The use of plant stress biomarkers in assessing the effects of desiccation in zygotic embryos from recalcitrant seeds: challenges and considerations.

Zygotic embryos from recalcitrant seeds are sensitive to desiccation. In spite of their sensitivity, rapid partial dehydration is necessary for their successful cryopreservation. However, dehydration to water contents (WCs) that preclude lethal ice crystal formation during cooling and rewarming generally leads to desiccation damage. This study investigated the effects of rapid dehydration on selected stress biomarkers (electrolyte leakage, respiratory competence, rate of protein synthesis, superoxide production, lipid peroxidation, antioxidant activity and degree of cellular vacuolation) in zygotic embryos of four recalcitrant-seeded species. Most biomarkers indicated differences in the levels of stress/damage incurred by embryos dried to WCs < and >0.4 g·g(-1) , within species; however, these changes were often unrelated to viability and percentage water loss when data for the four species were pooled for regression analyses. Dehydration-induced electrolyte leakage was, however, positively related with percentage water loss, while biomarkers of cellular vacuolation were positively related with both percentage water loss and viability. This suggests that electrolyte leakage and degree of cellular vacuolation can be used to quantify dehydration-induced stress/damage. Biomarkers such as superoxide production, whilst useful in establishing the nature of the dehydration stress incurred may not be able to distinguish the effects of different WCs/drying times. Irrespective of which biomarker is used, the data suggest that understanding differences in desiccation sensitivity across recalcitrant-seeded species will remain a challenge unless these biomarkers are related to a generic desiccation stress index that integrates the effects of percentage water loss and drying time.

[Physiological and biochemical change of Paris seed in after-ripening during variable temperature stratification].

In order to explore the dormancy physiological and biochemical mechanism of Paris seeds, the seed embryo growth courses, and the dynamic change of 5 enzymes, include SOD, POD, CAT, MDH, G-6-PDH were measured during variable temperature stratification. The results indicated that Paris seeds embryo grew quickly after 40 d in warm-stratification (18 ± 1) °C, at the meantime the metabolic activity was significantly strengthened. These facts showed that Paris seeds turned into physiological after-ripening process. After 60-80 d, the morphological embryo after-ripping process basically completed, and the following cold-stratification (4 ± 1) °C furthered Paris seed to finish physiological after-ripening. After 40 d, the activity of MDH decreased while G-6-PDH increased significantly. This showed that the main respiratory pathway of seed changed from TCA to PPP, which benifited breaking seed dormancy. In the whole period of stratification process, the activity variation of SOD and CAT was insignificantly and the activity of POD was enhanced significantly after shifting the seed in cold stratification process. This showed that SOD, CAT had no direct effects on breaking Paris seed dormancy but keeping the seed vigor, while the POD might involve in the process of Paris seed dormancy breaking.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

KEY MESSAGE: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: challenges and considerations for cryopreservation.

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. However, the variable post-cryo success achieved with the excised zygotic explants traditionally used for cryopreservation has been a concern for some time. Differential drying rates amongst explants of different species, uneven drying amongst explants within a batch of seeds and uneven drying across tissues within individual embryos could be contributory factors to this variable success and these phenomena form the foci of the present study. Using zygotic explants from a range of recalcitrant-seeded species, which included sub-tropical dicotyledonous trees and sub-tropical monocotyledonous geophytes, the study showed that embryo morphology and anatomy are critical determinants of the drying characteristics of the different tissues composing the explant and hence, post-cryo survival. The results suggest that the rates of drying of explants to water contents (WCs) in the theoretically optimal range for successful cryopreservation are species-specific, and that more rapid drying rates may promote post-cryo survival. However, the large variation in WC amongst individual explants in bulk samples challenges the selection of the theoretically optimum WC for cryopreservation. As a consequence of differential drying rates across the different tissues composing explants, either lethal ice crystal damage or desiccation damage may sometimes be likely in tissues responsible for the onwards development of the embryo. Drying times for cryopreservation of such explants should, therefore, be selected on the basis of WC of segments containing root or shoot meristem, rather than embryo bulk WC. Drying intensity and duration also interact with explant morphology and embryo/axis size and anatomy to bring about - or preclude - post-cryo survival.

Cryo-tolerance of zygotic embryos from recalcitrant seeds in relation to oxidative stress--a case study on two amaryllid species.

Oxidative stress is a major component of cryoinjury in plant tissues. This study investigated the ability of recalcitrant (i.e. desiccation sensitive) Amaryllis belladonna L. and Haemanthus montanus Baker zygotic embryos to survive cryopreservation, in relation to oxidative stress. The study also investigated whether glycerol cryoprotection promoted embryo post-cryo survival by protecting enzymic antioxidant activities. Zygotic embryos excised from hydrated stored seeds were subjected to various combinations of rapid dehydration (to < or >0.4 g g⁻¹ [dmb]), cryoprotection (with sucrose or glycerol), and cooling (either rapidly or slowly), and were thereafter assessed for viability, extracellular superoxide (·O2⁻) production, lipid peroxidation (TBARS) and antioxidant enzyme activities. Short-term hydrated storage of whole seeds was accompanied by ·O2⁻ production and lipid peroxidation, but ·O2⁻ levels were lower than in dehydrated and cooled embryos and viability was 100%, possibly associated with the high activities of certain antioxidant enzymes. Partial dehydration and cryoprotection (in H. montanus only) increased ·O2⁻ production (especially in cryoprotected-dried embryos) and was associated with some viability loss, but this was not correlated with enhanced lipid peroxidation. Cooling was generally accompanied by the greatest increase in ·O2⁻ production, and with a decline in viability. In A. belladonna only, post-cryo TBARS levels were generally higher than for fresh and pre-conditioned embryos. Partial dehydration and cooling decreased antioxidant activities, but these were consistently less severe in glycerol cryoprotected-dried, as opposed to non-cryoprotected-dried embryos. Post-cryo viability retention for glycerol cryoprotected-dried embryos was significantly higher than for non-cryoprotected-dried embryos, possibly facilitated by relatively low post-drying TBARS levels and high post-drying and post-rewarming activities of some antioxidant enzymes in the former. Pre-conditioning treatments such as glycerol cryoprotection, when used in combination with partial drying, may enhance post-cryo viability retention in recalcitrant zygotic embryos by protecting the activities of certain antioxidant enzymes during pre-conditioning for, and after retrieval from, cryostorage.

Effects of cryopreservation of recalcitrant Amaryllis belladonna zygotic embryos on vigor of recovered seedlings: a case of stress 'hangover'?

Cryopreservation is the most promising long-term storage option for recalcitrant (i.e. desiccation-sensitive) seed germplasm; however, its effects on the vigor of recovered seedlings are unclear. This study looked at the vigor of seedlings recovered from partially dried (D) and cryopreserved (C) recalcitrant zygotic embryos (ZEs) of Amaryllis belladonna. Seedlings recovered from fresh (F), D- and C-embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water deficit (S), followed by 3 days of re-watering. Seedling vigor was assessed in terms of physiological and growth responses to the imposed water stress. Compared with F-embryos, partial dehydration and cryopreservation reduced the number of embryos that produced seedlings, as well as the subsequent in vitro biomass of these seedlings. DW- and CW-seedlings (i.e. seedlings recovered from dried and cryopreserved ZEs that were watered for 12 days) exhibited lower CO(2)-assimilation rates and abnormal root growth. Stomatal density was also lower in C-seedlings. DS- and CS-seedlings were exposed to persistent low leaf water and pressure potentials and unlike FS-seedlings, displayed signs of having incurred damage to their photosynthetic machinery. CS-seedlings were less efficient at adjusting leaf water potential to meet transpirational demands and more susceptible to persistent turgor loss than DS- and FS-seedlings. DS-seedlings performed slightly better than CS-seedlings but drought-induced seedling mortality in both these treatments was higher than FS-seedlings. These results suggest that seedlings recovered from partially dried and cryopreserved embryos were less vigorous and more susceptible to hydraulic failure than those from fresh ZEs.

Somatic embryogenesis in in vitro culture of Leucojum vernum L.

Procedures for somatic embryogenesis (SE) in in vitro culture of spring snowflake have been developed from different types of explants like scales and leaves isolated from bulbs, ovaries and fruits. Various plant growth regulators were tested including a cytokinin--benzyladenine (BA) and various concentrations of the exogenous auxins 3,6-dichloro-2-methoxybenzoic acid (Dicamba), 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (Picloram). Fruit explants, cultured on medium containing Picloram and BA, ensured the highest percentage of callusing and such calli were most efficient in inducing somatic embryos. The addition of abscisic acid (ABA) in combination with polyethylene glycol (PEG) stimulated somatic embryo maturation. Torpedo-stage embryos developed into plants in the presence of BA and 1-naphthaleneacetic acid (NAA). The formation and growth of adventitious bulbs required that the plantlets be chilled at 5 degrees C in the dark for 6 weeks. After chilling, the bulbs grew well in darkness 25 degrees C. High sucrose concentration in the medium was necessary for obtaining large bulbs.

A key role for floral scent in a wasp-pollination system in Eucomis (Hyacinthaceae).

BACKGROUND AND AIMS: Floral scent may play a key role as a selective attractant in plants with specialized pollination systems, particularly in cases where floral morphology does not function as a filter of flower visitors. The pollination systems of two African Eucomis species (E. autumnalis and E. comosa) were investigated and a test was made of the importance of scent and visual cues as floral attractants. METHODS AND KEY RESULTS: Visitor observations showed that E. autumnalis and E. comosa are visited primarily by pompilid wasps belonging to the genus Hemipepsis. These wasps carry considerably more Eucomis pollen and are more active on flowers than other visiting insects. Furthermore, experiments involving virgin flowers showed that these insects are capable of depositing pollen on the stigmas of E. autumnalis, and, in the case of E. comosa, pollen deposited during a single visit is sufficient to result in seed set. Experimental hand-pollinations showed that both species are genetically self-incompatible and thus reliant on pollinators for seed set. Choice experiments conducted in the field and laboratory with E. autumnalis demonstrated that pompilid wasps are attracted to flowers primarily by scent and not visual cues. Measurement of spectral reflectance by flower petals showed that flowers are cryptically coloured and are similar to the background vegetation. Analysis of headspace scent samples using coupled gas chromatography-mass spectrometry revealed that E. autumnalis and E. comosa scents are dominated by aromatic and monoterpene compounds. One hundred and four volatile compounds were identified in the floral scent of E. autumnalis and 83 in the floral scent of E. comosa, of which 57 were common to the scents of both species. CONCLUSIONS: This study showed that E. autumnalis and E. comosa are specialized for pollination by pompilid wasps in the genus Hemipepsis and achieve specialization through cryptic colouring and the use of scent as a selective floral attractant.

Temperature requirements for seed germination and seedling development determine timing of seedling emergence of three monocotyledonous temperate forest spring geophytes.

BACKGROUND AND AIMS: The optimal period for seedling emergence depends on factors such as habitat preference, life cycle and geographical distribution. This research was performed to clarify the role of temperature in regulating processes leading to seedling emergence of the European continental Scilla bifolia and the Atlantic Narcissus pseudonarcissus and Hyacinthoides non-scripta. METHODS: Experiments in natural conditions were performed to examine the phenology of embryo growth, seed germination in the soil and seedling emergence. Effects of temperature conditions on embryo growth, seed germination, seedling growth and leaf formation were studied in temperature-controlled incubators. KEY RESULTS: In nature, embryo growth of all three species was initiated from the moment the seeds were dispersed in spring and continued during summer. A sequence of high temperature followed by a lower temperature was required to complete embryo growth and initiate germination. Seeds of H. non-scripta and N. pseudonarcissus germinated in autumn once they attained the critical E:S ratio, while seeds of S. bifolia started germinating when temperatures were low in winter. Seedlings developed normally, but slowly, only when placed in low temperature conditions (5 or 10 degrees C), resulting in a time lag between the moment of radicle protrusion and seedling emergence in the field. CONCLUSIONS: A continuous development of the embryo and seedlings of the three species was observed from the moment the seeds were dispersed until seedlings emerged. A sequence of high summer temperatures followed by decreasing autumn and winter temperatures was required for all developmental processes to be completed. Although a time lag occurs between radicle protrusion and seedling emergence, the term 'epicotyl dormancy' does not apply here, due to the absence of a period of developmental arrest. Timing of first seedling emergence differed between the three species and could be related to differences in geographical distribution.

Does inbreeding avoidance maintain gender dimorphism in Wurmbea dioica (Colchicaceae)?

The maintenance of females in gender dimorphic populations requires that they have a fitness advantage to compensate for their loss of male reproductive function. We assess whether inbreeding avoidance provides this advantage in two subdioecious Wurmbea dioica populations by estimating seed production, outcrossing rates and inbreeding depression. Fruiting males produced less than half as many seeds as females, owing to low outcrossing rates and early acting inbreeding depression. Inbreeding coefficients of fruiting males demonstrated that progeny were more inbred than their parents, implying that few selfed progeny reach maturity, as confirmed by inbreeding depression estimates that exceeded 0.85. In a glasshouse experiment, open-pollinated females exhibited a fitness advantage of 3.7 relative to fruiting males, but when we increased fruiting male outcrossing rate, female advantage was only 1.4. This reduced advantage is insufficient to maintain females if nuclear genes control sex. Thus, inbreeding avoidance could maintain females at high frequencies, although this is contingent upon high frequencies of fruiting males, which can be altered by environmentally determined gender plasticity.

Embryoidogenesis and plant regeneration from leaf tissue of Gloriosa superba.

The induction, maturation and germination of embryoids from leaf tissue of Gloriosa superba L. were developed by exploiting solid and liquid culture. Nodular calli were obtained from SH medium supplemented with 2,4-D and 2iP. In solid culture, the nodular calli when transferred to 2,4-D along with glycerol gave the best response (68.4 %) in embryoid induction after 20 days. After two subcultures at 7-day intervals in a medium with thiamine instead of glycerol, the embryoids matured. When mature embryoids were transferred to BAP and IBA medium, they gave rise to plantlets with single shoots and roots. In liquid culture, the medium supplemented with NAA and L-glutamine with continuous agitation, the embryoidogenic calli produced embryoids (85 %) after 21 days. The mature embryoids began to turn green and produced shoots and elongated "radicles" after 35 days.

Evaluation of BC2 progenies derived from 3x-2x and 3x-4x crosses of Lilium hybrids: a GISH analysis.

An allotriploid (ALA, 2n=3 x=36) BC(1) plant was obtained by backcrossing a diploid F(1) interspecific hybrid (LA, 2n=2 x=24), derived from a Lilium longiflorum (L genome) and an Asiatic hybrid (A genome), to the latter parent. This allotriploid was backcrossed to a diploid Asiatic hybrid (2n=2 x=24) and to an allotetraploid (LLAA, 2n=4 x=48) LA hybrid. A total of 25 plants of these crosses were examined for ploidy level, and 12 individuals were analyzed for their genome constitution through genomic in situ hybridization (GISH). In most cases the progenies from the triploid-diploid (3 x-2 x) crosses consisted of aneuploids. Further more, there was evidence for the formation of near-haploid (x=12+2) to triploid (3 x=36) gametes in the allotriploid BC(1) plant. The progenies of triploid-tetraploid (3 x-4 x) cross also consisted of mostly aneuploids but in this case the triploid female parent had contributed predominantly near-triploid (2n) gametes for the origin of BC(2) progenies. The different ploidy levels observed between 3 x-2 x and 3 x-4 x crosses are possibly caused by preferential fertilization or survival resulting in a different ratio of chromosome numbers between the embryo and endosperm. Though Lilium has a tetrasporic, eight-nucleate type of embryo sac formation (Fritillaria type), the observed difference between the progeny types in 3 x-2 x and 3 x-4 x crosses is comparable to that of observed in monosporic eight nucleate types (Polygonum type) that predominate in most genera of Angiosperms. An important feature of the genome constitution of the progenies was that the homoeologous recombinant chromosomes were transmitted intact from BC(1) to BC(2) progenies in variable numbers. In addition, there was evidence for the occurrence of new homoeologous recombinations in the triploid BC(1). Of the two euploid BC(2) plants one had originated through the parthenogenetic development of a 2n egg and the other had originated through indeterminate meiotic restitution (IMR).

[Complementary nuclear DNA in embryo sac cells of Haemanthus albiflos and Ornithogalum caudatum]. / Dopolnitel'naia iadernaia DNK v kletkakh zarodyshevykh meshkov Haemanthus albiflos, Ornithogalum caudatum.

Cytophotometric measurements of Feulgen-DNA were carried out in the nuclei of embryo sac cells of Haemanthus albiflos and Ornithogalum caudatum. It was found that the nuclei of the egg system, haploid in the number of chromosomes, became polyploid in DNA amount at the final stages of gametophyte development.

Somatic embryogenesis in Asparagus officinalis L. cv. Argenteuil: interactions between genotype, explant type and growth regulators on callus induction, growth and embryogenic differentiation.

Callus induction, growth and embryogenic differentiation were studied in two selected genotypes of Asparagus officinalis L. cv. Argenteuil, to develop a protocol for somatic embryogenesis. Two experiments were carried out. In the first, four explant types (spear sections and lateral buds, both of in vivo and in vitro origin), and three levels of 2,4-D (1.5, 5 and 10 mg.l-1) and two of KIN (0 and 1 mg.l-1) added to MS basal medium, were tested during the three initial culture stages (90 d period). All factors were included in one factorial design to statistically analyze interaction effects. Interactions between genotype, explant type and time of culture initiation were significant for callus induction and growth during the two initial culture stages. The addition of kinetin enhanced callus induction and growth, and high levels of 2,4-D depressed callus growth. The average embryogenic differentiation was < 2% overall (11% with the best factor combination). In the second experiment, another explant type (bud clusters) was tested; the average embryogenic differentiation was 1.9% (best combination: 5.6%). For the two genotypes studied, the best factor combinations were the culture of in vivo lateral buds or bud clusters on MS basal medium with 1.5 mg.l-1 2,4-D and 1 mg.l-1 KIN.

The "gaseous" environment in sealed BRIC-100VC canisters flown on 'Mir' with embryogenic daylily cell cultures.

As part of the "Cellular Mechanisms of Spaceflight-Specific Stress to Plants" experiment, nine BRIC (Biological Research in Canisters) 100VC canisters, each containing four 100 mm dia polycarbonate petri dishes with embryogenic daylily (Hemerocallis sp.) cultures, were launched on 12 Jan 97 (STS-81), transferred to 'Mir' and returned on 24 May 97 (STS-84). Pre-flight, flight and ground control data for temperature, relative humidity, CO2 and ethylene in the BRIC canisters are presented.

Somatic embryos of daylily in space.

Poor growth and nuclear abnormalities observable in some space-grown plants have been hypothesized as due to a combination of factors such as degree of development, the specific way the plants are grown and the way they experience multiple stresses, some of which are space-specific. Data from a 132-day experiment on 'Mir' using embryogenic cell cultures of daylily (Hemerocallis) allow seemingly contradictory evidence from earlier Shuttle missions to be harmonized: a) the more developed an embryo the less likely it is to suffer catastrophic cell stress during growth, whereas the less developed it is, the greater its vulnerability; (b) the extent to which the stress becomes manifest is also dependent on the extent of pre-existing stresses imposed by suboptimal growing conditions; (c) an appropriate, albeit undesirable, 'stress match' with other non-equilibrium determinants, much like a 'tug of war', can result in genomic variations in space. It is not understood what is/are the feature(s) of the space environment that cause the various cell division perturbations but they have not yet been mimicked on earth. The stress symptoms were found only in space materials and, as predicted, they were most frequently encountered in smaller, less-developed materials grown under non-optimized conditions. It is concluded that, while any substantial deviation from 'optimum' can be a 'stress', spaceflight subjects vulnerable materials to cell division or DNA-repair stress(es) that appear distinctive, but remain elusive so far. Fastidiously-controlled growing environments must be devised to resolve the matter of direct versus indirect effects of space. On a practical level, it is predicted that adapting plant biotechnologies to space conditions will not be a casual matter. Grant Numbers: NAG21026.