Direct shoot and cormlet regeneration from leaf explants of ‘Silk’ banana (AAB)

Summary Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shool formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indolc-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacctic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, ie.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.     

Phragmites australis root secreted phytotoxin undergoes photo-degradation to execute severe phytotoxicity

Our study organism, Phragmites australis (common reed), is a unique invader in that both native and introduced lineages are found coexisting in North America. This allows one to make direct assessments of physiological differences between these different subspecies and examine how this relates to invasiveness. Recent efforts to understand plant invasive behavior show that some invasive plants secrete a phytotoxin to ward-off encroachment by neighboring plants (allelopathy) and thus provide the invaders with a competitive edge in a given habitat. Here we show that a varying climatic factor like ultraviolet (UV) light leads to photo-degradation of secreted phytotoxin (gallic acid) in P. australis rhizosphere inducing higher mortality of susceptible seedlings. The photo-degraded product of gallic acid (hereafter GA), identified as mesoxalic acid (hereafter MOA), triggered a similar cell death cascade in susceptible seedlings as observed previously with GA. Further, we detected the biological concentrations of MOA in the natural stands of exotic and native P. australis. Our studies also show that the UV degradation of GA is facilitated at an alkaline pH, suggesting that the natural habitat of P. australis may facilitate the photo-degradation of GA. The study highlights the persistence of the photo-degraded phytotoxin in the P. australis''s rhizosphere and its inhibitory effects against the native plants.Key words: ultraviolet, gallic acid, mesoxalic acid, novel weapons, invasive species, Phragmites australis     

Food-grade chemical and biological agents permeabilize red beet hairy roots,assisting the release of betalaines

Hairy root cultures of red beet, Beta vulgaris L., were permeabilized under the functions of food-grade chemical and biological agents cetyl trimethylammonium bromide (CTAB), Triton X-100, Tween-80, Lactobacillus helveticus, Saccharomyces cereviseae, and Candida utilis, as well as cell fractions of L. helveticus, for the recovery of betalaines with or without oxygen stress. Tween-80 (0.15%), Triton X-100 (0.2%), and CTAB (0.05%), in combination with oxygen stress, released 45%, 70%, and 90% pigment into the medium, respectively, with significantly lesser levels in agitated cultures receiving similar treatments. The release was rapid (1 h) in CTAB treatment with a much slower release in Tween-80. CTAB (0.002%) was found to be also useful in effluxing betalaines (80%) from hairy roots grown in a bubble column reactor. Viability of permeabilized hairy roots, tested on agar medium, was not affected by any level of CTAB treatment and was significantly retarded at higher levels of Triton X-100 and Tween-80. An altogether new approach of pigment release using biological agents such as live cells of food-grade microbes was used where C. utilis, L. helveticus, and S. cereviseae released 60%, 85%, and 54% betalaines, respectively, in 24 h, though lower level treatments also released similar levels of pigment by 48 h. Dried whole cell powder of L. helveticus, its total insoluble carbohydrate, and free lipid fractions released 10%, 0%, and 85% pigment, respectively. An extended study with a bubble column reactor using the free lipid fraction of L. helveticus showed 50% and 84% pigment release in 8 and 12 h, respectively, exhibiting good viability when plated on agar medium. Even in the bioreactor, replenishment of medium 8 h after treatment with free lipid of L. helveticus allowed regrowth of hairy roots. The high level of pigment release recorded here, using CTAB or lipid of L. helveticus, appears useful for developing processes for in situ recovery of betalaines. The live microbes, applicable only for batch cultures, are expected to impart improved sensory/nutraceutical effects to the recovered pigment and hence may add value to the product receiving the red beet pigment thus produced.     

Cyanogenic pseudomonads influence multitrophic interactions in the rhizosphere

In the rhizosphere, plant roots cope with both pathogenic and beneficial bacterial interactions. The exometabolite production in certain bacterial species may regulate root growth and other root-microbe interactions in the rhizosphere. Here, we elucidated the role of cyanide production in pseudomonad virulence affecting plant root growth and other rhizospheric processes. Exposure of Arabidopsis thaliana Col-0 seedlings to both direct (with KCN) and indirect forms of cyanide from different pseudomonad strains caused significant inhibition of primary root growth. Further, we report that this growth inhibition was caused by the suppression of an auxin responsive gene, specifically at the root tip region by pseudomonad cyanogenesis. Additionally, pseudomonad cyanogenesis also affected other beneficial rhizospheric processes such as Bacillus subtilis colonization by biofilm formation on A. thaliana Col-0 roots. The effect of cyanogenesis on B. subtilis biofilm formation was further established by the down regulation of important B. subtilis biofilm operons epsA and yqxM. Our results show, the functional significance of pseudomonad cyanogenesis in regulating multitrophic rhizospheric interactions.     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Native Plant and Microbial Contributions to a Negative Plant-Plant Interaction

A number of hypotheses have been suggested to explain why invasive exotic plants dramatically increase their abundance upon transport to a new range. The novel weapons hypothesis argues that phytotoxins secreted by roots of an exotic plant are more effective against naïve resident competitors in the range being invaded. The common reed Phragmites australis has a diverse population structure including invasive populations that are noxious weeds in North America. P. australis exudes the common phenolic gallic acid, which restricts the growth of native plants. However, the pathway for free gallic acid production in soils colonized by P. australis requires further elucidation. Here, we show that exotic, invasive P. australis contain elevated levels of polymeric gallotannin relative to native, noninvasive P. australis. We hypothesized that polymeric gallotannin can be attacked by tannase, an enzymatic activity produced by native plant and microbial community members, to release gallic acid in the rhizosphere and exacerbate the noxiousness of P. australis. Native plants and microbes were found to produce high levels of tannase while invasive P. australis produced very little tannase. These results suggest that both invasive and native species participate in signaling events that initiate the execution of allelopathy potentially linking native plant and microbial biochemistry to the invasive traits of an exotic species.Invasive weeds are a major source of agricultural costs due to reduced productivity and the labor expended for weed control. In addition, the extensive use of herbicides to control weed populations has undesirable environmental consequences. Therefore, understanding mechanisms that facilitate exotic plant dispersal and displacement of natives in new ranges is critical to predicting and controlling invasions and may yield insights into the ecological processes that govern homeostasis and perturbation in natural plant communities.Phragmites australis (Cav.) Trin ex. Steud. (common reed) has been present in the United States for at least 10,000 years as a major component of mixed tidal wetland plant communities (Saltonstall, 2002). However, over the past 200 years its distribution and abundance has expanded rapidly and it is now considered one of the most aggressive invasive species in marsh communities in North America. Chloroplast DNA analysis has shown that 13 native North American Phragmites haplotypes exist, while invasive populations possess a single chloroplast DNA haplotype (M) that is also widespread in Europe and Asia (Saltonstall, 2002). These data are supported by nuclear microsatellite DNA analysis (Saltonstall, 2003) and morphological differences that distinguish native, noninvasive from exotic, invasive Phragmites in North America (Saltonstall et al., 2004). When grown under the same conditions, exotic Phragmites has significantly higher aboveground and belowground biomass than native Phragmites (Vasquez et al., 2005; Saltonstall and Stevenson, 2007), and this pattern is typically observed under field conditions as well although exceptions exist (League et al., 2006; Meadows and Saltonstall, 2007). Unfortunately today, only remnant native P. australis populations remain along the Atlantic Coast of North America, indicating the near total displacement of native populations by exotic P. australis.Various hypotheses have been forwarded to explain the rapid invasion of P. australis, of which human activities, stress regimes, and hydrologic disturbances have received the greatest attention (Chambers et al., 1999). Compared to invasion in terrestrial ecosystems, invasiveness in marsh communities is less well documented and it is still not clear how environmental factors relate to the establishment of specific dominant marsh species. Although allelopathy has been superficially suggested as the main displacing mechanism in P. australis (Kaneta and Sugiyama, 1972; Drifmeyer and Zieman, 1979), there has been minimal success in characterizing the responsible allelochemical. Interestingly, three triterpenoids (β-amacin, taraxerol, and taraxerone) and a flavone (tricin) have been identified from aerial portions of P. australis (Kaneta and Sugiyama, 1972; Drifmeyer and Zieman, 1979). Regrettably, none of these identified chemicals were tested for possible allelopathic activity.Previously, we showed that a root exudate component of P. australis roots inhibits seedling growth, and that production of the exudates is higher in the invasive P. australis haplotype (Rudrappa et al., 2007). The active fraction of this exudate was found to be composed of gallic acid (3,4,5-trihydroxybenzoic acid). Gallic acid is toxic to a variety of weeds, crop plant species, and the model plant species Arabidopsis (Arabidopsis thaliana; Rudrappa et al., 2007; Rudrappa and Bais, 2008). Our published results also show the persistence of gallic acid in soil extracts from P. australis-invaded fields, which validates our in vitro results and strongly supports the idea that P. australis'' invasive behavior may partly be due to the exudation of gallic acid in the soil/marsh (Rudrappa et al., 2007). Our studies concur with the earlier established reports of phytotoxicity and persistence of gallic acid in soil (Weidenhamer and Romeo, 2004).Biochemically, the transition from simple galloylglucoses to complex gallotannins is marked by addition of further galloyl moieties to the pentagalloylglucose (Niemetz and Gross, 2005). It is now known that free gallic acid is released from complexed gallotannins by simple hydrolysis reactions, wherein a tannase activity breaks gallate ester to form free gallic acid, ellagic acid, and Glc (Mahoney and Molyneux, 2004). Treatment of fungal tannase from Aspergillus flavus results in hydrolysis of pellicle-localized gallotannin to form gallic acid, and ellagic acid as two phenolic components (Mahoney and Molyneux, 2004). As gallic acid is often complexed as gallotannins (Niemetz and Gross, 2005), we speculated that plant- or microbial-derived tannase may facilitate free gallic acid release in salt marsh soils.Aside from allelopathy, invasive plants may deleteriously affect interactions between rhizospheric microbial communities and native plant species (Klironomos, 2002; Wardle et al., 2004; Callaway et al., 2008) to promote their expansion in new ranges. One specific example is the disruption of interactions between native species and their arbuscular mycorhizae, upon which the native species rely for nutrient acquisition (Stinson et al., 2006). Another recent study suggests that the recruitment or establishment of an altered soil microbial community may negatively impact the ability of native species to survive in the same soils (Batten et al., 2008). Evidences suggest that soil biota have several effects on the success of invasive plants and the interactions are based in part on the biochemistry, i.e. novel biochemical weapons (Callaway and Ridenour, 2004). However, to our knowledge, no previous studies have directly tested whether P. australis or any other exotic plant may exploit the biochemical potential of native plant and microbial communities to release a phytotoxin (gallic acid) from a relatively benign precursor (gallotannin) in the rhizosphere. This report presents evidence that links native plant and microbial biochemistry to the invasive traits of an exotic species.     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

Causes and consequences of plant-associated biofilms

The rhizosphere is the critical interface between plant roots and soil where beneficial and harmful interactions between plants and microorganisms occur. Although microorganisms have historically been studied as planktonic (or free-swimming) cells, most are found attached to surfaces, in multicellular assemblies known as biofilms. When found in association with plants, certain bacteria such as plant growth promoting rhizobacteria not only induce plant growth but also protect plants from soil-borne pathogens in a process known as biocontrol. Contrastingly, other rhizobacteria in a biofilm matrix may cause pathogenesis in plants. Although research suggests that biofilm formation on plants is associated with biological control and pathogenic response, little is known about how plants regulate this association. Here, we assess the biological importance of biofilm association on plants.     

Root-secreted malic acid recruits beneficial soil bacteria

Beneficial soil bacteria confer immunity against a wide range of foliar diseases by activating plant defenses, thereby reducing a plant's susceptibility to pathogen attack. Although bacterial signals have been identified that activate these plant defenses, plant metabolites that elicit rhizobacterial responses have not been demonstrated. Here, we provide biochemical evidence that the tricarboxylic acid cycle intermediate L-malic acid (MA) secreted from roots of Arabidopsis (Arabidopsis thaliana) selectively signals and recruits the beneficial rhizobacterium Bacillus subtilis FB17 in a dose-dependent manner. Root secretions of L-MA are induced by the foliar pathogen Pseudomonas syringae pv tomato (Pst DC3000) and elevated levels of L-MA promote binding and biofilm formation of FB17 on Arabidopsis roots. The demonstration that roots selectively secrete L-MA and effectively signal beneficial rhizobacteria establishes a regulatory role of root metabolites in recruitment of beneficial microbes, as well as underscores the breadth and sophistication of plant-microbial interactions.