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.     

Polyembryony in non-apomictic citrus genotypes

Background and Aims

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in Citrus sp. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x × 4x hybridizations and in vitro culture of isolated nucelli. The origin of the plants arising from the aforementioned processes remains unclear.

Methods

The genetic structure (ploidy and allelic constitution with microsatellite markers) of plants obtained from polyembryonic seeds arising from 2x × 4x sexual hybridizations and those regenerated from nucellus culture in vitro was systematically analysed in different non-apomictic citrus genotypes. Histological studies were also conducted to try to identify the initiation process underlying polyembryony.

Key Results

All plants obtained from the same undeveloped seed in 2x × 4x hybridizations resulted from cleavage of the original zygotic embryo. Also, the plants obtained from in vitro nucellus culture were recovered by somatic embryogenesis from cells that shared the same genotype as the zygotic embryos of the same seed.

Conclusions

It appears that in non-apomictic citrus genotypes, proembryos or embryogenic cells are formed by cleavage of the zygotic embryos and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as a result of two different mechanisms that prevent the dominance of the initial zygotic embryo.     

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n = 30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4 ± 0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2 ± 1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6 ± 15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4 ± 5.3) was higher (P < 0.05) than the MOET method (6.7 ± 3.7). However, pregnancy rates were lower (P < 0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P > 0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.     

Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80°C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0°C and 25°C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.     

Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

<Emphasis Type="Italic">In vitro</Emphasis> germination of zygotic embryos excised from cryopreserved endocarps of queen palm (<Emphasis Type="Italic">Syagrus romanzoffiana</Emphasis> (Cham.) Glassman)

Queen palm (Syagrus romanzoffiana [Cham.] Glassman) is a palm species best known as an ornamental tree for urban landscaping, but recently, it has been evaluated as a potential crop for biofuel production. The objective of the present work was to establish a cryopreservation technique for queen palm to ensure long-term conservation of this species. The cryopreservation protocol consisted of direct immersion in liquid nitrogen (LN) of whole endocarps with water contents ranging from 5.5 to 10.9%, followed by slow thawing at room temperature (25 ± 2°C) excision and in vitro culture of zygotic embryos. Viability of zygotic embryos isolated from endocarps with different water contents was evaluated before (control) and after freezing in LN using in vitro culture on Woody Plant Medium (WPM) medium. Germination percentages of zygotic embryos isolated from endocarps stored in LN varied from 84 to 93%, whereas those isolated from controls ranged from 55 to 71%. Germination rates were significantly higher for zygotic embryos excised from cryopreserved endocarps. The water content of control or frozen endocarps did not have a significant effect on germination percentages of zygotic embryos. Zygotic embryos excised from endocarps following cryopreservation in liquid nitrogen developed into normal plantlets after in vitro culture. The technique tested is simple, efficient, and can be used in plant gene banks as a routine approach for long-term conservation of queen palm germplasm.     

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix?×?marschlinsii)

Background and Aims In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question.Methods Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis.Key Results Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg^–1 f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g^–1 d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap.Conclusions In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.     

Effect of maturation duration on desiccation tolerance in hybrid larch (Larix×leptoeuropaea dengler) somatic embryos

Summary Cotyledonary somatic embryos ofLarix &#x00D7; leptoeuropaea that developed after various maturation times on media containing abscisic acid showed different frequencies of conversion into plants. Drying of these somatic embryos under high relative humidity (RH) before germination improved plantlet recovery and eliminated differences in the performance of somatic embryos matured for different times. However, dehydration of somatic embryos under 98% RH to a water content below that of zygotic embryos excised from mature seeds (0.97 and 1.36 g H₂O/g dry weight, respectively) showed a strong positive correlation between longer maturation time and desiccation tolerance. Drying somatic embryos at 4&#x00B0; C under 59% RH for 1 wk resulted in desiccation to a water content of 0.30 g H₂O/g dry weight, which was the closest to the hydration state of zygotic embryos in dried, stored seeds (0.20 g H₂O/g dry weight). Under this condition, only somatic embryos matured for 5 wk germinated and produced plantlets at a relatively high frequency (73 and 41%, respectively).     

The effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of <Emphasis Type="Italic">Amaryllis belladonna</Emphasis>

Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and <0.4 g g⁻¹, respectively. In contrast, glycerol cryoprotection, which promoted retention of generally normal ultrastructure and also counteracted any increase in the degree of vacuolation, was associated with 100% and 90% survival of embryos at the higher and lower water contents. After exposure to liquid nitrogen (LN), ultrastructural irregularities were minimal in rapidly cooled glycerol-cryoprotected embryos, at water content <0.4 g g⁻¹, which showed 70% survival after retrieval from cryogenic conditions. At the other extreme, no embryos survived LN exposure when sucrose cryoprotected. The study relates the cumulative effects of subcellular abnormality and declining viability, in relation to experimental parameters for cryopreservation.     

A novel Glycine soja tonoplast intrinsic protein gene responds to abiotic stress and depresses salt and dehydration tolerance in transgenic Arabidopsis thaliana

Tonoplast intrinsic protein (TIP) is a subfamily of the aquaporin (AQP), also known as major intrinsic protein (MIP) family, and regulates water movement across vacuolar membranes. Some reports have implied that TIP genes are associated with plant tolerance to some abiotic stresses that cause water loss, such as drought and high salinity. In our previous work, we found that an expressed sequence tag (EST) representing a TIP gene in our Glycine soja EST library was inducible by abiotic stresses. This TIP was subsequently isolated from G. soja with cDNA library screening, EST assembly and PCR, and named as GsTIP2;1. The expression patterns of GsTIP2;1 in G. soja under low temperature, salt and dehydration stress were different in leaves and roots. Though GsTIP2;1 is a stress-induced gene, overexpression of GsTIP2;1 in Arabidopsis thaliana depressed tolerance to salt and dehydration stress, but did not affect seedling growth under cold or favorable conditions. Higher dehydration speed was detected in Arabidopsis plants overexpressing GsTIP2;1, implying GsTIP2;1 might mediate stress sensitivity by enhancing water loss in the plant. Such a result is not identical to previous reports, providing some new information about the relationship between TIP and plant abiotic stress tolerance.     

Thalassia testudinum seedling responses to changes in salinity and nitrogen levels

The dominant seagrass in Florida Bay, Thalassia testudinum Banks ex König, is a stenohaline species with optimum growth around marine salinity (30-40 PSU). Previous studies have examined the responses of mature short shoots of T. testudinum to environmental stresses. Our goal was to assess responses of seedlings to changes in water chemistry in Florida Bay that might occur as part of the Comprehensive Everglades Restoration Plan (CERP). Specifically, we examined seedling survival, growth, photosynthesis, respiration and osmolality in response to hypo- and hyper-salinity conditions, as well as possible synergistic effects of depleted and elevated ammonium concentrations. The study was conducted in mesocosms on T. testudinum seedlings collected during August 2003 near Florida Bay. Hyper- and hypo-saline conditions were detrimental to the fitness of T. testudinum seedlings. Plants at 0 and 70 PSU exhibited 100% mortality and a significant decrease in survival was observed in the 10, 50 and 60 PSU treatments. Increased levels of ammonium further decreased growth in the lower salinity treatments. Seedlings in 30 and 40 PSU had the greatest growth. Quantum yield and relative electron transport rate, measured using PAM fluorometry, showed a decrease in photosynthetic performance on either side of the 30-40 PSU optimum. Tissue osmolality decreased significantly with decreased salinity but tissue remained consistently hyperosmotic to the media across all salinity treatments. Maintaining negative water potential and allocating more energy to osmoregulation may decrease the productivity of this species in salinity-stress conditions. Our results suggest that the salinity-tolerance limits of this seagrass at the seedling stage are not as broad as those reported for mature plants. Increased fresh water inflow, especially if co-occurring with an increase in water-column ammonium, could negatively affect successful recruitment of T. testudinum seedlings in northern regions of Florida Bay.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Somatic embryogenesis and cryostorage of eastern hemlock and Carolina hemlock for conservation and restoration

Key message

Embryogenic cultures of eastern and Carolina hemlocks could be initiated, and somatic embryos and plantlets produced using standard conifer protocols and media. Embryogenic hemlock cultures were cryostored and recovered.

Abstract

Eastern hemlock (Tsuga canadenesis) and Carolina hemlock (Tsuga caroliniana) are threatened with extirpation from their native ranges in eastern North America by the introduction of the hemlock woolly adelgid (HWA; Adelges tsugae), an exotic insect pest that has already killed millions of hemlock trees. Efforts to conserve and restore these members of the Pinaceae could be greatly enhanced by the availability of an in vitro propagation system. We conducted experiments to initiate embryogenic cultures from eastern and Carolina hemlock zygotic embryos at different stages of development using three media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BA). Cone collection date, medium and source tree had significant effects on induction of embryogenic tissue from zygotic embryo explants of both species, which ranged as high as 52 % for eastern hemlock and 17 % for Carolina hemlock. Embryogenic hemlock cultures could be cryostored using a protocol employing sorbitol and DMSO, and recovered following several months of frozen storage. Transfer of embryogenic tissue from proliferation media containing 2, 4-D and BA to a Litvay medium with abscisic acid promoted the development of somatic embryos, which were stimulated to mature by slow drying under semi-permeable plastic film. Embryos moved to an imbibition-germination medium without plant growth regulators and incubated in the light elongated and subsequently germinated. A small number of germinated embryos survived transfer to ex vitro conditions and grew into somatic seedlings. The embryogenesis and cryostorage systems developed in the study are already being integrated with hemlock breeding efforts to develop clones with resistance or tolerance to HWA.     

Water relation parameters of embryogenic cultures and seedlings of larch

Changes in the water relations parameters of developing somatic embryogenic and xygotic European larch (Larix decidua) were studied. Water release curves were generated by suspending tissue samples over unsaturated NaCl solutions until they reached vapor equilibration with the surrounding air. Twenty solutions were used whose water potentials ranged from −0.05 to −10 MPa. Water release curves were obtained by plotting paired values of tissue relative water content (RWC) and solution potential. Curves were derived for embryonic larch at various stages of development and for hypocotyls and roots from germinated zygotic and somatic embryos. The ability to resist dehydration increased markedly with development. Stage 1 tissue, which consisted of clusters of loosely associated nonchlorophyllous cells, had extremely low bulk elastic modulus (ε) (1.91 MPa) and apoplastic water content (A) (0.023), relatively high osmotic potential (Ψ_π) (−0.53 MPa), and lost turgor at 0.56 RWC. In contrast, mature embryoids with primary roots, hypocotyl, and cotyledons (stage 3) had an almost 4-fold increase in A (0.089), significantly higher ε (3.49 MPa), and lower Ψ_π (−0.88 MPa) and lost turgor at 0.66 RWC. Hypocotyl tissue from germinated somatic embryos lost turgor at 0.74 RWC and had higher ε, A, and solute accumulation than pregerminated tissue. Hypocotyl tissue resisted dehydration more strongly than root tissue, and differences between root and hypocotyl water relation parameters were more pronounced in xygotic than in somatic seedlings. Highest dehydration resistance was in zygotic hypocotyls. The characterization of the water relations of tissue cultures should allow the development of more consistent and reliable desiccation protocols to induce maturation of embryos and produce synchronously germinating seed.     

Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures

A proliferating embryonal-suspensor mass (ESM) was initiated from immature embryos of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), 4–5 weeks after fertilization, on modified MS medium with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and N⁶-benzylaminopurine (BAP). ESMs were maintained for over 6 months as cell suspension cultures on modified DCR media with low 2,4-D and with kinetin and BAP. The development of individual somatic embryos was initiated in suspension culture by the gradual reduction of plant growth regulators and by addition of abscisic acid. The early stages of zygotic embryogenesis in Douglas-fir are unique among conifers and cleavage polyembroyogenesis is unknown. In somatic embryogenesis, characteristic stages of zygotic embryonic development were recapitulated and complete embryos were recovered by inhibiting cleavage polyembryony with abscisic acid and culturing individual embryos without growth regulators. Histological examination confirmed bipolar organization of somatic embryos. While conversion is low, plantlets with multiple cotyledons have been transferred to soil and continue to grow with production of a new shoot.     

Effects of Desiccation, Medium Osmolarity and Abscisic acid on the Maturation of Hevea brasiliensis Somatic Embryos

The effects of desiccation of Hevea somatic embryos and of sucroseand ABA concentrations in the maturation medium on their germinabilitywere investigated. Conversion into plant, water and histochemicalstatus of somatic embryos were compared systematically to thoseof the zygotic embryos used as reference. Slow desiccation ormaturation on 351 mol m^–3 sucrose supplemented with 1mmol m^–3 ABA strongly improved germinability and conversionof embryos into plants. The combination of the two treatmentswas the most effective, increasing the germination frequencyby 3·7 and plant conversion by 6·6 in clone PR107. Each of these two treatments increased the vigour of somaticembryos, stimulated the formation of root and shoot meristemsand the synthesis and accumulation of starch and protein reserves.At the end of maturation, the Hevea somatic embryos bore ananatomical and histochemical resemblance to mature zygotic embryos.Likewise, the two treatments brought the water status of somaticembryos closer to that of the mature zygotic embryos, but withoutachieving a perfect match. Optimization of the successful conversioninto plants may require full acquisition of this water status. Key words: ABA, embryo maturation, Hevea, somatic embryogenesis, water status     

Rate of dehydration,state of subcellular organisation and nature of cryoprotection are critical factors contributing to the variable success of cryopreservation: studies on recalcitrant zygotic embryos of <Emphasis Type="Italic">Haemanthus montanus</Emphasis>

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g⁻¹ dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g⁻¹ showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g⁻¹, embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g⁻¹, lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g⁻¹—in which the degree of vacuolation remained moderate—were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.     

In vitro zygotic embryo germination and propagation of an endangered Boswellia serrata Roxb., a source of boswellic acid

This study aims to establish an efficient protocol for development of seedlings of an endangered medicinally important forest tree Boswellia serrata Roxb., for mass plantation and consistent supply of salai guggul. The green mature fruits served as source of seeds. The excised green zygotic embryos were cultured on Gamborg (B5), McCown and Loyd (WPM) and Schenk and Hildebrandt (SH) media fortified with different concentration of sucrose and on Murashige and Skoog (MS) medium containing 3 % sucrose, polyvinylpyrrolidone (PVP) (0–300 mg l^−l), Gibberellic acid (GA₃), Indoleacetic acid (IAA), Naphthaleneacetic acid (NAA), Indole-3-Butyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4 D) and 6-benzylaminopurine (BA) or kinetin (Kin) individually. The highest frequency of embryo germination (96 %) and conversion into seedling was obtained on MS medium containing 3 % sucrose together with 200 mg l^−l PVP; other media were either inferior or induced abnormalities in the seedlings including callus formation from the zygotic embryos. Fully developed seedlings could be successfully established in soil with about 94 % survival. The embryos from mature dry seeds did not respond for germination in any of the experiments. In conclusion, selection of zygotic embryo from green mature seeds and their in vitro germination is important for propagation of B. serrata.     

Conditional knock-out reveals that zygotic vezatin-null mouse embryos die at implantation

Vezatin, a protein associated to adherens junctions in epithelial cells, is already expressed in mouse oocytes and during pre-implantation development. Using a floxed strategy to generate a vezatin-null allele, we show that the lack of zygotic vezatin is embryonic lethal, indicating that vezatin is an essential gene. Homozygous null embryos are able to elicit a decidual response but as early as day 6.0 post-coitum mutant implantation sites are devoid of embryonic structures. Mutant blastocysts are morphologically normal, but only half of them are able to hatch upon in vitro culture and the blastocyst outgrowths formed after 3.5 days in culture exhibit severe abnormalities, in particular disrupted intercellular adhesion and clear signs of cellular degeneration. Notably, the junctional proteins E-cadherin and β-catenin are delocalized and not observed at the plasma membrane anymore. These in vitro observations reinforce the idea that homozygous vezatin-null mutants die at the time of implantation because of a defect in intercellular adhesion. Together these results indicate that the absence of zygotic vezatin is deleterious for the implantation process, most likely because cadherin-dependent intercellular adhesion is impaired in late blastocysts when the maternal vezatin is lost.     

Dehydrin genes and their expression in recalcitrant oak (<Emphasis Type="Italic">Quercus robur</Emphasis>) embryos

In this work, three dehydrin genes, QrDhn1, QrDhn2, QrDhn3, were isolated from recalcitrant oak (Quercus robur). Their expression pattern was analyzed in both zygotic and somatic embryos as well as in vegetative tissues exposed to different kinds of abiotic stresses including desiccation, osmotic stress, and chilling. The QrDhn1 gene encoding for Y_nSK_n type dehydrin was expressed during later stages of zygotic embryo development but in somatic embryos only when exposed to osmotic or desiccation stress. In contrast, the other two oak dehydrin genes encoding for putative K_n type dehydrins were expressed only in somatic embryos (both not-treated and osmotically stressed) and leaves of oak seedlings exposed to desiccation. Behavior of these genes suggests that different dehydrins are involved in processes of seed maturation and response to altered osmotic (water status) conditions in somatic embryos. Revealing further members of dehydrin gene family in recalcitrant oak might contribute to clarify non-orthodox seed behavior as well as identify mechanisms contributing to desiccation tolerance in plants.