Characterization of Protein Synthetic Changes in a Desiccation-Tolerant Fern, Polypodium virginianum. Comparison of the Effects of Drying, Rehydration and Abscisic Acid

The desiccation-tolerant fern, Polypodium virginianum, was ableto lose more than 60% of its fresh weight during periods ofprolonged water stress and fully recover during periods of wateravailability. Rehydration from the air-dry state occurred rapidly(within 24 h) with a concurrent initiation of protein synthesis.A low-molecular-weight doublet was synthesized during waterstress and was stable for at least 24 h after rehydration. Theseproteins were expressed also when the fully hydrated frondswere subjected to exogenous ABA even though drying resultedin a 10-fold reduction in ABA content. The large subunit ofRubisco was synthesized during drying, but despite temporaryaccumulation, the de novo synthesized component was degradedduring rehydration. During rehydration from the air-dry state,unique rehydration-specific polypeptides were produced. Thesepolypeptides were synthesized at different water contents andtimes of rehydration, and were transiently expressed. This transientexpression may mean that they are only necessary during theearly stages of rehydration when the rapid initiation of physiologicaland repair processes is essential. The response of this fernto desiccation has features which are similar to those reportedfor desiccation-tolerant mosses, and others which are reminiscentof desiccation-tolerant angiosperms. Key words: Desiccation-tolerance, fern, Polypodium virginianum, protein synthesis, abscisic acid     

Drought Tolerance in Two Mosses: Correlated with Enzymatic Defence Against Lipid Peroxidation

Drought-induced changes in the activities of superoxide dismutase(SOD) and catalase, level of lipid peroxidation, and membranepermeability (solute leakage) have been studied in two mosses,the drought-tolerant Tortula ruralis and the drought-sensitiveCratoneuron filicinum. In T. ruralis the activities of SOD andcatalase increase during slow drying. The level of lipid peroxidationconsequently declines. On subsequent rehydration the enzymeactivities decline and the level of lipid peroxidation risesgradually to normal levels. The leakage of preloaded ⁸⁶Rb onrehydration of slowly dried T. ruralis is similar to that inturgid moss, i.e. leakage of about 20% of tissue ⁸⁶Rb. WhenT. ruralis is subjected to rapid drying there is no change inthe enzyme activities or in lipid peroxidation. However, whenthis moss is rehydrated there is a large immediate increasein lipid peroxidation. Half of the tissue ⁸⁶Rb is leaked intothe bathing medium during the first hour of rehydration. Butwithin the next hour, when SOD and catalase activities haveincreased to high levels, lipid peroxidation quickly declinesto a level lower than that in the turgid control moss, and the⁸⁶Rb leaked earlier is partly reabsorbed indicating that membranerepair is well underway. On prolonged rehydration the enzymeactivities decline and the level of lipid peroxidation risesgradually to reach normal levels found in control turgid moss.In the case of drought-sensitive C. filicinum the activitiesof SOD and catalase decline during drying as well as duringsubsequent rehydration. There is a rapid increase in lipid peroxidationduring rehydration and most of the preloaded ⁸⁶Rb leaks intothe bathing medium irreversibly. The changes in lipid peroxidationduring drying and subsequent rehydration of both the mossesappear to coincide in time with the reported changes in O₂ uptake,indicating that the drought-induced membrane damage may be dueto free radical-induced lipid peroxidation which is known torequire active O₂ uptake. Furthermore, there appears to be agood correlation between an ability of the tissue to controllipid peroxidation and its ability to retain solutes. It issuggested that ability of plant tissues to mobilize enzymaticdefence against uncontrolled lipid peroxidation may be an importantfacet of their drought tolerance.     

Stability and Synthesis of Phospholipids during Desiccation and Rehydration of a Desiccation-Tolerant and a Desiccation-Intolerant Moss

The fatty acid composition of the phospholipids from the desiccation-tolerant moss Tortula ruralis (Hedw.) Gaertn, Meyer and Scherb and the desiccation-intolerant moss Cratoneuron filicinum has been determined. No changes in composition occur in either moss as a consequence of rapid drying, but, after slow drying, there is a decline in some unsaturated fatty acids. Upon rehydration of T. ruralis after slow drying, these acids decline further; however, within 105 minutes, they regain the same levels as those in undesiccated controls. A smaller and more transient decline occurs after rapid desiccation. Most phospholipid unsaturated fatty acids decrease during rehydration of C. filicinum, and their levels are not recovered. After both rapid and slow drying of T. ruralis, acetate and glycerol are incorporated into the phospholipid fraction, although de novo synthesis, alone, might not account for the increase in unsaturated fatty acids upon rehydration. Very little acetate or glycerol is incorporated during rehydration of C. filicinum. Loss of unsaturated fatty acids from the phospholipids of T. ruralis does not appear to be associated with increased lipoxygenase activity. Furthermore, there is little correlation between the extent of peroxidation of fatty acids due to desiccation and changes in the phospholipid fraction.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: III. INSOLUBLE PROTEIN SYNTHETIC PATTERN CHANGES WITHIN THE ENDOSPERM OF Ricinus communis L. SEEDS

Kermode, A. R., Gifford, D. J. and Bewley, J. D. 1985. The roleof maturation drying in the transition from seed developmentto germination. III. Insoluble protein synthetic pattern changeswithin the endosperm of Ricinus communis L. seeds.—J.exp. Bot. 36: 1928–1936. Immature seeds of Ricinus communisL. cv. Hale (castor bean) removed from the capsule at 30 or40 days after pollination (DAP) can be induced to germinateby being subjected to drying. This desiccation–inducedswitch from development to germination is mirrored by a change,upon subsequent rehydration, in the pattern of insoluble proteinsynthesis within the endosperm storage tissue. During normaldevelopment from 25–40 DAP there is rapid synthesis ofthe insoluble (11S) crystalloid storage protein. At later stagesof development (45 and 50 DAP), crystalloid protein synthesisdeclines markedly and synthesis of new insoluble proteins commences.Following premature drying at 30 or 40 DAP, the pattern of insolubleprotein synthesis upon rehydration is virtually identical tothat following imbibition of the mature seed. Proteins synthesizedduring normal late development (at 45 and 50 DAP) are producedup to 48 h after imbibition; a subsequent change in the patternof insoluble protein synthesis occurs between 48 and 72 h. Thus,in contrast to the rapid switch in the pattern of soluble proteinsynthesis induced by drying, insoluble protein syntheses withinthe endosperm are redirected towards those uniquely associatedwith a germination/growth programme only after a considerabledelay following mature seed imbibition, or following rehydrationof the prematurely dried seed. Nevertheless, these results supportour contention that drying plays a role in the suppression ofthe developmental metabolic programme and in the permanent inductionof a germination/growth programme. Key words: Desiccation, crystalloid storage proteins, castor bean, seed development, seed germination     

Plant Desiccation and Protein Synthesis : VI. Changes in Protein Synthesis Elicited by Desiccation of the Moss Tortula ruralis are Effected at the Translational Level

Upon rehydration of the moss Tortula ruralis following desiccation at a rapid or slow rate, there is increasing utilization of newly synthesized-poly(A)⁺ RNA for protein synthesis. Initially, poly(A)⁺ RNA conserved in the dry moss is associated with polysomes, but by 2 hours of rehydration there is an overwhelming recruitment of newly synthesized poly(A)⁺ RNA, at the expense of conserved messages. In rehydrated moss, there is a marked synthesis in vivo of new proteins, which are separable by two-dimensional electrophoresis, and identifiable by fluorography. These new proteins, termed rehydration proteins, are synthesized after both rapid and slow desiccation, but their synthesis persists longer after rapid desiccation. The protein patterns obtained following in vitro translation of bulk RNA from hydrated, desiccated, and rehydrated moss were qualitatively identical. Thus the differences in protein patterns observed in vivo must result from preferential selection of specific mRNAs from the same pool, which is indicative of control of protein synthesis at the translational level. The implications of these observations in relation to the response of the moss to drying in its natural environment are discussed.     

Desiccation of Phaseolus vulgaris Seeds During and Following Germination, and its Effect Upon the Translatable mRNA Population of the Seed Axes

Misra, S. and Bewley, J. D. 1986. Desiccation of Phaseolus vulgansseeds during and following germination, and its effect uponthe translatable mRNA population of the seed axes.—J.exp. BoL 37: 364–374. After imbibition and germination, seeds of P. vulgaris passfrom a stage where they are insensitive to desiccation to astage where they are sensitive. Desiccation of seeds duringthe sensitive stage results in an almost total impairment ofprotein synthesis upon subsequent rehydration. Seeds desiccatedduring the desiccation-tolerant stage, however, resume proteinsynthesis at almost control levels. The protein patterns obtained following in Vitro translationof bulk RNA from fresh imbibed, desiccated, and desiccated-rehydratedseed axes were qualitatively similar at 5 HAI (the desiccation-tolerant stage). The drying treatment resulted in increasedintensity of extant proteins at 5 and 12 HAI. At 12 HAI (thetransition stage between the desiccation-tolerant and desiccation-intolerantphases) desiccation and subsequent rehydration triggered synthesisof a unique set of proteins-the rehydration proteins. At 20HAI (the desiccation-intolerant stage) desiccation resultedin an overall decline in the intensity of proteins synthesizedin vitro. Also the rehydration proteins were not synthesizedin response to a drying and rehydration treatment at this time. Key words: Seed germination, desiccation, mRNA, in vitro translation, Phaseolus vulgaris     

The Effect of Drying Rate on the Survival of Three Desiccation-tolerant Angiosperm Species

The effect of drying rate on the survival of three angiospermresurrection plants, Craterostigma wilmsii (homoiochlorophyllous),Xerophyta humilis (poikilochlorophyllous) and Myrothamnus flabellifolius(homoiochlorophyllous) was examined. All species survived slowdrying, but only C. wilmsii was able to survive rapid drying.C. wilmsii was rapidly able to induce protection mechanismssuch as folding of cell walls to prevent mechanical stress andcurling of leaves to minimize light stress, and thus survivedfast drying. Rapid drying of X. humilis andM. flabellifoliusappeared to allow insufficient time for complete induction ofprotection mechanisms. In X. humilis, there was incomplete replacementof water in vacuoles, the photosynthetic apparatus was not dismantled,plasma membrane disruption occurred and quantum efficiency ofphotosystem II (F_V/F_M) did not recover on rehydration. Rapidlydried leaves of M. flabellifolius did not fold tightly againstthe stem and F_V/F_Mdid not recover. Ultrastructural studies showedthat subcellular damage incurred during drying was exacerbatedon rehydration. The three species co-occur in environments inwhich they experience high desiccation pressures. C. wilmsiihas few features to retard water loss and thus the ability forrapid induction of subcellular protection is vital to survival.X. humilis and M. flabellifolius are able to retard water lossand protection is acquired relatively slowly. Copyright 1999Annals of Botany Company Chlorophyll fluorescence, Craterostigma wilmsii, drying rate, Myrothamnus flabellifolius, resurrection plant, ultrastructure, Xerophyta humilis.     

Glutathione Status and Protein Synthesis during Drought and Subsequent Rehydration in Tortula ruralis

Glutathione status and its relationship to protein synthesis during water deficit and subsequent rehydration have been examined in the drought-tolerant moss, Tortula ruralis. During slow drying there is a small decrease in total glutathione but the percentage of oxidized glutathione (GSSG) increases. During rapid drying there is little change in total glutathione but a small increase in GSSG. On rehydration of slowly dried moss, GSSG rapidly declines to normal level. But when rapidly dried moss is rehydrated, there is an immediate, sharp increase in GSSG as a percentage of total glutathione. After 2 hours of rehydration GSSG starts declining and reaches a normal level in about 6 hours. When an increasing degree of steady state water deficit is imposed on the moss tissue with polyethylene glycol 6000, there is a progressive decrease in protein synthesis but an increase in oxidized glutathione. When 5 millimolar GSSG is supplied exogenously during rehydration of rapidly dried or slowly dried moss, protein synthesis is strongly inhibited. In vitro protein synthesis supported by moss mRNA is also inhibited by more than 85% by 150 micromolar GSSG. The role of glutathione status in water deficit-induced inhibition of protein synthesis is discussed.     

Rates of Drying and Survival of Rhizobium meliloti Strains During Storage at Different Relative Humidities

An investigation was made of the survival of six strains of Rhizobium meliloti filtered on membrane filters and held in atmospheres of controlled relative humidities (RH) of from 0 to 100% at 30°C in the presence of air. The rate of water loss in the desiccator was determined by the humidity-controlling solution used. Drying was accelerated by a mild evacuation of the desiccator during the drying step. Survival rates of R. meliloti strains were much higher after slow drying to 0% RH than immediately after rapid drying. Fast drying (drying period less than 3.4 h) was shown to adversely affect the tolerance to storage at all RH values tested (no survival after 2 to 5 days of storage). When survival during storage was measurable (after slow drying), the optimum RH values for storage were 43% for strains A145 and Wu498, 22 to 43% for strains RCR2011, Wu499, and Ar16, and 83% for strain RCR2004. The most favorable drying periods were 8, 9.2, 14.2, and 50.1 h for the subsequent storage of strain RCR2011 at RH values of 0, 22, 43, and 83%, respectively. The damaging effects of rapid drying on the tolerance of strain RCR2011 to storage at different RH values could be prevented either by rehydration and subsequent slow redrying or incomplete rapid drying followed by slow drying. It is suggested that R. meliloti strains are susceptible to desiccation stresses. However, the quantitative differences among strains appear to be large enough to permit selection with regard to tolerance to desiccation and storage in dried states.     

Desiccation and the Control of Expression of {beta}-Phaseolin in Transgenic Tobacco Seeds

Drying of seeds at certain stages prior to maturation, i.e.premature desiccation, will terminate synthetic events uniqueto development, for example, storage protein synthesis, andinitiate processes associated with germination. In this studywe have investigated the role of desiccation in the expressionof a storage protein gene, ß-phaseolin, to determineif such a developmentally-regulated gene remains sensitive todrying when controlled by a promoter that has no known sensitivityto this treatment. We compared, in transgenic tobacco seeds,the effects of maturation and premature drying on the expressionof a full ß-phaseolin gene, and ß-phaseolingenes driven by a cauliflower mosaic virus 35S promoter withor without an alfalfa mosaic virus (AMV) 5' untranslated leadersequence. The results indicate that the ß-phaseolinpromoter is directly down-regulated by desiccation during maturationand, although activated during the drying phase of a prematuredesiccation event, it is not active upon rehydration or imbibition.The 35S promoter is down-regulated also by both maturation dryingand premature desiccation but unlike the ß-phaseolinpromoter it is reactivated upon rehydration or imbibition. Key words: Desiccation, ß-phaseolin, gene regulation, Phoseolus vulgaris, seed development     

Abscisic Acid Enhances the Ability of the Desiccation-Tolerant Fern Polypodium virginianum to Withstand Drying

Detached fronds of Polypodium virginianum L. survived loss of65–70% of their fresh weight over 10 d of slow-drying.Drying over silica gel resulted in a faster rate of water loss,to a lower fresh weight, which the fronds did not survive. Whenfronds were incubated in abscisic acid for 24 h prior to silica-drying,the amount of water lost was reduced, resulting in survivalof the fronds upon subsequent rehydration. Incubation in abscisicacid for at least 18 h was necessary for survival. Fronds inwhich the final fresh weight after drying was below a criticalamount (i.e. to less than 25% original fresh weight) did notsurvive. A reasonable correlation could be drawn between electrolyteleakage upon rehydration and survival of somedesiccation treatments,although this was not always clear-cut, especially in frondsincubated in abscisic acid for an insufficient time to ensuresurvival. Several polypeptides were synthesized during slow-and silica-drying, and in response to abscisic acid. No novelpolypeptides were identified that were unique to the desiccationregimes which resulted in survival. Nor did ABA induce specificproteins in fronds desiccated after preincubation in this regulatorfor 24 h. Key words: Desiccation-tolerance, abscisic acid, protein synthesis, fern, Polypodium virginianum     

Dehydration rate and time of desiccation affect recovery of the lichenic algae <Emphasis Type="Italic">Trebouxia erici</Emphasis>: alternative and classical protective mechanisms

The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F′_o) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q _E), whereas after fast dehydration it was photoinhibition (q _I). Although q _E seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.     

Respiration in Relation to Adenosine Triphosphate Content during Desiccation and Rehydration of a Desiccation-tolerant and a Desiccation-intolerant Moss

O₂ consumption by the desiccation-tolerant moss Tortula ruralis and the desiccation-intolerant Cratoneuron filicinum increased markedly during the latter stages of desiccation. ATP content of the mosses during desiccation was not correlated with O₂ consumption, but was influenced by the rate at which the mosses lost water. The more rapid the water loss, the more ATP that was present in the dry mosses. The pattern of O₂ consumption on rehydration also was influenced by the previous rate of desiccation. After rapid desiccation of T. ruralis O₂ consumption upon rehydration was considerably elevated, and for up to 24 hours. After very slow desiccation the elevation was small and brief. Normal O₂ consumption did not occur in C. filicinum after rapid desiccation, but did so within a few hours of rehydration after slower speeds of drying. ATP levels in T. ruralis returned to normal within 5 to 10 minutes of rehydration. In C. filicinum, increases in ATP were closely correlated with O₂ consumption. These observations are considered to be related to differential damage caused to mitochondria and to cellular integrity by different speeds of water loss. The desiccation-tolerant moss appears to be able to repair the severe damage imposed by rapid desiccation whereas the desiccation-intolerant moss cannot.     

Drought Stress, Enzymes of Glutathione Metabolism, Oxidation Injury, and Protein Synthesis in Tortula ruralis

The activities of glutathione reductase (EC 1.6.4.2), glutathione peroxidase (EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) were found to increase during slow drying or during rehydration following rapid drying of the drought-tolerant moss Tortula ruralis. Little change was observed in the activity of malate deydrogenase (NAD⁺ oxidoreductase, EC 1.1.1.37) during dehydration or subsequent rehydration. When the tissue was treated with cycloheximide, actinomycin D, or cordycepin, the increase in the activities of glutathione reductase and glutathione S-transferase was largely prevented while effect on glutathione peroxidase was much smaller. Concomitantly, oxidized glutathione (GSSG) as percentage of total glutathione increased. GSSG level was correlated positively with the levels of lipid peroxidation and solute leakage and negatively with the rate of protein synthesis. The results show that GSSG level is a good indicator of oxidation stress and provide support to the suggestion that GSSG mediates, at least in part, the drought stress-induced inhibition of protein synthesis.     

Effects on Phytochrome Controlled Germination Produced by Far-Red Irradiation of Seeds Before and During Rehydration

Seeds irradiated with red light and then re-dried will respondto this light treatment on subsequent rehydration in the dark.If such high-Pfr seeds are irradiated in the dry state withfar-red light immediately before rehydration the percentagegermination is significantly reduced in the case of Plantagomajor and Sinapis arvensis but increased in Bromus steriliswhere Pfr inhibits germination. This effect of far-red lightcan be reversed by red light despite the fact that red lightalone has no effect on dry seed. This is due to the interconversionof Pfr and the red light absorbing phytochrome intermediatecomplex meta-Fa. If there is a delay between far-red irradiationand rehydration of Sinapis seeds, the inhibitory effect of thefar-red irradiation becomes progressively less the longer thedelay. This reduction in effectiveness of far-red is interpretedin terms of a dark reversal of meta-Fa to Pfr with a half-lifeof about 4–6 h. The reappearance of Pfr is either veryslow or docs not occur in dehydrated Plantago seeds, as far-redtight given 96 h prior to hydration is just as inhibitory asfar-red light given immediately before hydration. Meta-Fa doesappear to revert to Pfr in darkness in Bromus seeds, but onlyvery slowly. The rapid increase in effectiveness of red irradiationduring rehydration of high-Pfr Plantago seeds suggests that,in this species, the pre-treatment used in preparation of high-Pfrseeds may increase the receptivity or amount of the Pfr reactionpartner. Key words: Phytochrome intermediates, Seeds, Germination     

Plant Desiccation and Protein Synthesis : V. Stability of Poly (A) and Poly (A) RNA during Desiccation and Their Synthesis upon Rehydration in the Desiccation-Tolerant Moss Tortula ruralis and the Intolerant Moss Cratoneuron filicinum

Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)⁻ RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)⁺ RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)⁻ RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)⁺ RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples.

Poly(A)⁻ RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)⁺ RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)⁺ RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)⁺ RNA for protein synthesis. The rate of synthesis of poly(A)⁺ RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)⁺ RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance.

     

Factors Inducing Successful Anhydrobiosis in the African Chironomid Polypedilum vanderplanki: Significance of the Larval Tubular Nest

The African chironomid Polypedilum vanderplanki exhibits anhydrobiosis,i.e., the larvae can survive complete desiccation. Recoveryrate and trehalose content were investigated in larvae desiccatedslowly or at a rate more than 3 times faster. Upon slow desiccation(evaporation rate 0.22 ml day^–1) larvae synthesized 38µg trehalose/individual before complete desiccation, andall of them recovered after rehydration, whereas larvae thatwere dehydrated quickly (evaporation rate 0.75 ml day^–1)accumulated only 6.8 µg trehalose/individual and noneof them revived after rehydration. In the pools that are theirnatural habitat P. vanderplanki larvae make tubes by incorporatingdetritus or soil with their sticky saliva. This tubular structureis a physical barrier not only to protect the larva from naturalenemies but also induces successful anhydrobiosis by reducingthe dehydration rate. When larvae were dehydrated with 100 µldistilled water (DW) in soil tubes, they accumulated 37 µgtrehalose/individual and more than half of them could reviveafter rehydration, whereas larvae without tubes accumulatedlower level of trehalose and none recovered after rehydration.     

Dried Cryopreserved Somatic Embryos of Two Picea Species Provide Suitable Material for Direct Plantlet Regeneration and Germplasm Storage

The present study aimed to develop a cryopreservation methodfor long-term storage of mature somatic embryos of Picea spp.The effects of drying rate, embryo water content prior to cryopreservation,thawing rate, and rehydration on survival of cryopreserved somaticembryos were investigated. Emphasis was placed on the capacityof cryopreserved somatic embryos to germinate and regenerateplantlets directly, or to reinduce embryogenic tissue for newembryo production. Firstly, a slow drying rate at 97 or 88%relative humidity (RH) was needed to achieve high germination(96.7–100%) and high plantlet conversion rates (26.7–46.7%)(not different from controls). Secondly, somatic embryos hadto reach a water content of 0.23 g H₂O g^-1d.wt (48 h of desiccationat 97% RH) before immersion in liquid nitrogen to germinateat high frequency (93.8%). Thawing techniques had no effecton embryo survival. Dried and cryopreserved somatic embryosof Picea can also be used to reinduce embryogenic tissue andstart new embryo production. Best reinduction frequency (66.7%)was obtained from cryopreserved embryos dried at 97% RH andrehydrated at 100% RH for 12 h prior to reinduction. No differencein embryo production was noticed between the parent line (1stembryogenic cycle) and the sub-lines (2nd embryogenic cycle).Second generation embryos germinated and regenerated into plantletsat rates similar to controls. The optimal cryopreservation methodwas successfully applied to severalP. mariana and P. glaucagenotypes. Copyright 2000 Annals of Botany Company Picea mariana(Mill) B.S.P., Picea glauca(Moench) Voss, desiccation, embryo water content, thawing, rehydration, embryogenic tissue, genotypes, conifers, clonal propagation