Cryopreservation of Citrus aurantifolia seeds and embryonic axes using a desiccation protocol

The desiccation and freezing tolerance of seeds, with and without testas, and embryonic axes of Citrus aurantifolia were investigated. Seeds were desiccated with silica gel, under the laminar air flow cabinet or by placing them on a laboratory bench. Whatever the desiccation method employed, survival before and after cryopreservation was higher for seeds without testas. When freezing intact seeds, the highest survival percentage (41.3 %) was achieved after desiccation to 7.3 % moisture content (fresh weight basis) on the laboratory bench. Survival of seeds cryopreserved without testas could reach up to 85 % after desiccation under the laminar air flow cabinet or on the laboratory bench, corresponding to moisture contents of 7.1 and 4.5 %, respectively. After desiccation with silica gel, survival reached a maximum of 60.0 %, for a seed moisture content of 3.3 %. Survival of control embryonic axes was high (80-100 %) whatever the sucrose concentration in the preculture medium and the duration of the desiccation period. After cryopreservation, no survival was noted with embryonic axes, which had not been precultured nor desiccated. Survival of non-desiccated embryonic axes after cryopreservation increased progressively in line with increasing sucrose concentrations in the preculture medium, from 7.5 % with 0.1 M sucrose to 77.5 % with 0.7 M sucrose. Survival of desiccated and cryopreserved embryos was always high, whatever the preculture treatment and desiccation period, ranging from 55.8 % to 92.5 %.     

Ammonium nitrate in the culture medium influences regeneration potential of cryopreserved shoot tips of Holostemma annulare

Influence of NH4NO3 in the pre-freeze and post-freeze culture medium and 2 or 30 day preconditioning in the presence of 0.5 M sucrose on regeneration of shoot tips of Holostemma annulare following cryopreservation using an encapsulation-dehydration protocol was studied. A long preconditioning phase of 30 days significantly reduced tissue water and improved post-freeze recovery of shoot tips. Under the long preconditioning treatment, Murashige & Skoog (MS) medium free of NH4NO3 (MS-3) allowed maximum regeneration (59%) of liquid nitrogen (LN) exposed shoot tips with less frequency of callusing (10.4%) after 45 days of post-freeze culture. Corresponding desiccated control shoot tips showed 85-90% regeneration. A 3.75 mM NH4NO3 concentration (MS-4) favoured 72-89% and 43-47% regeneration after desiccation and LN exposure respectively. The standard MS medium with 20.6 mM NH4NO3 (MS-1) allowed poor regeneration after desiccation (39-53%) as well as LN exposure (8-23%). The study reveals the importance of reducing ammonium nitrate in the culture medium to get maximum recovery of cryopreserved shoot tips of Holostemma annulare.     

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique

Cryopreservation of ovules and somatic embryos from several genotypes of citrus was achieved using the encapsulation-dehydration technique. Survival of cryopreserved ovules was occasional and erratic after different pregrowth conditions in liquid medium with 0.75M, 1M or up to 1.25M sucrose. An efficient cryopreservation protocol was established for somatic embryos derived from two embryogenic sources (ovules and cut thin layer explants from stigma, style and ovaries). High survival rates (75-100%) were consistently obtained after 1 day pregrowth in 0.75M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in liquid nitrogen. The histological study showed that embryos subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation.     

Zygotic and nucellar embryo survival following dehydration and cryopreservation of citrus intact seeds

A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16 percent (C. sinensis) or 10 percent (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50 percent after drying to 15 percent moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93 percent germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.     

Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature

Seeds of Dactylorhiza fuchsii (common spotted orchid) and Anacamptis morio (green-winged orchid) were encapsulated in alginate beads with hyphae of the basidomycete fungus Ceratobasidium cornigerum. Pre-treatment of beads for 18 h with sucrose at an optimum concentration of 0.75 M decreased the desiccation rate in a flow of sterile air (c. 23 degree C, 30% RH) and increased seed and fungal survival after up to 16 h drying. Pre-treated and 16-h dried beads were transferred to cryo-vials and subsequently stored at a range of low temperatures for up to 30 d. Neither embryo growth of both orchids nor fungal development was detrimentally affected by 1 d storage at -196 degree C when the beads were pre-dried to c. 20% moisture content. Encapsulated D. fuchsii seed and compatible fungus had < 5% and < 45% viability when beads of the same moisture content were stored for 1 d at -20 degree C and -70 degree C respectively. In contrast, viability of the seed and the fungus remained unchanged during 30 days storage at -196 degree C but was progressively lost at 16 degree C over the same interval. The results indicate opportunities for the use of simultaneous cryopreservation as a conservation tool for diverse taxa.     

Cryopreservation of winter-dormant apple buds: II - tissue water status after desiccation at -4C and before further cooling

The established protocol for the cryopreservation of winter-dormant Malus buds requires that stem explants, containing a single, dormant bud are desiccated at -4 degree C, for up to 14 days, to reduce their water content to 25-30 percent of fresh weight. Using three apple cultivars, with known differences in response to cryopreservation, the pattern of evaporative water loss has been characterised, including early freezing events in the bud and cortical tissues that allow further desiccation by water migration to extracellular ice. There were no significant differences between cultivars in this respect or in the proportions of tissue water lost during the desiccation process. Differential Scanning Calorimetry (to -90 degree C) of intact buds indicated that bud tissues of the cultivar with the poorest response to cryopreservation had the highest residual water content at the end of the desiccation process and froze at the highest temperature.     

Narrowing oF the critical hydration window for cryopreservation of Salix caprea seeds following ageing and a reduction in vigour

We investigated the effects of desiccation, rehydration and cryopreservation on the viability of seeds of a wild mountain species and seven clones of Salix caprea L. Seeds responded differently to all treatments depending on clone, seed initial moisture content (MC) and seed vigour. Fresh seeds of two randomly selected clones tolerated desiccation to MC 8.5-9.6 % FW (0.09-0.11 g water per g dry mass. g/gdw) without any noticeable loss in viability and were successfully cryopreserved at MCs ranging from 8.5 to 23.4 % (0.09-0.30 g/gdw). Storage at 5 degree C for approximately 10 weeks significantly reduced the viability of seed lots of a wild species and of three S. caprea clones, whilst viability of seeds of four other clones remained unaffected. Since all clones tested were genetically derived from one tree, this variation is unlikely to be of maternal origin. Most probably paternal x environmental factors have influenced seed behavior during desiccation and storage. As viability decreased due to partial ageing, seeds became more susceptible to desiccation stress. When seeds of three clones were cryopreserved, the hydration window for survival was wider for highly vigorous seeds (c. 0.05-0.28 g/gdw) than for seeds with intermediate vigour (c. 0.10-0.24 g/gdw) and low vigour (c. 0.20-0.37 g/gdw). Rehydration to MC above 0.15 g/gdw improved germination of low vigour seeds, both in controls and after cryopreservation. In contrast, cryopreservation of high vigour seeds rehydrated to MCs above 0.11 g/gdw resulted in a sharp decrease in normal seedling production. Whilst no effect of cryogenic temperature on germination and normal seedling production was observed when seeds of seven clones were cryopreserved within their hydration windows, the results indicate the need to account for seed lot vigour when designing cryopreservation protocols.     

Acquisition of cryotolerance in maize embryos during seed development

A desiccation-based cryopreservation protocol was employed to study the development of cryotolerance and desiccation tolerance in maize embryos from 23 to 50 days after pollination (DAP). Tolerances were acquired gradually and concomitantly. Maize embryos had low desiccation tolerance at 23 DAP when assessed by survival (embryo elongation) and emergence (root and shoot growth) after dehydration. Desiccation tolerance increased progressively, reached its maximum at 38 DAP, and remained constant afterwards. Cryotolerance, assessed by survival and emergence of post-thaw embryos, however, was nil until 26 DAP. Embryos at 29 DAP withstood cryoexposure within a very narrow moisture range only. Throughout development cryotolerance increased gradually, reached a maximum at 44 DAP and then remained at this level. The correlation between moisture content and cryopreservation success was notably influenced by the maize embryo's development stage. As seeds developed, the moisture content allowing 90% dehydrated embryos to survive and to emerge decreased, while the upper moisture content allowing 50% post-thaw embryos to survive and to emerge increased. Moisture contents of c. 14% allowed no less than 50% post-thaw embryos to emerge at the later development stages (e.g. c. 44 DAP); but no embryos within the same moisture range survived cryoexposure at 29 DAP, although they could withstand desiccation to this moisture level without impairment of survival and emergence. The relationship between cryotolerance and desiccation tolerance during maize seed development is discussed.     

Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut

This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93 % and 100 % of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24 % (on a fresh weight basis), and some 63 % subsequently developed as whole plants. Desiccation to moisture contents less than 19 % produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6-8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25 % before storage in LN, the embryogenesis resumption level after thawing was 33 %. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68 %.     

Desiccation and freezing tolerance of embryonic axes from Citrus sinensis [L.] osb. pretreated with sucrose

Embryonic axes of Citrus sinensis L. were successfully cryopreserved. While fully hydrated unfrozen axes germinated 100%, survival decreased as axes water content dropped, and total loss of viability was observed when the water content dropped to 0.04 and 0.10 mg H2O/mg dry mass, for axes without and with sucrose preculture, respectively. Fully hydrated axes did not survive exposure to liquid nitrogen. Highest seedling recovery (93-100%) for untreated axes was observed at 0.26 to 0.15 mg H2O/mg dry mass. Differential scanning calorimetry revealed the presence of broad melting peaks in fully hydrated embryonic axes. The size of the melting peak diminished as water was removed by desiccation. Minimum melting of water was observed at the point axes survived cryopreservation. Occurrence of a glass transition upon warming was not a condition for axes to survive liquid nitrogen exposure. In untreated axes, glucose, increased with desiccation to 0.2 mg H2O/mg dry mass, and decreased as the axes were desiccated to lower water contents. Fructose and sucrose levels did not increase when untreated samples were desiccated for the same periods of time. Raffinose and stachyose levels decreased as untreated and precultured embryonic axes were desiccated. In sucrose precultured axes, sucrose and fructose levels increased when they were dehydrated, reaching maximum levels at 0.2 mg H2O/mg dry mass. Tissue glucose did not change significantly with desiccation. Raffinose and stachyose levels dropped as precultured embryonic axes were dried.     

Cryopreservation of mango (Mangifera indica L.) embryogenic cultures

Three techniques were compared for cryopreserving embryogenic masses (EMs) sampled from mango (Mangifera indica L.) cv. Zihua embryogenic cultures: (i) encapsulation/dehydration; (ii) pregrowth/dehydration; and (iii) vitrification. In all experiments, EMs were sampled from embryogenic cultures during their exponential growth phase and pretreated for 24 h on solid medium containing 0.5 M sucrose before freezing. No recovery was achieved after cryopreservation using the encapsulation/dehydration technique, whatever the moisture content (fresh weight basis) of EMs, which ranged from 78.3% without dehydration to 40.8% after 6 h dehydration. With the pregrowth plus dehydration technique, limited recovery (8.3%) was achieved after desiccation of EMs for 1 h, to 58.5% MC. Using the vitrification technique, recovery ranged from 94.3% after treatment of EMs with the PVS3 vitrification solution for 20 min (EM moisture content of 34.7%) to 10.9% after a 120 min treatment with the vitrification solution (EM moisture content of 26.0%).     

Cryopreservation of black iris (Iris nigricans) somatic embryos by encapsulation-dehydration

Somatic embryos of black iris (Iris nigricans) were cryopreserved using encapsulation-dehydration. Embryos size of 2-4 mm gave the highest survival after cryopreservation. Preculturing embryos on medium containing 0.75 M sucrose for 3 d at 22 degree C, then at 30 degree C for 1 d ensured maximum survival (60%). Viable embryos were brown in color after cryopreservation and during early recovery while dead ones where light brown or creamy. The first sign of regrowth was noted after 15 d. The final regrowth percentage of living embryo was 90% after 35 d. Only 10% of the embryos in all treatments showed secondary embryogenesis. Direct sowing in vivo of cryopreserved embryos was not successful in achieving germination.     

The pertinence of expression of heat shock proteins (HSPs) to the efficacy of cryopreservation in HELAs

HELAs (Hela cells, passed cells of human cervical carcinoma) were heat or cold treated (named heat or cold shock) and then resumed normal culture for 2, 4 or 8 hours respectively. The expressions of heat shock protein 70 (HSP70) and 90 (HSP90) of the HELAs were measured by Northern and Western blotting. HELAs after 4-hour culture were exposed to or cryopreserved with different concentration of dimethyl sulfoxide (Me2SO, 2.5%, 5%, 10%, 15% and 20% respectively, V/V). Meanwhile, the HELAs after different culture time (2, 4 and 6 hours of culture) were cryopreserved with 5% Me2SO. After exposure or cryopreservation, the number of live HELAs was counted and the survival rate was calculated. The results showed that heat shock increased the expression of HSP70 and HSP90 of HELAs, while cold shock decreased the expression of the two proteins. When the concentrations of Me2SO were 10%, 15% and 20%, the survival rates of HELAs after exposure to Me2SO or cryopreservation were much lower than those when the concentrations were small. The survival rates of the heat shocked HELAs were significantly higher than those of the cold shocked and control HELAs. After cryopreservation with 5% Me2SO, the survival rate of heat shocked HELAs group with 2 hours culture time was the lowest among all the groups of HELAs with different cultural time. From the results of this study, we conclude that the expressions of HSP70 and HSP90 in HELAs increased significantly after heat shock, while cold shock decreased the expressions of these two proteins. The over-expressions of HSPs in the heat shocked HELAs could protect the cells from both injury caused by potential toxicity of high concentrations of Me2SO and cryoinjury caused by the freeze-thawing/cryopreservation procedure.     

Cryopreservation of Quercus suber and Quercus ilex embryonic axes: in vitro culture,desiccation and cooling factors

This study examines different factors included in the cryopreservation protocols for Quercus ilex and Q. suber embryonic axes. In vitro incubation temperature played an important role in the appropriate development of Q. ilex axes, as 15 degrees C was superior to 25 degrees C. Q. suber axes proved to be more sensitive to desiccation and cooling. Poor survival (35%) was observed when axes were included into cryovials and then in liquid nitrogen, and none when immersed in sub-cooled liquid nitrogen (-210 degrees C). Q. ilex axes showed poorly organised development in vitro (c. 50% of non-cooled axes showed shoot development). However, c. 80% survival was observed after cryopreservation (either in liquid nitrogen or sub-cooled liquid nitrogen at 0.34 g water / g dry weight), of which c. 15% showed shoot development.     

Cryopreservation, encapsulation and promotion of shoot production of embryonic axes of a recalcitrant species Ekebergia capensis, Sparrm

A study on zygotic axes of the recalcitrant seeds of Ekebergia capensis compared two cryopreservation methods, partial desiccation, and encapsulation-dehydration, and also investigated a method to promote shoot production. High (80 percent) survival (assessed as root production) was obtained after direct immersion into liquid nitrogen of axes rapidly dehydrated by flash drying for 20 min to a water content about 0.4 g water per g dry mass. In contrast, no survival at all was obtained of axes that were first encapsulated, then desiccated for three hours to the same water concentration as those fast-dried, and then placed in a cryovial and immersed in liquid nitrogen. Axes encapsulated after cryopreservation germinated both in vitro and in soil, and could be stored at room temperatures for several weeks while maintaining germinability, thus producing synseeds capable of distribution. However, shoot production after cryopreservation was seldom observed. The inclusion of the plant growth regulator, N6-benzyl adenine (BA) in the MS-based recovery medium promoted vigorous multiple shoot formation. Microscopical examination of embryos of E. capensis revealed that the cotyledonary insertions were contiguous with the shoot apex, leading to the conclusion that injury to, and ultimate necrosis of, the apical meristem following severing of these connections was a primary cause of the observed lack of, or poor, shoot development in excised axes (whether cryopreserved or not). The study demonstrated that it may be possible to resolve two of the problems facing attempts at cryopreservation of axes of recalcitrant seeds; lack of shoot production and difficulty of distribution of cryopreserved material for re-introduction.     

Cryopreservation of chayote (Sechium edule JACQ. SW.) zygotic embryos and shoot-tips from in vitro plantlets

This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM were successfully cryopreserved using a vitrification technique. Optimal conditions included the culture of mother-plants for 22 days on medium containing 0.3 M sucrose, culture of excised apices on the same medium for 1 day, loading of apices for 20 min with 2M glycerol + 0.4M glycerol, treatment with a series of diluted PVS2 solution (60 % PVS2 followed by 80 % PVS2 solution for 15 min (cultivar Cocoro Blanco [CB]) or 30 min (cultivars CN and Cl) at each concentration), rapid freezing and thawing, washing of shoot-tips with a 1.2 M sucrose solution, followed by recovery on media with progressively decreasing sucrose concentrations until the standard concentration of 0.1 M was reached. The highest survival percentages achieved ranged between 17 and 38 %, depending on the cultivar.     

Seed cryopreservation and longevity of two Salix hybrids

The effects of desiccation and storage temperature on the viability and longevity of willow seeds was investigated using two hybrids, Salix rehderiana x (Salix x capreola) [cross 458] and Salix x sericans x Salix viminalis [cross 512]. Freshly harvested seed of both crosses survived silica gel drying down to c. 3 to 5% moisture content. Hybrid 458 seed stored in liquid nitrogen (-196C) for 3 d retained viability when equilibrated to < or = 45% RH (pre-storage), showed slightly reduced survival at 65% RH and exhibited no survival at > or =82% RH. The level of survival after 68 d for seeds pre-equilibrated to either c. 10 or 65% (5 or 10% moisture) and stored at four temperatures was -196C > -20C > 2C > 16C. At all temperatures, drier seed stored better than wetter seed. For hybrid 512, seed longevity at 20C > 40C > 60C, and a 10% fall in pre-storage seed RH resulted in a c. 2-fold increase in longevity at each storage temperature. The response of hybrid willow seeds to desiccation and cooling raises possibilities for the long-term seed conservation of Salix species by cryopreservation.     

Acquisition and loss of cryotolerance in Livistona chinensis embryos during seed development

Changes in desiccation tolerance and cryotolerance of chinese fan palm (Livistona chinensis [Jacq.] R. Br.) Embryos were studied during seed development from 15 to 45 weeks after flowering (WAF). Acquisition and then progressive loss in both desiccation tolerance and cryotolerance was observed within this period. Survival (apparent elongation of embryos) and emergence (formation of root and/or shoot) of embryos following dehydration increased progressively with development of seeds until 33 WAF, and then decreased up to 45 WAF. Similar changes occurred in the minimum moisture content at which 90% of embryos survived or emerged. Cryotolerance of embryos was nil at the early stages of seed development, until 21 WAF. Embryos acquired slight cryotolerance at 23 WAF and cryotolerance increased gradually from 27 to 36 WAF, then decreased by 45 WAF. Survival and emergence of post-thaw embryos were closely related to their moisture contents prior to freezing. However, this correlation between cryopreservation and moisture content was notably influenced by the embryos' developmental stage. Embryos at stages with greater cryotolerance gave higher post-thaw survival and emergence at a given moisture content, and the moisture content range allowing embryos to avoid cryo-damage was widened at both the lower and upper limits. Greater than 50% post-thaw emergence was observed only in embryos with moisture contents below 20% (fresh weight) at developmental stages between 27 and 36 WAF, although more than 90% of embryos could be dehydrated to < 20% moisture contents without loss in survival and emergence as early as 21WAF. Nearly 80% embryos could be dehydrated safely to 20% moisture content as late as 45 WAF.     

Cryopreservation of dormant herbaceous peony (Paeonia lactiflora pall.) shoot-tips by desiccation

A simplified technique for the cryoprotection of dormant shoot-tips was developed for the germplasm conservation of herbaceous peony (Paeonia lactiflora Pall.). The highest post-thaw regrowth level was obtained from shoot-tips desiccated by air drying for 5 h. The post-thaw regrowth of P. lactiflora. var. 'Mikang' was the highest (74.9 percent) among the five genotypes tested. The regeneration level after cryopreservation was highest (66approximate74%; average of 70 percent) for dormant shoot-tips collected from November to February. Shoot tips which were dissected from non-dormant buds in late March, however, showed very low post-thaw regrowth. Post-thaw regrowth of shoot-tips was promoted in MS medium containing 1 mg 1(-1) of gibberellic acid (GA3) and 0.5 mg 1(-1) of N(6)-benzyladenine (BA). The elongated shoots were rooted on a 1/4 MS medium containing 0.1 mg 1(-1) of beta-naphthalene acetic acid (NAA). In addition, random amplified polymorphic DNAs (RAPDs) assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the peony dormant shoot-tips. This cryopreservation method appears to be promising for the conservation of herbaceous peony germplasm.     

The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa

The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) DMSO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (approximately 5 degrees C/min(-1)) and high cooling rates (approximately 35 degrees C/min(-1)). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0 +/- 2.5%, 15.3 +/- 1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 +/- 1.2%, 26.8 +/- 2.5%, P<0.02, respectively), 3. glycerol was more effective than DMSO with fast cooling (18.3 +/- 2.2%, 12.6 +/- 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 +/- 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as cryoprotectants and not as exogenous energy substrates.