Ultrasound, as a hypomethylating agent, remodels DNA methylation and alters mRNA transcription in winter wheat (Triticum aestivum L.) seedlings.

Any treatment that affects seed germination and seedling development is of paramount importance from an agricultural point of view since they are critical prerequisites for successful crop production. In present study, we have examined the after-effect of ultrasonication (at 30 kHz, 70 W for 5 min) of winter wheat (Triticum aestivum L. cv. SE15) seeds on the early seedling growth and development, and accompanying changes in the DNA methylation and transcriptomic pattern in 7-day-old seedlings. We used mRNA-sequencing and whole genome bisulfite sequencing (WGBS) to identify significantly differentially expressed genes (DEGs), significantly differently methylated regions (DMRs) and genes (DMGs). Ultrasonication of seeds did not alter the germination rate but increased both the length and weight of roots and shoots of 7-day-old seedlings significantly by 23%-68% and 16%-28%, respectively. Analyzing the expression intensity of 107,891 genes, significantly differentially expressed sequences related mainly to starch biosynthesis, IAA biosynthesis, photosynthesis and TCA cycle pathways. The same pathways were also affected by DNA-methylation changes. DNA hypomethylation occurred in the global methylation profile after ultrasound treatment altering the accessibility of some genes for transcription. Transcriptomic changes suggested alterations in the crosstalk between IAA and sucrose signaling, enhancement of growth processes, and increased activity of nuclear transcription factor stimulating the transcription of genes having CCAAT motif in the promoter. In the present first whole genome level study, we have identified seed ultrasonication as a priming technique that can act as a hypomethylating agent and thereby is able to modify the mRNA transcription allowing enhanced seedling growth.

Shoot tip necrosis of in vitro plant cultures: a reappraisal of possible causes and solutions.

MAIN CONCLUSION: Shoot tip necrosis is a physiological condition that negatively impacts the growth and development of in vitro plant shoot cultures across a wide range of species. Shoot tip necrosis is a physiological condition and disorder that can arise in plantlets or shoots in vitro that results in death of the shoot tip. This condition, which can spread basipetally and affect the emergence of axillary shoots from buds lower down the stem, is due to the cessation of apical dominance. STN can occur at both shoot multiplication and rooting stages. One of the most common factors that cause STN is nutrient deficiency or imbalance. Moreover, the presence or absence of plant growth regulators (auxins or cytokinins) at specific developmental stages may impact STN. The cytokinin to auxin ratio within an in vitro plant can be modified by varying the concentration of cytokinins used in the culture medium. The supply of nutrients to in vitro shoots or plantlets might also affect their hormonal balance, thus modifying the occurrence of STN. High relative humidity within culture vessels and hyperhydricity are associated with STN. An adequate supply of calcium as the divalent cation (Ca2+) can hinder STN by inhibiting the accumulation of phenolic compounds and thus programmed cell death. Moreover, the level of Ca2+ affects auxin transport and ethylene production, and higher ethylene production, which can occur as a result of high relative humidity in or poor ventilation of the in vitro culture vessel, induces STN. High relative humidity can decrease the mobility of Ca2+ within a plant, resulting in Ca2+ deficiency and STN. STN of in vitro shoots or plantlets can be halted or reversed by altering the basal medium, mainly the concentration of Ca2+, adjusting the levels of auxins or cytokinins, or modifying culture conditions. This review examines the literature related to STN, seeks to discover the associated factors and relations between them, proposes practical solutions, and attempts to better understand the mechanism(s) underlying this condition in vitro.

mRNA transcription profile of potato (Solanum tuberosum L.) exposed to ultrasound during different stages of in vitro plantlet development.

KEY MESSAGE: In response to an ultrasound pulse, several hundred DEGs, including in response to stress, were up- or down-regulated in in vitro potato plantlets. Despite this abiotic stress, plantlets survived. Ultrasound (US) can influence plant growth and development. To better understand the genetic mechanism underlying the physiological response of potato to US, single-node segments of four-week-old in vitro plantlets were subjected to US at 35 kHz for 20 min. Following mRNA purification, 10 cDNA libraries were assessed by RNA-seq. Significantly differentially expressed genes (DEGs) were categorized by gene ontology or Kyoto Encyclopedia of Genes and Genomes identifiers. The expression intensity of 40,430 genes was studied. Several hundred DEGs associated with biosynthesis, carbohydrate metabolism and catabolism, cellular protein modification, and response to stress, and which were expressed mainly in the extracellular region, nucleus, and plasma membrane, were either up- or down-regulated in response to US. RT-qPCR was used to validate RNA-seq data of 10 highly up- or down-regulated DEGs, and both Spearman and Pearson correlations between SeqMonk LFC and RT-qPCR LFC were highly positive (0.97). This study examines how some processes evolved over time (0 h, 24 h, 48 h, 1 week and 4 weeks) after an abiotic stress (US) was imposed on in vitro potato explants, and provides clues to the temporal dynamics in DEG-based enzyme functions in response to this stress. Despite this abiotic stress, plantlets survived.

In vitro tissue culture of apple and other Malus species: recent advances and applications.

MAIN CONCLUSION: Studies on the tissue culture of apple have allowed for molecular, biotechnological and applied breeding research to advance. In the past 8 years, over 100 papers advancing basic biology, genetic transformation and cryobiology have emerged. Apple (Malus × domestica Borkh.; Rosaceae) is an important fruit crop grown mainly in temperate regions of the world. In vitro tissue culture is a biotechnological technique that has been used to genetically improve cultivars (scions) and rootstocks. This updated review presents a synthesis of findings related to the tissue culture of apple and other Malus spp. between 2010 and 2018. Increasingly complex molecular studies that are examining the apple genome, for example, in a bid to identify the cause of epigenetic mutations and the role of transposable elements in this process would benefit from genetically stable source material, which can be produced in vitro. Several notable or curious in vitro culture methods have been reported to improve shoot regeneration and induce the production of tetraploids in apple cultivars and rootstocks. Existing studies have revealed the molecular mechanism underlying the inhibition of adventitious roots by cytokinin. The use of the plant growth correction factor allows hypothetical shoot production from leaf-derived thin cell layers relative to conventional leaf explants to be determined. This updated review will allow novices and established researchers to advance apple and Malus biotechnology and breeding programs.

The biotechnology (genetic transformation and molecular biology) of Bixa orellana L. (achiote).

MAIN CONCLUSION: Genetic transformation allows for greater bixin or norbixin production in achiote. Knowledge of genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants. Annatto is a natural dye or coloring agent derived from the seeds, or their arils, of achiote (Bixa orellana L.), and is commercially known as E160b. The main active component of annatto dye is water-insoluble bixin, although water-soluble norbixin also has commercial applications. Relative to other antioxidants, bixin is light- and temperature stable and is thus safe for human consumption. Bixin is, therefore, widely applied as a dye and as an antioxidant in the medico-pharmaceutical, food, cosmetic, and dye industries. Even though bixin has also been isolated from leaves and bark, yield is lower than from seeds. More biotechnology-based research of this industrial and medicinal plant is needed. Building on provisional genetic transformation studies, it would be advantageous to transform genes that could result in greater bixin or norbixin production. Reliable protocols for the extraction of bixin and norbixin, as well as deeper knowledge of the genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants.

Notices and Policies for Retractions, Expressions of Concern, Errata and Corrigenda: Their Importance, Content, and Context.

A retraction notice is an essential scientific historical document because it should outline the reason(s) why a scientific manuscript was retracted, culpability (if any) and any other factors that have given reason for the authors, editors, or publisher, to remove a piece of the literature from science's history books. Unlike an expression of concern (EoC), erratum or corrigendum, a retraction will usually result in a rudimentary vestige of the work. Thus, any retraction notice that does not fully indicate a set of elements related to the reason and background for the retraction serves as a poor historical document. Moreover, poorly or incompletely worded retraction notices in fact do not serve their intended purpose, i.e., to hold all parties accountable, and to inform the scientific and wider public of the problem and reason for the paper's demise. This paper takes a look at the definitions and the policies of clauses for retractions, EoCs, errata and corrigenda in place by 15 leading science, technology and medicine (STM) publishers and four publishing-related bodies that we believe have the greatest influence on the current fields of science, technology and medicine. The primary purpose was to assess whether there is a consistency among these entities and publishers. Using an arbitrary 5-scale classification system, and evaluating the different categories of policies separately, we discovered that in almost all cases (88.9 %), the wording used to define these four categories of polices differs from that of the Committee on Publication Ethics (COPE), which is generally considered to be the guiding set of definitions in science publishing. In addition, as much as 61 % deviation in policies (wording and meaning), relative to COPE guidelines, was discovered. When considering the average pooled deviation across all categories of policies, we discovered that there was either no deviation or a small deviation, only in the wording, in the definition of policies when compared to the COPE guidelines in 1 out of 3 ethical bodies, and in 40 % (6 out of 15) STM publishers. Moderate deviation from the COPE guidelines was detected in 26.7 % of STM publishers and one ethical body but a large deviation in one ethical body and 20 % of STM publishers was observed. Two STM publishers (13.3 %) did not report any information about these policies. Even though in practice, editors and publishers may deviate from these written definitions when dealing with case-by-case issues, we believe that it is essential, to serve as a consistent guide for authors and editors, that the wording be standardized across these entities. COPE and these entities also have the responsibility of making it clear that these definitions are merely suggestions and that their application may be subjected to subjective interpretation and application.

Fortifying the Corrective Nature of Post-publication Peer Review: Identifying Weaknesses, Use of Journal Clubs, and Rewarding Conscientious Behavior.

Most departments in any field of science that have a sound academic basis have discussion groups or journal clubs in which pertinent and relevant literature is frequently discussed, as a group. This paper shows how such discussions could help to fortify the post-publication peer review (PPPR) movement, and could thus fortify the value of traditional peer review, if their content and conclusions were made known to the wider academic community. Recently, there are some tools available for making PPPR viable, either as signed (PubMed Commons) or anonymous comments (PubPeer), or in a hybrid format (Publons). Thus, limited platforms are currently in place to accommodate and integrate PPPR as a supplement to traditional peer review, allowing for the open and public discussion of what is often publicly-funded science. This paper examines ways in which the opinions that emerge from journal clubs or discussion groups could help to fortify the integrity and reliability of science while increasing its accountability. A culture of reward for good and corrective behavior, rather than a culture that protects silence, would benefit science most.

Advances in Dendrobium molecular research: Applications in genetic variation, identification and breeding.

Orchids of the genus Dendrobium are of great economic importance in global horticultural trade and in Asian traditional medicine. For both areas, research yielding solid information on taxonomy, phylogeny, and breeding of this genus are essential. Traditional morphological and cytological characterization are used in combination with molecular results in classification and identification. Markers may be useful when used alone but are not always reliable in identification. The number of species studied and identified by molecular markers is small at present. Conventional breeding methods are time-consuming and laborious. In the past two decades, promising advances have been made in taxonomy, phylogeny and breeding of Dendrobium species due to the intensive use of molecular markers. In this review, we focus on the main molecular techniques used in 121 published studies and discuss their importance and possibilities in speeding up the breeding of new cultivars and hybrids.

Methods for genetic transformation in Dendrobium.

KEY MESSAGE: The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

Multiple Authorship in Scientific Manuscripts: Ethical Challenges, Ghost and Guest/Gift Authorship, and the Cultural/Disciplinary Perspective.

Multiple authorship is the universal solution to multi-tasking in the sciences. Without a team, each with their own set of expertise, and each involved mostly in complementary ways, a research project will likely not advance quickly, or effectively. Consequently, there is a risk that research goals will not be met within a desired timeframe. Research teams that strictly scrutinize their modus operandi select and include a set of authors that have participated substantially in the physical undertaking of the research, in its planning, or who have contributed intellectually to the ideas or the development of the manuscript. Authorship is not an issue that is taken lightly, and save for dishonest authors, it is an issue that is decided collectively by the authors, usually in sync with codes of conduct established by their research institutes or national ministries of education. Science, technology and medicine (STM) publishers have, through independent, or sometimes coordinated efforts, also established their own sets of guidelines regarding what constitutes valid authorship. However, these are, for the greater part, merely guidelines. A previous and recent analysis of authorship definitions indicates that the definitions in place regarding authorship and its validity by many leading STM publishers is neither uniform, nor standard, despite several of them claiming to follow the guidelines as set forward by the International Committee of Medical Journal Editors or ICMJE. This disparity extends itself to ghost and guest authorship, two key authorship-related issues that are examined in this paper to assess the extent of discrepancies among the same set of STM publishers and what possible influence they might have on publishing ethics.

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development.

The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.

Dendrobium micropropagation: a review.

Dendrobium is one of the largest and most important (ornamentally and medicinally) orchid genera. Tissue culture is now an established method for the effective propagation of members of this genus. This review provides a detailed overview of the Dendrobium micropropagation literature. Through a chronological analysis, aspects such as explant, basal medium, plant growth regulators, culture conditions and final organogenic outcome are chronicled in detail. This review will allow Dendrobium specialists to use the information that has been documented to establish, more efficiently, protocols for their own germplasm and to improve in vitro culture conditions based on the optimized parameters detailed in this review. Not only will this expand the use for mass propagation, but will also allow for the conservation of important germplasm. Information on the in vitro responses of Dendrobium for developing efficient protocols for breeding techniques based on tissue culture, such as polyploidization, somatic hybridization, isolation of mutants and somaclonal variants and for synthetic seed and bioreactor technology, or for genetic transformation, is discussed in this review. This is the first such review on this genus and represents half a decade of literature dedicated to Dendrobium micropropagation.

Asymbiotic in vitro seed propagation of Dendrobium.

The ability to germinate orchids from seeds in vitro presents a useful and viable method for the propagation of valuable germplasm, maintaining the genetic heterogeneity inherent in seeds. Given the ornamental and medicinal importance of many species within the genus Dendrobium, this review explores in vitro techniques for their asymbiotic seed germination. The influence of abiotic factors (such as temperature and light), methods of sterilization, composition of basal media, and supplementation with organic additives and plant growth regulators are discussed in context to achieve successful seed germination, protocorm formation, and further seedling growth and development. This review provides both a basis for the selection of optimal conditions, and a platform for the discovery of better ones, that would allow the development of new protocols and the exploration of new hypotheses for germination and conservation of Dendrobium seeds and seedlings.

In vitro conservation of Dendrobium germplasm.

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation-dehydration, encapsulation-vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation-vitrification and encapsulation-dehydration are the most used protocol, while PLBs represent the main explant explored.

Meta-Topolin as an Effective Benzyladenine Derivative to Improve the Multiplication Rate and Quality of In Vitro Axillary Shoots of Húsvéti Rozmaring Apple Scion.

In vitro mass propagation of apple plants plays an important role in the rapid multiplication of genetically uniform, disease-free scions and rootstocks with desired traits. Successful micropropagation of apple using axillary shoot cultures is influenced by several factors, the most critical of which is the cytokinin included in the culture medium. The impact of medium composition from single added cytokinins on shoot proliferation of apple scion Húsvéti rozmaring cultured on agar-agar gelled Murashige and Skoog medium fortified with indole butyric acid and gibberellic acid was investigated. The optimum concentration for efficient shoot multiplication differs according to the type of cytokinin. The highest significant multiplication rate (5.40 shoots/explant) was achieved using 2.0 µM thidiazuron while the longest shoots (1.80 cm) were observed on the medium containing benzyladenine at a concentration of 2.0 µM. However, application of either thidiazuron or benzyladenine as cytokinin source in the medium resulted in shoots of low quality, such as stunted and thickened shoots with small leaves. In the case of benzyladenine riboside, the 8 µM concentration was the most effective in increasing the multiplication rate (4.76 shoots/explant) but caused thickened stem development with tiny leaves. In the present study, meta-topolin was shown to be the most effective cytokinin that could be applied to induce sufficient multiplication (3.28 shoots/explant) and high-quality shoots along with shoot lengths of 1.46 cm when it was applied at concentrations of 4 µM. However, kinetin was the least active cytokinin; it practically did not induce the development of new shoots. The superior cytokinin for in vitro axillary shoot development of apple scion Húsvéti rozmaring with high-quality shoots was the meta-topolin, but it may be different depending on the variety/genotype under study.

Vegetal memory through the lens of transcriptomic changes - recent progress and future practical prospects for exploiting plant transcriptional memory.

Plant memory plays an important role in the efficient and rapid acclimation to a swiftly changing environment. In addition, since plant memory can be inherited, it is also of adaptive and evolutionary importance. The ability of a plant to store, retain, retrieve and delete information on acquired experience is based on cellular, biochemical and molecular networks in the plants. This review offers an up-to-date overview on the formation, types, checkpoints of plant memory based on our current knowledge and focusing on its transcriptional aspects, the transcriptional memory. Roles of long and small non-coding RNAs are summarized in the regulation, formation and the cooperation between the different layers of the plant memory, i.e. in the establishment of epigenetic changes associated with memory formation in plants. The RNA interference mechanisms at the RNA and DNA level and the interplays between them are also presented. Furthermore, this review gives an insight of how exploitation of plant transcriptional memory may provide new opportunities for elaborating promising cost-efficient, and effective strategies to cope with the ever-changing environmental perturbations, caused by climate change. The potentials of plant memory-based methods, such as crop priming, cross acclimatization, memory modification by miRNAs and associative use of plant memory, in the future's agriculture are also discussed.

Expression responses of XTH genes in tomato and potato to environmental mechanical forces: focus on behavior in response to rainfall, wind and touch.

Rainfall, wind and touch, as mechanical forces, were mimicked on 6-week-old soil-grown tomato and potato under controlled conditions. Expression level changes of xyloglucan endotransglucosylase/hydrolase genes (XTHs) of tomato (Solanum lycopersicum L. cv. Micro Tom; SlXTHs) and potato (Solanum tuberosum L. cv. Desirée; StXTHs) were analyzed in response to these mechanical forces. Transcription intensity of every SlXTHs of tomato was altered in response to rainfall, while the expression intensity of 72% and 64% of SlXTHs was modified by wind and touch, respectively. Ninety-one percent of StXTHs (32 out of 35) in potato responded to the rainfall, while 49% and 66% of the StXTHs were responsive to the wind and touch treatments, respectively. As previously demonstrated, all StXTHs were responsive to ultrasound treatment, and all were sensitive to one or more of the environmental mechanical factors examined in the current study. To our best knowledge, this is the first study to demonstrate that these ubiquitous mechanical environmental cues, such as rainfall, wind and touch, influence the transcription of most XTHs examined in both species.

Deep inside the epigenetic memories of stressed plants.

Recent evidence sheds light on the peculiar type of plant intelligence. Plants have developed complex molecular networks that allow them to remember, choose, and make decisions depending on the stress stimulus, although they lack a nervous system. Being sessile, plants can exploit these networks to optimize their resources cost-effectively and maximize their fitness in response to multiple environmental stresses. Even more interesting is the capability to transmit this experience to the next generation(s) through epigenetic modifications that add to the classical genetic inheritance. In this opinion article, we present concepts and perspectives regarding the capabilities of plants to sense, perceive, remember, re-elaborate, respond, and to some extent transmit to their progeny information to adapt more efficiently to climate change.

Optimizing Medium Composition and Environmental Culture Condition Enhances Antioxidant Enzymes, Recovers Gypsophila paniculata L. Hyperhydric Shoots and Improves Rooting In Vitro

Gypsophila paniculata L. is one of the most important commercial cut flowers worldwide. The plant is sterile and propagated mainly by in vitro culture techniques. However, hyperhydricity hinders its micropropagation and increases mortality during ex vitro acclimatization. Hyperhydric shoots of G. paniculata were proliferated from nodal explants on MS medium without growth regulators that contained 30 g L-1 sucrose, and gelled with 6.0 g L-1 agar. Medium components and environmental culture conditions were optimized to revert hyperhydricity in G. paniculata microshoots and develop an efficient micropropagation protocol for commercial production. Multiple shoots with high quality were successfully regenerated on MS medium fortified with potassium and ammonium nitrate at full concentration, 2.0 mg L-1 paclobutrazol, solidified with 9.0 g L-1agar in Magenta boxes of 62.87 gas exchange/day and incubated under light density of 60 µmol m-2s-1. We recorded 4.33 shoots, 40.00 leaves, 6.33 cm, 2.50 g and 95.00% for number of shoots/explant, number of leaves/shoot, shoot length, shoot fresh weight and normal shoots percentage, respectively. Well-rooted plantlets of G. paniculata were developed from the reverted microshoots, with the rooting percentage (95.00%) on MS medium augmented with 1.0 mg L-1 IBA in Magenta boxes of 62.87 gas exchange/day and 60 µmol m-2s-1 light density. In vitro-rooted plantlets exhibited reduced electrolyte leakage, and enhanced antioxidant enzymes activity of peroxidase, catalase, and polyphenol oxidase due to good ventilation at the highest gas exchange rate of the culture vessels.