Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Improvement of somatic embryogenesis inFeijoa sellowiana berg (Myrtaceae) by manipulation of culture media composition

Summary Somatic embryos could be induced from the cotyledons of zygotic embryos from immature fruits ofFeijoa sellowiana Berg (Feijoa) in the presence of a wide range of concentrations of fructose, glucose, maltose, and sucrose. Mannitol or sorbitol alone were ineffective. The highest frequencies of induction (99%) and the greatest number of somatic embryos per explant (134) were obtained with 0.4M fructose and 0.3M sucrose, respectively. This sucrose concentration also showed greater induction capacity than equimolar combinations of its monosaccharide constituents combined. Somatic embryo development was arrested at the globular stage at concentrations higher than 0.5M of all the sugars tested. When transferred to solid germination medium containing 2.0 mg/liter (5.77μM) gibberellic acid, 0.5 mg/liter (2.32μM) kinetin, and 0.029M sucrose, somatic embryos formed under 0.3 or 0.4M sucrose had better germination capacity than those induced under lower (0.1 and 0.2M) concentrations, as assessed by the frequency of explants presenting germinated embryos and by the number of plants obtained from those explants. On liquid media of similar composition somatic embryos did not germinate. Our data suggest that high (0.3 to 0.4M) carbohydrate levels improve somatic embryogenesis by acting both as carbon source and as osmotic regulator.     

Occurrence of somaclonal variation among somatic embryo-derived white spruces (Picea glauca,Pinaceae)

Four different variegata phenotypes were identified among 2270 white spruce plants [Picea glauca (Moench.) Voss.] produced over a period of 2 yr from the time of induction of embryogenic tissues. The four variegated plants differed from each other in the extent and distribution of chlorophyll-deficient needles. Light microscopy showed that variegated leaves of a selected variant consisted of a chimeral mixture of green and white cells. Electron microscopy showed that cells in completely white needles had large nuclei with predominant euchromatin, lacked large cytoplasmic vacuoles, and harbored vacuolized plastids with aberrant morphologies. Various observations suggest that the recovered variegata phenotypes reflect some kind of genetic instability of either chloroplastic or nuclear genomes. To elucidate the genetic basis of these variegata phenotypes in white spruce, three out of the four variants were subjected to randomly amplified polymorphic DNA (RAPD) analysis. Out of more than 250 RAPD markers screened, only one correlated with white needles of variegated plants. The nucleotide sequence of this DNA fragment showed no homology with any known gene, but the amplified sequence appears most likely of nuclear origin.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Maternal control of cold and desiccation tolerance in eggs of the band-legged ground cricket Dianemobius nigrofasciatus in relation to embryonic diapause

Cold and desiccation tolerance was investigated in the eggs of the band‐legged ground cricket Dianemobius nigrofasciatus in relation to embryonic diapause. Diapause eggs were more tolerant to both desiccation and cold than non‐diapause eggs. In addition, diapause‐destined eggs on day zero (0–12 h after being laid) already showed high tolerance to these stresses before entering diapause. This clearly indicates that stress tolerance, like diapause, is controlled by photoperiod, but is not directly associated with diapause itself. Because the acquisition of stress tolerance predates the onset of diapause, it is plausible that diapause programming during some period before the onset of diapause is involved in the acquisition of stress tolerance. Weights and sizes were nearly identical in short‐day and long‐day eggs until day five. Sorbitol, a major sugar alcohol in eggs of D. nigrofasciatus, was accumulated at the same level in short‐day and long‐day eggs on days zero and five. These results indicate that the surface‐to‐volume ratio as well as the accumulation of sugar alcohol is not involved in the acquisition of stress tolerance. Maternal factors are clearly involved in the acquisition of stress tolerance in D. nigrofasciatus eggs, but the physiological mechanisms underlying the tolerance are still unclear.     

Changes in starch and soluble sugars in relation to the acquisition of desiccation tolerance during maturation of Brassica campestris seed

Abstract. We investigated the onset of desiccation tolerance in developing embryos of Brassica campestris seeds and possible correlated ultrastructural modifications in the radiele cells. Since the acquisition of desiccation tolerance is a long asynchronous process which took 9 d to be achieved, we determined criteria allowing us to separate freshly intact harvested seeds into desiccation intolerant and desication tolerant batches that differed in age by only 2 d. No particular structural modifications were found except a strong depletion of intraplastidial starch (-90%) coincident with the appearance of stachyose and an increase of sucrose (+30%) on the acquisition of desiccation tolerance. As we did not observe an increase of lipid reserves as a consequence, we suggest that these metabolic events can be a key factor towards the acquisition of desiccation tolerance.     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

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

Summary Cotyledonary somatic embryos ofLarix × 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° 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).     

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

Summary The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (±)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.     

Manipulating the desiccation tolerance and vigor of dry somatic embryos of Medicago sativa L. with sucrose,heat shock and abscisic acid

Summary The effects of sucrose concentration in the maturation medium in combination with a heat shock treatment at 36°C were investigated in an attempt to improve the vigor of seedlings grown from dry somatic embryos of alfalfa (Medicago sativa L.). Callus was formed from petiole expiants and dispersed in liquid suspension medium in the presence of 5 M 2,4-D. The cell suspension was sieved to synchronize embryo development. The 200 – 500 m fraction was plated on embryo development medium without 2,4-D, grown for 14 days, and transferred to maturation medium. With 3% sucrose in the maturation medium, the somatic embryos germinated precociously and were unable to survive desiccation. At higher sucrose concentrations, germination was delayed and the embryos continued to accumulate dry matter. After 13 days on 6% sucrose medium (27 days after sieving), the somatic embryos were tolerant of drying to 12% moisture without exposure to exogenous ABA. Heat shock, which presumably stimulates endogenous ABA synthesis, improved the desiccation tolerance of somatic embryos if applied prior to day 27 after sieving, but its effects were minimal after day 27. High sucrose concentrations up to 9% in the maturation medium were optimal during the first 8 days on maturation medium (days 14 to 22 after sieving), but a lower concentration (6%) was optimal during the later stages of embryo maturation (days 22 to 30 after sieving). The inclusion of 10^–5 M ABA in the maturation medium with 6% sucrose further improved embryo quality if applied approximately 20 days after sieving.     

Redox changes during cold acclimation affect freezing tolerance but not the vegetative/reproductive transition of the shoot apex in wheat

Cold acclimation is necessary for winter wheat (Triticum aestivum L.) to achieve its genetically determined maximum freezing tolerance, and cold also fulfils the vernalisation requirement. Chromosome 5A is a major regulator of these traits. The aim of the present study was to discover whether changes in the half‐cell redox potential of the glutathione/glutathione disulphide (GSH/GSSG) and ascorbate/dehydroascorbate (AA/DHA) couples induced by cold acclimation are related to freezing tolerance and vernalisation requirement in a specific genetic system including chromosome 5A substitution lines. The amounts of H₂O₂ and AA, and the AA/DHA ratio showed a rapid and transient increase in the crown of all genotypes during the first week of acclimation, followed by a gradual increase during the subsequent 2 weeks. The amount of GSH and its ratio compared to GSSG quickly decreased during the first day, while later these parameters showed a continuous slow increase. The H₂O₂, AA and GSH concentrations, AA/DHA and GSH/GSSG ratios and the half‐cell reduction potential of the GSH/GSSG couple were correlated with the level of freezing tolerance after 22 days at 2 °C; hence these parameters may have an important role in the acclimation process. In contrast to H₂O₂ and the non‐enzymatic antioxidants, the lipid peroxide concentration and activity of the four antioxidant enzymes exhibited a transient increase during the first week, with no significant difference between genotypes. None of the parameters studied showed any relationship with the vegetative/generative transition state monitored as apex morphology and vernalisation gene expression.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

The sensitivity of transgenic spruce (Picea glauca (Moench) Voss) cotyledonary somatic embryos and somatic seedlings to kanamycin selection

Transformed white spruce cultures containing immature Stage I somatic embryos, were developed after particle bombardment of somatic embryos with pBI 426, carrying an expression cassette with a gus::nptII fusion gene. These Stage I cultures did not show tolerance to kanamycin concentrations above 3 to 5 mg l–1, although assays for GUS and NPTII showed that functional enzymes were present in the transgenic tissue. Embryonic liquid suspension cultures were initiated from this transformed tissue. After treatment on agar-solidified maturation medium with 48 m (±)-ABA high numbers of Stage III (cotyledonary) somatic embryos were produced. When subjected to an embryogenesis re-induction process with 2,4-D and BA, these Stage III embryos produced a new generation of Stage I embryogenic tissues which could tolerate 5--10 mg l–1 kanamycin. Stage III somatic embryos could alternatively be placed onto germination medium for the development of somatic seedlings. When germinated in the presence of 20 mg l–1 kanamycin, 77% of inoculants were resistant. The stability of integration of the gus::nptII fusion gene in the genome of white spruce Stage III somatic embryos and somatic seedlings was confirmed through Southern blot analysis     

Developing sporophytes transition from an inducible to a constitutive ecological strategy of desiccation tolerance in the moss Aloina ambigua: effects of desiccation on fitness

Background and Aims Two ecological strategies of desiccation tolerance exist in plants, constitutive and inducible. Because of difficulties in culturing sporophytes, very little is known about desiccation tolerance in this generation and how desiccation affects sexual fitness.Methods Cultured sporophytes and vegetative shoots from a single genotype of the moss Aloina ambigua raised in the laboratory were tested for their strategy of desiccation tolerance by desiccating the shoot–sporophyte complex and vegetative shoots at different intensities, and comparing outcomes with those of undried shoot–sporophyte complexes and vegetative shoots. By using a dehardened clonal line, the effects of field, age and genetic variance among plants were removed.Key Results The gametophyte and embryonic sporophyte were found to employ a predominantly inducible strategy of desiccation tolerance, while the post-embryonic sporophyte was found to employ a moderately constitutive strategy of desiccation tolerance. Further, desiccation reduced sporophyte fitness, as measured by sporophyte mass, seta length and capsule size. However, the effects of desiccation on sporophyte fitness were reduced if the stress occurred during embryonic development as opposed to postembryonic desiccation.Conclusions The effects of desiccation on dehardened sporophytes of a bryophyte are shown for the first time. The transition from one desiccation tolerance strategy to the other in a single structure or generation is shown for only the second time in plants and for the first time in bryophytes. Finding degrees of inducible strategies of desiccation tolerance in different life phases prompts the formulation of a continuum hypothesis of ecological desiccation tolerance in mosses, where desiccation tolerance is not an either/or phenomenon, but varies in degree along a gradient of ecological inducibility.     

In vivo characterization of the effects of abscisic acid and drying protocols associated with the acquisition of desiccation tolerance in alfalfa (Medicago sativa L.) somatic embryos

Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some tolerance to desiccation at the cotyledonary stage of development (22 d after plating), additional culturing in 20 microm abscisic acid (ABA) for 8 d induced greater desiccation tolerance, as determined by increased germination. Compared with fast drying, slow drying of the ABA-treated embryos improved desiccation tolerance. However, slow drying of non-ABA-treated embryos led to the complete loss of germination capacity, while some fast-dried embryos survived. An electron paramagnetic resonance spin probe technique and in vivo Fourier transform infrared microspectroscopy revealed that cellular membrane integrity and a-helical protein secondary structure were maintained during drying in embryos cultured in media enriched with 20 microM ABA, but not in embryos cultured in the absence of ABA. Slow-dried, non-ABA-treated embryos had low oligosaccharide to sucrose ratios, an increased proportion of beta-sheet protein secondary structures and broad membrane phase transitions extending over a temperature range of more than 60 degrees C, suggestive of irreversible phase separations. The spin probe study showed evidence of imbibitional damage, which could be alleviated by prehydration in humid air. These observations emphasize the importance of appropriate drying and prehydration protocols for the survival and storage of somatic embryos. It is suggested that ABA also plays a role in suppressing metabolism, thus increasing the level of desiccation tolerance; this is particularly evident under stressful conditions such as slow drying.