The antagonistic effect and mechanisms of Bacillus amyloliquefaciens DGA14 against anthracnose in mango cv. ‘Carabao’

Bacillus amyloliquefaciens strain DGA14 was tested for in vitro antagonism towards Colletotrichum gloeosporioides, a causal pathogen of anthracnose in mango cv. ‘Carabao’. DGA14 produced extracellular metabolites in solid and liquid media that suppressed the growth of C. gloeosporioides. The cells of DGA14 were often observed adjacent to the pathogen so affecting its spore germination and mycelium development. DGA14 colonised mango fruit 48 h after artificial inoculation and persisted 14 days after storage at 18–20°C. On fruit surfaces, DGA14 attached and produced dents to spores of C. gloeosporioides. Dipping mangoes in aqueous cell suspension (10⁸ mL L^?1) of DGA14 significantly decreased the incidence of anthracnose as compared to untreated fruit.     

Agrobacterium tumefaciens– mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos

Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l^–1 picloram and 50 mg l^–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l^–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively. Received: 2 June 1997 / Revision received: 26 September 1997 / Accepted: 11 October 1997     

Stage-specific protein regulation during somatic embryo development of Carica papaya L. ‘Golden’

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.     

Temporary immersion systems (RITA®) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production

Somatic embryogenesis in cork oak (Quercus suber L.) is an efficient tool that allows the production of large number of embryos from selected quality and productive trees. Temporary immersion systems (TIS) are an alternative to semi-solid or liquid culture that combine the advantages of liquid culture and avoid the associated problems. Parameters that affect the TIS multiplication efficiency of Q. suber L. embryogenic cultures were evaluated. Immersion frequencies of 1 min every 6 or 4 h increased the fresh weight 3.7 or 7.5-fold compared with an immersion frequency of 1 min every 12 h or cultures on semi-solid medium, respectively. The cellular fate of embryogenic cultures was also affected by the immersion frequency, 1 min every 6 h was the best for mass propagation of proliferative developmental stages (embryogenic calli and embryo clusters) while 1 min every 4 h promoted the formation of single, fully developed cotyledonary embryos. An initial amount of 1.5 g fresh weight of proliferative tissues produced the best results in RITA^® containers while 0.5 g of embryogenic callus was the best for semi-solid cultures.     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv. ‘Dwarf Cavendish’ (Musa AAA): effect of spermidine on transformation efficiency

An efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of the important banana cultivar ‘Dwarf Cavendish’ (Musa AAA). A high embryogenic response was obtained in 1.36 % of immature male flower explants. Once embryogenic structures were transferred to liquid medium, embryogenic cell suspensions (ECSs) with high regeneration capacity were obtained. ECSs were incubated under different conditions with Agrobacterium tumefaciens strain EHA101 harboring vector pFAJ3000 that contains pNos-nptII-tOcs and p35S-uidAintron-t35S expression cassettes. The effect of spermidine and infection time on transformation efficiency was examined. The highest efficiency was obtained when ECSs were infected for 6 h, in medium supplemented with 200 μM acetosyringone and 1.0 mM spermidine, with more than 600 independent lines/~50 mg FW of settled cells. Spermidine showed an enhancing effect, increasing significantly the transient Gus expression and the number of transformed embryo colonies and regenerated plants in comparison with the same treatments without this polyamine. This is the first report showing efficient Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in the ‘Dwarf Cavendish’ banana cultivar.     

The antagonistic action of Trichoderma harzianum strain DGA01 against anthracnose-causing pathogen in mango cv. ‘Carabao’

The mechanisms of control and efficacy of Trichoderma harzianum strain DGA01 against anthracnose-causing pathogen Colletotrichum gloeosporioides in mango cv. ‘Carabao’ were examined. The action of DGA01 towards C. gloeosporioides was mycoparasitism and production of metabolites. DGA01 parasitised the pathogen by coiling its mycelia and spores on both artificial media and mango fruit surfaces. DGA01 was a parasitic necrotroph capable of killing C. gloeosporioides in 14 days of coexistence in artificial media. Dipping fruit in conidial suspension (10⁶?mL?L^?1) of DGA01 significantly decreased the incidence of anthracnose as compared to untreated fruit. Reduction in anthracnose severity was 87.90% showing high antagonistic potential of DGA01 in vivo.     

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L^?1, and for the ovary, it is MS + picloram 1.5 mg L^?1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L^?1 + TDZ 0.1 mg L^?1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.     

Somatic embryogenesis in conifers—A case study of induction and development of somatic embryos in Picea abies

Vegetatively propagated material offers many advantages over seed material in forest tree breeding research and in reforestation programmes. Evidence is accumulating to suggest that using somatic embryos in forestry is a viable option. However, before somatic embryos can be used optimally in forestry, basic research aimed at increasing the number of responsive genotypes as well as the age of the primary explant is needed. This in turn requires the establishment of a basic understanding of the physiological and molecular processes that underlie the development of somatic embryos. The functions of genes and their developmental and tissue specific regulation are studied using transient and stable transformation techniques.The process of somatic embryogenesis can be divided into different steps: (1) initiation of somatic embryos from the primary explant, (2) proliferation of somatic embryos, (3) maturation of somatic embryos and (4) plant regeneration. Cortical cells in the primary explant are stimulated to go through repeated divisions so that dense nodules are formed from which somatic embryos differentiate. The first formed somatic embryos continue to proliferate and give rise to embryogenic cell lines. Embryogenic cell lines of Picea abies can be divided into two main groups A and B, based on morphology, growth pattern and secretion of proteins. Our results suggest that extracellular proteins play a crucial role in embryogenesis of Picea abies. Somatic embryos from group A can be stimulated to go through a maturation process when treated with abscisic acid. Mature somatic embryos can develop into plants.Abbreviations ABA abscisic acid - BA N⁶-benzyladenine - 2,4-D dichlorophenoxy acetic acid     

The effect of cytokinins on shoot proliferation,biochemical changes and genetic stability of Rhododendron ‘Kazimierz Odnowiciel’ in the in vitro cultures

Plant Cell, Tissue and Organ Culture (PCTOC) - Shoot proliferation is a very important micropropagation phase, decisive for economic efficiency of this method for a given taxon. To obtain a high...     

Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos

Summary Somatic proembryos of mango (Mangifera indica L. cv. Hindi) were co-cultivated withAgrobacterium tumefaciens strain A208 harboring pTiT37-Se::pMON 9749 (9749 ASE). Transformed somatic proembryos capable of growing on selection medium containing 200 μg/ml kanamycin produced the characteristic indigo blue precipitate in the presence of 5-bromo-4-chloro-3-glucuronic acid. These proembryos were chimeral consisting of transformed (blue) and nontransformed (yellow/white) cells. A stepwise selection strategy was found necessary to eliminate chimeras. a) Initial screening at 200 μg/ml kanamycin to enable growth of transformed cells, b) further screening at 400 μg/ml kanamycin to reduce chimeras, and c) recovery of pure transformed clones of proembryos in liquid selection medium with 100 μg/ml kanamycin. The integration of the NPT II and GUS genes into mango genome was confirmed by Southern hybridization.     

Somatic embryogenesis of Pinus rigida?×?P. taeda and the relationship between the initiation of embryogenic tissue and zygotic embryo development

The pitch-loblolly pine hybrid (Pinus rigida × P. taeda) has useful characteristics of the parents, but its exploitation is hindered by restrictions of conventional breeding and propagation methods. This study was undertaken to establish an effective in vitro system for propagating pitch-loblolly hybrid pine through somatic embryogenesis and to unravel the relationship between the efficiency of embryogenic tissue initiation and zygotic embryo development. To initiate embryogenic tissue, megagametophytes of developing seeds were used as explants. Seeds were collected weekly, examined, and tested during June and July 2004. The medium and seed collection date were the most important factors for the successful somatic embryogenesis of P. rigida × P. taeda. Five embryogenic lines were obtained using a modified P. taeda basal medium, and the highest initiation rate was 0.55%, for seeds collected in 2 weeks, between July 3 and 16. Histological observation revealed that zygotic embryos of those seeds were mostly at the proembryonic stage or in transition to precotyledonary stages. For the successful maturation of somatic embryos, abscisic acid and gellan gum were needed in the medium. The results show that, although further tests and development are required, somatic embryogenesis could provide a viable option for propagating P. rigida × P. taeda hybrids.     

Effect of α-linolenic acid on oocyte maturation and embryo development of prepubertal sheep oocytes

The purpose of this study was to evaluate the effect of omega-3 α-linolenic acid (ALA) added to the IVM medium on embryo development of prepubertal sheep oocytes. Experiment 1 investigated the effect of ALA at different concentrations (0 [control], 50, 100, and 200 μM) and DMSO (100 μM) in IVM media on cumulus cell expansion and oocyte nuclear maturation and on synthesis of prostaglandins (PGE2 and PGF2α). Experiment 2 investigated the effects of ALA at different concentrations in the IVM medium on oocyte fertilization, cleavage, and developmental potential to blastocyst stage and changes in estradiol and progesterone concentrations in the spent IVM media. IVM oocytes were fertilized with frozen-thawed spermatozoa capacitated in a serum-free sperm medium. Presumptive zygotes were cultured 8 days in synthetic oviductal fluid (SOF) medium without serum. Blastocyst quality was assessed by counting total cell number and the number of apoptotic cells using Hoechst and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Nuclear maturation of oocytes and the number of fully expanded cumulus cells were reduced after treatment with 200 μM of ALA compared with other groups (P ≤ 0.05). Supplementation with ALA increased both PGE2 and PGF2α concentrations in the spent media (P ≤ 0.05). No differences were observed in blastocyst development among control (12.2%) and 50, 100, and 200 μM ALA groups (6.9%, 11.5% and 14.0%, respectively). However, the total cell number (46.50 ± 5.85, 67.94 ± 6.71, 45.20 ± 6.37, and 59.80 ± 5.51, respectively; P ≤ 0.05) and apoptotic cell number (6.45 ± 0.89, 2.48 ± 0.81, 4.02 ± 1.15, and 3.67 ± 1.15, respectively; P ≤ 0.05) were significantly improved. After IVM, estradiol concentration was lower and progesterone concentration was higher in ALA groups compared with the control group (P ≤ 0.05). In conclusion, these results revealed that ALA affects prepubertal sheep embryo quality associated with alteration of releasing reproductive hormones.     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

The effect of aging on brain barriers and the consequences for Alzheimer’s disease development

Life expectancy has increased in most developed countries, which has led to an increase in the proportion of elderly people in the world’s population. However, this increase in life expectancy is not accompanied by a lengthening of the health span since aging is characterized with progressive deterioration in cellular and organ functions. The brain is particularly vulnerable to disease, and this is reflected in the onset of age-related neurodegenerative diseases such as Alzheimer’s disease. Research shows that dysfunction of two barriers in the central nervous system (CNS), the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier (BCSFB), plays an important role in the progression of these neurodegenerative diseases. The BBB is formed by the endothelial cells of the blood capillaries, whereas the BCSFB is formed by the epithelial cells of the choroid plexus (CP), both of which are affected during aging. Here, we give an overview of how these barriers undergo changes during aging and in Alzheimer’s disease, thereby disturbing brain homeostasis. Studying these changes is needed in order to gain a better understanding of the mechanisms of aging at the brain barriers, which might lead to the development of new therapies to lengthen the health span (including mental health) and reduce the chances of developing Alzheimer’s disease.     

In vitro conservation of oil palm somatic embryos for 20 years on a hormone-free culture medium: characteristics of the embryogenic cultures, derived plantlets and adult palms

This study was conducted over a period of 20 years, to assess the problems involved in developing subcultures over a very long period, of oil palm (Elaeis guineensis Jacq.) somatic embryos which were maintained in vitro on a Murashige and Skoog mineral-based culture medium, without growth regulators. Analysis of the proliferation rate of the embryogenic cultures, along with the survivability of the regenerated plantlets after their transfer into soil and of the flowering of the derived adult palms has been conducted for cultures maintained in vitro during 1 to 20 years. From the ninth year of maintenance, the tissue quality of the somatic embryos gradually began to decline. However, after more than 20 years, 30% of the 20 clones tested still continued to proliferate satisfactorily on the same maintenance medium, keeping their multiplication potential intact. Even though a depressive effect of the age of the lines has been observed on the survival capacity of plants under natural conditions, it is noteworthy that among the clones originating from 20-year-old cultures only eight of them (40%) have exhibited the “mantled” floral abnormality. Different hypotheses concerning the origin of the disruptions observed on the in vitro cultures, plantlets and adult palms that occur over a very long period of in vitro conservation are discussed.     

The effect of β-bungarotoxin, or geniculate ganglion lesion on taste bud development in the chick embryo

Chick taste bud (gemmal) primordia normally appear on embryonic day (E) 16 and incipient immature, spherical-shaped buds at E17. In ovo injection of β-bungarotoxin at E12 resulted in a complete absence of taste buds in lower beak and palatal epithelium at developmental ages E17 and E21. However, putative gemmal primordia (solitary clear cells; small, cell groupings) remained, lying adjacent to salivary gland duct openings as seen in normal chick gemmal development. Oral epithelium was immunonegative to neural cell adhesion molecule (NCAM) suggesting gemmal primordia are nerve-independent. Some NCAM immunoreactivity was evident in autonomic ganglion-like cells and nerve fibers in connective tissue. After unilateral geniculate ganglion/otocyst excision on E2.5, at developmental ages E18 and posthatching day 1, ∼12% of surviving ipsilateral geniculate ganglion cells sustained ∼54% of the unoperated gemmal counts. After E18, proportional stages of differentiation in surviving developing buds probably reflect their degree of innervation, as well as rate of differentiation. Irrespective of the degree of geniculate ganglion damage, the proportion of surviving buds can be sustained at the same differentiated bud stage as on the unoperated side, or may differentiate to a later bud stage, consistent with the thesis that bud maturation, maintenance, and survival are nerve-dependent.     

Initiation and maintenance of long term somatic embryogenesis from anthers and ovaries of Vitis longii ‘Microsperma’

Anthers and ovaries of Vitis longii Microsperma produced embryogenic callus when cultured on solidified Murashige and Skoog medium with 5M 2,4-dichlorophenoxyacetic acid (2,4-D) and 1M benzyladenine (BA). The initial callus was short-lived. However, long-term embryogenesis from callus was maintained through serial transfers by careful selection of clustered embryos with subtending callus. Alternatively, long term culture maintenance was through secondary embryogenesis which occurred directly from previously formed embryos on medium lacking growth regulators. Somatic embryos were white, exhibited frequent pluricotyly and tended to be larger than zygotic embryos. Histology of embryogenic callus demonstrated the presence of lipid-like substances and abundant starch. Somatic embryos were attached to callus by narrow to wide suspensor-like structures and possessed typical epidermal, cortical, and vascular tissue. Embryo cells contained abundant lipid-like accumulations but no starch. Embryos germinated when placed on medium containing 1M BA and produced plants of normal appearance.