Relation between phytohormone level and vascular bridge differentiation in graft union of explanted internode autografting

Development of the graft union of explanted internode autografting of cucumber ( Cu-cumis sativus Linn.) cultured in vitro is regulated by plant hormones exogenously added to the media. The levels of auxin (indole-3-acetic acid, IAA) and zeatin plus zeatin riboside (Z+ ZR) in graft unions and in 4 parts of the graft union have been analyzed by ELISA assay.

Immunohistochenlical localization of IAA in graft union of explanted internode grafting

The dynamic changes of IAA in graft union of explanted internode autografting of Cucumber (Cucumis sativus Linn.) have been investigated using the immunohistochemical localization technique. It is shown that the efficiency of IAA fixation using lyophilization-gas fixation is higher than that using liquid chemical fixation. In contrast to few silver particles and no significant changes during the development of graft union cultured in hormone-free medium, more silver particles in graft union and significant changes of IAA related to graft union development have been found when graft union was cultured in medium supplemented with appropriate hormones. The fixation procedure of plant hormones and the roles of IAA in graft union are discussed.

Optimal transcorneal electrical stimulation parameters for preserving photoreceptors in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a hereditary retinal disease that affects rod and cone photoreceptors,leading to progressive photoreceptor loss.Previous research supports the beneficial effect of electrical stimulation on photoreceptor survival.This study aims to identify the most effective electrical stimulation parameters and functional advantages of transcorneal electrical stimulation(tcES)in mice affected by inherited retinal degeneration.Additionally,the study seeked to analyze the electric field that reaches the retina in both eyes in mice and post-mortem humans.In this study,we recorded waveforms and voltages directed to the retina during transcorneal electrical stimulation in C57BL/6J mice using an intraocular needle probe with rectangular,sine,and ramp waveforms.To investigate the functional effects of electrical stimulation on photoreceptors,we used human retinal explant cultures and rhodopsin knockout(Rho^(-/-))mice,demonstrating progressive photoreceptor degeneration with age.Human retinal explants isolated from the donors’eyes were then subjected to electrical stimulation and cultured for 48 hours to simulate the neurodegenerative environment in vitro.Photoreceptor density was evaluated by rhodopsin immunolabeling.In vivo Rho^(-/-)mice were subjected to two 5-day series of daily transcorneal electrical stimulation using rectangular and ramp waveforms.Retinal function and visual perception of mice were evaluated by electroretinography and optomotor response(OMR),respectively.Immunolabeling was used to assess the morphological and biochemical changes of the photoreceptor and bipolar cells in mouse retinas.Oscilloscope recordings indicated effective delivery of rectangular,sine,and ramp waveforms to the retina by transcorneal electrical stimulation,of which the ramp waveform required the lowest voltage.Evaluation of the total conductive resistance of the post-mortem human compared to the mouse eyes indicated higher cornea-to-retina resistance in human eyes.The temperature recordings during and after electrical stimulation indicated no significant temperature change in vivo and only a subtle temperature increase in vitro(~0.5-1.5°C).Electrical stimulation increased photoreceptor survival in human retinal explant cultures,particularly at the ramp waveform.Transcorneal electrical stimulation(rectangular+ramp)waveforms significantly improved the survival and function of S and M-cones and enhanced visual acuity based on the optomotor response results.Histology and immunolabeling demonstrated increased photoreceptor survival,improved outer nuclear layer thickness,and increased bipolar cell sprouting in Rho^(-/-)mice.These results indicate that transcorneal electrical stimulation effectively delivers the electrical field to the retina,improves photoreceptor survival in both human and mouse retinas,and increases visual function in Rho^(-/-)mice.Combined rectangular and ramp waveform stimulation can promote photoreceptor survival in a minimally invasive fashion.

Postnatal development of rat retina:a continuous observation and comparison between the organotypic retinal explant model and in vivo development

The organotypic retinal explant culture has been established for more than a decade and offers a range of unique advantages compared with in vivo experiments and cell cultures.However,the lack of systematic and continuous comparison between in vivo retinal development and the organotypic retinal explant culture makes this model controversial in postnatal retinal development studies.Thus,we aimed to verify the feasibility of using this model for postnatal retinal development studies by comparing it with the in vivo retina.In this study,we showed that postnatal retinal explants undergo normal development,and exhibit a consistent structure and timeline with retinas in vivo.Initially,we used SOX2 and PAX6 immunostaining to identify retinal progenitor cells.We then examined cell proliferation and migration by immunostaining with Ki-67 and doublecortin,respectively.Ki-67-and doublecortin-positive cells decreased in both in vivo and explants during postnatal retinogenesis,and exhibited a high degree of similarity in abundance and distribution between groups.Additionally,we used Ceh-10 homeodomain-containing homolog,glutamate-ammonia ligase(glutamine synthetase),neuronal nuclei,and ionized calcium-binding adapter molecule 1 immunostaining to examine the emergence of bipolar cells,Müller glia,mature neurons,and microglia,respectively.The timing and spatial patterns of the emergence of these cell types were remarkably consistent between in vivo and explant retinas.Our study showed that the organotypic retinal explant culture model had a high degree of consistency with the progression of in vivo early postnatal retina development.The findings confirm the accuracy and credibility of this model and support its use for long-term,systematic,and continuous observation.

Low-molecular-weight fucoidan inhibits the proliferation of melanoma via Bcl-2 phosphorylation and PTEN/AKT pathway

Fucoidan,a sulfate polysaccharide obtained from brown seaweed,has various bioactive properties,including anti-inflammatory,anti-cancer,anti-viral,anti-oxidant,anti-coagulant,anti-thrombotic,anti-angiogenic,and anti-Helicobacter pylori properties.However,the effects of low-molecular-weight fucoidan(LMW-F)on melanoma cell lines and three dimensional(3D)cell culture models are not well understood.This study aimed to investigate the effects of LMW-F on A375 human melanoma cells and cryopreserved biospecimens derived from patients with advanced melanoma.Ultrasonic wave was used to fragment fucoidan derived from Fucus vesiculosus into smaller LMW-F.MTT and live/dead assays showed that LMW-F inhibited cell proliferation in both A375 cells and patientderived melanoma explants in a 3D-printed collagen scaffold.The PTEN/AKT pathway was found to be involved in the anti-melanoma effects of fucoidan.Western blot analysis revealed that LMW-F reduced the phosphorylation of Bcl-2 at Thr 56,which was associated with the prevention of anti-apoptotic activity of cancer cells.Our findings suggested that LMW-F could enhance anti-melanoma chemotherapy and improve the outcomes of patients with melanoma resistance.

Punch‑excised explants of bovine mammary gland to model early immune response to infection

Background Mammary gland(MG)infections(mastitis)are frequent diseases of dairy cows that affect milk quality,animal welfare and farming profitability.These infections are commonly associated with the bacteria Escherichia coli and Staphylococcus aureus.Different in vitro models have been used to investigate the early response of the MG to bacteria,but the role of the teat in mastitis pathogenesis has received less attention.In this study,we used punch-excised teat tissue as an ex vivo model to study the immune mechanisms that arise early during infection when bacteria have entered the MG.Results Cytotoxicity and microscopic analyses showed that bovine teat sinus explants have their morphology and viability preserved after 24 h of culture and respond to ex vivo stimulation with TLR-agonists and bacteria.LPS and E.coli trigger stronger inflammatory response in teat when compared to LTA and S.aureus,leading to a higher produc-tion of IL-6 and IL-8,as well as to an up-regulation of proinflammatory genes.We also demonstrated that our ex vivo model can be applied to frozen-stored explants.Conclusions In compliance with the 3Rs principle(replacement,reduction and refinement)in animal experimenta-tion,ex vivo explant analyses proved to be a simple and affordable approach to study MG immune response to infec-tion.This model,which better reproduces organ complexity than epithelial cell cultures or tissue slices,lends itself particularly well to studying the early phases of the MG immune response to infection.

Study on Tissue Culture of Japanese Honeysuckle ( Lonicera japonica )

[Objectives]This study was conducted to increase the reproduction coefficient of Japanese honeysuckle(Lonicera japonica)to keep the character of optimal benign.[Methods]The young leaves of medicinal Japanese honeysuckle were selected as explants,and MS was used as the basic culture medium.Suitable culture concentrations and conditions were screened through different concentration gradients of growth regulators and cytokinin.[Results]As the concentration of 6-BA in the culture medium increased,the browning rate increased,and the browning phenomenon occurred earlier.On the contrary,a lower concentration of 6-BA was suitable for the differentiation and growth of young leaves,and the browning response was slow.However,if the cultivation time was too long and the materials were not transferred in a timely manner,browning would also occur.The optimal combination of levels was obtained through a 3×3 orthogonal experiment(three parallel groups for each of 6-BA and NAA).The culture conditions included a constant temperature of 26℃and light intensity of 1200 lx.The optimal medium for inducing callus proliferation was MS+6-BA 0.5 mg/L+NAA 0.5 mg/L;and the optimal medium for inducing bud differentiation was MS+6-BA 1.0 mg/L+NAA 0.1 mg/L.[Conclusions]This study provides a theoretical basis for accelerating the development of the honeysuckle industry.

Effects of Different Sample Parts and Sterilization Methods on Contamination and Survival Rates of Shoots with Buds in Chinese Chestnut

In this study,we investigated the preferable explant types and sterilization method for tissue culture in Chinese chestnut,in order to provide a technical support for the asexual reproduction of Chinese chestnut.The base,middle and apex parts of annual shoots with buds in Chinese chestnut were sampled and cut to 2-3 cm stem segments each with one bud,then sterilized orderly with different duration in 2%NaClO plus 0.1%HgCl_(2)solutions.The results indicated that the duration of 2%NaClO for 20 min+0.1%HgCl_(2)for 15 min exerted an effective disinfection property on the middle parts of annual shoots with buds,and achieved a contamination rate under 5%and a survival rate over 90%.

Biotechnological Assessment of Suaeda arcuate under in Vitro Conditions

This article presents data on the introduction of in vitro culture and microclonal propagation of plants identified in the group of hyperhalophytes belonging to the species Suaeda arcuata Bunge. This study was carried out to optimize the composition of nutrient media for the main stages of reproduction in vitro, as well as studies on the rooting and adaptation of regenerants for species of the genus Suaeda obtained from axillary or apical buds, but more often from stem segments with a node. In this work, hormones of the cytokinin and auxin series, or a combination of them, were added to the nutrient environment for growth activation. The analyzing of microplants for the content of soluble in water B vitamins was carried out. As a result of the research, it was found that intact Suaeda arcuata plants in their natural habitats produce a large amount of soluble in water vitamins: riboflavin—0.062% and thiamine up to 0.006%. And in regenerated plants obtained on media without hormones, the content of vitamins was: B2 0.053%, B1 0%, respectively, and with a combination of 1/2 MS + 1 mg/l 6-BAP + 0.3 mg/l IAA + 2, 4-D, the content of vitamins varied as follows: riboflavin—0.059%, folic acid—0.030%, and thiamine was not detected. The cultivation of regenerates on the environment 1/2 MS + 1 mg/l 6-BAP + 0.3 mg/l IAA + 2,4-D showed the best effect on the growth of regenerants, created the possibility of obtaining the maximum amount of biomass and accumulation of B vitamins.

Jonzac Thermal Spring Water Reinforces Skin Barrier Function of Human Skin and Presents a Soothing and Regenerating Effect

The skin is a formidable physical and biological barrier which communicates continuously with the outside of the body. And the stratum corneum, the outermost layer of human epidermis, plays a central role in the interaction between the cutaneous tissue and the external environment. The horny layer, and more generally the whole skin layers, avoid the penetration of harmful exogenous agents, produce molecules named anti-microbial peptides which impact the composition of the cutaneous microbiota, regulate the internal corporal temperature, avoid the water loss from the inside of the body and constitute an incredible efficient anti-oxidant network. Nevertheless, nowadays, the skin is more and more solicited by the different elements of the cutaneous exposome, including atmospheric pollution and solar radiations, which can cause a dramatic acceleration of the skin ageing process. As a consequence, due to the multifunctional protective role of the skin, during the recent decade the cosmetic industry invested massively in the development of new raw materials and end-products (dermo-cosmetics) able to preserve an optimal state of the skin regarding the external environment. Based on their physical-chemical properties thermal spring waters, which are extremely rich in inorganics ions, are interesting and powerful candidates to be part, as integral component, of new efficient dermo-cosmetic formulations dedicated to protect the skin from the external stimuli. The aim of the present work was to investigate and characterize the activity of Jonzac thermal spring water on the skin. Using different models, we proved for the first time that Jonzac thermal spring water reinforces the barrier function of the skin by modulating the expression of key markers including filaggrin and human beta defensin 2 on ex vivo human skin. The ex vivo and in vivo hydration activity, by Raman spectroscopy and corneometry respectively, has been also demonstrated. We have also shown that Jonzac thermal spring water ameliorates significantly the cutaneous microrelief in vivo. To conclude, we characterize the soothing effect of Jonzac thermal spring water by the analysis of histamine release in Substance P treated skin explants and by measuring the redness of the skin following UV exposure of the skin in vivo. We observed that both parameters decreased following a preventive treatment of the skin with Jonzac thermal spring water. Taken together our results indicate that Jonzac thermal spring water is a promising and powerful dermo-cosmetic which can be used to preserve an optimal state of the cutaneous tissue.

Establishment of high frequency shoot regeneration system in Himalayan poplar(Populus ciliata Wall. ex Royle) from petiole explants using Thidiazuron cytokinin as plant growth regulator

Populus species are important resources for industry and in scientific study on biological and agricul- tural systems. Our objective was to enhance the frequency of plant regeneration in Himalayan poplar （Populus ciliata wall. ex Royle）. The effect of TDZ alone and in combi- nation with adenine and NAA was studied on the regen- eration potential of petiole explants. The explants were excised from Himalayan poplar plants grown in glass- houses. After surface sterilization the explants were cul- tured on shoot induction medium. High percentage shoot regeneration （86 %） was recorded on MS medium sup- plemented with 0.004 mg L-1 TDZ and 79.7 mg L-1 adenine. The regenerated shoots for elongation and multi- plication were transferred to MS ＋ 0.5 mg L-1 BAP ＋ 0.2 mg L-1 IAA ＋ 0.3 mg L-1 GA3. Root re- generation from shoots developed in vitro was observed on MS medium supplemented with 0.10 mg L-1 IBA. Hi- malayan poplar plantlets could be produced within 2 months after acclimatization in a sterile mixture of sand and soil. We developed a high efficiency plant regeneration protocol from petiole explants of P. ciliata.

Investigation on the Morphological Characteristics of Dendrobium officinale Plantlets Propagated from Different Explants

[ Objective] This study aimed to investigate the differences in morphological characteristics of Dendrobium officinale plantlets propagated from different explants. [ Method] Randomly 1 000 D. offtcinale plantlets propagated via different regeneration pathways were selected for morphological investigation and classification. [ Result] D. officinale plantlets propagated from stem segment explants exhibited highly consistent morphological characteristics, while those propagated from seed explants exhibited a variety of morphological characteristics. [ Conclusion] Therefore, using seed explants for regeneration can effectively broaden the germplasms resources of D. officinale.

In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments ofNewhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growthregulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation isbeneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formationefficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L-1 BAPand 0.5 mg L-1 NAA. ABA at 0.2 mg L-1 positively affected the bud formation efficiency, which amounted to 8.5 buds perinternodal segment in the presence of BAP at 1.0 mg L-1. The adventitious shoots successfully rooted and weretransferred to the soil.

Differentiation potential of human adipose tissue derived stem cells into photoreceptors through explants culture and enzyme methods

AIM： To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells （ADSCs） generated by enzyme （EN） and explant （EX） culture methods.METHODS： We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescent （IMF） staining.RESULTS： Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17th day and then the rhodopsin mRNA was detected on the 24th day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31st day. EX orbital ADSCs expressed rhodopsin protein on the 24th day, while EN orbital ADSCs expressed rhodopsin protein on the 31st day. CONCLUSION： Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method.

Effect of cytokinins on in vitro multiplication of Sophora tonkinensis

Objective:To determine the effects of different cytokinins at various concentrations onin vitro shoot multiplication of an important medicinal plant.Methods:Nodal explants(1.5-2.0 cm)of Sophora tonkinensiswere used.Multiple shoots were induced from nodal explants cultured onthe Murashige and Skoog(MS)medium supplemented with 0.0,0.5,1.0,2.0,4.0,8.0,or 16.0μmol2-isopentyladenine(2iP),N6 benzyladenine,kinetin or thiadiazuron.Results:Among the fourinvestigated cytokinins,2iP showed the best response for shoot multiplication.Maximum shootinduction(75%)was achieved on the MS medium supplemented with 2.0μmol 2iP,with a meannumber of 5.0 shoots per explant.In comparison to other cytokinins tried,2iP showed the highestshoot elongation with a mean shoot length of 4.8 cm.Root initiation was observed within 15 dwithin the transfer of shoots onto the MS basal medium,and the rooting percentage was 100%with a mean number of 5.4 roots per shoot and root length of 6.2 cm over a period of 4 weeks.Thehealthy plants,hardened and transferred to a greenhouse for proper acclimatization,exhibited100%survival.Conclusions:It can be summarized that 2iP is the optimal plant growth regulatorforSophoramultiplication.

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1(file # 864/2016-PR) on September 14, 2016.

An Improved System for Shoot Regeneration from Stem Explants of Lombardy Poplar (Populus nigra L. var. italica Koehne)

We developed a system for the regeneration of Lombardy poplar (Populus nigra L. var. italica) shoots from internodal stem explants. Using this system, shoots regenerated from 87% of the stem explants placed on Murashige and Skoog (MS) medium supplemented with 0.1 mg/L indole-3-acetic acid and 0.5 mg/L benzylaminopurine without undergoing callus formation. About 80% of the in vitro regenerated shoots developed roots on MS medium supplemented with 0.5 mg/L indole-3-butyric acid and 0.02 mg/L 1-naphthylacetic acid. Well-rooted seven-to eight-week-old regenerated plants could be transferred to soil for further growth and the survival rate of such plants after three weeks was 88%. The protocol presented here is simple and economical because it does not rely on pre-incubation in callus induction medium or repeated subculture in shoot induction medium containing trans-zeatin, an expensive substance. The in vitro regeneration system presented here could be used for evaluation of radiation sensitivity for Lombardy poplar tissues.

Differential Expression of Genes during Somatic Embryogenesis in Upland Cotton

Somatic embryogenesis in upland cotton isstrongly genotype-dependent,which is a troublein cotton genetic engineering.Cloning genesrelated to somatic embryogenesis and thenintroducing the gene into mainly cultivatedvarieties would be greatly helpful for cottonimprovement by gene transfer.To study

The Influence of Endogenous Hormones on Culture Capability of Different Explants in Rice

The endogenous hormones (EHs) content of different explants (anther, young panicle, young embryo and mature embryo) and calli with different culture capability were analyzed by means of high performance liquid chromatography (HPLC). The results showed that the contents and ratio of endogenous hormones were one of the key factors affecting callus induction frequencies (CIF) and green plantlet differentiation frequencies (GPDF). The influence of endogenous hormones of different explants on CIF represented as: Zoatin ribosile (ZR) showed negative effect, indole-3-acetic acid (IAA) did positive effect, and gibberellic acid (GA) did negative effect except for mature embryos. The influence of endogenous hormones on green plantlet differentiation frequency (GPDF) showed: IAA and GA were negative effect; abscisic acid (ABA) and zeatin+ zeatin riboside (Z+ZR) were positive effect. The mixture ratio of endogenous hormones played a role on CIF and GPDF. IAA/Z+ZR had a positive effect on CIF, and there was a notable positive correlation between Z+ZR/ IAA and GPDF, so was between ABA/IAA and GPDF.

Influence of cytokinins, basal media and pH on adventitious shoot regeneration from excised root cultures of Albizia lebbeck

A highly reproducible and efficient in vitro shoot regeneration system was developed in a potential medicinal plant, Albizia lebbeck using root explants. Root explants from 15 day-old-aseptic seedlings were cultured on Murashige and Skoog （MS） medium supplemented with different concentrations （0.5, 2.5, 5.0, 7.5 and 10.0 μM） of 6-Benzyladenine （BA）, Kinetin （Kn）, 2-Isopentenyl adenine （2-iP） singly as well as in combination with α-Naphthalene acetic acid （NAA） （0.1, 0.5, 1.0, 1.5 and 2.0 μM）. The highest rate of shoot multiplication （16.0 ± 1.87 for the average shoot number and 5.16 ± 0.38 cm for shoot length） was achieved on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA. The effects of medium type, medium strength, pH and subculture on shoot induction and proliferation were also tested. An average of 21.6±2.87 shoots per explants could be obtained following this protocol. Rooting was achieved on microshoots using half strength MS medium with 2.0 μM Indole-3-butyric acid （IBA） after four weeks of culture. The in vitro raised healthy plantlets were successfully established in earthen pots containing garden soil and grown in greenhouse with 〉80% survival rate.