Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Ethylene and polyamine interactions in morphogenesis of Passiflora cincinnata: effects of ethylene biosynthesis and action modulators, as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (""Edison de Paula"", EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch`s (VS 50) and Guillard & Ryther`s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L(-1)) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.

Desiccation tolerance in Pleurostima purpurea (Velloziaceae)

Previous works suggested that Pleurostima purpurea (Velloziaceae-Barbacenioideae) shows a remarkable capacity to endure desiccation of its vegetative tissues. P. purpurea occurs in monocotyledons mats on soil islands in the Pao de Acucar (Sugar Loaf) one of the most recognizable rock outcrops of the world, in Rio de Janeiro, southeastern Brazil. Mats of P. purpurea occur in cliffs by the sea some meters above the tidal zone. Although living in rock outcrops almost devoid of any soil cover, P. purpurea seems to occur preferably on less exposed rock faces and slightly shady sites. Usually, less extreme adaptations to drought would be expected in plants with the habitat preference of P. purpurea. Relying on this observation, we argue if a combination of different strategies of dealing with low water availability can be found in P. purpurea as on other desiccation tolerant angiosperms. This study aims to examine the occurrence of desiccation tolerant behavior in P. purpurea together with the expression of drought avoidance mechanisms during dehydration progression. For this, it was analyzed the gas exchanges, leaf pigments and relative leaf water content during desiccation and rehydration of cultivated mature individuals. P. purpurea behaved like typical drought avoiders under moderated drought condition with stomatal closure occurring around a relative leaf water content up to 90%. During this process, it was observed a delay in the leaf relative water content (RWC(leaf)) decrease comparing to the plant-soil relative water content (RWC(plant-soil)). As soil dehydration worsened, gas exchanges restrictions progressed until a lack of activity which characterizes anabiosis. The loss of chlorophyll occurs before the end of total dehydration, characterizing the presence of poikilochlorophylly. The chlorophyll degradation follows the RWC(leaf) decrease, which achieved the minimum average value of 17% without incurring in leaf abscission. The chlorophyll re-synthesis seems to start well after the full rehydration of the leaf. During all of this process, carotenoid content remained stable. These results are coherent with a combination of drought avoidance and desiccation tolerance in P. purpurea which seems to be coherent with the amplitude of water availability in the rock outcrop habitat where it occurs, suggesting that the periods of water availability are sufficiently long for the success of the costly desiccation tolerant behavior but too short to make a typical drought avoider species win the competition for exploring the rock outcrop substrate where P. purpurea occurs.

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination. Methods Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC. Key Results During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied. Conclusions The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.

Preparation of Achiral and Chiral (E)-Enaminopyran-2,4-diones and Their Phytotoxic Activity

A short and efficient approach to a range of new chiral and achiral functionalized (E)-enaminopyran-2,4-diones starting with commercially available dehydroacetic acid is described. The phytotoxic properties of these (E)-enaminopyran-2,4-diones were evaluated by their ability to interfere with the growth of Sorghum bicolor and Cucumis sativus seedlings. A different sensitivity of the two crops was evident with the (E)-enaminopyran-2,4-diones. The most active compounds were also tested against two weeds, Ipomoea grandifolia and Brachiaria decumbens. To the best of our knowledge, this is the first report describing enaminopyran-2,4-diones as potential plant growth regulators.

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

Characterization of the Wheat Grain PKABA1-Interacting Protein TaWD40

Abscisic acid (ABA)-mediated gene expression is a critical component of plant responses to this important hormone, which affects plant growth, development, and responses to environmental stresses. Plant responses to ABA are mediated by a number of factors including PKABA1, an ABA induced protein kinase involved in ABA-suppressed gene expression in cereal grains, and TaWD40, which has previously been shown to physically interact with PKABA1. A full-length 1.9 kb TaWD40 cDNA, CK210682, was sequenced as part of this project. Based on the deduced protein sequence, it is thought that TaWD40 may belong to the family of E3 ubiquitin ligases, possibly targeting PKABA1 for destruction. Construction of expression plasmids for overproduction of the TaWD40 polypeptide in E. coli is currently underway. The TaWD40 cDNA has been successfully amplified from the source plasmid and inserted into an intermediate plasmid, pCR2.1. The TaWD40 cDNA is currently being cloned from the pCR2.1 intermediate plasmid into two different expression vectors, pRSET-A and pMAL-c2x, for future protein production and purification.

Efeitos da aplicação de herbicidas sobre a eficiência fotoquímica em plantas de soja convencional e geneticamente modificada

Objetivou-se, neste trabalho, avaliar o efeito da aplicação de herbicidas em pós-emergência sobre a eficiência fotoquímica de duas cultivares de soja (M-SOY 7908 RR e M-SOY 8001). O delineamento experimental utilizado foi em blocos ao acaso, com quatro repetições. Os tratamentos constaram da aplicação de herbicidas isolados e em misturas: lactofen, glyphosate, lactofen + chlorimuron-ethyl, chlorimuron-ethyl + imazethapyr, chlorimuron-ethyl + bentazon, glyphosate + imazethapyr, lactofen + chlorimuron-ethyl + imazethapyr e lactofen + chlorimuron-ethyl + imazethapyr/haloxyfop-methyl. Além disso, foram mantidas duas testemunhas sem aplicação de herbicida. As avaliações foram realizadas aos 4; 11; 18; 25 e 32 dias após aplicação dos herbicidas (DAA) no primeiro ano, e aos 6, 14, 21, 28 e 35 DAA, no segundo. A cultivar M-SOY 8001 foi suscetível à aplicação dos herbicidas, principalmente às misturas contendo lactofen + chlorimuron-ethyl e lactofen + chlorimuron-ethyl + imazethapyr + haloxifop-methyl, os quais provocaram redução no rendimento quântico máximo do PSII (Fv/Fm). As duas cultivares apresentaram suscetibilidade aos herbicidas quando tratados com lactofen aplicado isolado e nas misturas lactofen + chlorimuron-ethyl, chlorimuron-ethyl + imazethapyr, lactofen + chlorimuron-ethyl + imazethapyr e lactofen + chlorimuron-ethyl + imazethapyr + haloxifop-methyl com redução nos teores de clorofila, estimados pelo Medidor Portátil de Clorofilas. Os danos foram reversíveis, desaparecendo gradativamente com a idade das plantas.

Estimativa da área foliar de Synedrellopsis grisebachii usando método não destrutivo

A área foliar é uma das principais características usadas para avaliar o crescimento vegetal. O objetivo desta pesquisa foi determinar uma equação matemática para estimar a área foliar de Synedrellopsis grisebachii - uma importante planta daninha no Brasil - a partir de dimensões lineares dos limbos foliares. Duzentas folhas foram medidas em comprimento (C), largura máxima (L) e área foliar real (AF). Os dados de AF e CxL foram submetidos à análise de regressão linear, determinando-se uma equação matemática para estimar a área foliar da espécie. A correlação entre os valores de área foliar real e estimada foi significativa. Portanto, a área foliar de S. grisebachii pode ser estimada satisfatoriamente pela equação: AF = 0,730829(C×L).

The Role of Phytochrome in Stress Tolerance

It is well-documented that phytochromes can control plant growth and development from germination to flowering. Additionally, these photoreceptors have been shown to modulate both biotic and abiotic stress. This has led to a series of studies exploring the molecular and biochemical basis by which phytochromes modulate stresses, such as salinity, drought, high light or herbivory. Evidence for a role of phytrochromes in plant stress tolerance is explored and reviewed.

Estimativa da área foliar de crotalaria juncea l. a partir de dimensões lineares do limbo foliar

Knowledge of the leaf area plant are needed for agronomic and physiological studies involving plant growth. The aim of this study was to obtain a mathematical model using linear measures of leaf dimensions, which will allow the estimation of leaf area of Crotalaria juncea L. Correlation studies were conducted involving real leaf area (Sf) and leaf length (C), maximum leaf width (L) and the product between C and L. All tested models (linear, exponential or geometric) provided good estimation of leaf area (above 87%). The better fit was attained using linear model, passing or not through the origin. From a practical viewpoint, it is suggested to use the linear model involving the C and L product, using a linear coefficient equal to zero. Estimation of leaf area of Crotalaria juncea L. can be obtained using the model Sf = 0.7160 x (C*L) with a determination coefficient of 0.9712.

Desenvolvimento e composição mineral do picão-preto sob diferentes níveis de pH

Visando estudar a tolerância do picão-preto (Bidens pilosa) a diferentes níveis de pH do substrato, conduziu-se um ensaio em condições de câmara de crescimento e utilizando-se a técnica de hidroponia, no qual três plântulas foram transplantadas para recipientes contendo solução nutritiva de Hoagland & Arnon, com pH ajustado para 3,5, 4,0, 5,0, 6,0, 7,0 e 8,0. O delineamento experimental utilizado foi o inteiramente casualizado com três repetições. Ao final do período experimental, 42 dias após o transplante, observou-se que as plantas que se desenvolveram nos pHs 3,5 e 8,0 apresentaram-se menores, com menor comprimento e volume do sistema radicular, menor número de folhas vivas e, consequentemente, menor peso de matéria seca das folhas, caule e raízes. Os teores de N e S nas raízes diminuiram com o aumento do pH do substrato, enquanto os de Ca e Mg aumentaram e os de K e P diminuiram no pH 5,0 a 6,0. No caule, apenas o teor de Mg foi afetado pelo pH, mostrando resposta positiva. Na folha obteve-se os maiores teores de P, S, Ca e Mg quando a planta se desenvolveu em pH 6,0. Não se constatou diferença significativa entre os efeitos dos pHs 5,0, 6,0 e 7,0 nos parâmetros avaliados, sendo que foi no pH 6,0 que se estimou, através de análise de regressão, melhor desenvolvimento do picão-preto. Concluiu-se que o picãopreto apresenta melhor desenvolvimento em pHs próximo ao neutro, com certa tolerância a pHs extremos (3,5 e 8,0).

Estimativa da área foliar de plantas daninhas de ambiente aquático: Pistia stratiotes

A área foliar é uma das principais características para avaliar o crescimento vegetal. Objetivou-se neste trabalho determinar uma equação matemática para estimar a área foliar de Pistia stratiotes a partir de dimensões lineares dos limbos foliares. A pesquisa foi desenvolvida na Universidade Estadual Paulista, Jaboticabal-SP, Brasil. Cem folhas, coletadas no ambiente natural, foram eletronicamente medidas em comprimento (C), largura máxima (L) e área foliar (AF). Os dados de AF e C × L foram submetidos à análise de regressão linear, determinando-se uma equação matemática para estimar a área foliar da espécie. A análise de variância sobre a regressão linear e a análise de correlação entre os valores de área foliar e estimada foram significativas (p < 0,01). A área foliar de P. stratiotes pode ser estimada pela equação: AF = 0,79499 (CL).