The issue of finding plants suitable for phytoremediation of inorganic contaminants can be addressed through the preparation of genetically modified plants with an increased metal accumulation potential. A HisCUP gene, which encodes for a yeast metallothionein fused with a polyhistidine tail (His), was chosen for preparation of two plant vectors. These two plant vectors were constructed and a HisCUP gene expression was subsequently investigated. We firstly prepared a vector pNOV2819/RbcS/HisCUP which enabled selection on a mannose medium and contained the HisCUP gene under an inducible Rubisco promoter. Secondly, we designed a vector pGreen0029/35S/HisCUP which enabled selection of plants on a medium with kanamycin and carried the HisCUP gene under a constitutive CaMV 35S promoter. The transient expression of the HisCUP gene in tobacco plants was confirmed at RNA and protein levels for both constructs. The relative expression of the HisCUP gene was determined by semi-quantitative real-time PCR; a higher expression was detected for the vector pNOV2819/RbcS/HisCUP.

Chloride channels (CLCs) play pivotal roles in plant development and anion transport. However, little research has been conducted about the CLC in fruit-bearing plants. Here we provide an insight into the evolution and expression patterns of CLC gene family members in various tissues of trifoliate orange [Poncirus trifoliata (L.) Raf.] and their responses to several treatments. Genome-wide analysis identified six PtrCLC genes. The predicted proteins had similar numbers of amino acids, but shared a low sequence identity. Phylogenetic analysis revealed that PtrCLC were classified into two separate subgroups, and PtrCLC4 and PtrCLC6 in subgroup II were more closely related to bacterial CLCs. Sequence comparison with EcCLCA from Escherichia coli reveals that PtrCLC showed amino acid divergence in anion selectivity of CLC proteins. Real time qPCR analysis shows that PtrCLC genes, particularly PtrCLC6, preferentially expressed in leaves. Nitrogen deficiency irreversibly inhibited expression of PtrCLC genes except for PtrCLC1. In contrast, NaCl stress profoundly induced expression of PtrCLC genes, particularly PtrCLC2 and PtrCLC4, both of which were also upregulated by ABA treatment. The results presented here provide a solid foundation for a future functional research on citrus CLC genes.

The antioxidative system was studied during the development of pea plants. The reduced glutathione (GSH) content was higher in shoots than in roots, but a greater redox state of glutathione existed in roots compared with shoots, at least after 7 d of growth. The 3-d-old seedlings showed the highest content of oxidised ascorbate (DHA), which correlated with the ascorbate oxidase (AAO) activity. Also, the roots exhibited higher DHA content than shoots, correlated with their higher AAO activity. The activities of antioxidant enzymes were much higher in shoots than in roots. Ascorbate peroxidase (APX) activity decreased during the progression of growth in both shoots and roots, whereas peroxidase (POX) activity strongly increased in roots, reflecting a correlation between POX activity and the enhancement of growth. Catalase activity from shoots reached values nearly 3 or 4-fold higher than in roots. The monodehydroascorbate reductase (MDHAR) activity was higher in young seedlings than in more mature tissues, and in roots a decrease in MDHAR was noticed at the 11^th day. No dehydroascorbate reductase (DHAR) was detected in roots from the pea plants and DHAR values detected in seedlings and in shoots were much lower than those of MDHAR. In shoots, GR decreased with the progression of growth, whereas in roots an increase was seen on the 9^th and 11^th days. Finally, superoxide dismutase (SOD) activity increased in shoots during the progression of growth, but specific SOD activity was higher in roots than in shoots.

Roles of an altered porphyrin biosynthesis and antioxidants in protection against chilling and heat stresses were evaluated in rice (Oryza sativa L.). When exposed to the same exposure time (6 or 30 h), heat-stressed (45 °C) plants exhibited a less oxidative stress as indicated by a lower dehydration, ion leakage, and H₂O₂ production compared to chilling-stressed (4 °C) plants. Malondialdehyde production also increased after a mild chilling stress, whereas it increased only after a long-term heat stress. The content of protoporphyrin IX, Mg-protoporphyrin IX and its methyl ester, and protochlorophyllide drastically declined under both the stresses, particularly under the long-term heat stress. Greater increases in catalase and peroxidase activities in heat-stressed plants indicate more cofactors supplied for hemoproteins compared to those of chilling-stressed and untreated control plants. Intermediates of carotenoid biosynthesis, zeaxanthin and antheraxanthin, also increased under the chilling and heat stresses. In comparison to chilling-stressed plants, heat-stressed plants were more efficient in porphyrin scavenging and antioxidant enzyme responses, which may play crucial roles in plant protection under temperature stress, thereby suffering less from oxidative stress.

The effect of benzothiadiazole (BTH) and arbutin (ARB) on sugar metabolism and plant fitness in cucumber growing hydroponically in media with different doses of NO₃ ^- and urea as nitrogen sources (100 % NO₃ ^-, 75 % NO₃ ^- + 25 % urea, and 50 % NO₃ ^- + 50 % urea) was studied on the 7^th and 14^th day after the treatment. The glucose, sucrose, and chlorophyll (Chl) content, acid and alkaline invertases and lactate dehydrogenase activities, as well as leaf area of the 3^rd and 5^th leaves were determined. Urea changed the plant sugar metabolism in a dose-, time- and leaf-age-dependent manners and influenced a cucumber response to the BTH and ARB treatments. The BTH caused a significant cessation of growth, a decrease in Chl content, a reduction of leaf area, and an enhancement of lactate dehydrogenase and alkaline invertase activities. In the older leaves of the BTH-treated plants, a high accumulation of glucose and sucrose was found. At the lower dose of urea, the metabolic changes were limited. In the ARB-treated plants, the Chl content remained unchanged in all the nitrogen variants. In these plants, decrease in glucose and sucrose content and in both invertase activities was observed mainly in younger leaves of the plants grown on the high dose of urea. The ARB improved the fitness of the cucumber plants grown in the presence of urea.

The major capsid protein L1 of human papillomavirus type 16 (HPV16) was transiently expressed in tobacco (Nicotiana benthamiana and Nicotiana tabacum) and tomato (Lycopersicon esculentum) leaves using Agrobacterium tumefaciens. The expression vector pTV00 was derived from tobacco rattle virus (TRV). The highest L1 expression 15 μg g^-1(f.m.) was achieved when the coding sequence of L1 was optimized for expression in humans that caused an increase of the guanine and cytosine (GC) content from 38.2 % in wild type HPV16 to 64.1 % in optimized sequence. L1 monomers readily self-assembled into capsomeres and further into virus like particles (VLPs). Immunological characterization and electron microscopy showed that 89 % of L1 retained VLP structure also in extracts prepared from freeze-dried leaves. Plant expressed L1 in crude extracts was highly immunogenic without any additional adjuvant as vaccinated mice developed strong humoral and cellular immune response, comparable to that elicited by purified VLPs derived from insect cells. Further, the induced antibodies effectively neutralized infection of 293TT cells with pseudovirions. This finding demonstrates that the TRV expression system is comparable to other plant expression systems and due to the broad host range of TRV is particularly attractive when expression in plants with low content of toxic alkaloids is desired. Moreover, a monoclonal anti-L1 antibody E2 raised in the course of immunization with crude extract from freeze-dried leaves expressing L1 is specific preferentially against HPV VLPs and could be used in direct ELISA for monitoring of VLPs assembly and VLP purification protocols.

The expression of MdSAMDC2 gene, which encodes S-adenosylmethionine decarboxylase (SAMDC) in apple, was up-regulated by low temperature, salt and drought stresses. To identify its in vivo biological functions in the responses to stresses, the promoter region of MdSAMDC2 was isolated and characterized by analyzing the cis-acting regulatory elements and GUS reporter gene by an Agrobacterium-mediated transformation. GUS activity was enhanced upon salt and cold stresses, indicating that MdSAMDC2 promoter region controls gene transcription under stresses. In parallel, several lines of the transgenic tobacco plants over-expressing MdSAMDC2 were obtained. The contents of three polyamines greatly increased in the transgenic lines compared with the non-transgenic WT control. Upon exposed to low temperature (4 °C), salt (150 and 250 mM NaCl) and osmotic (20 % polyethylene glycol) stresses, transgenic plants produced more free polyamines and more active antioxidative enzymes such as superoxide dismutase and catalase than the WT control. Meanwhile, malondialdehyde content, an indicator for membrane lipid peroxidation, decreased in transgenic plants relative to the WT control. Thus, over-expression of MdSAMDC2 in tobacco conferred tolerance to stresses.

Axillary buds from adult field-grown plants of Lavandula dentata L. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growth. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with a combination of 2.2 μM of benzyladenine and 2.5 μM indole-3-butyric acid. The best condition for rooting was MS medium plus 2.5 μM naphthaleneacetic acid. Rooted plantlets were successfully transferred to soil. Short-term culture derived plants (6 month) exhibited a normal development, but a low frequency of not heritable morphological changes were detected in long term culture derived plants (more than 1 year).

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.

We have applied a simple method for evaluation of gfp gene expression in plants using a CCD camera and computerized processing of images. Transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transfer of plasmid T-DNA bearing a m-gfp5-ER sequence governed by the 35S promoter together with the nptII selectable marker gene. Presence of the gfp gene in plants was confirmed by a polymerase chain reaction method. Mean brightness values measured using image analysis software showed differences between transgenic and control plants and suggest the possibility of rapid selection of transgenic individuals among regenerants and their progenies.

Susceptibility of C. rubrum to Agrobacterium-mediated transformation was demonstrated by inoculating the petioles of in vitro grown plants with A. rhizogenes strain A4M70GUS. Hairy roots were produced in 8 % of explants. They were isolated and maintained on plant growth regulator-free solid or liquid half-strength Murashige and Skoog medium for two years. Hairy root fresh mass increased 30 - 90 folds when grown in liquid medium, which was superior to solid medium, where most of the hairy roots produced calli. When these calli were grown on medium supplemented with 0.5 mg dm-3 thidiazuron, embryo-like structures were obtained. Transgenic status of long-term callus and hairy root cultures was confirmed by histochemical GUS assay, by PCR specific to the uidA, rolA&B and ags genes and by Southern hybridization.

A modified plant 7SL RNA gene from Arabidopsis thaliana designated AHIIA63M was introduced into potato plants via Agrobacterium-mediated transformation. No transgenic plants could be obtained using pGPTV-based binary vectors where AHIIA63M gene driven by polIII promoter was located close to the polII promoter of the selection gene. Special binary vectors with matrix attachment region (MAR) elements had to be used for transformation to insulate polII and polIII promoters within T-DNA. The level of AHIIA63M RNA in transgenic plants was lower than the levels of transcripts of transgenes driven by RNA polymerase II. The level of AHIIA63M transcript in transgenic potato plants was tissue specific. The highest expression was detected in roots and gynoecium and the lowest in tubers. Moreover, non-specific promoter activity within the MAR element was revealed. This activity contributed to AHIIA63M transcription. This is the first report of expression of a modified 7SL RNA gene in transgenic plants and promoter activity within the MAR element.

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5-6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm^-3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.

Cotyledonary leaves of 9-d-old tomato (Lycopersicon esculentum Mill.) were co-cultivated with Agrobacterium tumefaciens GV 3101 harboring binary vector pBI101 containing kanamycin resistance gene (npt II) as selection marker. Murashige and Skoog (MS) inorganic salts with Gamborg's B5 vitamins supplemented with optimized concentrations of zeatin riboside and indole-acetic acid resulted in enhanced regeneration efficiency. Under optimized conditions of plant regeneration, transformation frequency in cvs. Pusa Ruby, Pusa Uphar and DT-39 was greater than 37 %. Transformed shoots were selected on kanamycin medium and the presence of the transgene in the primary transformants was confirmed by PCR. Integration of the npt II gene in the tomato genome was further confirmed by Southern blot analysis. RT-PCR analysis using neomycin phospho-transferase (npt II) gene-specific primers confirmed the expression of the transgene in transgenic plants. Transformed plants were successfully transferred to phytotron, where these plants grew to maturity and produced flowers and fruits.

High rates of homologous recombination (HR) in comparison to other plants make the moss Physcomitrella patens an attractive model organism for genetic studies as well as biotechnological applications. We describe a simple protocol for the efficient biolistic transformation of protonemal tissue with minimum tissue handling steps. The transformation efficiency depends on the biolistic conditions. The bombardment of tissue with 1 μm gold particles yielded between 20 and 40 stable transformants per 1 μg of DNA. Transformation with circular plasmids generates higher frequencies of random transgene integration, whereas linear plasmids are more efficient in generating gene-targeted insertions.

Protoporphyrinogen oxidase (Protox) in the porphyrin pathway is the target site of the peroxidizing herbicides such as carfentrazone-ethyl and oxyfluorfen. In an attempt to develop herbicide-resistant plants, transgenic rice plants were generated via expression of herbicide-insensitive Bacillus subtilis Protox gene fused to the transit sequence for targeting to the plastid using Agrobacterium-mediated gene transformation. Homozygous transgenic rice lines of T₃ generation selected by hygromycin resistance test were examined if they are resistant to the herbicides carfentrazone-ethyl and oxyfluorfen. The homozygous transgenic lines had single copy insertion of B. subtilis Protox gene into their genomes and express its mRNA. Compared to wild-type rice, the transgenic lines were less susceptible to the herbicides when examined with respect to growth, electrolyte leakage, chlorophyll loss and lipid peroxidation. The in vitro Protox activities in transgenic lines were about 56 % higher than those in wild-type rice. With 10 µM concentration of the herbicides in the enzyme assays, Protox activities in transgenic lines were similar to those in non-inhibited wild-type rice. Less amount of protoporphyrin IX was accumulated in transgenic lines than in wild-type rice upon the treatment of the herbicides at 10 µM concentration. Our results indicated that expression of B. subtilis Protox gene was stably transmitted into T₃ rice plants and reduced their sensitivity to carfentrazone-ethyl and oxyfluorfen.

Reproducible and high-frequency transgenic plant regeneration from callus and embryo axes of four different genotypes of chickpea (Cicer arietinum) was achieved after Agrobacterium-mediated transformation. Three different strains of Agrobacterium (EHA105, AGL1 and LBA4404) harboring the binary vector pCAMBIA1301 containing β-glucuronidase (GUS) and hygromycin phosphotransferase (hpt) genes under the control of a CaMV35S promoter were used. The highest number of transgenic plants was obtained from cotyledonary node-derived calli of genotype Pusa-256. A highly efficient rooting was achieved on Murashige and Skoog medium supplemented with indole-3-butyric acid. The stable integration of the gene was confirmed by molecular analyses of the transformed plants. Inheritance of GUS and hpt gene was followed through two generations and they showed the expected 3:1 inheritance.

The δ-aminolevulinic acid synthase (ALA-S) is an enzyme which catalyzes the synthesis of δ-aminolevulinic acid (ALA). The Bradyrhizobium japonicum ALA-S coding sequence lacking plastidal transit sequence was introduced into the rice genome (C line). The transgenic lines, C4 and C5, were compared with the transgenic lines expressing TALA-S gene with plastidal transit sequence (P line) to investigate whether the plastidal sequence affects the targeting capacity of B. japonicum ALA-S gene and the ALA-synthesizing capacity in rice plants. The B. japonicum ALA-S mRNA was expressed efficiently in C lines and the protein was localized in the stroma of chloroplasts regardless of the transit sequence as in P lines. The resulting transgenic plants, C line, had similar levels of ALA-S activity, ALA, protoporphyrin IX and chlorophylls, compared to those of P lines. In response to irradiance of 350 μmol m^-2 s^-1, transgenic lines C4 and C5 displayed the characteristic phenotypes of photodynamic damage, i.e., decreases in photosynthetic parameter F_v/F_m, as in P5 and P14 lines, whereas wild type did not. These results indicate that the lack of the plastidal transit sequence influences neither chloroplast translocation of B. japonicum ALA-S nor ALA-synthesizing capacity in the transgenic rice.

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.

Unfavourable environment brings many kinds of stresses to plants. To survive such stresses, efficient resistance is required for the plants. Multifunctional genes enable the cross-talk among the various abiotic stress resistance systems. This paper reviews the action mechanisms of multifunctional genes. These genes can be classified into three groups: genes encoding diverse proteins through mRNA splicing (e.g. AOX in rice); genes like BADH, P5CS and HAV that control drought, salinity, osmotic and heat stress resistance; and a gene family, for example AQP, controlling transport of many compounds including water and nutrients. These genes participate in signal sensing and transduction, transcriptional regulation and functional gene activation during stress resistance induction. Furthermore, it should be noted that, under abiotic stresses, the regulation cascades are mutually interdependent and there also exists a close correlation between those cascades and normal plant growth and development.

Transgenic tobacco (Nicotiana tobacum) plants carrying the gene coding for potato virus A (PVA) non-structural P3 protein were prepared by inoculation with Agrobacterium tumefaciens. Seeds from self-pollinated flowers (T₁ generation) were collected. To estimate the effectiveness of vertical transfer of the introduced gene and usefulness of respective plant lines for further experiments, the T₁ generation was characterized by testing its ability to grow in the presence of kanamycin (Km) and by PCR of both neomycin phosphotransferase (nptII) and PVA P3 genes. Eight and ten of 29 lines showed Mendelian segregation of Km-resistant phenotype 3:1 and ≥15:1, respectively, the T₁ of eleven lines showed low Km resistance. Selected PCR-positive lines were tested for the presence of P3 mRNA. In most cases the transgene transcription was dependent on the presence or absence of Km in the plant growth medium. Prepared transgenic plants were furthermore tested for sensitivity to PVA and potato virus Y (PVY) infection. All of them showed identical symptom development as the non-transgenic control plants.

The DIANTHIN gene encoding a ribosome-inactivating protein (RIP) from Dianthus caryophyllus L. was tested for negative selection in tobacco and rice. Tobacco leaf discs and scutellum-derived callus of rice were transformed with Agrobacterium tumefaciens strain LBA4404 (pSB1, pJAS1). pJAS1 harbors the DIANTHIN gene under the control of the CaMV 35S promoter. Tobacco transformation efficiency, in comparison to pCAMBIA1301, was reduced by 87 % in pJAS1-transformed leaf discs. The DIANTHIN gene proved to be completely toxic to tobacco as all the recovered hygromycin-resistant transgenic plants harbored truncated T-DNAs with deletions of the DIANTHIN gene. Transformation of the DIANTHIN gene under a Mungbean yellow mosaic virus (MYMV)-inducible promoter did not cause any toxicity in tobacco as reflected by the recovery of transgenic tobacco plants with the complete DIANTHIN gene. Transformation efficiency of pJAS1 did not decline in rice. Interestingly, all transgenic rice plants harbored the complete DIANTHIN gene and expressed the gene. The T₁ transgenic lines showed reduction of sheath blight symptom in the range of 29 to 42 %. The difference in the sensitivity to DIANTHIN between tobacco and rice provides a new direction to study the mechanisms underlying RIP toxicity in plants.

This communication examines the role of small heat shock proteins (sHsps) targeted to mitochondria (Mt) and endoplasmic reticulum (ER) in tomato plants (Lycopersicon esculentum Mill.) under heat stress. Genetic response of transgenic and wild type plants varied under optimum, moderately elevated and elevated temperature. In optimum temperature higher biomass was recorded in wild type than the transgenic lines, whereas in moderately elevated temperature biomass increased in Mt-sHsp line. Also, net photosynthetic rate (P_N) increased in Mt-sHsp line in both the elevated temperatures, though higher in moderately elevated. Cell membrane stability (CMS) improved in all the lines after exposure to elevated temperatures, but always remained higher in transgenic lines. Transgenic lines expressed sHsps in different temperature regimes in both vegetative and reproductive parts, while wild type expressed such proteins only after 1 h of heat shock.

CDPKs (calcium-depended protein kinases) are of great importance for the activation of defense reactions in plants. In this study, we aimed to find a connection between CDPK expression and increased salt tolerance in Panax ginseng. Treatment of P. ginseng cell cultures with W7 (CDPK protein inhibitor) showed that CDPK proteins were necessary for salt tolerance. Expression of PgCDPK1c, PgCDPK2c and PgCDPK4a was significantly increased in the cells treated with 60 mM NaCl compared to control cells, whereas expression of PgCDPK1b and PgCDPK3a was decreased. In the NaCl-treated cells, new CDPK transcripts also appeared (PgCDPK3c, PgCDPK4as). We also used rolC and rolB transformed cultures and the effects of the rol genes on CDPK expression were similar to the effects of salt stress: they caused a significant increase in the expression of PgCDPK1c, PgCDPK2c, and PgCDPK4a and decreased expression of PgCDPK3a, in addition to the appearance of the "short" CDPK transcripts.

Drought is one of the main environmental stresses and many investigators identified beneficial effects of both silicon and selenium on plant growth and development. To examine the effects of Si and Se on rice (Oryza sativa L.) responses to drought, two cultivars Giza 177 and IET 1444 pretreated with 1.5 mM Si or 0.03 mM Se were then exposed to a water stress until leaf rolling was observed. The enhanced growth of Se or Si pre-treated plants was associated with a significant increase in the content of proline and glycine betaine in both shoots and roots. Furthermore, the transcription factors (TFs), dehydration responsive element-binding protein DREB2A, and NAC5 [no apical meristem (NAM), Arabidopsis thaliana activating factor (ATAF), and cup-shaped cotyledon (CUC)] were over-expressed in the drought stressed rice shoots. Notably, a pretreatment with either Se or Si significantly enhanced the expression of both TFs, DREB2A, NAC5, as well as the expression of the ring domain containing OsRDCP1 gene and some drought specific genes, such as OsCMO coding rice choline monooxygenase and dehydrin OsRAB16b. Expression of TFs and the studied genes was markedly enhanced in the Si-stressed shoots of cv. IET 1444 which favors its drought tolerance.

Durum wheat (Triticum turgidum L. var. durum) is mainly produced under rainfed but often sub-optimal moisture conditions in the Mediterranean basin. A set of 114 durum wheat recombinant inbred lines (RILs) developed from the cross of cultivars Omrabi5 × Belikh2 were tested for the ability to tolerate moisture deficiency at the germination and early seedling growth stage. The stress was imposed by exposing the germinating grain to 12 % polyethylene glycol. It induced a measurable reduction in root length, shoot length, and the percentage of normal seedlings. The germination and seedling growth of Belikh2 were more strongly inhibited than those of Omrabi5, and both parents were outperformed by > 50 % of the RILs. A quantitative trait locus (QTL) analysis was carried out by first assembling a linkage map from 265 informative microsatellites. Composite interval mapping revealed nine QTL spread over seven chromosomes. Five of these were associated with coleoptile length, and one of the five explained nearly 29 % of the relevant phenotypic variance. The coleoptile length was significantly correlated with the seedling growth, plant height, and thousand kernel mass derived from field-grown plants of the same RIL population.

In vitro flowering protocols were developed for a limited number of early flowering pea (Pisum sativum L.) cultivars. This work was undertaken to understand the mechanisms regulating in vitro flowering and seed-set across a range of pea genotypes. Its final goal is to accelerate the generation cycle for faster breeding novel genotypes. We studied the effects of in vivo and in vitro applications of the antigibberellin Flurprimidol together with radiation of different spectral compositions on intact plants, plants with the meristem removed, or excised shoot tip explants. Based on our results, we present a simple and reliable system to reduce generation time in vitro across a range of pea genotypes, including mid and late flowering types. With this protocol, more than five generations per year can be obtained with mid to late flowering genotypes and over six generations per year for early to mid flowering genotypes.

The MYB, MYC, and WD40 transcription factors (TFs) are known to regulate the expression of structural biosynthetic genes at different steps depending on the plant species. In this work, we used an agroinfiltration protocol with Tobacco rattle virus (TRV) constructs containing partial sequences from MYB or WD40 for virus-induced gene silencing (VIGS) to demonstrate their participation in the regulation of anthocyanin biosynthesis in chilli pepper (Capsicum eximium) fruits. The accumulation of anthocyanins in chilli pepper fruits of plants transformed with TRV2-MYB and TRV2-WD40 constructs was significantly reduced compared to the control or empty TRV2-transformed plants. A significant reduction in gene expression of both TFs was also detected. The expressions of the chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), and UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT) genes were decreased in the plants transformed with the TRV2-MYB construct but not the transcription of flavanone 3-hydroxylase (F3H). When chilli pepper plants were infected with the TRV2-WD40 construct, a significant reduction in CHS, F3H, F3'5'H, DFR and 3GT expression, but not in CHI in the fruits was observed.

In order to study chloroplast biogenesis, we chose natural variegated Epipremnum aureum (golden pothos) and regenerated pale yellow, variegated and green plants from all three types of tissue explants. The percentage of three types of regenerated shoots from three different explants was very close. Regenerated plants have been maintained for a year and show no sign of a colour switch. By comparing their protein profiles, two major differences between pale yellow and green plants were observed at the 15 and 40 to 50 kDa proteins. Moreover, pale yellow plants had unexpected high molecular mass proteins (greater than 60 kDa). Both variegated and green plants had more chlorophyll (Chl) a than Chl b, the ratios were about 1.46 and 1.93, respectively. In contrast, the pale yellow plants not only had less total Chl, but also the reduction of Chl a was much greater than Chl b, resulting in a higher content of Chl b than Chl a. Microscopic analysis revealed that pale yellow plants contained predominantly undeveloped chloroplasts with low Chl contents, even though their mesophyll cells were similar to green and variegated plants. PCR amplification of chloroplast DNA with 14 universal chloroplast primers did not reveal any difference among these regenerated plants.

In this work, the response of the halophytic shrub Prosopis strombulifera to lowering an osmotic potential (Ψ_o) to -1.0, -1.9, and -2.6 MPa generated by NaCl, Na₂SO₄, and the iso-osmotic combination of them was studied at 6, 12, and 24 h after reaching such values in the growing media. By analyzing the content of abscisic acid (ABA) and related metabolites and transpiration rates, we observed that ABA content varied depending on type of salt, salt concentration, organ analyzed, and age of a plant. ABA content in leaves was much higher than in roots, presumably because of rapid biosynthesis and transport from roots. Leaves of Na₂SO₄-treated plants had the highest ABA content at Ψ_o -2.6 MPa (24 h) associated with sulfate toxicity symptoms. Significant content of ABA-glucose ester (ABA-GE) was found in both the roots and leaves, whereas only low content of phaseic acid (PA) and dihydrophaseic acid (DPA). The roots showed high ABA-GE accumulation in all treatments. The highest content of free ABA was correlated with ABA-GE glucosidase activity. The results show that ABA-GE and free ABA work together to create a specific stress signal.