Thidiazuron-induced high-frequency direct shoot organogenesis of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L.

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l⁻¹ activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m⁻² s⁻¹). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m⁻² s⁻¹ and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m⁻² s⁻¹. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m⁻² s⁻¹) tested. Intercellular CO₂ concentration (C _i) and the ratio of intercellular CO₂ concentration to ambient CO₂ (C _i/C _a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.     

The influence of explant orientation and contact with the medium on the pathway of shoot regeneration in vitro in epicotyl cuttings of Troyer citrange

The effect of orientation as regards to gravity, and that of contact with the medium of culture, on shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Citrus sinensis (L.) Osbeck×Poncirus trifoliata (L.) Raf.) have been separated. The shoot regeneration pathway was not affected by the orientation of the explants as regards to gravity, and was determined by explant polarity and the contact with the culture medium. At the apical edge of the explants, the contact with the medium shifted the pathway of shoot regeneration from a direct one to an indirect one, with formation of a callus. This callus formation was cytokinin-dependent, but the change in the pathway of organogenesis was not caused by the increase in cytokinin availability resulting from the contact with the medium. In contact with the media, regeneration at the basal edge of the explants occurred through an indirect pathway after callus formation. No regeneration occurred, at the basal edge, if the contact with the media was prevented. The orientation of the explants as regards to gravity affected shoot formation through the direct pathway of organogenesis. The number of buds differentiated, and that of growing shoots increased when the orientation of the explants departed from the vertical upright position.     

Primary culture and histological characterization of phagocytic cells from rainbow trout Oncorhynchus mykiss

In this study the pronephros of rainbow trout Oncorhynchus mykiss was explanted and cultured. The morphology of cultured cells suggested they were leukocyte derived. To confirm this, cells were incubated with non-opsonized fluorescent beads and human apoptotic polymorphonuclear leukocytes (PMN) to establish that they were capable of phagocytosis and that they became activated following exposure to apoptotic bodies. The cells were studied using light microscopy, transmission electron microscopy and a live cell observer system. Cells in culture were also stained with phalloidin to identify actin reorganization following activation. Cultured cells ingested the inert beads and apoptotic PMNs and c . 80% of cells became activated following exposure to the apoptotic PMN, evidenced by enhanced filopodial extensions. This methodology may play a role in future studies, in particular, the competence of macrophages following exposure to radiation can now be tested.     

Plant regeneration from leaf explants of Rhodiola fastigiata

Summary An efficient plant regeneration protocol for rapidly propagating Rhodiola fastigiata (Hk. f. et Thoms.) S.H.FU, a traditional Chinese medicinal plant, was developed. Shoot organogenesis occurred from the leaf explants inoculated on medium with appropriate supplements of plant growth regulators. Up to 5.3 shoots formed per leaf explant cultured on a medium containing 13.32 μM 6-benzylaminopurine (BA) and 0.54 μM 1-naphthaleneacetic acid (NAA). Regenerated shoots formed complete plantlets on a medium containing 1.48 μM indole-3-butyric acid (IBA), and mature plants were established, acclimatized, and thrived in greenhouse conditions. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite Chinese medicinal plant.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

Solanum Nigrum is a Model System in Plant Tissue and Protoplast Cultures

Solanum nigrum is a model system especially for newcomer to the subject of plant tissue culture. Shoot culture has been easily established from shoot cutting of germinated seeds on Gamborg (B5), or Murashige and Skoog (MS) medium without phytohormones. Direct regeneration was possible using basal media B5, B5C (B5 supplemented with 5 % coconut endosperm milk), Schenk and Hildebrandt (SH), and MS, leaf, stem, shoot tip as explants, cytokinins benzylaminopurine (BAP) or kinetin (KIN) at concentrations from 0.25 to 2 mg dm^–3, and different light treatments (dark, dim and normal light). The best culture condition for shoot formation was the culture of stem internode segments on B5 medium supplemented with 0.5 mg dm^–3 BAP at 16-h photoperiod (irradiance of 100 µmol m^–2 s^–1). Also, root formation was possible under different culture conditions. The best culture condition was the culture of microshoot segments on half strength MS medium supplemented with 1 mg dm^–3 isobutyric acid. Induction of callus formation from young and mature tissues on MS medium supplemented with 0.5 mg dm^–3 BAP, 0.1 mg dm^–3 2,4-dichlorophenoxyacetic acid and 1 mg dm^–3 naphthalene acetic acid, and subsequent plant regeneration on B5C medium supplemented with 0.5 mg dm^–3 BAP was easy. Regeneration of protoplasts isolated from shoot tips and fully expanded leaves was also simple. Finally, the transfer of rooted plantlets to the soil was successful.     

竹节海棠叶外植体脱分化启动的细胞学观察

竹节海棠叶外植体接种于MS+6-BA1ppm+NAA0.1ppm培养基上。外植体脱分化启动过程中,表皮及叶肉细胞主要以劈裂的无丝分裂方式进行分裂:最初核延伸为纺锤形,核仁大而明显,使整个核的轮廓呈“眼”状;随着核的中部出现裂缝,核断开成为两部分,稍后,由原来的母细胞形成两个子细胞。由于核分裂前向细胞中央移动的距离及断裂时断裂面的不同,从而造成细胞团内细胞大小悬殊及分裂面严重混乱的不等分裂现象。文中对栅栏组织细胞脱分化启动后重复进行无丝分裂形成梯状细胞团的现象也进行了讨论。     

Optimization of bud induction in cotyledonary explants of Pinus canariensis

A method is described for using liquid pulsing as an alternative to the conventional induction protocol for Pinus canariensis. Using Day 0 and Day 3 explants, the best exposure time was 8 h and 4 h respectively, in a non-buffered 100 M N⁶-benzyladenine solution, followed by culture on half-strength Bornman's medium containing 3% sucrose and 0.8% Difco Bacto^R agar. With this procedure, 97% of the cotyledonary explants produced about 14 buds/explant. These results were comparable to a 14-day induction period on full-strength Bornman's medium containing 10 M N⁶-benzyladenine.     

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP¹ and Plp-GFP mice², respectively.Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact³.Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings³. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins⁴. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative⁵. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist''s technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species⁶.In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation^7,8 has proven useful for studies of NMJ development, physiology and pathology⁹. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.     

Enhancement of Neurite Outgrowth and Aspartate-Glutamate Uptake Systems in Retinal Explants Cultured with Chick Gizzard Extract

Chicken gizzard extract promoted a long and radially directed neurite outgrowth from retinal explants of 8-day-old chick embryo in cultures of 2–3 days. The neurite outgrowth from retinal explants cultured in the absence of gizzard extract was short and restricted to the explant perimeter. The neurite outgrowth promoted by gizzard extract depended strictly on several factors. (a) Fetal calf serum and polycationic substratum were required in this culture system, (b) Pretreatment of the polyornithine-coated substratum with gizzard extract allowed the retinal explants to extend neurites even in the absence of gizzard extract in the medium. (c) Maximal neurite outgrowth was observed in retinal explants dissected from 8-day embryos, but thereafter the explants’response to gizzard extract rapidly declined and was almost lost at the 12th day. As a biochemical parameter of differentiation of cultured neuroretina, uptake systems for neurotransmitter candidates were examined in homogenates of retinal explants cultured in the absence or presence of gizzard extract. After 3 days in culture with gizzard extract, the uptake increased for aspartate and glutamate 1.6- to 1.8-fold and for γ-aminobutyric acid to a lesser degree when examined at a concentration for high-affinity uptake (10^-6M). In contrast, the uptake capacity for glycine, choline, and dopamine was not altered in explants cultured with or without gizzard extract. Kinetic analysis showed that the enhanced capacity to accumulate aspartate was not due to an alteration of K^m, but to an increase of V^max. The results suggest that one or several factors in chick gizzard muscle promote not only neurite outgrowth but also the aspartate-glutamate uptake systems in the developing neuroretina, probably related to ganglion cells.