Anatomy and photosystem II activity of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis> as affected by 1-naphthaleneacetic acid

Auxins are one of the main regulators of in vitro plant growth and development. However, the mechanisms, by which auxins, such as 1-naphthaleneacetic acid (NAA), affect in vitro root and leaf anatomy and photosystem function, remain unclear. Accordingly, the aim of the present study was to analyze the effect of different NAA concentrations on the anatomy and photosynthetic performance of in vitro-propagated Aechmea blanchetiana and to determine whether such a treatment affects micropropagated plants after acclimatization. In vitro-established A. blanchetiana plants were transferred to culture media that contained 0, 2, 4, or 6 μM NAA, and after 50 d, they were transplanted into plastic seedling trays with a commercial substrate and cultivated for 60 d in a greenhouse. The plants were evaluated after a 50-d in vitro NAA exposure (growth traits, chlorophyll α fluorescence, and root and leaf anatomy) and after 60 d of acclimatization in the greenhouse (root and leaf growth). Changes induced by NAA in root anatomy might improve uptake of minerals and sugars from the medium, thereby increasing the in vitro growth. In the leaves, the lowest chlorenchyma thickness and sclerenchyma area were observed in plants grown without NAA, and NAA exposure also improved photosystem II activity. The highest ex vitro growth rate was observed for plants that were propagated with 4 μM NAA. Therefore, the use of NAA during in vitro propagation can improve the anatomical and physiological quality of A. blanchetiana plants, as well as to improve ex vitro transfer.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Lignan accumulation in two-phase cultures of <Emphasis Type="Italic">Taxus</Emphasis> x <Emphasis Type="Italic">media</Emphasis> hairy roots

The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cultures of KT and ATMA hairy root lines were supplemented with precursors: coniferyl alcohol (CA 1, 10 or 100 µM) and/or l-phenylalanine (100 µM PHEN) and/or methyl jasmonate (100 µM MeJa). Moreover the two-phase in vitro cultures supported with perfluorodecalin (PFD) as a gas carrier and in situ extrahent were used. The hairy root lines differed in lignan production profiles. In the control untreated cultures KT roots did not accumulate secoisolariciresinol and lariciresinol while ATMA roots did not accumulate matairesinol. In ATMA roots the treatment with CA (1 or 10 µM) resulted in the production of lariciresinol and secoisolariciresinol whereas solely lariciresinol was present after 100 µM CA application. Elicitation with 1 µM CA and MeJa yielded with hydroxymatairesinol aglyca and lariciresinol glucosides with their highest content 37.88 and 3.19 µg/g DW, respectively. The stimulatory effect of simultaneous treatment with 1 µM CA, PHEN and MeJa on lignan production was observed when the cultures were supplemented with PFD-aerated or degassed. In ATMA root cultures these applied conditions were the most favourable for matairesinol content which amounted to 199.86 and 160.25 µg/g DW in PFD-aerated and PFD-degassed supported cultures, respectively. In KT root cultures solely, hydroxymatairesinol and coniferin/CA content was enhanced with their highest yield 59.29 and 134.60 µg/g DW in PFD-aerated and PFD-degassed cultures, respectively.     

<Emphasis Type="Italic">In vitro</Emphasis> morphogenesis and micropropagation of <Emphasis Type="Italic">Aechmea ramosa</Emphasis> var. <Emphasis Type="Italic">ramosa</Emphasis> Mart. ex Schult. f. (Bromeliaceae) from leaf explants

Aechmea ramosa Mart. ex Schult. f. is an endemic bromeliad of the Brazilian Atlantic Forest. The current habitat degradation of this hotspot biome threatens this species, which besides having an important ecological role, is also of invaluable ornamental interest. Plant tissue culture has been used in mass propagation and conservation of various bromeliads. We have established a micropropagation protocol for A. ramosa var. ramosa using leaf explants grown in MS medium supplemented with 2 μM of 1-naphthaleneacetic acid (NAA) and 2 μM of 6-benzylaminopurine (BAP) that showed higher values of shoot induction. NAA and BAP are associated with the production of proteins involved in stress response modulation, metabolic activity, and cell division, the latter being involved in inducing the differentiation of competent cells. After 120 d of culture, each explant presented 28.9 shoots with an average size of 27.8 mm, with no variation in either Stomatal Index or density of the regenerated shoots. Plantlets measuring above 15-mm height were successfully acclimatized, presenting 100% survival rate. Thus, this protocol can be used for mass propagation of A. ramosa, and to supply demand for the market of ornamental plants. Furthermore, it represents an important tool for the conservation of this species and maintenance of an in vitro germplasm.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Peculiarities of Regeneration and Genetic Variability of <Emphasis Type="Italic">Crambe koktebelica</Emphasis> and <Emphasis Type="Italic">Crambe tataria</Emphasis> Plants in vitro

The regenerative capability of three types of explants was studied on media with different compositions of growth regulators with the purpose of selecting optimal conditions of fast reproduction of endangered Crambe species that could be used as a relevant source of genetic material for the improvement of industrially valuable plants. PCR-analysis of genotypes of C. koktebelica and C. tataria plants was conducted to identify the influence of in vitro cultivation on the genetic stability of plants. The highest regeneration rates were observed with the use of petiole explants on MS medium with BA and NAA. The absence of somaclonal variability in C. koktebelica and C. tataria in vitro regenerated plants was demonstrated.     

Production of <Emphasis Type="Italic">cis</Emphasis>-Vaccenic Acid-oriented Unsaturated Fatty Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesel is produced worldwide as an alternative energy fuel and substitute for petroleum. Biodiesel is often obtained from vegetable oil, but production of biodiesel from plants requires additional land for growing crops and can affect the global food supply. Consequently, it is necessary to develop appropriate microorganisms for the development of an alternative biodiesel feedstock. Escherichia coli is suitable for the production of biodiesel feedstocks since it can synthesize fatty acids for lipid production, grows well, and is amenable to genetic engineering. Recombinant E. coli was designed and constructed for the production of biodiesel with improved unsaturated fatty acid contents via regulation of the FAS pathway consisting of initiation, elongation, and termination steps. Here, we investigated the effects of fabA, fabB, and fabF gene expression on the production of unsaturated fatty acids and observed that the concentration of cis-vaccenic acid, a major component of unsaturated fatty acids, increased 1.77-fold compared to that of the control strain. We also introduced the genes which synthesize malonyl-ACP used during initiation step of fatty acid synthesis and the genes which produce free fatty acids during termination step to study the effect of combination of genes in elongation step and other steps. The total fatty acid content of this strain increased by 35.7% compared to that of the control strain. The amounts of unsaturated fatty acids and cis-vaccenic acid increased by 3.27 and 3.37-fold, respectively.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.