Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Genetic transformation of a hepatoprotective plant, <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid, catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacterium-mediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β-glucuronidase encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots, which rooted to produce 13 complete transgenic plants.     

Comparison of tolerance of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Vigna radiata</Emphasis> to cadmium

The effect of different cadmium concentrations (6–120 μM) on Hill reaction activity (HRA) of isolated chloroplasts, contents of chlorophylls (Chls) and carotenoids (Cars), and Cd uptake and accumulation in plant organs of Indian mustard (Brassica juncea L. cv. Vitasso) and mung bean [Vigna radiata (L.) Wilczek] were determined. The Cd stress inhibited photochemical activity of isolated chloroplasts of both species and in both tested developmental stages. On the basis of EC₅₀ values, the mung bean showed a higher sensitivity to Cd treatment than Indian mustard. The higher sensitivity of both species was determined in the earlier than in the older developmental stage. The leaves of Cd-treated plants possessed lower contents of Chls and Cars in both species and the negative effect increased with Cd concentration. A difference between species was also found in Cd uptake and accumulation. In both species, Cd was accumulated more in roots than in shoots, with higher accumulation in Indian mustard than in mung bean.     

Overexpression of <Emphasis Type="Italic">Petunia SOC1</Emphasis>-like Gene <Emphasis Type="Italic">FBP21</Emphasis> in Tobacco Promotes Flowering Without Decreasing Flower or Fruit Quantity

FBP21 is one of the SOC1-like genes isolated from Petunia hybrida. Based on sequence analysis, FPB21 is suggested to have a role in promoting flowering. In this study, FBP21 was expressed in a tobacco host plant under the control of the CaMV 35S promoter. Our results showed that the transgene accelerated flowering, i.e. the transgenic plants flowered just 3 months after germination, in comparison to the wild-type tobacco which flowered after 5 months. Plant morphology was also affected, with the transgenic tobacco plants developing at least five robust lateral branches, while the control plants generally had just three. Total leaf area was significantly reduced in the transgenic tobacco compared to wild-type tobacco. By contrast, there was no significant difference between transgenic and control plants for the total number of flowers or fruits. Thus, the flower or fruit yield expressed per unit leaf area was higher in transgenic tobacco than in wild-type plants. Semi-quantitative RT-PCR analysis indicated that overexpression of FBP21 in tobacco resulted in the up-regulation of some flowering-related genes. The results of this study in tobacco indicate that the Petunia FBP21 gene may permit the engineering of early-flowering and short-growth habits without compromising flower or fruit yields.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

Over-expression of <Emphasis Type="Italic">ThpI</Emphasis> from <Emphasis Type="Italic">Choristoneura fumiferana</Emphasis> enhances tolerance to cold in Arabidopsis

Thermal hysteresis proteins (Thps) known as antifreeze proteins for their antifreeze activity, depress the freezing point of water below the melting point in many polar marine fishes, terrestrial arthropods and plants. For the purpose of breeding cold-resistant plants, we designed to introduce the Thp gene into the plants. The physiological and biochemical effect of high-lever expression of the modified Choristoneura fumiferana Thp (ThpI) in Arabidopsis thaliana plants was analyzed. Under low temperature stress, the ThpI transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the ThpI over-expressing plants was confirmed by the changes of electrolyte leakage activity, malonyldialdehyde and proline contents. These results preliminarily showed that the Thp possibly be used to enhance the low temperature-tolerant ability of plants.     

Rice Gene <Emphasis Type="Italic">OsDSR-1</Emphasis> Promotes Lateral Root Development in <Emphasis Type="Italic">Arabidopsis</Emphasis> Under High-Potassium Conditions

Rice gene Oryza sativa Drought Stress Response-1 (OsDSR-1) was one of the genes identified to be responsive to drought stress in the panicle of rice at booting and heading stages by both microarray and quantitative real-time PCR analyses. OsDSR-1 encodes a putative calcium-binding protein, and its overexpression in Arabidopsis rendered transgenic plants to produce much shorter lateral roots (LRs) than wild-type (WT) plants in the medium supplemented with abscisic acid (ABA), suggesting that OsDSR-1 may act as a positive regulator during the process of ABA inhibition of LR development. No significant difference was observed in the total LR length between WT and transgenic plants in the media with the increase of only osmotic stress caused by NaCl, LiCl, and mannitol, while transgenic Arabidopsis seedlings appeared to produce larger root systems with longer total LR lengths under high-potassium conditions than WT seedlings. Further analysis showed that external Ca²⁺ was required for the production of larger root systems, indicating that the promotion by OsDSR-1 of the LR development of transgenic Arabidopsis seemed to occur in a Ca²⁺-dependent manner under high-potassium conditions. We propose that OsDSR-1 may function as a calcium sensor of the signal transduction pathway controlling the LR development under high-potassium conditions.     

Peat-Based Inoculum of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> Supplemented with Xanthan Gum

The addition of xanthan to high water retention capacity peat (HWRC) inoculants did not show differences on the survival of Bradyrhizobium japonicum E109. In low water retention capacity peats (LWRC) however, xanthan increased the survival of B.japonicum significantly. Xanthan showed the best effect at 0.1 g/l for B. japonicum, in contrast to Sinorhizobium fredii USDA205 where the concentrations evaluated (0–1.0 g/l) did not affected significantly its survival. Nevertheless, when the symbiotic performance on soybean was evaluated, the presence of 0.1 g xanthan/l increased the nodule number for both strains.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Effect of ectopic expression of the eutypine detoxifying gene <Emphasis Type="Italic">Vr</Emphasis>-<Emphasis Type="Italic">ERE</Emphasis> in transgenic apple plants

The development of alternative selection systems without antibiotic resistance genes is a key issue to produce safer and more acceptable transgenic plants. Eutypine is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine, which is detoxified in mung bean (Vigna radiata) by the gene Vr-ERE. Many phytotoxic compounds containing an aldehyde group can act as substrates for the Vr-ERE enzyme. The aim of the present work was to evaluate the effects of the overexpression of Vr-ERE in transgenic apple plants, as a first step towards the development of an alternative selection system. Viable transgenic apple clones expressing Vr-ERE were produced from the cultivar Greensleeves under kanamycin selection. Although the Vr-ERE transgene was normally expressed at the RNA and protein levels, the increase in aldehyde reductase activity tested on a range of potential substrates was very low in these clones. None of them revealed a significant increase in tolerance to toxic aldehydes compared to their non-transgenic control. This work with transgenic apple plants overexpressing the detoxifying gene Vr-ERE illustrates some of the difficulties in developing an alternative selection pressure.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Rhizosphere competent <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in the management of <Emphasis Type="Italic">Heterodera cajani</Emphasis> on sesame

Biological control of the cyst forming nematode Heterodera cajani was studied on sesame using plant growth promoting rhizobacteria (PGPR) Pseudomonas aeruginosa LPT3 and LPT5. Based on plant growth promoting attributes, two fluorescent pseudomonads, LPT3 and LPT5 were evaluated for their efficacy against cyst forming nematode Heterodera cajani that parasitize Sesamum indicum. Pseudomonas aeruginosa LPT5 produced IAA, HCN, chitinase, glucanase and siderophore, and also solubilized inorganic phosphate in vitro. Moreover, LPT5 resulted in mortality of second stage juveniles of H. cajani, which was 13% higher as compared to P. aeruginosa LPT3. Interestingly, when both strains were inoculated together for the management of H. cajani on Sesamum indicum the population of H. cajani was reduced significantly, in field trial. Approximately 60% reduction in cyst and juveniles population was recorded with LPT5 coated seeds, while LPT3 resulted in 49% reduction in cyst and juvenile population as compared to control. Plants grown with seeds bacterized with LPT5 and reduced doses of urea, diammonium phosphate (DAP), muriate of potash (K) and gypsum gave maximum increase in yield, in comparison to that of plants raised under the influence of recommended or full doses of the chemical fertilizers. Pseudomonas aeruginosa LPT5 also showed excellent root colonization.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

Glutathione-mediated Antioxidative Mechanisms in Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Cells in Response to Cadmium Stress

Cadmium (Cd) homeostasis and detoxification in sunflower (Helianthus annuus L.) cells differing in Cd sensitivity/tolerance were studied by analyzing the glutathione-mediated antioxidant mechanism vis-à-vis phytochelatin biosynthesis in vitro. Calluses exposed to Cd-shock/-acclimatization (150μM) were assayed for oxidative stress, reduced glutathione (GSH), glutathione disulfide (GSSG), phytochelatins (PCs) and reactive oxygen species (ROS). Although Cd did not induce any oxidative stress in Cd-tolerant callus (TCd), it generated oxidative stress in Cd-shock callus (SCd) both in terms of lipid peroxidation and protein oxidation. GSH/GSSG ratio remained similar to control values in the cadmium-acclimatized calluses. However, after acute treatment, there was a decline in both GSH and GSSG levels in SCd with concomitant reduction in the GSH/GSSG ratio. Analysis of PCs was performed using HPLC and mass spectrometry methods. PC concentration in TCd were approximately twice those that in SCd, showing in both cases a 1:2:1 relative proportion for PC n = 2 (PC2): PC n = 3 (PC3): PC n = 4 (PC4). Calluses growing in the presence of Cd developed an increased resistance to paraquat oxidative stress generation. These results indicated that PCs synthesis was an important mechanism for Cd detoxification in sunflower calluses, but the capacity to grow in the presence of Cd is related to the tissues ability to maintain high intracellular levels of GSH.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration from shoot apices and gene transfer by particle bombardment in <Emphasis Type="Italic">Jatropha curcas</Emphasis>

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.     

Gynogenesis in the vine cacti <Emphasis Type="Italic">Hylocereus</Emphasis> and <Emphasis Type="Italic">Selenicereus</Emphasis> (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.     

Geometric characters of the radius and tibia in <Emphasis Type="Italic">Macaca mulatta</Emphasis> and <Emphasis Type="Italic">Macaca fascicularis</Emphasis>

We performed comparative analyses of four cross-sections of the distal radius and tibia in two species of macaque to clarify the relationships between bone morphology and locomotor type. The lengths of bones and five bone geometric properties in each section were examined and compared separately in both female and male Macaca mulatta and Macaca fascicularis. In M. mulatta, there were no significant gender-specific differences in either the radius or the tibia. In contrast, the radius and tibia of male M. fascicularis had greater geometric parameters in the 20% and 40% positions relative to the 5% and 10% positions from the distal end than those of their female counterparts. The radius and tibia of M. mulatta were relatively longer than those of M. fascicularis, and the sectional parameters of the tibia of M. mulatta were relatively larger than those of M. fascicularis. Standardization of the log-transformed bone length between the species revealed larger radial cortical bone areas in M. fascicularis. In contrast, there were minimal differences in the tibial cortical bone areas between the two species. This study suggests that the observed distinctions in bone geometry in female and male M. fascicularis may be due to gender-specific differences in the muscle weights of the forearm and calf, which may underlie the divergence in the leaping abilities of females and males of this species. Taken together, these results of interspecies comparisons may be related to the fact that arboreal primates such as M. fascicularis undergo compressive mechanical stress due to the forelimb lead that occurs as the animal descends a sloping trunk or bridges a tree gap downward, while terrestrial primates such as M. mulatta move on nearly flat substrates. Differences in fore- and hind-limb bone properties between the two species are discussed with regard to functional morphology and locomotor type.