Alfalfa Somatic Embryos Research Articles

The Role of Endogenous Ethylene in Carbohydrate Metabolism of Medicago sativa L. Somatic Embryos in Relation to Their Regenerative Ability

Effects of the ethylene biosynthesis inhibitors salicylic acid (SA) and aminoethoxyvinylglycine (AVG) on germination of Medicago sativa L. somatic embryos and their conversion to seedlings in relation to carbohydrate content and α-amylase activity were studied. Both SA, an inhibitor of ACC oxidase, and AVG, an inhibitor of ACC synthase, when present in the regeneration medium (0.1 and 1 μM) were found to drastically reduce the embryo germination rate. In addition, SA and AVG were found to almost completely or completely, respectively, arrest the process of embryo conversion to seedlings. The inhibitory effects of SA and AVG on germination and conversion may indicate that the processes required endogenous ethylene. AVG and SA clearly slowed down starch disappearance during the 48-h imbibition in the regeneration medium prior to radicle elongation, which was correlated with inhibition of the activity of α-amylase, an enzyme responsible for starch hydrolysis. It is probable that ethylene may activate α-amylase in the germinating alfalfa somatic embryos. In contrast, the disappearance of soluble sugars in the embryos in the presence of the inhibitors tested was accelerated. The disappearance of soluble sugars (to null or almost null) in embryos was faster in the presence of SA in the regeneration medium after 24 and 48 h compared to the disappearance rate with AVG present in the medium. Only glucose was present after a 48-h incubation in the regeneration medium in the presence of the two ethylene biosynthesis inhibitors, in contrast to the control embryos in which glucose was not detected.

Regulation of Medicago sativa L. somatic embryos regeneration by gibberellin A3 and abscisic acid in relation to starch content and α-amylase activity

Somatic embryo quality is an important factor decisive for the efficiency of somatic embryogenesis. Addition gibberellic acid (GA3) at a concentration of 1 μM to germination medium improved the regeneration of alfalfa somatic embryos. Inhibitory effect of ancymidol, an inhibitor of gibberellin biosynthesis, on germination and conversion may indicate that those processes require endogenous GAs. Since fluridone, an ABA biosynthetic inhibitor, at a concentration of 1 μM, had a slight stimulatory effect on germination of somatic embryos, it may be presumed that embryos contain a too high level of residual ABA after maturation phase (20 μM ABA is used at that phase). The observed improvement of regeneration of somatic embryos by GA3 was correlated with acceleration of starch hydrolysis through α-amylase activity enhancement by GA3. In contrast, the inhibitory effect of ABA on the above processes was probably related to inhibition of α-amylase activity and, in consequence, to delayed starch hydrolysis. It is suggested that α-amylase activity can be considered a good marker of the quality of Medicago sativa L. somatic embryos.

Induction of raffinose oligosaccharide biosynthesis by abscisic acid in somatic embryos of alfalfa ( Medicago sativa L.)

Raffinose oligosaccharides (ROs) accumulate during late seed development in orthodox seeds, just after the levels of the phytohormone abscisic acid (ABA) rise during mid seed development. To elucidate a possible relationship between ABA and ROs, alfalfa somatic embryos (SE) were treated with exogenous ABA in a range of 50 nM to 50 μM. Only trace amounts of ROs are found in SE, providing an elegant experimental system to study the hormonal regulation of RO biosynthesis. Compared to controls without ABA, a significant accumulation of all ROs (galactinol, raffinose and stachyose) could be observed, even at the lowest ABA concentration used. At the highest ABA concentration, the amount of galactinol, raffinose and stachyose accumulated was three-, four- and seven-fold higher than that of controls, respectively. This accumulation was accompanied by a three-fold increase in galactinol synthase (GLS) activity, while the levels of raffinose synthase (RFS) and stachyose synthase (STS) activities remained almost constant. We therefore conclude that exogenous ABA induces the accumulation of ROs in alfalfa SE.

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.

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajecarska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l−1 sucrose, 1 g l−1 yeast extract and 0.05 mg l−1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l−1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or β-glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.

Soluble saccharides and cyclitols in alfalfa ( Medicago sativa L.) somatic embryos, leaflets, and mature seeds

Soluble carbohydrates were identified and quantified during development, maturation and desiccation of somatic embryos of alfalfa ( Medicago sativa L.) and compared to soluble carbohydrates in leaflets and mature seeds, to relate changes in soluble carbohydrates to maturation events. Somatic embryos have elevated levels of sucrose. However, in contrast to mature seeds, alfalfa somatic embryos do not accumulate d-pinitol or the galactosyl derivatives of d-pinitol such as galactopinitol A, galactopinitol B and ciceritol. Lower levels of stachyose accumulate during maturation of somatic embryos, but stachyose increases to levels in mature seeds during desiccation of somatic embryos. When stachyose accumulation is limited in somatic embryos, galactinol and digalactosyl myo-inositol increase. Reducing sugars decline to low levels during desiccation of somatic embryos and sucrose: oligosaccharide ratio decreases from 2.7 to 0.9, approaching the ratio 0.2 to 0.3 in mature dry seeds. Except for the lack of pinitol and galactosyl pinitols, changes in soluble carbohydrates during the maturation and desiccation of alfalfa somatic embryos are typical of changes occurring in mature seeds that have been reported to be associated with desiccation tolerance and storability.

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

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.

Germination of Alfalfa ( Medicago sativa L.) Seeds and Desiccated Somatic Embryos : I. Mobilization of Storage Reserves

Desiccated somatic embryos lack the vigor that is accociated with seeds, and this reduces the feasibility of using artificial seeds as a means of plant propagation. Although desiccated somatic embryos of alfalfa (Medicago sativa L.) are characterized by faster imbibition than in seeds, radicle and shoot emergence are slower. To determine the physiological basis for this low vigor, the utilization of storage proteins and carbohydrates was compared between alfalfa somatic embryos and seeds during the first days after imbibition. Mature somatic embryos contained comparable quantities of storage proteins and carbohydrates to seeds, and these reserves were rapidly and extensively degraded within a few days of imbibition. Therefore, low vigor does not appear to be a consequence of inadequate or unavailable storage reserves. However, the hydrolysis products of the storage reserves, especially the free amino acids asparagine and glutamine, were lower in the seedlings from somatic embryos than seeds. Therefore, although the storage reserves in the dry somatic embryos were quantitatively similar to seeds and although they were rapidly hydrolysed after imbibition, the relatively low quantity of amino acids and sugars available for biosynthetic activity in these seedlings may limit their vigor.

Regulation of starch and protein accumulation in alfalfa ( Medicago sativa L.) somatic embryos

Compared to seeds, somatic embryos accumulated relatively low levels and different types of storage carbohydrates. The regulation of starch accumulation was studied to determine its effects on desiccation tolerance and vigor of dry somatic embryos. Somatic embryos of Medicago sativa are routinely matured through three phases: 7 days of development; 10 days of phase I maturation, a rapid growth phase; and 10 days of phase II maturation, a phase leading to the acquisition of desiccation tolerance. The control of starch deposition was investigated in alfalfa somatic embryos by manipulating the composition of the phase I maturation medium with different levels of sucrose, abscisic acid, glutamine and different types of carbohydrates and amino acids. After phase II maturation, mature somatic embryos were collected for desiccation and subsequent conversion, or for biochemical analyses. Starch deposition occurred primarily during phase I maturation, and variations in the composition of this medium influenced embryo quality, storage protein and starch accumulation. A factorial experiment with two levels of glutamine × three levels of sucrose showed that increasing the sucrose concentration from 30 to 80 g/l increased embryo size and starch content, but had minimal effect on accumulation of storage proteins; glutamine also increased embryo size, but decreased starch content and increased accumulation of the high salt soluble S-2 (medicagin) storage proteins. ABA did not influence any of the parameters tested when included in phase I maturation at concentration up to 10 μM. Replicating sucrose with maltose, glucose, or glucose and fructose did not alter embryo size or starch accumulation (mg/g fresh weight), but replacement with fructose alone reduced embryo size, and replacement with glucose alone reduced germination. Suplementation with the amino acids, asparagine, aspartic acid and glutamine increased seedling vigor, but decreased the starch content of embryos. The data indicate that starch accumulation in somatic embryos is regulated by the relative availability of carbon versus nitrogen nutrients in the maturation medium. The quality of mature somatic embryos, determined by the rate of seedling development (conversion and vigor), correlated with embryo size, storage protein and free amino acid but not with starch. Therefore, further improvements in the quality of somatic embryo may be achieved through manipulation of the maturation medium in order to increase storage protein, but not starch deposition.

Effect of nutrition of maturation of alfalfa ( Medicago sativa L.) somatic embryos

The low vigour of seedlings grown from somatic embryos limits their use as artificial seeds. It was postulated that seedlings vigour could be enhanced by improving the nutrition of the somatic embryo during maturation, and thereby increasing deposition of storage carbohydrates and proteins. The maturation of alfalfa ( Medicago sativa L.) somatic embryos after the torpedo stage of development was experimentally divided into two phases, I and II. Rapid growth occured during maturation phase I and acquisition of desiccation tolerance occured during maturation phase II. Several experimental approaches were applied to maturation phase I to manipulate embryo quality, as assessed by germination, conversion to seedlings, seedlings vigour and level of storage proteins: (1) plating density, (2) frequent transfer of embryo cultures to fresh media, (3) medium volume and (4) supplementation with 0–125 mM glutamine. Since glutamine had been previously shown to enhance synthesis of storage protein in these somatic embryos, experiments 2 and 3 were conducted in a factorial experimental design with 50 mM glutamine supplement. Elevated nutrient levels by all of these approaches increased embryo size, raised total protein content of individual alfalfa somatic embryos, enhanced conversion to seedlings, and increased the vigour of those seedlings. Glutamine had two additional effects; it increased the levels of free amino acids and increased the ratio of S2/S1 proteins. The S2 protein fraction contains the 11S medicagin storage protein which is synthesized in low amounts in somatic embryos. Therefore, glutamine seems to have a regulatory role as well as a nutritive role in storage protein synthesis. Optimal embryo quality would be achieved with low plating densities, and frequent transfer to large volumes of fresh nutrient media and supplementation with glutamine during maturation.

Maturation of alfalfa ( Medicago sativa L.) somatic embryos by abscisic acid, sucrose and chilling stress

The vigor of seedlings from dry somatic embryos of alfalfa ( Medicago sativa L.) is considerably less than the vigor of those from truw seeds, hypothetically as the result of low levels of storage proteins, an inhibitory effects of the ABA in the maturation medium, or injury from the desiccation process. In an attempt to increase vigor, three different treatments, abscisic acid (ABA), osmotic stress (0.4 M sucrose) and chilling (4°C), were applied during the final stage of embryo maturation as signals to induce the expression of desiccation tolerance in the developing embryos. Somatic embryos were induced on medium containing 2,4-D and were transferred to hormone-free medium where they developed to the torpedo stage. Storage protein synthesis occurred after transfer of the embryos to maturation phase I medium containing 146 mM sucrose and 50 mM glutamine. Subsequently, in maturation phase II, somatic embryos were transferred to fresh medium, and exposed to one of the above inductive treatments for 3, 12 or 19 days. Somatic embryos were then dried at a relative humidity of 43%. Desiccated embryos were rehydrated by placing them directly on agar medium with no added nutrients and scored for quality based on germination after 7 days and leaf development (conversion) after 14 days. The highest somatic embryo quality was obtained after 3 days on maturation medium with sucrose or ABA; longer durations reduced both germination and conversion. In contrast, 19 days of maturation at 4°C was required for the somatic embryos to express significant levels of desiccation tolerance. A positive correlation was observed between the embryo's total protein content and their quality. SDS-PAGE indicated that the low molecular weight (LMW) proteins in the S-1 fraction (low salt soluble) and the S-2 (high salt soluble) storage protein, medicagin, declined in the somatic embryos after 3 days on maturation medium containing ABA or sucrose, whereas their levels continually increased during the chilling treatment. These results are discussed in relation to somatic embryo quality, desiccation tolerance and accumulation of storage proteins during the maturation of alfalfa somatic embryos.

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.

Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.)

During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media, yet that protein is not evident on stained gels until day 10. Thus, both transcriptional and posttranscriptional events appear to be important in determining the protein complement of these seed tissues. On the basis of storage protein and mRNA accumulation, mature (14 days) somatic embryos most closely resemble stage VI zygotic embryos. The results of the developmental comparison also suggest that the patterns of synthesis of the individual storage proteins (7S, 11S, or 2S) are regulated independently of each other during embryogenesis in alfalfa.

Field planting of alfalfa artificial seeds

Encapsulated somatic embryos (artificial seeds) and naked (uncoated) somatic embryos of alfalfa (Medicago sativa L.) were planted directly into the field to demonstrate the feasibility of using artificial seeds for direct sowing. Various row coverings that provided protection for the somatic embryos during conversion (plant formation) in the field and encapsulation methods were investigated. The highest conversion obtained in the field was 25% when naked somatic embryos were planted under the protective covering of inverted styrofoam cups. In comparison, 60% conversion was obtained when embryos were planted in potting mix in a growth chamber. Somatic embryos encapsulated by the thin-coat method converted at 23% under cups in the field and 40% in potting mix in the growth chamber. Naked somatic embryos had an average of 13 and 9% conversion in the field under plastic and cloth coverings, respectively, whereas encapsulated embryos converted at 5 and 14%, respectively. Direct-planted embryos (no row covering) converted at 1% in the field. Successful conversion of coated and naked somatic embryos planted in the field supports the concept of artificial seeds serving as a substitute for natural seeds.

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.)

Microspore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.

Alfalfa heat shock genes are differentially expressed during somatic embryogenesis

We have isolated two cDNA clones (Mshsp18-1; Mshsp18-2) from alfalfa (Medicago sativa L.) which encode for small heat shock proteins (HSPs) belonging to the hsp17 subfamily. The predicted amino acid sequences of the two alfalfa proteins are 92% identical and a similar degree of homology (90%) can be detected between Mshsp18-2 and the pea hsp17. In comparison to various members of small HSPs from soybean amino acid sequence similarities of 80-86% were identified. The alfalfa HSPs share a homologous stretch of amino acids in the carboxy terminal region with hsp22, 23, 26 from Drosophila. This region contains the GVLTV motif which is characteristic of several members of small HSPs. At room temperature alfalfa hsp18 mRNAs were not detectable in root and leaf tissues but northern analysis showed a low level of expression in microcallus suspension (MCS). The transcription of Mshsp18 genes is induced by elevated temperature, CdCl2 treatment and osmotic shock in cultured cells. In alfalfa somatic embryos derived from MCS a considerable amount of hsp18 mRNA can be detected during the early embryogenic stages under normal culture conditions. The differential expression of these genes during embryo development suggests a specific functional role for HSPs in plant cells at the time of the developmental switch in vitro.

Maturation and greenhouse planting of alfalfa artificial seeds

Conversion (plant production) was obtained from direct-planting alfalfa somatic embryos and encapsulated somatic embryos (artificial seeds) of alfalfa into a growth chamber and greenhouse. The embryos were planted in a commercial soil potting mix under nonsterile conditions in a manner similar to zygotic seed. Embryo maturation with abscisic acid (ABA), prior to planting, gave 48% conversion in soil under growth chamber conditions. Under greenhouse conditions, 64% conversion was obtained when humidified air was used to prevent soil surface drying. Previously, conversion in soil was between 0–6% without the ABA maturation treatment. The replacement of ABA with mannitol or combinations of mannitol and ABA during maturation resulted in lower conversion in the growth chamber than with ABA alone. ABA may be promoting the accumulation of embryo storage reserves such as proteins and carbohydrates for growth after planting in the soil environment.

Fifty years on: molecular biology's hall of fame

Genetic material is absorbed onto the surface of minute tungsten beads. The beads are then explosively discharged against plant tissue and, in some instances, penetrate individual cells. Some cells are stably transformed and the GUS gene has been expressed after such treatment. This method of transformation does not rely on sophisticated plant protoplast systems and the species to be transformed need not be susceptible to biological transforming agents such as Agrobacterium tumefaciens. The chief requirements for transformation are that the bombarded cells be regenerable and that the inserted genetic material be compatible with the recipient cell genome. Thus, particle bombardment is potentially significant as a 'generic' approach to plant transformation, having application to a wide range of species and, generally, requiring readily available regeneration systems (i.e. from tissue). Synthetic seeds The plant division meeting ended with a special symposium on synthetic seeds. Synthetic seed technology seeks to use somatic embryos as high efficiency clonal propagules. Synthetic seeds would be useful for clonal production of hybrid strains, for the rapid multiplication of genetically engineered seed-propagated crops and for germplasm preservation, particularly for clonally propagated plants. One major line of research involves conferring desirable seed-like qualities, such as the existence of a reversible dehydrated resting phase, on somatic embryos. McKersie et al. (University of Guelph, Guelph) reported that plants could be regenerated from alfalfa somatic embryos stored in a dehydrated state at ambient temperatures for up to eight months. Such a quiescent resting phase will be necessary for synthetic seed of most crops to be commer-

Somatic embryogenesis from cell cultures of medicago sativa L. II. the interaction of amino acids with ammonium

We report that the proline-enhanced regeneration of alfalfa somatic embryos is dependent upon ammonium ion. Optimization of regeneration occurs when 100 mM proline and 25 mM ammonium is added to the embryogenesis medium. Glutamine was the only amino acid capable of stimulating somatic embryogenesis which was not dependent on ammonium for its effect in alfalfa. Glutamine could substitute for ammonium in proline-stimulated embryogenesis. These results are discussed in light of earlier results of amino acid additions in the somatic embryogenesis of carrot and cotton.