Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Secondary metabolite profiling of the model legume <Emphasis Type="Italic">Lotus japonicus</Emphasis> during its symbiotic interaction with <Emphasis Type="Italic">Mesorhizobium loti</Emphasis>

Plant secondary metabolites, particularly flavonoids, are key components in the early stages of nitrogen-fixing symbiosis. Despite their importance, the endogenous secondary metabolites involved in symbiosis have not yet been identified in the model legume Lotus japonicus. We therefore determined changes in the secondary metabolic profile of Lotus japonicus roots in response to its symbiont. Analysis of the root secondary metabolite profiles 1 week after inoculation with Mesorhizobium loti revealed quantitative changes in the level of 14 phenolic peaks when compared with non-inoculated control plants. These changes affected compounds from most phenolic classes, possibly resulting from interconversion between classes since the total phenolic level remained constant. In addition, the use of 2 M. loti strains differing only in their capacity to synthesise Nod factor revealed that, although Nod factor signalling induced accumulation of a specific subset of 4 phenolic peaks, most changes were induced in response to both rhizobial strains.     

<Emphasis Type="Italic">MAX4</Emphasis> gene is involved in the regulation of low inorganic phosphate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MAX4 gene has been shown to be involved in the regulation of shoot branching in Arabidopsis (Arabidopsis thaliana). However, little is known about the role of MAX4 gene in low inorganic phosphate (Pi) stress response in Arabidopsis. Here we showed that MAX4 gene is involved in the regulation of low Pi stress response in Arabidopsis. MAX4 gene was repressed by low Pi stress, and the max4 mutants showed lower anthocyanin content and longer primary root length. In addition, max4 mutant plants also displayed altered root architecture such as increased root-to-shoot ratio, lower lateral root number and root hair density compared with wild-type plants under low Pi stress. Higher total Pi contents were detected in shoots and roots of max4 plants than those of wild-type plants when subjected to low Pi stress, which was associated, at least in part, with increase in expression of WRKY75 as well as AtPT1 and AtPT2 genes encoding high-affinity Pi transporters. Taken together, all these results suggest that MAX4 gene mediates low Pi stress response, at least in part, by regulating the expression of WRKY75 as well as AtPT1 and AtPT2 genes.     

<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.     

Effect of <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> on <Emphasis Type="Italic">in vitro</Emphasis> pineapple plants’ growth during acclimatization

Pineapple is one of the most important tropical fruits, but the availability of planting material is insufficient to agricultural demands. Therefore, several pineapple micropropagation protocols have been developed. However, acclimatization of in vitro plants continues to take a prolonged period. Biofertilizers have been found as safe alternatives to improve the agricultural performances of many crops. This study highlights some of the effects of the application of Azotobacter chroococcum (INIFAT5 strain) on in vitro pineapple plants during acclimatization. The bacteria were sprayed immediately after transplanting to the ex vitro environment; the plants were then sprayed every 4 wk. A control group of plants was established. Subsequently, after 5 mo, the evaluated variables included fresh and dry plant weight, plant height (cm), and root length (cm). The anatomy of middle-aged leaves and roots was also studied: transversal thickness and width of cuticle, epidermis, hypodermis, aquiferous parenchyma, and photosynthetic parenchyma. Thickness of root exoderm, external cortex, internal cortex, and stele were also evaluated. In general, the INIFAT5 strain improved the plant development. Results showed that the bacteria significantly provoked changes in the plant fresh weight, the thickness of the leaf abaxial and adaxial cuticles, and the root exoderm width. Contrastingly, A. chroococcum did not affect the thickness of the leaf photosynthetic parenchyma.     

Isolation of a <Emphasis Type="Italic">Ve</Emphasis> homolog, <Emphasis Type="Italic">mVe1</Emphasis>, and its relationship to verticillium wilt resistance in <Emphasis Type="Italic">Mentha longifolia</Emphasis> (L.) Huds

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2–99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.     

Simultaneous Expression of <Emphasis Type="Italic">Spinacia oleracea</Emphasis> Chloroplast Choline Monooxygenase (CMO) and Betaine Aldehyde Dehydrogenase (BADH) Genes Contribute to Dwarfism in Transgenic <Emphasis Type="Italic">Lolium perenne</Emphasis>

Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) catalyze the first and second steps in the biosynthesis of glycine betaine in betaine-accumulating plants. Over-expression of the Spinacia oleracea chloroplast choline monooxygenase (SoCMO) and betaine aldehyde dehydrogenase (SoBADH) genes has not been reported in Lolium perenne. In this investigation, the SoCMO and SoBADH genes have been used to generate transgenic L. perenne plants via particle bombardment. Transgenic plants have been confirmed with PCR, Southern blot, and Northern blot analyses. Enhanced salt stress tolerance has been observed from SoBADH–SoCMO transgenic L. perenne plants. The dwarf phenotype was first observed 3 months after transgenic plants were established in soil and was to be stably inherited. Height of transgenic plants was decreased by 63% compared to the control. Measurement of endogenous GAs content demonstrated that the content of endogenous GA1 was decreased by 75.2%, and the content of endogenous GA4, GA12, GA19, and GA53 of transgenic plants was increased by 200%, 221%, 105%, and 108%, respectively, compared to the control plants. Dwarf trait of SoBADH–SoCMO transgenic L. perenne plants can be recovered by application of exogenous GAs. These results demonstrated that simultaneous expression of the SoCMO and SoBADH genes enhanced salt stress tolerance and induced dwarfism in transgenic L. perenne. Dwarfism induced by expression of the SoCMO and SoBADH genes was associated with synthesis of endogenous GAs and it could be recovered by application of exogenous GAs. This is the first report on dwarfism induced by expression of the SoCMO and SoBADH genes in a species in turfgrass.     

<Emphasis Type="Italic">Rhizobia species</Emphasis>: A Boon for “Plant Genetic Engineering”

Since past three decades new discoveries in plant genetic engineering have shown remarkable potentials for crop improvement. Agrobacterium Ti plasmid based DNA transfer is no longer the only efficient way of introducing agronomically important genes into plants. Recent studies have explored a novel plant genetic engineering tool, Rhizobia sp., as an alternative to Agrobacterium, thereby expanding the choice of bacterial species in agricultural plant biotechnology. Rhizobia sp. serve as an open license source with no major restrictions in plant biotechnology and help broaden the spectrum for plant biotechnologists with respect to the use of gene transfer vehicles in plants. New efficient transgenic plants can be produced by transferring genes of interest using binary vector carrying Rhizobia sp. Studies focusing on the interactions of Rhizobia sp. with their hosts, for stable and transient transformation and expression of genes, could help in the development of an adequate gene transfer vehicle. Along with being biologically beneficial, it may also bring a new means for fast economic development of transgenic plants, thus giving rise to a new era in plant biotechnology, viz. “Rhizobia mediated transformation technology.”     

Feeding Ecology of <Emphasis Type="Italic">Trachypithecus pileatus</Emphasis> in India

We observed a group of capped langurs for 12 mo in the Pakhui Wildlife Sanctuary, Arunachal Pradesh, India. We recorded the time of feeding on different food plant species, food categories, and the feeding heights of monkeys in trees. Capped langurs spent 68% of their feeding time on leaves, 16% on flowers, and 16% on fruits. Feeding on leaves was consistently high (p < 0.01) during the year, with the highest feeding in May (85%) and the lowest in January (47%). The seasonal difference in feeding on leaves is significant (p < 0.05): it was higher in summer and during monsoon. The feeding time on flowers was maximal (35%) in March and that on fruits and seeds was minimal (38%) in January. Langurs ate 52 plant species throughout the year. The largest number of plants (6) were species of Moraceae, and langurs spent more feeding time (20%) on them alone. The number of plants eaten per month varied significantly (p < 0.05). Langurs ate Gmelina arborea, Albizzia lucida, Ficus glomereta, and Makania micrantha throughout the year. They spent 44% of their feeding time in terminal canopies and their average feeding height was 30–35 m. This is the first study to examine the feeding ecology of capped langurs and provides baseline data for the species.     

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.     

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.     

Photosynthetic and transpiration responses of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. plants to <Emphasis Type="Italic">ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

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.     

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.     

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.