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 Explants Sterilization and Callus Induction of Aquilegia oxysepala

[Objective] The aim was to study the explants sterilization and callus induction of Aquilegia oxysepala.[Method] the seeds of Aquilegia oxysepala were sterilized by different kinds and concentrations of disinfectants,and the pollution rate and pollution speed were investigated so as to find the best way to build sterile seedling setup.Taking the roots,stem segments and leaves of the sterile seedlings from Aquilegia oxysepala seeds as explants,the optimum explants and medium were screened by adding MS basic medium with different hormone proportions.[Result] The best germicidal treatment was as follows：explants were soaked in 75% alcohol for 30 s firstly,washed by sterile water for 5 times,then soaked in 0.2% mercuric chloride liquid for 2 min,finally washed by sterile water for 5 times again.The sterilization treatment could get the lowest pollution rate,the highest germinating capacity and the best sterile seedling.Roots were the optimum explants for the callus induction of Aquilegia oxysepala,meanwhile the optimal medium was MS＋0.6 mg/L 2,4-D＋0.5 mg/L 6-BA.[Conclusion] The research provides technical support for the large scale production of Aquilegia oxysepala and also makes a contribution to the medicinal and ornamental value of Aquilegia oxysepala.

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.

Different Explants of Lilium lancifolium Have Different Potential to Differentiate and Regenerate in Tissue Culture

[Objective] The aim was to investigate differences in differentiation and regeneration of the explants from different parts of Lilium lancifolium（Yixing Lily） in tissue culture.[Method] The different parts of scale,leaf and root of Yixing Lily were cultured as explants on MS basic medium supplemented with different concentrations of plant growth regulators,so as to compare their capacity to differentiate and regenerate.[Result] The explants had different potential to differentiate（scale root leaf）.The capacity of different scale parts to differentiate was the lower part middle partupper part;the capacity of different leaf parts to differentiate was the leaf base middle part leaf tip;the capacity of different root parts to differentiate was the root base root tip middle part.[Conclusion] Tissue culture could be well applied in propagation of Yixing Lily.

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.

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.

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.

Effect of peripheral nerve on the neurite growth from retinal explants in culture

The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined. Cultures of retinal explants, and co-cultures of retinal explants and PN were performed using chick retinal basement membrane (BM) as substrate. The presence of PN increases the number and length of neurite outgrowth. In addition, a high proportion of neurites situated close to PN tend to grow towards it. Since there was no contact between retinal ex-plants and PN, we suggest that PN might secrete diffusible substances to attract the neurites to grow towards it.

Plant Regeneration from In Vitro Cultured Hypocotyl Explants of Euonymus japonicus Cu zhi

Adventitious shoots were successfully regenerated from hypocotyl explants of in vitro cultures of Euonymus japonicus Cu zhi. Hypocotyl slices were cultured on Murashige and Skoog （MS） and B5 basal medium supplemented with varied concentration of different plant growth-regulators, e.g., α-naphthaleneacetic acid （NAA） and indole-3-butyric acid （IBA） in combination with 6-benzylaminopurine （6-BA） and kinetin. The study showed that shoots could be directly regenerated from hypocotyl explants without the intervening callus phase; MS medium was more suitable for adventitious shoots regeneration. The ability of hypocotyls segments to produce shoots varied depending upon their position on the seedlings. The highest regeneration rate was obtained with hypocotyl segments near to the cotyledon cultured on MS basal medium supplemented with 1.5 mg L^-1 6-BA and 0.05 mg L^-1 NAA （63.64%）. The regenerated shoots were readily elongated on the same medium as used for multiplication and rooted on half-strength MS basal medium supplemented with 1.0 mg L^-1 IBA and 100 mg L^-1 activated carbon. After being transferred to greenhouse conditions, 96% of the plantlets were successfully acclimatized. This regeneration system is applied for genetic transformation now.

Plantlet regeneration from mature zygotic embryos andembryonic explants of masson pine (Pinus massoniana Lamb.)

Excised zygotic embryos, cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones. BA (1.0 mg/ L) in combination with NAA (0.05 mg/L)in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos, but most of them were formed at the tips of embryonic cotyledons. Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L. Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal (0.5%). Root initiation was achieved with full or half strength DCR inedium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L. Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated (3-20h) with BA 50-100 mg/L, followed by transfer to hormone-free DCR medium. The maximum number of shoots obtained per explant within six months was 33.

Efficient Disinfection of Explants in the Tissue Culture of Chinese Orchids

Explants,namely the seeds (Cymbidium faberi) and buds (Cymbidium goeringii) of local Hunan orchids were disinfected with 75% ethanol, 0.1% mercuric chloride,and 10% sodium hypochlorite in six different disinfection treatments. Results showed that The treatment with 75% ethanol (contact time: 45 s) and 0.1% mercuric chloride (contact time: 8 min) proved to be the most effective one in disinfecting the explants of Cymbidium faberi seeds.The combination of 75% ethanol (contact time: 45 s) and 0.1% mercuric chloride (contact time: 5 min) provided the optimal disinfection effects when disinfecting Cymbidium goeringii buds.In general, the disinfection effect of mercuric chloride was found to be superior to that of sodium hypochlorite,but care should be taken with the disinfection time to avoid damage to the explants.

Study on Initial Explants Induction of Litsea cubeba

[ Objectlve] The aim of this study was to explore an explants induction method for L. cubeba. [ Method ] Different types of L. cubeba stem segments were collected at different time as explants materials for induction and culture. [ Result ] The results showed that sterilization with 0. 1% HgC12 for 10 min achieved better effects, with an explants contamination rate of dO. 0% and a mortality rate of 15.0% ; stem segments of the 2na -5th buds below the terminal bud were better explants materials for initial induction; the best sampling time was January, with an induction rate as high as 81.1%, while the contamination rate was only 11.7% ; modified MS ＋ 1.0 mg/L of 6-BA ＋ 0.2 mg/L of NAA was the optimal medium for explants induction of L. cubeba. [ Conclusion] Semi-lignified stem segments of L cubeba collected in January, sterilized with 0.1% HgCI2 for 10 min and cultured in modified MS ＋ 1.0 mg/L of 6-BA ＋ 0.2 mg/L of NAA a- chieved better induction effects on L. cubeba seedlings.

In vitro Propagation of Walnut （Juglans regia） by Nodal Explants

Micropropagation techniques were set up for walnut. Many plantlets were obtained by culturing node explants in MS medium,with 30 g/L sucrose, 7 g/L agar, and different concentrations of BA or Kin alone and different concentrations of BA ＋ NAA or BA ＋ Kin. BA at concentration （2 mg L-1 BA） gave the best results in percentage response and number of shoots/plant （100%, 2.84） respectively. While, Kin at 3 mg L-1 gave the highest average shoot length （2.81 cm）. However, MS medium supplemented with 2.5 mg L-1 ＋ 0.5 mg L-1 Kin gave the best results compared with all treatment shoot/explants and the highest number of leaves/explants （3.7, 8.2） respectively. While the highest length of shoot （4.7 cm） in MS medium supplemented with 3 mg L-1 Kin. and a high rooting percentage up to 60% at MS medium supplemented with 1 mg L-1 IBA （Indole-3-butyric acid） was obtained.

A Spectrophotometric Analysis of Human Osteoarthritic Cartilage Explants Subjected to Specific Capacitively Coupled Electric Fields

We have previously shown that capacitively coupled electrical stimulation of either normal bovine articular chondrocytes or osteoarthritic human articular cartilage explants resulted in up-regulation of cartilage matrix gene expression and down-regulation of metalloproteinase gene expression. In addition, collagen and proteoglycan protein levels were also elevated. To determine visually the effect of specific electric fields on modifying cartilage structure, freshly harvested human full-thickness osteoarthritic cartilage explants were stimulated in the absence or presence of interleukin-1β, an inflammatory cytokine, and were examined photographically and spectrophotometrically. Hexosamine and hydroxyproline contents were also determined. Spectrophotometric analysis was used to quantify any changes in the depth of defects in the cartilage ranging from surface level (red-colored) to the deepest affected layer (blue-colored). Interleukin-1β treatment alone caused significant additional cartilage erosion. Electrical stimulation alone resulted in significant decreases in the cartilage defects. Electrical stimulation in the presence of interleukin-1β resulted in a small, but significant, surface improvement. Meta-analysis also confirmed a significant increase in the hexosamine and hydroxyproline contents (indicating matrix deposition). It was concluded that an appropriate electric field could modify osteoarthritic lesions in full-thickness cartilage plugs by increasing matrix production and/or by decreasing matrix destruction. Furthermore, it appears that spectrophotometric analysis is a relatively easy method for quantifying the “filling in” or healing of articular cartilage defects.

Influence of Ethylene Inhibitor Silver Nitrate on Direct Shoot Regeneration from in Vitro Raised Shoot Tip Explants of Sphaeranthus indicus Linn.—An Important Antijaundice Medicinal Plant

In the present investigation, an attempt has been made to study the influence of ethylene inhibitor silver nitrate on direct shoot regeneration in Sphaeranthus indicus, an important antijaundice medicinal plant, by using in vitro raised shoot tip explants. The effect of various concentrations of kinetin, BAP (0.5 - 3.0 mg/l), and NAA (0.1 - 0.5 mg/l) along with AgNO3 (0.1 - 1.0 mg/l) was studied. Among the combinations tested MS medium augmented with kinetin (1.0 mg/l), NAA (0.1 mg/l) and AgNO3 (0.4 mg/l) was found to be optimum for production of multiple shoots (34.3 ± 0.36). Addition of AgNO3 to the media not only increases shoot number in all the concentrations tested but also shoot length. AgNO3 at the concentration of 0.4 mg/l produced 35% more number of multiple shoots when compared to multiple shoots (10.8 ± 0.12) produced in control. In the present study by the addition of ethylene inhibitor silver nitrate and growth regulators, more number of multiple shoots (three folds) and shoot length was observed compared to control. These in vitro raised shoots were transferred to the rooting medium containing different concentrations of auxins such as NAA and IAA along with AgNO3 (0.1 - 0.6 mg/l). Better rooting response (21.6) was observed on NAA (2.0 mg/l) and AgNO3 (0.4 mg/l) containing media. The healthy rooted plantlets were transferred to polybags containing soil and vermiculate in 1:1 ratio for hardening. Finally the hardened plants were transferred to field environment for utmost survivability.

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.

Tissue Culture Responses from Different Explants of Rice

Different culture explants, including anther, young panicle, young embryo, and mature embryo, from 19 rice varieties were used for callus induction and green plantlet differentiation. The culture efficiency differed significantly among the four types of explants, and varied from genotype to genotype. Callus induction frequency presented significantly positive correlation each between anther and young panicle, anther and mature embryo, and young panicle and young embryo. Green plantlet differentiation showed no relationship between different types of explants. In addition, no relationship was found between callus induction frequency and green plantlet differentiation frequency.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants

Objective:To develop the reproducible in vitro propagation protocols for the medicinally important plants viz.,Achyranthes aspera(A.aspera)L.and Achyranthes bidentata(A.bidentata)Blume using nodal segments as explants.Methods:Young shoots of A.aspera and A.bidentata were harvested and washed with running tap water and treated with 0.1%bavistin and rinsed twice with distilled water.Then the explants were surface sterilized with 0.1%(w/v)HgCl_2 solutions for I min.After rinsing with sterile distilled water for 3-4 times,nodal segments were cut into smaller segments(1 cm)and used as the explants.The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog(MS)medium supplemented with 3%sucrose,0.6%(w/v)agar(HiMedia,Mumbai)and different concentration and combination of 6-benzyl amino purine(BAP),kinetin(Kin),naphthalene acetic acid(NAA)and indole acetic acid(IAA)for direct regeneration.Results:Adventitious proliferation was obtained from A.aspera and A.bidentata nodal segments inoculated on MS basal medium with 3%sucrose and augmented with BAP and Kin with varied frequency.MS medium augmented with 3.0 mg/L of BAP showed the highest percentage(93.60±0.71)of shootlets formation for A.aspera and(94.70±0.53)percentages for A.bidentata.Maximum number of shoots/explants(10.60±0.36)for A.aspera and(9.50±0.56)for A.bidentata was observed in MS medium fortified with 5.0 mg/L of BAP.For A.aspera,maximum mean length(5.50±0.34)of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A.bidentata(5.40±0.61)was observed in the very same concentration.The highest percentage,maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of 1BA.Seventy percentages of plants were successfully established in polycups.Sixty eight percentages of plants were well established in the green house condition.Sixty five percentages of plants were established in the field.Conclusions:The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A.aspera and A.bidentata.The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can he easily adopted for commercial large scale cultivation.

An Efficient Plant Regeneration System for Different Explants of Rare and Endangered Plants in Mussaenda anomala

To establish an efficient regeneration method for the rare and endangered plant Mussaenda anomala to address problems regarding its reproductive obstacles and scarce populations.In this study,the terminal buds,axillary buds,stem segments with two axillary buds,stem segments with two axillary buds and one terminal bud,and leaves of M.anomala were used as explants.The effects of different explants and disinfection methods,plant growth regulators and substrates on plant regeneration were explored.The following results were obtained:(1)The terminal bud was a suitable explant for M.anomala tissue culture,and the disinfection method utilized was treatment with 0.2%HgCl2 for 8 min.(2)Initiate medium:MS basic medium supplemented with 0.5 mg/L 6-BA and 0.2 mg/L IBA for the high germination rate(100%)and the maximum bud height(1.70 cm)of the terminal bud.(3)Proliferation medium:MS basic medium supplemented with 3.0 mg/L 6-BA and 0.2 mg/L IBA for a high proliferation rate(96%)and proliferation time(6.0)of terminal buds.(4)Proliferation medium supplemented with 0.7 mg/L GA3 significantly increased the bud heights of multiple buds.(5)Rooting medium:MS basic medium supplemented with 0.5 mg/L IBA and 0.5 mg/L IAA for a high rooting rate(88%),root number(12.0)and root length(5.07 cm).(6)The optimal substrate for seedling acclimation and transplanting was perlite:vermiculite(1:1),which resulted in the highest survival rate(97%)and plant height(5.89 cm),as well as better growth potential for seedlings.The surfaces of M.anomala explants are densely covered with trichome,which increased the difficulty of disinfection;the plant growth regulators directly affected the growth and development in the regeneration process of M.anomala,and the substrate significantly affected the survival rate and height growth for seedling acclimation.

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.