Eny Iochevet Segal Floh

Effect of plant growth regulators on the cellular growth and levels of intracellular protein, starch and polyamines in embryogenic suspension cultures of Pinus taeda

Somatic embryogenesis is the most important in vitro culture system for conifer propagation. However, Pinus taeda has been considered recalcitrant to somatic embryogenesis in commercial scale-up. The study of biochemical and physiological aspects of cell growth could lead to a better understanding of somatic embryogenesis in this species. In the present work, we investigated the cell growth dynamics, intracellular levels of proteins, starch and polyamines in suspension cultures of Pinus taeda established in plant growth regulator-free medium (BM0) and in medium supplemented with 2 μM 2,4-dichlorophenoxyacetic acid, 0.5 μM 6-benzylaminopurine and 0.5 μM Kinetin (BM2). Cell cultures growing in BM0 medium showed an increase in the sedimented cell volume from 3.77 to 17.73 ml after 24 days of culture. Those cultured in BM2 medium showed an increase in the sedimented cell volume from 4.23 to 25.17 ml after 20 days of culture. Intracellular proteins levels increased during the exponential growth phase and starch levels decreased until the exponential phase, followed by a synthesis up to the stationary phase, in both BM0 and BM2 media. Highest putrescine levels occurred in cultures growing in BM0 medium and this was associated with the low cellular growth.

Polyamine effects on growth and endogenous hormones levels in Araucaria angustifolia embryogenic cultures

The aim of the present work was to determine the effects of exogenous polyamines (PAs) supplemented into a BM medium free of plant growth regulators (PGR), on the endogenous PAs, IAA and ABA contents and growth of A. angustifolia embryogenic cultures. The addition of PAs to the PGR-free BM medium, enhanced the growth of embryogenic cultures. PAs endogenous metabolism was significantly affected by exogenous PAs. PAs supplemented to a PGR-free BM medium, mainly Put and Spm, increased the endogenous IAA and ABA levels, showing a direct relationship -between PAs levels and ABA accumulation. These results direct new strategies to further improve A. angustifolia somatic embryogenesis, especially during the maturation phase, by culture medium manipulation using exogenous PAs.

Plant growth regulators and amino acids released by Azospirillum sp. in chemically defined media

Aims: To investigate the ability of Azospirillum sp., a facultative endophitic diazotrophic bacterium, to release plant growth regulators (PGR) such as polyamines, ethylene, indoleacetic acid and amino acids in both combined‐N and N‐free cultures.

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aimsEndophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants.MethodsA total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique.ResultsThe putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA.ConclusionEndophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

SERK gene homolog expression, polyamines and amino acids associated with somatic embryogenic competence of Ocotea catharinensis Mez. (Lauraceae)

In the present work, we investigate the association of SERK gene homolog expression, polyamines (PAs) and amino acids related to putrescine synthesis (arginine and ornithine) and polyamines degradation (γ-aminobutiric acid) or S-adenosylmethionine synthesis (methionine), with the embryogenic competence in cell aggregates of Ocotea catharinensis Mez. (Lauraceae). Cell aggregates were cultivated during 7 days in woody plant medium (WPM) supplemented with 20 g l−1 sucrose, 22 g l−1 sorbitol, 400 mg l−1 glutamine and 2 g l−1 phytagel, and in Murashige and Skoog medium (MS) supplemented 20 g l−1 sucrose, 3 g l−1 activated charcoal, 2 g l−1Phytagel with and without 40 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). The cell aggregates cultivated in MS plus 2,4-D and in the WPM medium showed hybridization with a SERK gene homolog both in northern and in situ hybridization experiments. Cell aggregates cultivated in an MS basal medium, without 2,4-D, did not exhibit any hybridization signal to the SERK probe used, thus they were considered potentially non-embryogenic cells. In all three media only free polyamines were detected. The higher putrescine levels occurring in WPM callus were associated with a higher arginine and ornithine content, lower γ-aminobutiric acid level, and SERK homolog expression. Putrescine was also the major polyamine in the MS medium. In the MS plus 2,4-D medium, the levels of putrescine, spermidine and spermine were similar. Spermine exhibited similar and the lowest levels in all media. Spermidine intermediary levels occurred in the WPM and MS media. In cell aggregates methionine level was lowest in the MS plus 2,4-D medium, but similar in the MS and WPM media.

Phenylpropanoid derivatives and biflavones at different stages of differentiation and development of Araucaria angustifolia

Chemical investigations carried out with tissues at different developmental stages of Araucaria angustifolia established the presence of E and Z isomers of octadecyl p-coumarate and octadecyl ferulate in undifferentiated callus; in the seedling stems, the source of explants, three biflavones of the amentoflavone-type were isolated, whereas the diterpene, trans-communic acid, was obtained from the seedling roots. Adult stems accumulated the benzaldehydes, vanillin, p-hydroxybenzaldehyde and coniferaldehyde; the lignans, pinoresinol, eudesmin and lariciresinol; and the isoflavones, cabreuvine and irisolidone.

Biochemical changes during seed development in Pinus taeda L.

Amino acids, polyamines, 3-indoleacetic acid (IAA), abscisic acid (ABA), buffer-soluble protein and starch contents and dry matter accumulation were analyzed in megagametophytes containing developing embryos during seed development in Pinus taeda. The highest total amino acids and polyamine contents occurred at the cotyledonary stage, followed by a significant decrease in the mature seed. Free polyamines exhibited higher levels than conjugated ones, with putrescine being the predominant type until the cotyledonary stage, and spermidine at the mature seed stage. IAA content increased continually from the globular stage reaching the maximum at the cotyledonary stage, followed by a decrease in the mature seed. The highest ABA level occurred at the globular stage, followed by a continuous reduction until stabilization at the pre-cotyledonary stage. Buffer-soluble protein and starch contents, and dry matter increased progressively during development, reaching their maximum values at the mature stage.

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of Ocotea catharinensis

In this study we examined the effect of polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) on growth, morphology evolution, endogenous PAs levels and nitric oxide (NO) release in Ocotea catharinensis somatic embryo cultures. We observed that Spd and Spm reduced culture growth, permitted embryo morphogenetic evolution from the earliest to last embryo development stages, increased endogenous PAs levels, and induced NO release in O. catharinensis somatic embryos. On the other hand, Put had little effect on these parameters. Spd and Spm could successfully be used to promote somatic embryo maturation in O. catharinensis. The results suggest that Spd and Spm have an important role during the growth, development and morphogenetic evolution of somatic embryos, through alterations in the endogenous nitric oxide and PAs metabolism in this species.