De novo transcriptome analysis provides insights into formation of in vitro adventitious root from leaf explants of Arnebia euchroma.

BACKGROUND: Adventitious root formation is considered a major developmental step during the propagation of difficult to root plants, especially in horticultural crops. Recently, adventitious roots induced through plant tissue culture methods have also been used for production of phytochemicals such as flavonoids, anthocyanins and anthraquinones. It is rather well understood which horticultural species will easily form adventitious roots, but the factors affecting this process at molecular level or regulating the induction process in in vitro conditions are far less known. The present study was conducted to identify transcripts involved in in vitro induction and formation of adventitious roots using Arnebia euchroma leaves at different time points (intact leaf (control), 3 h, 12 h, 24 h, 3 d, 7 d, 10 d and 15 d). A. euchroma is an endangered medicinal Himalayan herb whose root contains red naphthoquinone pigments. These phytoconstituents are widely used as an herbal ingredient in Asian traditional medicine as well as natural colouring agent in food and cosmetics. RESULTS: A total of 137.93 to 293.76 million raw reads were generated and assembled to 54,587 transcripts with average length of 1512.27 bps and N50 of 2193 bps, respectively. In addition, 50,107 differentially expressed genes were identified and found to be involved in plant hormone signal transduction, cell wall modification and wound induced mitogen activated protein kinase signalling. The data exhibited dominance of auxin responsive (AUXIN RESPONSE FACTOR8, IAA13, GRETCHEN HAGEN3.1) and sucrose translocation (BETA-31 FRUCTOFURANOSIDASE and MONOSACCHARIDE-SENSING protein1) genes during induction phase. In the initiation phase, the expression of LATERAL ORGAN BOUNDARIES DOMAIN16, EXPANSIN-B15, ENDOGLUCANASE25 and LEUCINE-rich repeat EXTENSION-like proteins was increased. During the expression phase, the same transcripts, with exception of LATERAL ORGAN BOUNDARIES DOMAIN16 were identified. Overall, the transcriptomic analysis revealed a similar patterns of genes, however, their expression level varied in subsequent phases of in vitro adventitious root formation in A. euchroma. CONCLUSION: The results presented here will be helpful in understanding key regulators of in vitro adventitious root development in Arnebia species, which may be deployed in the future for phytochemical production at a commercial scale.

Barley Isolated Microspore Culture.

The production of doubled haploids (DHs) has proved to be a highly valuable tool to obtain new cultivars. Among the cereals, barley (Hordeum vulgare L.) is the most successful species in large-scale haploid production. Techniques employed for this purpose are based on either the gynogenetic or the androgenetic pathway. Interspecific cross with Hordeum bulbosum L., haploid gene inducer (the hap gene), ovary culture, anther culture (AC), and isolated microspore culture (IMC) are the most used methods. Among all of them, IMC is regarded as a particularly effective system owing to the great increase in green plant numbers per spike and also the higher induction of chromosome doubling when compared with other methods. Thus, IMC provides the best way to mass scale production of new varieties.

Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.

Bread Wheat Doubled Haploid Production by Anther Culture.

The use of doubled haploid (DH) plants in plant breeding programmes is the fastest route to release new varieties (4-6 years), allowing for a rapid response to end-user needs. Microspore embryogenesis is one of the most efficient methods for DH plant production in bread wheat. In this process, microspores triggered by a stress treatment or by application of bioactive compounds are reprogrammed to follow an embryogenic pathway that leads to the production of haploid or DH plants. In this chapter, we describe a protocol for anther culture of bread wheat. This protocol is based on an osmotic and starvation treatment of the anthers followed by the application of a microtubule disrupting agent. Anthers are cultured in an ovary pre-conditioned medium with mature ovaries from cv. Caramba. This protocol has been applied to a wide range of genotypes and F1s from bread and spelt wheat.

In Vitro Anther Culture for Doubled Haploid Plant Production in Spelt Wheat.

Doubled haploid (DH) plant production belongs to modern biotechnology methods of plant breeding. The main advantage of DH plant production methods is the development of genetically homozygous lines in one generation, whilst in conventional breeding programmes, the development of homozygous lines requires more generations. The present chapter describes an efficient protocol for DH plant production in spelt wheat genotypes using in vitro anther culture.

Triticale Isolated Microspore Culture for Doubled Haploid Production.

Here, we describe a method of triticale isolated microspore culture for production of doubled haploid plants via androgenesis. We use this method routinely because it is highly efficient and works well on different triticale genotypes. To force microspores into becoming embryogenic, we apply a 21-day cold pretreatment. The shock of cold facilitates redirecting microspores from their predestined pollen developmental program into the androgenesis pathway. Ovaries are included in our culture methods to help with embryogenesis, and the histone deacytelase inhibitor Trichostatin A (TSA) is added to further improve androgenesis and increase our ability to recover green doubled haploid plants.

Isolation of Staged and Viable Maize Microspores for DH Production.

Isolated microspore culture systems have been designed in maize by several groups, mainly from the late 1980s to early 2000s. However, even with optimized protocols, microspore embryogenesis induction has remained very dependent on the genotype in maize, with elite germplasm generally displaying no response or very low response. Yet, these last few years, significant progress has been accomplished in understanding and controlling microspore embryogenesis induction in model dicot and monocot species. This knowledge may be transferred to maize, and isolated microspore culture may gain new interest in this crop, at least for embryogenesis research. The methods we hereby present in detail permit the purification of 3-12 × 105 viable microspores per maize tassel, at the favorable stage for microspore embryogenesis. When cultured in appropriate liquid media, microspores from responsive genotypes give rise to androgenic embryos, which can then be regenerated into fertile doubled haploid plants.

Production of Haploid and Doubled Haploid Lines in Nut Crops: Persian Walnut, Almond, and Hazelnut.

This chapter deals with induction of haploidy via parthenogenesis in Persian walnut and via microspore embryogenesis in almond and hazelnut. Haploid induction through in situ parthenogenesis using pollination with irradiated pollen to stimulate the embryogenic development of the egg cell, followed by in vitro culture of the immature haploid embryos. Microspore embryogenesis allows the induction of immature pollen grains (microspores), to move away from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos in this case). Unlike other fruit crops (such as Citrus), regeneration of entire plants has not yet been obtained in our studied nut crops; however, it gives the methodology should be used to continue the roadmap.

High Chromosomal Stability and Immortalized Totipotency Characterize Long-Term Tissue Cultures of Chinese Ginseng (Panax Ginseng).

Chinese ginseng (Panax ginseng C. A. Meyer) is a highly cherished traditional Chinese medicine, with several confirmed medical effects and many more asserted health-boosting functions. Somatic chromosomal instability (CIN) is a hallmark of many types of human cancers and also related to other pathogenic conditions such as miscarriages and intellectual disabilities, hence, the study of this phenomenon is of wide scientific and translational medical significance. CIN also ubiquitously occurs in cultured plant cells, and is implicated as a major cause of the rapid decline/loss of totipotency with culture duration, which represents a major hindrance to the application of transgenic technologies in crop improvement. Here, we report two salient features of long-term cultured callus cells of ginseng, i.e., high chromosomal stability and virtually immortalized totipotency. Specifically, we document that our callus of ginseng, which has been subcultured for 12 consecutive years, remained highly stable at the chromosomal level and showed little decline in totipotency. We show that these remarkable features of cultured ginseng cells are likely relevant to the robust homeostasis of the transcriptional expression of specific genes (i.e., genes related to tissue totipotency and chromosomal stability) implicated in the manifestation of these two complex phenotypes. To our knowledge, these two properties of ginseng have not been observed in any animals (with respect to somatic chromosomal stability) and other plants. We posit that further exploration of the molecular mechanisms underlying these unique properties of ginseng, especially somatic chromosomal stability in protracted culture duration, may provide novel clues to the mechanistic understanding of the occurrence of CIN in human disease.

The potential of porcine ex vivo platform for intestinal permeability screening of FcRn-targeted drugs.

Conventionally, the intestinal permeability of drugs is evaluated using cell monolayer models that lack morphological, physiological and architectural features, as well as realistic neonatal Fc receptor (FcRn) expression. In addition, it is time-consuming, expensive and excessive to use a large number of mice for large-scale screening of FcRn-targeted candidates. For preclinical validation, it is critical to use suitable models that mimic the human intestine; the porcine ex vivo model is widely used for intestinal permeability studies, due to its physiological and anatomical similarities to humans. This study intended to analyze the potential to measure the intestinal permeability of FcRn-targeted substances using a porcine ex vivo platform, which is able to analyze 96 samples at the same time. In addition, the platform allows the screening of FcRn-targeting substances for transmucosal delivery, taking into consideration (cross-species) receptor-ligand binding kinetics. After analyzing the morphology of the porcine tissue, the FcRn expression across the gastrointestinal tract was verified. By studying the stomach, duodenum and jejunum, it was demonstrated that FcRn expression is maintained for up to 7 days. When evaluating the duodenum permeability of free engineered human albumin variants, it was shown that the variant with the mutation K573P (KP) is more efficiently transported. Given this, the porcine ex vivo platform was revealed to be a potential model for the screening of FcRn-targeted oral drug formulations.

Identification of lupeol produced by Vernonanthura patens (Kunth) H. Rob. leaf callus culture.

The lupeol detection in callus of Vernonanthura patens (Kunth) H. Rob. leaves is discussed. Leaf segments previously treated with sodium hypochlorite, ethanol, and distilled water were placed in MS basal medium (Murashige and Skoog) for 7 days. Next, callus induction were done in two complemented MS medium for 6 weeks. Then, callus propagation were performed in MS medium supplemented with 1.0 mg/L of benzylaminopurine (BAP) and 0.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) for 50 days. Fresh callus were extracted every 10 days in an ultrasonic bath using ethyl acetate (1.0 g/10 mL). The identification was carried out by Gas Chromatography-Mass Spectrometry (GC-MS) using selected ion monitoring (SIM) acquisition mode with characteristic ions of lupeol. The results obtained indicate the occurrence of lupeol in callus extract after twenty days of proliferation. These findings could be use in subsequent scale-up studies for biomass production containing this active compound in order to replace conventional methods.

Thyroxine restores severely impaired cutaneous re-epithelialisation and angiogenesis in a novel preclinical assay for studying human skin wound healing under "pathological" conditions ex vivo.

Impaired cutaneous wound healing remains a major healthcare challenge. The enormity of this challenge is compounded by the lack of preclinical human skin wound healing models that recapitulate selected key factors underlying impaired healing, namely hypoxia/poor tissue perfusion, oxidative damage, defective innervation, and hyperglycaemia. Since organ-cultured human skin already represents a denervated and impaired perfusion state, we sought to further mimic "pathological" wound healing conditions by culturing experimentally wounded, healthy full-thickness frontotemporal skin from three healthy female subjects for three days in either serum-free supplemented Williams' E medium or in unsupplemented medium under "pathological" conditions (i.e. hypoxia [5% O2], oxidative damage [10 mM H2O2], absence of insulin, excess glucose). Under these "pathological" conditions, dermal-epidermal split formation and dyskeratosis were prominent in organ-cultured human skin, and epidermal reepithelialisation was significantly impaired (p < 0.001), associated with reduced keratinocyte proliferation (p < 0.001), cytokeratin 6 expression (p < 0.001) and increased apoptosis (p < 0.001). Moreover, markers of intracutaneous angiogenesis (CD31 immunoreactivity and the number of of CD31 positive cells and CD31 positive vessel lumina) were significantly reduced. Since we had previously shown that thyroxine promotes wound healing in healthy human skin ex vivo, we tested whether this in principle also occurs under "pathological" wound healing conditions. Indeed, thyroxine administration sufficed to rescue re-epithelialisation (p < 0.001) and promoted both epidermal keratinocyte proliferation (p < 0.01) and angiogenesis in terms of CD31 immunoreactivity and CD31 positive cells under "pathological" conditions (p < 0.001) ex vivo. This demonstrates the utility of this pragmatic short-term ex vivo model, which recapitulates some key parameters of impaired human skin wound healing, for the preclinical identification of promising wound healing promoters.

Improved chemosensitivity following mucolytic therapy in patient-derived models of mucinous appendix cancer.

Abundant intraperitoneal (IP) accumulation of extracellular mucus in patients with appendiceal mucinous carcinoma peritonei (MCP) causes compressive organ dysfunction and prevents delivery of chemotherapeutic drugs to cancer cells. We hypothesized that reducing extracellular mucus would decrease tumor-related symptoms and improve chemotherapeutic effect in patient-derived models of MCP. Mucolysis was achieved using a combination of bromelain (BRO) and N-acetylcysteine (NAC). Ex vivo experiments of mucolysis and chemotherapeutic drug delivery/effect were conducted with MCP and non-MCP tissue explants. In vivo experiments were performed in mouse and rat patient-derived xenograft (PDX) models of early and late (advanced) MCP. MCP tumor explants were less chemosensitive than non-MCP explants. Chronic IP administration of BRO + NAC in a mouse PDX model of early MCP and a rat PDX model of late (advanced) MCP converted solid mucinous tumors into mucinous ascites (mucolysis) that could be drained via a percutaneous catheter (rat model only), significantly reduced solid mucinous tumor growth and improved the efficacy of chemotherapeutic drugs. Combination of BRO + NAC efficiently lyses extracellular mucus in clinically relevant models of MCP. Conversion of solid mucinous tumors into mucinous ascites decreases tumor bulk and allows for minimally invasive drainage of liquified tumors. Lysis of extracellular mucus removes the protective mucinous coating surrounding cancer cells and improves chemotherapeutic drug delivery/efficacy in cancer cells. Our data provide a preclinical rationale for the clinical evaluation of BRO + NAC as a therapeutic strategy for MCP.

Predicting Metabolism-Related Drug-Drug Interactions Using a Microphysiological Multitissue System.

Drug-drug interactions (DDIs) occur when the pharmacological activity of one drug is altered by a second drug. As multimorbidity and polypharmacotherapy are becoming more common due to the increasing age of the population, the risk of DDIs is massively increasing. Therefore, in vitro testing methods are needed to capture such multiorgan events. Here, a scalable, gravity-driven microfluidic system featuring 3D microtissues (MTs) that represent different organs for the prediction of drug-drug interactions is used. Human liver microtissues (hLiMTs) are combined with tumor microtissues (TuMTs) and treated with drug combinations that are known to cause DDIs in vivo. The testing system is able to capture and quantify DDIs upon co-administration of the anticancer prodrugs cyclophosphamide or ifosfamide with the antiretroviral drug ritonavir. Dosage of ritonavir inhibits hepatic metabolization of the two prodrugs to different extents and decreases their efficacy in acting on TuMTs. The flexible MT compartment design of the system, the use of polystyrene as chip material, and the assembly of several chips in stackable plates offer the potential to significantly advance preclinical substance testing. The possibility of testing a broad variety of drug combinations to identify possible DDIs will improve the drug development process and increase patient safety.

Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

In Vitro Micropropagation of Industrially and Medicinally Useful Plant Aloe trichosantha Berger Using Offshoot Cuttings.

This study was aimed to develop in vitro micropropagation protocol of Aloe trichosantha Berger using offshoots as explants. MS media supplemented with plant growth regulators helped explants develop shoots within about 14 to 17 days. The mean number of days to shooting has decreased from 16.8 ± 0.8 with 0.5/0.5 mg/L BAP/NAA supplement to 15.5 ± 0.5 with 2.0/0.5 mg/L BAP/NAA. While the mean shoot number has increased with increasing the concentration of BAP supplements, the reverse was true with mean shoot lengths, whereas supplement of 2.0/0.5 mg/L BAP/NAA has generated significantly more shoots (17 ± 3.8), and longer shoots were produced with the addition of 0.5/0.5 and 1.0/0.5 mg/L BAP/NAA. In regard to rooting, though higher concentrations of NAA have resulted in quick rooting, the rooting performance in terms of mean number and length of roots was better with low concentrations. All the plantlets subjected to greenhouse acclimatization in cocopeat have survived. Secondary acclimatization in composted and manured soil media has also resulted in 93 to 95% survival rate. Lighting conditions (nursery shade or direct sunlight) of secondary acclimatization did not lead to any difference in the survival rate of the plantlets.

Indian sarsaparilla, Hemidesmus indicus (L.) R. Br. ex Schult: tissue culture studies.

Hemidesmus indicus (L.) R. Br. ex Schult is commonly known as anantmul or Indian sarsaparilla. The roots of this plant, which display a wide range of medicinal, biological, and phytopharmaceutical properties, are used in the pharmaceutical and food industries. Conventionally, the plant is propagated by seed germination or vegetatively, but the efficacy of traditional methods has some limitations: plants derived from seed germination are prone to seed-borne diseases, or plantlet production using vegetative propagation is limited. In contrast, plant tissue culture allows for large-scale propagation and secondary metabolite production in vitro without sacrificing plants from their natural habitats. Many efforts have been made over 40 years of research to establish efficient micropropagation protocols to speed up cultivation of this plant, including callus-mediated in vitro propagation, somatic embryogenesis, and shoot multiplication using cotyledenory nodes, stem segments, shoot tips, and nodal explants. Among these explants, nodal explants are the most commonly used for H. indicus micropropagation. The application of adenine sulfate, citric acid, ascorbic acid, and arginine may be useful in preventing explant browning, premature leaf senescence, and shoot tip abscission during in vitro culture. This review provides insight into micropropagation, use of synthetic seeds for short-term germplasm preservation, and in vitro production of secondary metabolites such as 2-hydroxy-4-methoxybenzaldehyde, lupeol, vanillin, and rutin, from in vitro root and callus cultures. Furthermore, unexplored and possible innovative areas of research in Hemidesmus biotechnology are also discussed. KEY POINTS: • Hemidesmus indicus has multiple therapeutic applications. • H. indicus roots are used in confectionary and pharmacy. • This review comprehensively assesses H. indicus tissue culture. • Challenges and future research of H. indicus biotechnology are discussed.

Toxicity evaluation of water extract of tissue-cultured Taraxacum formosanum by acute, subacute administration, and Ames test

BACKGROUND: Taraxacum species (commonly known as dandelion) used as herbal medicine have been reported to exhibit an antiproliferative effect on hepatoma cells and antitumor activity in non-small-cell lung cancer cells. Although several investigations have demonstrated the safety of Taraxacum officinale, the safety of tissue-cultured plants of T. formosanum has not been assessed so far. Therefore, the present study examines the safety of the water extract of the entire plant of tissue cultured T. formosanum based on acute and subacute toxicity tests in rats, as well as the Ames tests. RESULTS: No death or toxicity symptoms were observed in the acute and subacute tests. The results of the acute test revealed that the LD50 (50% of lethal dose) value of the T. formosanum water extract for rats exceeded 5 g/kg bw. No abnormal changes in the body weight, weekly food consumption, organ weight, or hematological, biochemical, and morphological parameters were observed in the subacute toxicity test. Thus, the no observed adverse effect level (NOAEL) of T. formosanum water extract was estimated to be higher than 2.0 g/kg. Finally, the results of the Ames test revealed that T. formosanum water extract was not genotoxic at any tested concentration to any of five Salmonella strains. CONCLUSIONS: The water extract of tissue-cultured T. formosanum was non-toxic to rats in acute and subacute tests and exhibited no genotoxicity to five Salmonella strains.

Robotically handled whole-tissue culture system for the screening of oral drug formulations.

Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman's correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.

Cannabinoid receptor 2 agonist promotes parameters implicated in mucosal healing in patients with inflammatory bowel disease.

BACKGROUND: Cannabis benefits patients with inflammatory bowel disease (IBD). Cannabinoid receptors are expressed in gut immune cells and in epithelial cells of inflamed guts. Mucosal healing (MH) requires epithelial layer restoration. OBJECTIVE: To analyze the effects of CB2 agonist on parameters implicated in gut inflammation and MH. METHODS: Mucosal samples from areas of inflamed/uninflamed colon from 16 patients with IBD were cultured without/with cannabinoid receptor 2 (CB2) agonist (JWH-133, 10 µM, 6 hours (hr)), and analyzed for epithelial/stromal cell proliferation, apoptosis (secretome matrix metalloproteinase 9 (MMP9) activity, which impairs epithelial permeability) and interleukin-8 (IL-8) levels (n = 5-9). In addition, Caco-2 (colon carcinoma epithelial cells) were cultured with biopsy secretomes (48 hr), and analyzed for phenotype and protein markers of proliferation (proliferating cell nuclear antigen), autophagy (LC3IIB) and permeability (Zonula occludens-1) (n = 4-6). RESULTS: Uninflamed tissue had higher epithelial proliferation (Ki67: 50%↑, p < 0.05), and reduced secretome MMP9 activity and IL-8 levels (>50%↓, p < 0.05) compared to inflamed tissue. Treatment with CB2 agonist had no effect on epithelial apoptosis, but increased epithelial Ki67 expression (25%), and reduced secretome MMP9 and IL-8 levels in inflamed biopsies. Secretomes of CB2-treated biopsies increased Caco-2 number, migration, proliferating cell nuclear antigen and LC3IIB expression (all, p < 0.05), but had no effect on ZO-1. CONCLUSION: Using ex vivo and in vitro human models, we demonstrated that manipulating the cannabinoid system affects colon cells and secretome characteristics that facilitate MH in IBD.