The embryogenic calli (EC) were obtained from hypocotyl explants of groundnut (Arachis hypogaea L.) cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg dm^-3 6-benzylaminopurine (BAP). The EC were exposed to γ-radiation (10-50 Gy) or treated with 1-5 mM of ethyl methane sulphonate (EMS) or sodium azide (SA). The mutated EC were subcultured on embryo induction medium containing 20 mg dm^-3 2,4-D. Somatic embryos (SE) developed from these calli were transferred to MS medium supplemented with BAP (2.0 mg dm^-3) and 0.5 mg dm^-3 2,4-D for maturation. The well-developed embryos were cultured on germination medium consisting of MS salts with 2.0 mg dm^-3 BAP and 0.25 mg dm^-3 naphthaleneacetic acid (NAA). Well-developed plantlets were transferred for hardening and hardened plants produced normal flowers and set viable seeds. The fresh mass of the EC, mean number of SE per explant and regeneration percentage were higher at lower concentrations of mutagens (up to 30 Gy/3 mM). Some abnormalities in regenerated plants were observed, especially variations in leaf shape.

A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rooting) for in vitro propagation of Dracaena sanderiana Sander ex Mast was developed. Callusing was achieved from nodal stem segment explants treated with various concentrations of ethylmethane sulphonate (EMS) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1.5 g m^-3). A significant increase in callus induction percentage and biomass production was noticed from lower EMS treated lines (ET₁ and ET₂) comparatively to control and other (ET₃, ET₄ and ET₅) lines. Calli of ET₁ line showed high regeneration potential on MS medium with N ⁶-benzylaminopurine (BAP; 1.75 g m^-3). Length of microshoots, which was reduced by EMS, restored by addition of gibberellic acid (GA₃; 0.4 g m^-3). A marked increase in rooting with increasing EMS concentration was noticed on MS medium fortified with 3-indolebutyric acid (IBA; 1.5 g m^-3).

Effects of four amino acids, L-asparagine, L-cysteine, L-citrulline, and L-glutamine in different concentrations (0, 0.5, 1, and 2 mg dm^-3) combined with 2 mg dm^-3 indole-3-butyric acid, on in vitro rooting and biochemical constituents of cherry rootstocks CAB-6P (Prunus cerasus L.) and Gisela 6 (P. canescens × P. cerasus) were investigated. In CAB-6P, root number and root fresh mass (FM) were maximum at 0.5 mg dm^-3 cysteine. All amino acids reduced root length in CAB-6P and root number as well as root FM in Gisela 6. In Gisela 6, 0.5 mg dm^-3 asparagine or 2 mg dm^-3 glutamine reduced root length. In CAB-6P, 100 % rooting was achieved in the control and with 1 and 2 mg dm^-3 cysteine or 1 mg dm^-3 citrulline. In Gisela 6, the rooting percentage was maximum (76.92 %) with 0.5 mg dm^-3 asparagine. Callus FM in CAB-6P was the greatest at 1 mg dm^-3 and in Gisela 6 at 2 mg dm^-3 citrulline. Callusing was 100 % in the majority of treatments for CAB-6P and 92.31 % for Gisela 6 with 0.5 or 2 mg dm^-3 citrulline. Cysteine, citrulline, and glutamine diminished chlorophyll content in Gisela 6 whereas in CAB-6P all four amino acids hardly affected it. Carotenoid and porphyrin content in CAB-6P was decreased due to asparagine (0.5 or 1 mg dm^-3). Porphyrin content in CAB-6P was also reduced by adding 0.5 or 1 mg dm^-3 cysteine or 2 mg dm^-3 citrulline. In Gisela 6, all amino acids decreased carotenoid and porphyrin content. In CAB-6P, all treatments except 0.5 mg dm^-3 glutamine or 2 mg dm^-3 asparagine increased leaf sucrose content. In roots, both sucrose and proline content were increased only at 1 mg dm^-3 cysteine whereas in leaves only 0.5 mg dm^-3 asparagine caused a 3-fold increase in proline content. A decrease in root proline in CAB-6P was observed due to asparagine, citrulline, or glutamine. In Gisela 6, decreased leaf sucrose and proline content was recorded at 2 mg dm^-3 cysteine. All amino acids did not alter root sugar content remarkably whereas root proline content was raised by adding 0.5 mg dm^-3 glutamine or 1 mg dm^-3 cysteine.

A reliable protocol for plant regeneration from mature embryo derived calli of nine barley (Hordeum vulgare) cultivars has been developed. The auxins 2,4-dichlorophenoxyacetic acid, picloram and dicamba proved effective in inducing callus from mature embryos of most of the barley cultivars. The induced primary callus was loose, friable and translucent. It ultimately yielded creamy white and compact callus after 2 - 3 transfers on fresh medium of the same composition. Callus induction and regeneration capacity were highly cultivar dependent. Addition of a high concentration of picloram (4 mg dm^-3) promoted regeneration in 3 cultivars (Tallon, Grimmett and Sloop). In cv. Arapiles, abscisic acid and betaine were crucial in generating morphogenic callus from the mature embryos. Plants regenerated from these calli were hardy and developed roots readily when transferred to hormone free medium.

Cell lines able to grow on media containing 50, 100, 150 or 200 mM NaCl were established from potato callus cultures by direct recurrent selection or gradual selection. In callus subjected to direct selection only small clusters of cells survived on medium with 150 or 200 mM NaCl, whereas on 100 mM small cell portions appear necrotic. When cell lines were obtained by successive subcultures on media with increased concentrations of NaCl, salt-tolerant calli were more compact and developed a greenish colour free from necrotic areas. The response of calli lines grown on media with NaCl was compared to control line. The NaCl-tolerant calli showed a decrease in relative growth rate and water content, with higher reductions in the 150 mM tolerant callus. Lipid peroxidation was increased in 50 mM and 100 mM NaCl-tolerant calli, while in 150 mM tolerant callus remained similar to 100 mM values. There was a significant increase in ascorbic acid content in 100 mM and 150 mM NaCl-tolerant calli as compared to the 50 mM, that was two-fold the value found in the control. Also, the contents of soluble and insoluble proteins increased in salt-tolerant lines. SDS-PAGE of soluble proteins showed the synthesis of specific polypeptides in the presence of NaCl in culture medium and the synthesis of a new polypeptide.

Globally, drought is the main factor that reduces common bean yield. For this reason, breeding alternatives, such as molecular marker-assisted selection, that focus on various functional genes directly involved in the response to water stress, such as those encoding late embryogenesis abundant (LEA), early response to dehydration (ERD), and dehydrin proteins, have been implemented. The aim of this study was to identify differentially expressed genes of Phaseolus vulgaris in drought-tolerant cultivars Pinto Saltillo (PS) and Pinto Villa (PV), and drought-susceptible cultivars Bayo Madero (BM) and Canario 60 (C60) in vegetative and reproductive stages. Relative water content (RWC) in leaf tissue was measured. Twenty-eight P. vulgaris genes obtained from GenBank and from a subtractive suppressive library from the PS cultivar were analysed, and their expression profiles were examined by reverse transcription polymerase chain reaction (RT-PCR). Then, cDNA arrays were developed and hybridised to confirm expression which was finally validated by quantitative PCR (qPCR). The usefulness of the identified genes as selection criteria for the tolerance of different genotypes to drought was examined using cDNA arrays. Expression of 21 genes was induced by drought. The cDNA arrays confirmed that expression of 19 of these genes increased in the vegetative stage upon exposure to the drought, and a higher expression was observed in the reproductive stage compared with vegetative stage V4. Only five genes induced by the drought were found to have a lower expression in the susceptible cultivars compared with the tolerant ones. During recovery after the drought in the reproductive stage, 13 of the 21 induced genes remained transcriptionally active including LEA3 and dehydrin. The RWC during the drought in vegetative stage V4 decreased by about 55 % in all cultivars, but at the onset of flowering, it increased to 80 % in PV and PS. In contrast, in the susceptible cultivars, it remained at 55 %. Using qPCR validation, expression induction was confirmed in the drought-tolerant cultivars. Polyubiquitin2, LEA3, LEA4, and dehydrin were useful genes for selecting drought-tolerant genotypes under field conditions.

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^-1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.

Several studies have been performed to elucidate the role of brassinosteroids (BRs) in plant growth and development. However, information on the role of BR signaling in nutrient uptake is limited. This study explores the relationship between BRs and ammonium transporter 1 (AMT1) expression in Arabidopsis roots. We found that BR treatment reduced the expression of AMT1 genes and that a BR receptor BRI1 mutant bri1-5 reversed its BR-repressed expression. Furthermore, the BR signaling transcription factor, BES1, regulates AMT1 expression in roots. NH₄ ⁺-mediated repression of AMT1;1, AMT1;2, and AMT1;3 was suppressed in a gain-of-function BES1 mutant (bes1-D). This mutant was more sensitive to methyl-ammonium and contained a higher ammonium content compared to wild-type plants. However, BES1 failed to bind E-box elements present in the promoter region of the AMT1 genes. Furthermore, NH₄ ⁺-mediated glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) gene expressions were partially inhibited, and GS activity was slightly lower in the bes1-D mutant relative to that observed in wild-type En2 roots. NH₄ ⁺-mediated AMT1 suppressions are known to be caused by N-metabolites rather than NH₄ ⁺ itself, and glutamine application inhibited AMT1 expression in both En2 and bes1-D indicating that BES1 activation inhibited NH₄ ⁺-mediated GS/GOGAT induction, which might in turn inhibit AMT1 repression. In conclusion, the present study demonstrates that BR regulated nitrogen uptake and assimilation via the BR signaling pathway.

Somatic embryogenesis has been obtained in many citrus cultivars. However, culture synchronization is yet to be achieved and in the present work we evaluate the effect of desiccation, cold and size of cell clusters on embryo production efficiency from callus cultures of Citrus sinensis L. Osbeck, cv. Valencia. The results showed that sieving was effective in promoting somatic embryo synchronization, whether or not it was followed by cold or desiccation treatments. Histological and histochemical analyses are presented to characterize the structure of cell aggregates and protein accumulation.

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 µM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 µM indole-3-butyric acid (IBA) and 0.93 µM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.

Cytochrome P₄₅₀, CYP93A1, is involved in the synthesis of the phytoalexin glyceollin in soybean (Glycine max L. Merr). The gene encoding CYP93A1 has been used as defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To further understand the tissue-specific role of CYP93A1 in soybean defense, we analyzed the expression of this gene in mechanically damaged leaves and stems. In leaves, CYP93A1 was constitutively expressed; its expression did not change in response to mechanical damage. In stems, however, expression of CYP93A1 was induced as quickly as 4 h after mechanical damage and remained upregulated for at least 48 h. The induction of CYP93A1 was associated with the synthesis of glyceollins. In comparison to several other defense-related genes encoding cysteine protease inhibitors L1 and R1 and storage proteins vspA and vspB, CYP93A1 was the most strongly induced by stem wounding. The induction of CYP93A1 was observed only locally, not systemically. Similar stem expression patterns were consistently observed among three different soybean genotypes. The strong induction of CYP93A1 in mechanically damaged stems suggests an important role in the soybean stem defense response; therefore, this study expands the use of CYP93A1 as a defense response marker to stems, not just soybean cell cultures.

Plant regeneration through somatic embryogenesis of Areca catechu L. was established using leaf, root and stem segments as explants. Embryogenic callus was induced and maintained on medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,6-dichloro-2-methoxybenzoic acid (dicamba) at concentrations 2, 4, 6 and 8 mg dm^-3 in darkness. Somatic embryos were found on primary callus in the presence of 2 and 4 mg dm^-3 dicamba and during subculture on 2 - 8 mg dm^-3 2,4-D or 2 - 4 mg dm^-3 dicamba-containing media. Plantlet conversion from embryos was successfully achieved on growth regulator-free medium. The plants grew well when transplanted to containers in shaded greenhouse.

Arabidopsis shoots regenerate from root explants through a two-step process consisting of pre-incubation on an auxin-rich callus induction medium (CIM), followed by transfer to a cytokinin-rich shoot induction medium (SIM). The auxin receptor gene TIR1 was up-regulated when explants were transferred to SIM. The CIM pre-incubation is required for its up-regulation. The tir1-1, TIR1 knockdown mutant, reduced the efficiency of shoot regeneration in tissue culture, while its over-expression mutant significantly improved efficiency. TIR1 promoter::GUS fusion analysis demonstrated that TIR1 expression was in the shoot and the newly emerging leaves. After 10 d on SIM, several cytokinin related genes (CDKB1;1, CKS1, IPT4 and ARR15), which associate with shoot regeneration, were up-regulated in plants over-expressing TIR1 and some of these were down-regulated in the tir1-1 mutant. Thus, TIR1 appears to be involved in regulating shoot regeneration.

Leaves from 14-d-old Capsicum annuum L. cv. Anaheim seedlings were cultured on Murashige and Skoog (MS) medium containing different combinations of indole-3-acetic acid (IAA) and 6-benzyladenine (BA). After 3 months, cultures were transferred to new medium where BA was replaced with 9 μM isopentenyladenine (2iP) to enhance the growth of shoot buds. Developing shoots were elongated and rooted on MS medium enriched with 9 μM indole-3-butyric acid (IBA). All cultures were maintained in 250 cm³ baby jars covered with a clear polypropylene lid with or without microporous polypropylene membrane. Vessel type and plant growth regulators significantly affected callus morphogenic appearance, organogenesis and in vitro plantlet growth. Ventilated vessels supported photomixotrophic culture and improved regeneration and growth of plantlets. Higher plantlet dry mass and content of photosynthetic pigments, and lower stomatal density of plantlets grown in ventilated than in non-ventilated vessels facilitated ex vitro acclimation and growth.

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^-3 benzylaminopurine (BAP) + 0.25 mg dm^-3 indole-3-acetic acid (IAA). Subsequent transfer of 5 mm² callus pieces to MS medium supplemented with BAP (0.5 mg dm^-3) alone or in combination with IAA (0.25 mg dm^-3) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm^-3) + IAA (0.25 mg dm^-3) supported rapid shoot elongation. Shoot propagules subcultured to Gamborg's medium (B5) with 0.5 mg dm^-3 indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.

The effect of N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU-30) on the growth and content of endogenous cytokinins of adenine type in tobacco (Nicotiana tabacum L.) callus was investigated. Biomass accumulation in calli grown on Murashige and Skoog (MS) medium with 4PU-30 was higher than that on MS medium with kinetin. The obvious presence of isopentenyladenine type cytokinins and traces of zeatin type cytokinins supposes modification in the endogenous cytokinin metabolism of the tobacco callus grown on 4PU-30.

The halophyte Thellungiella salsuginea is a new model plants due to its small genome size, short life cycle, and copious seed production. Although T. salsuginea shares a high sequence identity with its close relative Arabidopsis thaliana, it shows a greater tolerance to salinity, drought, freezing, heat, and cold. To elucidate the mechanism of abiotic stress resistance in T. salsuginea, we characterized its cytosolic Apx1 gene (TsApx1) and established A. thaliana transgenic lines overexpressing TsApx1. Under 300 mM NaCl, the content of H₂O₂, malondialdehyde, and proline were lower and the activities of superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase were all higher in the transgenic plants overexpressing TsApx1 (35S:TsApx1-GFP) than in the wild-type plants. The atapx1 mutant plants of A. thaliana had a NaCl/mannitol-sensitive phenotype. The ectopic expression of TsApx1 in the atapx1 mutant effectively remedied the phenotype. These results suggest that TsApx1 plays an important role in scavenging reactive oxygen species in the cytoplasm under salinity or drought. Although TsApx1 in T. salsuginea was constantly expressed at a high level, this gene was clearly inducible. In summary, the high constitutive expression and rapid induction of TsApx1 may contribute to the tolerance to abiotic stresses in T. salsuginea.

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 µM sucrose, 0.8 % agar, 3.62 µM 2,4-dichlorophenoxy acetic acid and 2.22 µM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant^-1) was achieved on MS medium supplemented with 8.88 µM BA, 2.5 µM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 µM) favoured shoot elongation and indole 3-butyric acid (7.36 µM) induced rooting. Rooted plants were hardened and successfully established in soil.

Plant microRNAs (miRNAs) play important roles in regulating plant growth, development, and responses to abiotic stresses. In this study, 38 miRNAs (TaMIRs) from wheat (Triticum aestivum L.), 36 from the miRBase database, and two from our previous work were characterized and subjected to an expression pattern analysis under normal conditions and a drought stress. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and small RNA blot analyses revealed that two TaMIRs (TaMIR1120 and TaMIR1123) were root-predominant and two TaMIRs (TaMIR1121 and TaMIR1134) were leaf-predominant. Seven TaMIR precursors showed altered expressions after the drought; of these, TaMIR1136 was upregulated, whereas TaMIR156, TaMIR408, TaMIR1119, TaMIR1129, TaMIR1133, and TaMIR1139 were downregulated. These seven drought-responsive TaMIRs showed dose-dependent and typical temporal expression patterns during drought induction, and they gradually returned back under the normal growth conditions. The drought-responsive and the tissue-predominant TaMIRs had varying numbers of target genes. Randomly selected target genes exhibited opposite expression patterns to their corresponding TaMIRs suggesting that they were regulated by distinct TaMIRs through a post-transcriptional cleavage. The target genes regulated by drought-responsive and tissue-predominant TaMIRs are involved in various cellular processes, such as signal transduction, transcriptional regulation, primary and secondary metabolisms, development, and defense responses. These results provide a novel insight into the miRNA-mediated responses of wheat to drought stress.

Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange [Citrus sinensis (L.) Osbeck] was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harboring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron β-glucuronidase (GUS) gene and the Arabidopsis APETALA1 (AP1) gene. Transformation treatment was with inoculation time of 30 min, co-culture of 3 d at 23 °C and supplementation of the co-culture medium with 2 mg dm^-3 acetosyringone (AS). Kanamycin (50 mg dm^-3) was effective to inhibit the growth of non-transformed calluses while it did not affect the transformed ones. The total number of transformed callus lines was 7 with 100 % embryo induction. High efficient regeneration of the transgenic embryos (88 % with 4-5 shoots per embryoid) was realized within 3 months. Integration of the transgene into the citrus genome was confirmed by histochemical GUS staining, polymerase chain reaction (PCR) analysis with AP1-specific primer and Southern blot hybridization with a 712 bp PCR fragment of AP1 as the probe.

The GhMYB9 encodes a R2R3 MYB transcription factor in the upland cotton (Gossypium hirsutum L.) genome. Our studies show that GhMYB9 predominantly expressed in flowers and fibers. To gain a better understanding of its regulatory mechanism, we isolated the 5'-flanking region of GhMYB9 which was 1 487 bp in length. The cis-acting element prediction shows that this region contained the basic structure of the core promoter elements (TATA-box, CAAT-box) and the transcription start site (TSS). Other motifs, such as defense and stress responsiveness (TC-rich repeats), anaerobic induction (ARE), and MYB binding sites involved in drought-inducibility (MBS), were also found. Histochemical assay shows that the GhMYB9 promoter governed β-glucuronidase (GUS) expression mainly in seeds, fibers, and flowers of transgenic cotton. Also, the activity of the promoter was induced by auxin in fibers of transgenic cotton. This is consistent with its transcript abundance in different tissues. A further deletion analysis confirms that a promoter region from -1 231 to -860 was required for auxin response. Our findings provide a useful reference for the understanding of the transcriptional regulation mechanism of the GhMYB9 gene.

The aim of this work was to estimate genetic variability for in vitro culture response of recombinant inbred lines (RILs) of the genus Lycopersicon. The callus percentage (C), the regeneration percentage (R) and the productivity rate (PR) were evaluated 45 d after culture initiation in a set of 16 elite tomato RILs and their parents. The narrow sense heritability (h²) values were 0.38 ± 0.04 for C, 0.46 ± 0.04 for R, and 0.28 ± 0.03 for R, while the genetic correlation (r _g ) values were -0.96 ± 0.07 between C and R, 0.81 ± 0.14 between PR and R, and -0.79 ± 0.16 between PR and C. Three AFLP markers associated to the in vitro traits were identified.

The objective of the current study was to develop an efficient and reproducible protocol for plant regeneration using nodal (1.0-1.5 cm) explants excised from a field grown mature plant of Cassia occidentalis L. The highest shoot regeneration frequency (80 %) with a maximum number of shoots (11.66) and shoot length (3.83 cm) after eight weeks of culture were observed on a Murashige and Skoog (MS) medium amended with 5.0 μM 6-benzyladenine, 100 μM citric acid, and 1.0 μM α-naphthalene acetic acid. A half-strength MS medium supplemented with 1.5 μM indole-3-butyric acid proved best for the induction of maximum roots (8.33) per shoot. Plantlets with well-developed shoots and roots were successfully acclimatized in plastic pots containing sterile Soilrite under two irradiances of 50 and 300 μmol m^-2 s^-1 (LI and HI, respectively) in a culture room, and after transfer to the field, the survival rate was 70 %. A significant increase in chlorophyll, carotenoid, and malondialdehyde content was found during acclimatization under both the irradiances but higher under HI. Similarly, the activities of superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase increased more under HI. Plantlets acclimatized under HI exhibited a better growth than those under LI.

In many polar and alpine ecosystems, lichens of genus Umbilicaria represent dominant species forming community structure. Photosynthetic and spectral properties of the lichens may change rapidly according to an actual hydration status of their thalli. In this study, we investigated responses of photochemical reflectance index (PRI), normalized difference vegetation index (NDVI), effective quantum yield of photosynthetic efficiency of photosystem (PS) II (Φ_PSII), and several photosynthetic parameters derived from fast induction kinetics of chlorophyll fluorescence (OJIP) to controlled dehydration. We used U. arctica and U. hyperborea collected close to Nuuk, Greenland. In both the species, PRI showed a curvilinear increase with dehydration, i.e., a decreasing water potential (Ψ_w). The increase was apparent within Ψ_w range of 0 to -10 MPa. The PRI increase was less pronounced in U. arctica than in U. hyperborea. NDVI decreased with a progressive thallus dehydration in both the species, however, throughout Ψ_w range of 0 to -30 MPa, U. hyperborea had lower NDVI values than U. arctica. The relationship between Φ_PSII and Ψ_w resulted in a typical S curve. A critical Ψ_w at which photosynthetic processes were fully inhibited was -30 MPa in both the species, however, species-specific differences in the S curve shape were found. Analyses of photosynthetic parameters derived from OJIPs revealed that the absorption of radiation energy and a trapping rate increased with dehydration in active reaction centres of PS II, the number of which decreased with a more pronounced lichen thallus dehydration. It is concluded that U. arctica and U. hyperborea possess effective physiological mechanisms to maintain an effective photosynthesis when partly dehydrated (the Ψ_w range of 0 to -15 MPa). In spite of similar ecological niches that these two lichens occupy in nature, their spectral and photosynthetic properties differred.

Autophagy has been implicated as a cellular protein degradation process that is used to recycle cytoplasmic components under biotic and abiotic stresses and so restrict programmed cell death (PCD). In this study, we report a novel regulatory mechanism by which NADPH oxidase respiratory burst oxidase homolog D (RBOHD) regulated pathogen-induced autophagy and hypersensitive (HR) cell death. We found that the Pseudomonas syringae pv tomato bacteria DC₃000 expressing avrRps4 (Pst-avrRps4) induction of RBOHD-dependent reactive oxygen species (ROS) production promoted the onset of autophagy, whereas a pretreatment with an NADPH oxidase RBOHD inhibitor reversed this trend. The inhibitor significantly blocked pathogen-induced autophagosome formation and ROS increase. Moreover, we also show that in the wild-type and atrbohF mutant, Pst-avrRps4-induced cell death was limited, whereas in the case of the atrbohD mutant, the infection triggered a spreading-type necrosis. Our results demonstrate that the RBOHD-dependent ROS accumulation stimulated autophagosome formation and limited HR cell death.

Tospoviruses are devastating plant viruses causing severe economic losses in a diverse range of crops worldwide. Here, we describe the development and evaluation of an RNA interference (RNAi) broad-spectrum virus resistance strategy based on a unique and short hairpin-RNA-generating construct (pNhpRNA). This construct was designed from a region of the nucleocapsid gene (N) of Tomato spotted wilt virus (TSWV) that showed a high sequence identity to the corresponding region in the related species Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV). To test the effectiveness of the pNhpRNA construct, we developed a silencing reporter assay based on three fusion proteins in which the complete viral N gene sequence from each of the three tospoviruses was fused in frame to the green fluorescent protein (GFP) sequence. Co-agroinoculation of these constructs with pNhpRNA into leaves of Nicotiana benthamiana resulted in a strong silencing phenotype determined by GFP decay and suppression of the three N genes at the RNA and protein levels. To test the potential of the pNhpRNA construct to generate virus-resistant plants, we infiltrated the whole shoots of N. benthamiana with pNhpRNA. When these infiltrated plants were mechanically inoculated with the mentioned viruses 100, 70, and 60 % resistance phenotypes to TSWV, GRSV, and TCSV, respectively, were observed. The induction of a broad tospovirus resistance with a simple construct and a minimized off-target effect are the main contributions of pNhpRNA.

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.

Dune reed (DR) and swamp reed (SR) are two ecotypes of reed (Phragmites communis Trin.) that displayed differences in stress tolerance. To uncover the molecular basis for such difference, the effects of heat stress were studied using the calli derived from the two ecotypes. Heat stress caused increased ion leakage, inhibited growth, decreased cell viability, and elevated hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents in the calli of both ecotypes, but DR callus showed better heat tolerance than SR callus. In DR callus, heat stress caused significant increase in the endogenous ABA content but not in SR callus. Application of fluridone (an ABA synthesis inhibitor) aggravated the heat stress damages on the DR callus whereas it had only minimal impact on the SR callus. Exogenous application of ABA alleviated the heat stress symptoms in the calli of both ecotypes. ABA treatment increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase, and also decreased H₂O₂ and MDA contents. These results indicate that the ability of ABA synthesis under heat stress is a key factor attributing to the higher heat tolerance of DR than SR.

Primary leaf explants of aseptically grown seedlings of moth bean [Vigna aconitifolia (Jacq.) Marechal] immersed in water or not were treated in microwave oven (2450 MHz, 800 W cm^-2) for 1, 3, 5 and 7 s before culturing. Callusing and shoot emergence from these explants were enhanced up to microwave exposure lasted 5 s while longer treatment of water-immersed explants delayed callusing. One polypeptide (26.6 kD) was up regulated in the callus derived from microwave treatment in water-immersed explants. RAPD analysis detected alteration in DNA sequences due to microwave treatment in water-immersed explants for 7 s. The frequency of mutation was 1.6 % (4 bands out of 248) over all the cultures analyzed and the same was 13 % (4 bands out of 31), if amplicons generated at 7 s treatment alone were considered.

Hypericin, a naphthodianthrone, has been identified as the principal active compound found in St. John's wort (Hypericum perforatum L.). To generate a gene resource for hypericin and other valuable metabolites, we generated expressed sequence tags (ESTs) from H. erectum. Analyses of the ESTs enabled us to select three cDNAs, HeHyp1, HeHyp2, and HeHyp3, evidencing significant sequence homology to Hyp-1 that were involved in hypericin biosynthesis from H. erectum. The deduced amino acid sequence of HeHyp1 cDNA exhibits 95 % identity with Hyp-1. The HeHyp2 and HeHyp3 polypeptides also exhibit 81.1 % identity with Hyp-1. The transcripts of HeHyp1, HeHyp2, and HeHyp3 were detected in the root, stem, leaf, flower, and callus cells. Study using recombinant protein suggests that Hyp-1, HeHyp2, and HeHyp3 may be involved in the biosynthetic of hypericin or other emodin derivatives.