Cryopreservation in liquid nitrogen (LN) is relatively routine for many small, desiccation-tolerant (orthodox) seeds. Seeds of Pyrus species are considered orthodox but have not been evaluated for LN storage. Seeds of freshly collected P. communis L. (`Bosc') were evaluated for germinability and by TZ staining after exposure to four LN treatments: 1) direct immersion and direct removal; 2) direct immersion and 1 minute in LN vapor phase before removal; 3) 2 minutes in vapor phase before immersion and direct removal; and 4) 2 minutes in vapor phase before immersion and 1 minute in vapor phase before removal. Fresh `Bosc' seed viability evaluated by TZ and greenhouse germination tests remained high (83% to 100%) following four types of LN treatments, compared to the controls (77% to 87%). Differences in `Bosc' seed viability were small and TZ results showed no significant differences among the LN treatments. Direct LN immersion and removal resulted in significantly more greenhouse-germinated `Bosc' seeds than the other treatments and fewer control seeds germinated than any LN treated seeds. Fresh `Bosc' seed cryopreserved at 7.9% moisture exhibited high germinability by both TZ and germination tests. LN exposure caused no physical damage to the seeds. Chemical name used: 2,3,5-triphenyltetrazolium chloride (TZ).
Barbara M. Reed, Sara Schwanke, and Rebecca Shala
Derek N. Peacock and Kim E. Hummer
the assistance of Jeff Steiner for the liquid nitrogen protocol. Mention of a trademark or vendor does not imply endorsement of the products named nor criticism of similar products not mentioned. The cost of publishing this paper was defrayed in part
Cryopreservation using vitrification has been reported for several plant species. Shoot tips and vitrification solution were placed in semen straws and immersed in liquid nitrogen (LN). Cracking of the external glass occurred, but may be avoided by annealing slightly below the glass transition temperature before immersion. A varying percentage still cracked with some vitrification solutions. Rapid warming also can cause cracking. There is concern that cracking may reduce viability. Shoot tips from Mentha species were used to examine this problem. Glass cracking during either cooling or warming did not produce visible damage to shoot tips. Viability of shoot tips from tubes that cracked during cooling was not different from those that did not crack; however, shoot formation was slightly reduced. Cracking upon warming did not reduce viability nor shoot formation. Very slow warming reduced viability, but warming in either water or air (room temperature) gave higher levels of survival.
Derek N. Peacock and Kim E. Hummer
Many Rubus species have a seedcoat imposed exogenous dormancy. Our objective was to contrast the effect of liquid nitrogen (LN2), sulfuric acid (H2SO4), and an untreated control on seed germination of R. multibracteatus A. Leveille & Vaniot and R. parviflorus Nutt. and to determine if LN2 could be used as a mechanical scarifying agent for these species. Three replicates of 100 seeds of each species were treated with either three 3-min dips in LN2 with corresponding 10 min thaws or for 30 min with H2SO4 or were left untreated. The LN2 pretreatment did not significantly reduce the viability of R. multibracteatus or R. parviflorus as compared to the control. A random sample of germinated R. multibracteatus from the LN2 pretreatment showed normal seedling development upon planting. The H2SO4 pretreatment significantly increased germination percentages as compared to the control or LN2 pretreatment as well as for R. ursinus Cham. & Schldl. and R. eustephanus Focke ex Diels. The LN2 treatment did not significantly improve germination over the control group and therefore was not an effective scarifying agent as applied to R. multibracteatus or R. parviflorus. However, these two Rubus species were not damaged by repeated dips in LN2. Alternative LN2 pretreatments are being examined for their potential to improve Rubus germination further.
Maria M. Jenderek, Phil Forsline, Joseph Postman, Ed Stover, and David Ellis
plant germplasm collections. DBs are harvested from field-grown trees during winter dormancy. Single-node segments are slowly desiccated and slow cooled to –30 °C and then stored in liquid nitrogen vapor (LNV). To recover the tissue from the cryostorage
Wagner A. Vendrame, Virginia S. Carvalho, José M.M. Dias, and Ian Maguire
before immersion in liquid nitrogen (LN) for 48 h. Four controls were established. Control 1 consisted of fresh pollinia collected from one flower and immediately used to pollinate another flower with no subsequent LN. Control 2 consisted of desiccated
Natalia R. Dolce, Ricardo D. Medina, and María T. González-Arnao
germination compared with the control seeds. Fig. 2. Effect of desiccation duration (h) on survival after seed cryostorage (+liquid nitrogen) of seven South American Ilex species. Survival was assessed by in vitro culturing of intact seeds, bisected seeds
Jianying Gu, Michele Warmund, and Milon George
Floral buds of `Royalty' purple raspberry and `Heritage' red raspberry were used to develop a cryopreservation method without loss of viability. The effects of prefreezing (PF), cooling rate, thawing rate, and cold storage at -7°C were tested. No survival was observed in samples immersed directly in LN2 whereas `Heritage' and `Royalty' had 90 and 97% survival after holding the samples at -22°C (`Heritage') or -18°C (`Royalty') for one week before immersion in LN2. In all cases, fast thawing resulted in a higher survival rate than slow thawing. Rapid cooling rate decreased the buds survival in LN2, however the effect was diminished when the samples were stored at the PF temperature for one week. The effect of both thawing and PF storage became less critical with bud dehydration. Differential thermal analysis (DTA) was conducted on buds without any PF treatment and buds that were subjected to PF and cold storage. DTA samples that did not receive PF exhibited LTEs, while LTEs were absent in samples subjected PF for one week. Thus, the slow removal of intracellular water to extracellular ice appears to be associated with subsequent survival of Rubus buds in LN2.
Philip L. Forsline, Leigh E. Towill, John W. Waddell, Cecil Stushnoff, Warren F. Lamboy, and James R. McFerson
Clonally propagated crops, unlike seed-propagated crops, require intense and costly maintenance, generally in ex situ field gene banks. Consequently, large germplasm collections of tree species especially, are difficult to conserve in a well-replicated fashion and are vulnerable to damage from environmental stresses. Accordingly, long-term storage in liquid nitrogen presents a viable conservation alternative. To assess effectiveness of one approach to cryopreservation, dormant buds from 64 apple (Malus ×domestica Borkh. and other Malus spp.) accessions were collected and preserved in liquid nitrogen using a dormant-vegetative-bud method. Buds were retrieved from liquid nitrogen storage, rehydrated, and grafted onto rootstocks to determine survival. Mean recovery was 76% for 40 cold-hardy accessions, 66% for 20 moderately cold-hardy accessions, and 24% for four cold-tender accessions (range: 16% to 100%). Only four accessions had ≤25% recovery while 54 accessions had ≤50% recovery and 35 accessions had ≤75% recovery. No significant decline in recovery of these accessions by bud grafting occurred after 4 years of liquid nitrogen storage.
Seibi Oka, Harumi Yakuwa, Kimio Sate, and Takao Niino
Vegetative buds of pear (Pyrus serotina Rehder, cv. Senryo) associated with shoot tissue were frozen stepwise to – 40C, immersed in liquid N, and thawed either at 37C in water or at 0C in air. Survival and shoot formation on thawed material was higher when preliminary cooling lasted 12 to 48 h rather than 0.5 to 2 h. The proper thawing temperature depended on the degree of cooling before cryopreservation. A few whole plants were recovered from the cryopreserved buds.