded that, although methanol has been reported to improve several physiological features of C3 crops, our study suggested that it has little or no practical effect on grapes. More data on the determination of sugars in berries by HPLC will be discussed.
Alvan Gaus, Cecil Stushnoff and Ann McSay
Proper identification of apple rootstocks has always been a problem for nurseries and fruit growers. There needs to be a rapid, inexpensive, and repeatable protocol for identification of apple rootstocks. Fourier transform infrared spectroscopy (FTIR), an analytical chemical technique based on infrared laser characterization of molecular bonding energies for biochemical compounds, such as proteins, may provide an answer. Several rootstocks from the 1984 NC-140 apple rootstock trial were compared. Using a BioRad research spectrometer, spectra derived from 1000 scans per freeze dried sample were used to compare the rootstocks. Using Hit Quality Indices (HQI) generated by Lab Calc software, the rootstocks M.7 EMLA, B.9, and a seedling rootstock were compared with themselves, and each of the other two samples. A perfect match gives a HQI of zero. It was found that root cortex tissue could be used to separate these rootstocks from each other, but root xylem tissue was a poor tissue to use for identification.
Joyce C. Pennycooke, Ramarao Vepachedu, Cecil Stushnoff and Michelle L. Jones
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 22.214.171.124) 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.