Ammonium nitrate improves direct somatic embryogenesis and biolistic transformation of Triticum aestivum

Triticum aestivum is of major importance both nutritionally and economically. Introduction of new genes has been difficult to apply to elite wheat varieties mainly as a result of their recalcitrance to prerequisite tissue culture. We attempted to improve the frequency of wheat transformation by exposing plants to high level of ammonium nitrate. Our experiments showed that modification of the ammonium nitrate content in the direct somatic embryogenesis induction medium can increase the number of primary embryos produced over twofold in the elite hard red wheat cultivar Superb. The number of primary embryos that were capable of transitioning into shoot development also increased twofold. Biolistic transformation efficiency improved as much as sevenfold when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. This simple approach could become extremely useful for increasing transformation efficiency in wheat.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a ‘star-like’ morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.     

Characterization of a Putative Serk-Like Ortholog in Embryogenic Cell Suspension Cultures of Coffea arabica L.

The acquisition of embryogenic cell suspension cultures (ECS) has been the objective of studies on in vitro induction of somatic embryogenesis with biotechnological tools, due to the high efficiency of ECS as plant material for genetic transformation and large-scale production and cryopreservation of germplasm. The objective of this work was to identify and analyze one of the main gene families involved in somatic embryogenesis, somatic embryogenesis receptor-like kinase (SERK) in coffee (Coffea arabica L.). Coffee SERKs were identified by searching an EST (expression sequences tag) database generated by the Brazilian Coffee Genome Project starting from candidate sequences obtained from the NCBI database (National Center for Biotechnology Information) . In silico analysis and quantitative PCR results imply that the identified EST-contig C166 might directly be involved in somatic embryogenesis. The results suggest that C166 is the possible ortholog of SERK in C. arabica (CaSERK) and indicate that C166 might be a valuable bio-marker for ECS, and in that context can increase the methodological efficiency for ECS formation in C. arabica. Functional analysis of CaSERK with mutants of a more manageable species will lead to a better understanding of the molecular regulation as well as the specific functions of genes involved in somatic embryogenesis in coffee.     

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.     

Antioxidant Enzyme Activities during in vitro Morphogenesis of Gladiolus and the Effect of Application of Antioxidants on Plant Regeneration

Activity of antioxidant enzymes was evaluated during somatic embryogenesis and shoot organogenesis from cultured leaf segments of Gladiolus hybridus Hort. The effect of exogenous antioxidants on somatic embryogenesis and shoot organogenesis has also been monitored. Activity of superoxide dismutase (SOD) gradually increased during somatic embryogenesis. while activities of catalase (CAT) and peroxidase (POX) decreased. In contrast, increase in CAT and POX activity and a concomitant decrease in SOD activity were noted during shoot organogenesis. Exogenous application of antioxidants such as glutathione (GSH), α-tocopherol and ascorbate (AA) inhibited somatic embryogenesis but stimulated shoot organogenesis. The frequency of somatic embryogenesis increased with the addition of H₂O₂. However, H₂O₂ inhibited shoot organogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

An efficient system to produce transgenic plants via cyclic leave-originated secondary somatic embryogenesis in Rosa rugosa

An efficient and reproducible Agrobacterium-mediated transformation system via repetitive secondary somatic embryogenesis was developed for Rosa rugosa ‘Bao white’. Somatic embryogenesis was induced from in vitro-derived unexpanded leaflet explants on MS medium supplemented with 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L Kinetin and 30 g/L glucose. Secondary somatic embryos were successfully proliferated via cyclic secondary somatic embryogenesis on MS medium containing 1.0 mg/L 2,4-D, 0.01 mg/L 6-benzyladenine and 45 g/L glucose under light intensity of 500–1,000 lux. The highest germination rate (86.33 %) of somatic embryos was observed on 1/2-strength MS medium containing 1.0 mg/L BA. Relying on the repetitive secondary somatic embryogenesis and A. tumefaciens strain EHA105 harboring the binary vector pBI121, a stable and effective Agrobacterium-mediated transformation pattern was developed. The presented transformation protocol, in which somatic embryo clumps at globular stage (0.02–0.04 g) were infected by Agrobacterium for 60 min and co-cultivated for 2 days, and then selected under a procedure of 3 steps, were confirmed to be optional by GUS histochemical assay and Southern blot analysis. The procedure described here will be very useful for the introgression of desired genes into R. rugosa ‘Bao white’ and the molecular analysis of gene function.     

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.     

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.     

Characterization of genes associated with polyembryony and in vitro somatic embryogenesis in Citrus

Apomixis is a common reproduction system in the genus Citrus. To investigate the molecular mechanism of apomictic embryogenesis in Citrus, genes expressed specifically in an apomictic genotype were isolated by PCR-selected suppression subtractive hybridization with total RNAs obtained from the ovule at anthesis. Several genes showing conspicuously different expressions between polyembryonic (apomictic) and monoembryonic (nonapomictic) genotypes were selected, and their expression profiles during ovule development were analyzed in detail. This analysis identified two apomictic and three nonapomictic genotype-specific genes. Among the latter, msg-2 was highly expressed in the late stage of somatic embryogenesis. Specific expression during ovule development in monoembryonic cultivars and in the late stage of somatic embryogenesis indicated that msg-2 is not expressed in the initiation stage of polyembryogenesis and somatic embryogenesis, suggesting its role in suppressing initial cell formation of somatic embryos. The full-length complementary DNA of msg-2 contained small open reading frames in its sequence but showed no homology to functionally known genes in the public databases. As sequences similar to msg-2 were frequently found among Citrus expressed sequence tags, msg-2 may be associated with polyembryogenesis and somatic embryogenesis in a Citrus-specific manner.     

Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene

The Panax ginseng 2c3 embryogenic cell culture was earlier obtained by callus cell transformation with Agrobacterium rhizogenes rolC. Calcium channel blockers (LaCl₃, verapamil, and niflumic acid) reduced the production of somatic embryos in the 2c3 culture, implicating the Ca²⁺ signaling system in plant somatic embryogenesis. The protein kinase inhibitors W7 and H7 also decreased the yield of somatic embryos in the 2c3 culture. The total CDPK expression in the 2c3 culture was 1.2-to 1.5-fold lower than in a control callus culture as a result of a silencing of the genes belonging to the PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a) subfamilies. Expression of the PgCDPK2 subfamily genes (PgCDPK2b and PgCDPK2d) was increased. It was assumed that some genes of the PgCDPK1, PgCDPK2, and PgCDPK3 subfamilies were responsible for generation of embryogenic cells in the 2c3 culture. For the first time, rolC expression and embryogenesis were associated with changes in the expression of certain CDPK genes.     

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.