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In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

Several structural attributes associated with the buds of shoot apices of angiosperms have been related to protection both at the mechanical and chemical defense levels. Although several characteristics related to the protection of shoot buds are known in different taxa of Sapindales, their occurrence in the neotropical genus Homalolepis (Simaroubaceae) are still unknown. Homalolepis includes 28 species mostly with extra-Amazonian distribution, occurring mainly in the Brazilian domains of the “Cerrado” and the Atlantic Forest. Its species vary widely in terms of habit, many of which are considered adaptations to the environments in which they occur. In this study, usual light and scanning electron microscopy techniques were applied in order to analyze the morphoanatomical and histochemical characteristics associated with the protection of apical and lateral shoot buds in phanerophyte (H. arenaria Devecchi & Pirani (Devecchi & Pirani), H. glabra (Engl.) Devecchi & Pirani and H. insignis (A.St.-Hil. & Tul.) Devecchi & Pirani) and geophyte (H. pumila Devecchi & Pirani, H. salubris (Engl.) Devecchi & Pirani and H. suffruticosa (Engl.) Devecchi & Pirani) species. Glandular prophylls, cataphylls, suberized layer on the leaf primordium, colleters, non-glandular trichomes, as well as a secretory canal with precocious development in the leaf primordium are among the characteristics that can be considered strategies for the protection of shoot buds in the studied species. Such structures are related to physical (cataphyll and suberized layer) and chemical (prophylls and secretory canal) protection or both (trichomes), against desiccation or attack by herbivores.

The distribution of essential nutrients such as potassium (K), phosphorous (P), calcium (Ca), sulfur (S) and chlorine (Cl) within root tissues is crucial aspect of plant growth but nothing is known regarding this subject in seedlings of dicotyledons such as peanut (Arachis hypogaea). We used scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) and inductively coupled plasma optical emission spectrometry (ICP-OES) to analyze element distributions in developing root tissues. Distribution patterns and concentrations of endogenous K, P, S and exogenous Ca from seeds grown in water varied along the length of peanut radicle and were tissue specific. Semi-quantitative SEM/EDS data indicated 24 mM K in the cortex and 46 mM K in the stele at a distance of 5 mm from the root tip. The P concentration in the same region was 15 mM in cortex and 22 mM in stele. The concentration of K and P decreased in the more mature parts of the root. SEM–EDS element concentrations values were lower for K and higher for P when compared with the ICP-OES data of root segments. Peanut roots grown in 10 mM Ca(NO3)2 solution for hours showed tissue-specific Ca distribution at 25 mm from the root tip with the highest levels detected in portions of the cortex. The parent root cortex centrifugal to the tip of developing lateral root primordia had lower levels of calcium than could be detected by SEM/EDS.

Embryonic development of reproductive organs in rediae of the digenean Bunocotyle progenetica was studied using transmission electron microscopy. The germinal primordium becomes morphologically distinct in early embryos as a weakly separated cell mass with a forming cavity. It consists of undifferentiated, differentiating, and supporting cells. As embryos develop, the supporting cells form a wall around the enlarging cavity. Other cells of the germinal primordium are incorporated into the wall as solitary cells or as small cell aggregations. Those situated posteriorly give rise to an incipient germinal mass functioning during postembryonic development. Undifferentiated and differentiating cells in the middle and the anterior part of the primordium ensure a considerable growth of the cavity wall, which incorporates solitary germinal cells. In advanced embryonic rediae, these cells mature, cleave, and give rise to germinal balls, which enter the forming brood cavity. In the most mature embryonic rediae, all these early cercarial embryos reside in a brood cavity, which is lined by that time with a syncytium continuous with the supporting tissue of the incipient germinal mass. Based on our results and the literature data, we suggest that the morphogenesis of the reproductive apparatus of the daughter parthenitae in hemiuroid digeneans may be characterized by (1) emergence of an incipient brood cavity within the germinal primordium, (2) formation of the cavity lining from the cells of the germinal primordium, (3) fragmentation and uneven distribution of the germinal material of the germinal primordium around the cavity, and (4) an anticipatory development of some of this germinal material.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation. In Arabidopsis, xylem pole pericycle cells produce lateral roots. Here, two subfamilies of bHLH transcription factors are characterized. They confer to these pericycle cells the ability to re-activate cell division and initiate lateral roots.

Phyllotactic diversity and developmental transitions between phyllotactic patterns are not fully understood. The plants studied so far, such as Magnolia, Torreya or Abies, are not suitable for experimental work, and the most popular model plant, Arabidopsis thaliana, does not show sufficient phyllotactic variability. It has been found that in common verbena (Verbena officinalis L.), a perennial, cosmopolitan plant, phyllotaxis differs not only between growth phases in primary transitions but also along the indeterminate inflorescence axis in a series of multiple secondary transitions. The latter are no longer associated with the change in lateral organ identity, and the sequence of phyllotactic patterns is puzzling from a theoretical point of view. Data from the experiments in silico, confronted with empirical observations, suggest that secondary transitions might be triggered by the cumulative effect of fluctuations in the continuously decreasing bract primordia size. The most important finding is that the changes in the primary vascular system, associated with phyllotactic transitions, precede those taking place at the apical meristem. This raises the question of the role of the vascular system in determining primordia initiation sites, and possibly challenges the autonomy of the apex. The results of this study highlight the complex relationships between various systems that have to coordinate their growth and differentiation in the developing plant shoot. Common verbena emerges from this research as a plant that may become a new model suitable for further studies on the causes of phyllotactic transitions.

StCDPK2 is an early player in the salt stress response in potato plants; its overexpression promoted ROS scavenging, chlorophyll stability, and the induction of stress-responsive genes conferring tolerance to salinity. The salinity of soils affects plant development and is responsible for great losses in crop yields. Calcium-dependent protein kinases (CDPKs) are sensor–transducers that decode Ca2+ signatures triggered by abiotic stimuli and translate them into physiological responses. Histochemical analyses of potato plants harboring StCDPK2 promoter fused to the reporter gene β-glucuronidase (ProStCDPK2:GUS) revealed that GUS activity was high in the leaf blade and veins, it was restricted to root tips and lateral root primordia, and was observed upon stolon swelling. Comparison with ProStCDPK1:GUS and ProStCDPK3:GUS plants revealed their differential activities in the plant tissues. ProStCDPK2:GUS plants exposed to high salt presented enhanced GUS activity in roots which correlated with the numerous stress-responsive sites predicted in its promoter sequence. Moreover, StCDPK2 expression increased in in vitro potato plants after 2 h of high salt exposure and in greenhouse plants exposed to a dynamic stress condition. As inferred from biometric data and chlorophyll content, plants that overexpress StCDPK2 were more tolerant than wild-type plants when exposed to high salt. Overexpressing plants have a more efficient antioxidant system; they showed reduced accumulation of peroxide and higher catalase activity under salt conditions, and enhanced expression of WRKY6 and ERF5 transcription factors under control conditions. Our results indicate that StCDPK2 is an early player in the salt stress response and support a positive correlation between StCDPK2 overexpression and tolerance towards salt stress.

The initiation pattern of the floral organs of the Astragalus species displays uniqueness at the initial stages of ontogeny. However, its most common way is unidirectional initiation, which occurs from the abaxial to the adaxial sides. The overlapping of organs and the emergence of common primordia shortens the time of development and is of an evolutionary advantage to the genus. Astragalus species are constantly evolving, and they are bestowed with unique floral ontogeny that offers various advantages to these plants to survive and adapt to changing environmental conditions. The morphological features of the floral organ production of four Astragalus species were identical in the early and middle stages of development. During the middle and the late stages, some unique features such as rolled petals, sticky keel tips, diadelphous androecium were observed. Semi-circular receptacular nectaries were found in Astragalus henryi. Comparison of the inflorescence development between A. henryi and A. membranaceus revealed that A. membranaceus formed one terminal inflorescence, which developed into a raceme while A. henryi formed a variety of terminal inflorescences, branched by racemes to form panicles. Development of common primordia and organ overlap was also observed in both Oxytropis ochrocephala and Astragalus species. However, the most significant difference was observed in the keel petals of O. ochrocephala, which developed a beak at the later stages of development. The study unravels interesting relationships between four species of Astragalus and one species of Oxytropis. Study of floral ontogeny through series of sections using various microscopic techniques provided insights into the understudied floral traits of the genera. Such studies also need to be corroborated with other empirical evidence and analyses.

K, P, Cl, and Ca are distributed in tissue-specific patterns in Zea mays seedlings. These elements were mapped and analyzed using a relatively simple semi-quantitative technique, i.e., fast freezing, followed by freeze fracturing, then freeze drying, and finally scanning electron microscopy/energy-dispersive spectroscopy (SEM/EDS). In the radicle, endogenously derived (i.e., from seed) K and P transition from being homogenous in the apical meristem to tissue-specific in older regions. At 3 mm from the radicle apex, K concentration is approximately 40 mM in mid-cortex and decreases by approximately 50% at 15 mm. From 3 to 55 mm, P concentration in pericycle is approximately twice that found in adjacent regions. Ca is not detectable in younger portions of the radicle by SEM/EDS, but in older regions, it is present at 13 mM in mid-cortex. K concentration values of entire radicles analyzed with inductively coupled plasma optical emission spectrometry (ICP-OES) exceeded the SEM/EDS values. For Ca, the reverse was true. But, SEM/EDS analysis did not include several vascular tissues that contained high concentrations of K and low concentrations of Ca. The inception of lateral root primordia was accompanied by a localized decrease in Ca in cortical regions that were centrifugal to the primordium tip. A region of O-rich cells in endosperm was identified centripetal to the aleurone. These results indicate that (1) outer, mid-, and inner cortical regions, as well as the adjacent tissues, have distinct ion accumulation properties, and (2) ions are concentrated in some radicle tissues prior to development of Casparian strips.

Free-living filamentous thalli derived from an Analipus japonicus erect thallus were established in laboratory culture. The filamentous thalli were composed of slender filaments and densely pigmented oval or sub-oval cell masses. Temperature (5–20 °C) and irradiance (25–200 μmol photons m−2 s−1) effects on formation and growth of the thalli from three explant groups (isolated filaments, small cell masses, and large cell masses) were investigated for 15 days. These explants survived over the examined range. The isolated filaments had a high ability to form the thalli, and the isolated and new filaments formed filaments and cell masses. Meanwhile, the isolated and new cell masses formed only filaments, and their ability to grow and form the thalli decreased as they grew in size. Optimum culture conditions for formation and growth of the thalli from the isolated filaments, and the two cell mass growths were 15–20 °C and 100–200 μmol photons m−2 s−1. To produce erect thalli, the filamentous thalli were cultured on seedling threads for 6 weeks at 15 °C, 60 μmol photons m−2 s−1 and 20 cm s−1 water flow velocity. Erect axis density increased with increasing coverage of the filamentous thalli, and their cell masses (primordia of erect thallus holdfast) with mean area of about 140 × 103 μm2 began to produce erect axes. After 4 months of field cultivation, they grew into full-grown erect thalli of about 20 cm long. These findings show that the free-living filamentous thalli were applicable for stable seedling production of A. japonicus to be used in aquaculture.

We identified a RING-type E3 ligase (TaBAH1) protein in winter wheat that targets TaSAHH1 for degradation and might be involved in primordia development by regulating targeted protein degradation. Grain yield per spike in wheat (Triticum aestivum), is mainly determined prior to flowering during mature primordia development; however, the genes involved in primordia development have yet to be characterized. In this study, we demonstrated that, after vernalization for 50 days at 4 °C, there was a rapid acceleration in primordia development to the mature stages in the winter wheat cultivars Keumgang and Yeongkwang compared with the Chinese Spring cultivar. Although Yeongkwang flowers later than Keumgang under normal condition, it has the same heading time and reaches the WS9 stage of floral development after vernalization for 50 days. Using RNA sequencing, we identified candidate genes associated with primordia development in cvs. Keumgang and Yeongkwang, that are differentially expressed during wheat reproductive stages. Among these, the RING-type E3 ligase TaBAH1 (TraesCS5B01G373000) was transcriptionally upregulated between the double-ridge (WS2.5) stage and later stages of floret primordia development (WS10) after vernalization. Transient expression analysis indicated that TaBAH1 was localized to the plasma membrane and nucleus and was characterized by self-ubiquitination activity. Furthermore, we found that TaBAH1 interacts with TaSAHH1 to mediate its polyubiquitination and degradation through a 26S proteasomal pathway. Collectively, the findings of this study indicate that TaBAH1 might play a prominent role in post-vernalization floret primordia development.

In this study, we developed a repeatable in vitro micropropagation protocol for the medicinal plant Asparagus cochinchinensis, based on indirect organogenesis using leaf segments cut from seedlings of in vitro-germinated seeds. We obtained 85% callus induction from the leaf segments within 4–5 weeks when grown on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP, 1.0 mg/L) and 1-naphthaleneacetic acid (NAA, 0.5 mg/L). We found that MS media supplemented with Kn (1.0 mg/L) in combination with NAA (1.0 mg/L) and BAP (1.0 mg/L) combined with NAA (0.5 mg/L) were the most effective in promoting shoot regeneration, yielding plantlets with 6.72 and 6.48 shoots per culture, respectively. When cultured on PGR-free half-strength MS medium, regenerated plants developed root systems with an average 11.0 roots per shoot cluster and an average length of 36.14 mm at 9 weeks. During acclimatization, regenerated plantlets showed 96.4% survival and exhibited normal growth characteristics and morphology. We also made an attempt to directly regenerate A. cochinchinensis from shoot apices but it was futile. Histological analyses revealed the presence of crystal idioblasts in young leaves from the early stages of leaf differentiation. The leaf-based plant regeneration technique developed herein could be employed for large-scale propagation of the plants over a short time period, thereby substantially contributing to the germplasm preservation and rapid propagation of A. cochinchinensis. A repeatable in vitro micropropagation protocol for Asparagus cochinchinensis was developed based on indirect organogenesis. Histological analysis revealed crystal idioblasts for the first time in leaf primordia of this species.

Vegetative propagules play various important roles in lichen biology. We cultured soredia of Cladonia lichens in vitro and obtained three noteworthy results. Firstly, soredia are a beneficial source for the isolation of lichen symbionts. The mycobiont was obtained from 66% and the photobiont from 67% of the cultured soredia that were not contaminated. Secondly, the development of soredia followed a previously recognized pattern, arachnoid stage – soredium field – primordium, but a thalline structure was not achieved. We suggest that thallus formation in vitro is a question of favourable environmental factors, not partners compatibility. Finally, we discovered that fungi, other than the mycobiont, as well as airborne contaminants are dispersed together with lichen soredia. This is the first-ever report of such a phenomenon. The possible ecological consequences are discussed. Cystobasidiomycete yeasts were found among these fungi. We isolated representatives of three different lineages from a single thallus suggesting a low specificity for this association.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

Chinquapin (Castanea henryi) is a dual-purpose tree species in China valued for as a source of timber and starch. We investigated the effect of four cutting mediums (pure vermiculite; peat:river sand at 3:1 v/v; peat:krasnozem at 1:1 v/v; and pure krasnozem) and three stem cutting periods (March, May, and July) on rooting performance of C. henryi cuttings. Different cutting periods and cutting mediums greatly influenced the rooting rate of C. henryi, ranging from 3.35 to 77.31%. Principal component analysis indicated that the best combination of cutting period and cutting medium was semi-hardwood cuttings (May cuttings) + krasnozem. Histological evidence indicated that adventitious root initials were present by week 5–6, and that the site of root primordia initiation was observed in the vascular cambium. Stem anatomical structures observed at different periods indicated that a xylem/radius ratio of 29.90–37.42% and a fractured phloem fiber ring are indicative of rooting success. The relational model between rooting index and medium properties indicated that nutrient content and porosity significantly influenced callus production. However, pH strongly affected C. henryi root formation, with the Pearson correlation coefficients for May and July cuttings of − 0.856 and − 0.947, respectively. Our protocol is helpful to achieve mass clone propagation of improved C. henryi genotypes, thus overcoming a common hurdle in chinquapin breeding programs.

The root system of Arabidopsis thaliana comprises primary, lateral, and adventitious roots. Different types of roots are formed by diverse inductive cues and developmental programs. Here, we adopted the CRE/LOX system to trace cell lineage during the three types of root formation under the control of the promoter of WUSCHEL-RELATED HOMEOBOX5. The results show that the cells forming adventitious roots during de novo root regeneration from detached leaves and lateral roots from the primary root are descendants of the WOX5-expressing root primordium. During the post-embryonic growth of the primary root, some vascular and root cap cells are descendants of the WOX5-expressing stem cell niche in the root apical meristem. Overall, our data suggest that the CRE/LOX system is a useful tool to trace cell lineage in different types of root organogenesis.

Oversprouting is a poorly studied disease of guarana plant (Paullinia cupana var. sorbilis), a native species from Amazon Rainforest caused by Fusarium decemcellulare (FDC) that affects the plant growth and reproduction, severely compromising productivity. The sorbilis variety of guarana is anciently cultivated and is used today for the industry of soft drinks, cosmetic and pharmaceutical. Transcriptome, proteome, light and electron microscopy were used to compare symptomatic and asymptomatic tissues and reveal anatomical, histological and cytological alterations resultant from the disease. Disease symptoms described here include a marked reduction in the longitudinal axis and the formation of “capsules”, named as this for the first time here, which display hard thickened external walls and contain extremely malformed floral organs and/or poorly differentiated vegetative primordia when occurring isolate in individual branching points. The aggregate of multiple “capsules” in a same branch point produces galls. The production of indol-acetic acid (IAA) in vitro by F. decemcellulare was for the first time reported and the comparative transcriptome and preliminary proteome data from symptomatic and asymptomatic tissues, support the occurrence of hormonal imbalance identified through several plant hormone-related genes and proteins differentially expressed only in symptomatic tissues. Auxin signaling mechanism, as well as auxin-responsive genes associated to cell cycling, division and proliferation also occurred differentially in infected tissues. Oversprouting and the reduction of the longitudinal axis in malformed cells and organs are related to hormonal unbalance are discussed in this paper.

During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation.

The application of molecular breeding of eucalyptus is restricted by the difficulty of regeneration. One limitation of eucalyptus regeneration could be the production of excessive reactive oxygen species (ROS) during in vitro culture that greatly affects the shoot regeneration. NADPH oxidases, encoded by the respiratory burst oxidase homologue (rboh), and ascorbate peroxidases (APX) play an important role in the ROS metabolism. The study investigated the combined effect and preliminary mechanism on eucalyptus regeneration of N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU), 6-benzylaminopurine (BAP), and dimethylthiourea (DMTU) by inoculating Eucalyptus urophylla hypocotyls on SPCa medium supplemented with 0.57 µM indole-3-acetic acid (IAA) and various ratios of PBU and BAP. In the presence of 4.56 µM PBU alone, the rboh transcription levels were relatively low and the APX transcription levels were relatively high in the 2-week-old calli. However, the highest frequency of organogenic calli was induced by the combination of 3.99 µM PBU, 0.57 µM BAP and 0.57 µM IAA. DMTU, a ROS scavenger, was added in 10, 20, or 30 µM increments to the SPCa medium together with 3.99 µM PBU and 0.57 µM BAP to promote callus differentiation. The expression levels of all nine APX genes increased significantly in the calli following treatment with DMTU. The calli treated with 20 µM DMTU showed the highest APX transcriptional levels, which was consistent with the highest APX activity, and formed the most organogenic calli. During the subsequent process of adventitious shoot formation, the transcription levels of rboh1 showed a descending pattern. However, the transcription levels of rboh4, rboh5, and rboh6 showed an ascending pattern and peaked in the calli with visible shoot primordia. Specifically, the expression of rboh5 and rboh6 was 203.2 times and 573.9 times higher than that in the 2-week-old calli, respectively. Additionally, the calli with visible shoot primordia showed the highest H2O2 content. The results implied that control of oxidative stress by a combination of PBU, BAP, and DMTU enhances adventitious shoot formation in E. urophylla by affecting the expression of the rboh and APX genes and regulating ROS metabolism. The results would be helpful for an efficient regeneration protocol for eucalyptus. Cooperation of PBU, BAP, and DMTU enhances adventitious shoot formation in Eucalyptus urophylla by affecting the expression of rboh and APX genes.

A new fungal species constantly associated with hazelnut (Corylus avellana) fructification starting from its primordia is described. The fungus is associated with hazelnut fruit during all their developmental stages, being consistently more present in spring (March–June). A 4-year survey has been conducted, from young fruit formation to full kernel maturity including also the post-harvest phase, to collect fungi associated with damaged/discoloured kernels. A collection of 60 isolates of a new species has been obtained in this study, which is here described as Didymella corylicola sp. nov. Multi-locus phylogenies based on four genomic loci (nuITS and LSU rDNA, RPB2 and TUB2) in combination with morphological data confirmed the fungus to represent a new species of Didymella (Didymellaceae). The occurrence of D. corylicola sp. nov. might have an impact on the quality of hazelnut production by contributing to kernel defects.

Saffron is the world highest-priced spice because its production requires intensive hand labour. Reduce saffron production costs require containerised plant production under controlled conditions and expand the flowering period. Controlling the flowering process and identify the factors involved in saffron flowering is crucial to introduce technical improvements. The research carried out so far in saffron has allowed an extensive knowledge of the influence of temperature on the flower induction, but the molecular mechanisms controlling flowering induction processes are largely unknown. The present study is the first conducted to isolate and characterize a regulator gene of saffron floral induction the Short Vegetative Phase (SVP) gene, which represses the floral initiation genes in the temperature response pathway, which involved in saffron flower induction. The results obtained from both phylogenetic analysis and T-coffee alignment confirms that the isolated sequence belongs to the SVP gene clades of MADS-box gene family. Gene expression analysis in different developmental stages revealed the highest expression of SVP transcript (CsSVP) during the dormancy and the vegetative stages, but decrease when flower development initiated and it was the least in late September when flower primordia are developed. Furthermore, its expression increased in the apical bud when corms are storage at 9-10 ºC, thus inhibiting flower induction. Additionally, comparison of the CsSVP transcript in apical buds from big and small corms, differing in their flowering capacity, indicates that the CsSVP transcript is present only in vegetative buds. Taken together, these results suggested inhibitory role of the SVP gene.

Root branching is influenced by the soil environment and exhibits a high level of plasticity. We report that the radial positioning of emerging lateral roots is influenced by their hydrological environment during early developmental stages. New lateral root primordia have both a high degree of flexibility in terms of initiation and development angle towards the available water. Our observations reveal how the external hydrological environment regulates lateral root morphogenesis. Arabidopsis lateral root radial emergence is spatially constrained by the dual xylem poles in the main root. However, the asymmetric presence of water can modify the orientation of lateral roots at a very early stage.

The flower of Zingiber zerumbet is characterized by a distinctive labellum, a highly modified floral organ believed to be formed by the fusion of several infertile members of the androecial whorl (staminodes). Across the Zingiberaceae, the number of staminodes involved in labellum formation varies from two to four, and these are reflected in the number of lobes that comprise the mature labellum. Research on the flower development in Zingiberaceae has been limited to species with either no labellum lobes or species displaying a bilobed labellum. Zingiber zerumbet is a representative of the genus with a three-lobed labellum, and its flower development remains poorly understood at both morphological and molecular levels. This study aims to give a comprehensive description of its flower development and to identify potential genes related to flower development using morphological and genetic characterization. Our results show that floral organ initiation is sequential with the sepal whorl initiating first, followed by petal and inner androecium together, followed by outer androecium, and finally the initiation of the inferior gynoecium. The three-lobed labellum comprises four androecial members: Two abaxial inner androecial members fuse to form the single central lobe, and two adaxial outer androecial members individually form the two lateral lobes of the labellum. Two developmental stages (floral primordium and organ-differentiated flowers) were selected for transcriptome sequencing. Two-thousand and seventy-five transcription factors were identified. Seven boundary genes and seven organ-specific genes were also discovered. Our study provides fundamental information for further studies on the molecular mechanisms of flower development and evolution across the Zingiberaceae.

Amphioctopus fangsiao is an important fishery resource in northern coastal China, and there is an interest to develop its commercial cultivation under controlled conditions. In this study, we describe the embryonic development of A. fangsiao from eggs to hatchlings, focusing on the formation of the mantle, eyes and arms. Results indicate that this species has several favorable characteristics for aquaculture, such as a short incubation period and a large body size of hatchlings. During the organogenesis, the primordia of organs, including the optic sac and arm crown, first appeared at stage VIII; then the mantle primordium, eye primordia and arm crown become visible at stage IX–X, and the rudiment of embryo (mantle, eyes and arms) was completely outlined at stage XI. The retinal pigment appeared yellowish at stage X–XI and gradually deepens in pigmentation in the subsequent stages, turning from yellowish to yellow, red, dark-red and eventually black. At stage XV, the primary lid fold, with only a circular opening remaining, covered the eye, and the dorsal ocular edge did not reach the ventral side until hatching (day 1–7). Afterwards, about 24 days after hatching, both the dorsal and ventral sides of eyelid could contract freely, with an oval and elongate slit remaining that was a miniature version of the adult morphology. The chromatophores and ink sac appeared at stage XIV. These observations provide a foundation for the reproductive biology and aquaculture of A. fangsiao and also offer an important reference for the research of embryonic development in related species.

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding.

Background Liriodendron chinense is a distinctive ornamental tree species due to its unique leaves and tulip-like flowers. The discovery of genes involved in leaf development and morphogenesis is critical for uncovering the underlying genetic basis of these traits. Genes in the AP2/ERF family are recognized as plant-specific transcription factors that contribute to plant growth, hormone-induced development, ethylene response factors, and stress responses. Results In this study, we identified 104 putative AP2/ERF genes in the recently released L. chinense genome and transcriptome database. In addition, all 104 genes were grouped into four subfamilies, the AP2, ERF, RAV, and Soloist subfamilies. This classification was further supported by the results of gene structure and conserved motif analyses. Intriguingly, after application of a series test of cluster analysis, three AP2 genes, LcERF 94, LcERF 96, and LcERF 98, were identified as tissue-specific in buds based on the expression profiles of various tissues. These results were further validated via RT-qPCR assays and were highly consistent with the STC analysis. We further investigated the dynamic changes of immature leaves by dissecting fresh shoots into seven discontinuous periods, which were empirically identified as shoot apical meristem (SAM), leaf primordia and tender leaf developmental stages according to the anatomic structure. Subsequently, these three candidates were highly expressed in SAM and leaf primordia but rarely in tender leaves, indicating that they were mainly involved in early leaf development and morphogenesis. Moreover, these three genes displayed nuclear subcellular localizations through the transient transformation of tobacco epidermal cells. Conclusions Overall, we identified 104 AP2/ERF family members at the genome-wide level and discerned three candidate genes that might participate in the development and morphogenesis of the leaf primordium in L. chinense.

Background Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. Methods A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. Results At L3–L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal–ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. Conclusions A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 µl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species. Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.

The terrestrial carnivorous speciesUtricularia dichotomais known for a great phenotypic plasticity and unusual vegetative organs. Our investigation on 22 sources/populations revealed that after initiation of a leaf and two bladders on a stolon, a bud was formed in the proximal axil of the leaf, developing into a rosette with up to seven organs. The first two primordia of the bud grew into almost every possible combination of organs, but often into two anchor stolons. The patterns were generally not population specific. The interchangeability of organs increased with increasing rank in the succession of organs on stolon nodes. A high potential of switching developmental programs may be successful in a fluctuating environment. In this respect, we were able to show that bladders developed from anchor stolons experimentally when raising the water table. Anatomical structures were simple, lacunate and largely homogenous throughout all organs. They showed similarities with many hydrophytes, reflecting the plant’s adaptation to (temporarily) submerged conditions. The principal component analysis was used in the context of dynamic morphology to illustrate correlations between organ types in the morphospace ofU. dichotoma, revealing an organ specific patchwork of developmental processes for typical leaves and shoots, and less pronounced for a typical root. The concept and methods we applied may prove beneficial for future studies on the evolution of Lentibulariaceae, and on developmental morphology and genetics of unusual structures in plants.

Little is currently known about the interactions between structural and physiological factors in Neotropical fruit development. We sought to characterize fruit development and the dynamics of ABA biosynthesis during seed maturation in Caryocar brasiliense, a tree species endemic to the Cerrado biome of Brazil. Biometric evaluations were made of both the pericarp and seed during fruit development. ABA1, NCED4, and NCED9 expression were evaluated in embryonic tissues during seed maturation, as well as anatomical, histochemical, and ultrastructural evaluations, in vitro and ex vitro embryo cultures, and ABA quantifications. Fruit development demonstrated three stages: histodifferentiation (0–40 days after anthesis—DAA); endocarp maturation (40–60 DAA); and the conclusion of mesocarp and seed maturation (60–90 DAA). The embryos showed early degrees of differentiation: hypocotyl expansion, development of the leaf primordia, and vascularization. Seed maturation occurred (from 40 DAA) concomitantly with the development of endocarp aculei in four phases influenced by ABA dynamics. The beginning of reserve deposition and the acquisition of dehydration tolerance were related to increases in extra-embryonic ABA. ABA biosynthesis at the radicular pole was related to increased reserve deposition and dehydration tolerance (which was not associated with the occurrence of a desiccation phase). The maturation of C. brasiliense seeds is influenced by embryonic and extra-embryonic ABA, and the dormancy of the pyrenes results from the interaction of the effects of that hormone and pericarp tissue.

Adventitious roots (ARs) are important for the growth of plants and the improvement in their stress resistance and survival capacity. Although many genes have been confirmed to be involved in adventitious root (AR) formation in Arabidopsis and tomato plants, MADS-box genes have rarely been mentioned. Here, we isolated a MADS-box gene named SlMADS83, which may negatively regulate AR formation in tomato plants, as the number of the ARs formed in the transgenic lines in which the SlMADS83 gene was silenced by RNA interference (RNAi) was increased. The above phenotype was further confirmed by the analysis of the macroscopic, anatomical, and molecular features and related statistical data. Previous Studies have proven that auxin can stimulate early AR primordium initiation. Interestingly, in the RNAi transgenic lines, the concentration of auxin in the hypocotyl base was increased, resulting in early induction of AR primordia initiation, promoting the formation of ARs. Briefly, SlMADS83 may play an important role in AR formation.

International audience; Tissue morphogenesis emerges from coordinated cell shape changes driven by actomyosin contractions. Patterns of gene expression regionalize and polarize cell behaviours by controlling actomyosin contractility. Yet how mechanical feedbacks affect tissue morphogenesis is unclear. We report two modes of control over Rho1 and MyosinII activation in the Drosophila endoderm. First, Rho1/MyoII are induced in a primordium via localized transcription of the GPCR ligand Fog. Second, a tissue-scale wave of Rho1/MyoII activation and cell invagination progresses anteriorly. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocked acute Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. Last, we identify a cycle of 3D cell deformations whereby MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane, apical spreading, MyoII activation and invagination in the next row. Thus endoderm morphogenesis emerges from local transcriptional initiation and a mechanically driven wave of cell deformation.

Development in the colonial ascidian genus Diplosoma is unusual in producing two zooids already during the free-swimming larval period. The development of D. listerianum had been investigated in two studies. The present study reports embryonic development of a second species D. migrans. In D. migrans the pharynx of the oozooid develops from two separate primordia. The intestinal tract develops as well from two distinct primordia that fuse and subsequently connect to the prospective pharynx. The pharynx of the blastozooid develops from the paired epicardia of the oozooid. In the oozooid the primordia of the peribranchial sacs often derive from the endodermal wall of the prospective pharynx, but occasionally develop from thickenings of the ectodermal epidermis. No atrial siphon is formed; instead, the body wall retracts and the gill slits open directly to the exterior. In the oozooid the rectum develops from the initial prospective intestine, while the remaining intestine develops from a diverticulum of the stomach. In the blastozooid the main part of the intestinal tract develops from the initial prospective intestine, whereas the rectum develops as a diverticulum of the initial intestine. Due to this developmental mode, the intestinal tracts of oozooid and blastozooid are transitorily connected at the respective border of rectum and proximal part of posterior intestinal tract. The paired epicardia of the two zooids derive from the epicardial outgrowths of the pharyngeal primordia of the oozooid that are also the primordia of the heart and pericardia. Budding in the larva and the adult is highly similar, in particular concerning the development of a new thorax and the heart from paired epicardial Anlagen and the origin of the distal part of the posterior intestine. Discrepancies in the descriptions of the embryonic development of D. listerianum and differences to the development in D. migrans are discussed. The development of the pericardium-heart-complex from epithelially organized tissue constitutes the plesiomorphic condition within Didemnidae and Aplousobranchiata. The development from mesenchymatic cell masses is a secondarily derived simplification.

Made available in DSpace on 2019-10-06T15:48:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Mayacaceae are a small and monogeneric aquatic family including 4–6 species and with an unstable phylogenetic position within Poales. Therefore, the floral development and vasculature were studied in Mayaca fluviatilis and Mayaca sellowiana to better understand interfamilial relationships. In both species, the floral vascular system is organized in six central complexes from which the traces of all floral parts diverge. Both sepals and petals receive three traces, but in the sepals the median and lateral traces do not originate from the same vascular complex, whereas in the petals the median and lateral traces originate from the same complex. Both, ventral and dorsal carpellary traces, vascularize the ovary wall, ascending to the stigma. The sepals are the first organs to be initiated and have a rapid growth. Such structures exhibit glandular trichomes on their margins and remain united, protecting the inner whorls. After sepal development, the petals and the stamens of the outer whorl are initiated, followed by the gynoecium, which appears as an annular primordium. The petals have a markedly delay in development. In M. fluviatilis, the anthers may dehisce before anthesis and self-pollination occurs in the bud. The reduction (loss) of the inner stamen whorl and gynoecium development from an annular primordium link Mayacaceae to the cyperids (Poales). A high similarity in respect to floral vasculature was observed between Mayacaceae and Commelinaceae (Commelinales). Programa de Pós-Graduação em Ciências Biológicas (Biologia Vegetal) Departamento de Botânica Instituto de Biociências UNESP - Universidade Estadual Paulista, C. Postal 199 Programa de Pós-Graduação em Ciências Biológicas (Biologia Vegetal) Departamento de Botânica Instituto de Biociências UNESP - Universidade Estadual Paulista, C. Postal 199 CNPq: 168277/2014-0 CNPq: 301692/2010-6

Xanthoceras sorbifolium is apparently andromonoecious but exhibits a cryptically monoecious breeding system. Sexual differentiation in male and functional female flowers occurs 2 weeks before flowering. Individual trees of Xanthoceras sorbifolium bear male and morphologically bisexual flowers but functionally female flowers, and show labile sex expression. Investigations of the floral development are of significant value for understanding the breeding system and elucidating the systematic relationships. We studied floral development of male and bisexual flowers using scanning electron and light microscopy. The early stages of floral development were essentially the same, and all floral organ primordia were initiated in the two types of flowers of X. sorbifolium. Later, the stamens in bisexual flowers and the gynoecium in male flowers were aborted. We divided floral development into nine stages. Morphological differences between male and bisexual flowers appeared first at stage 8, when the style elongated obviously and the stigma papillae began to expand in the bisexual flowers but not in the male flowers. Ovule development was arrested shortly after formation of megaspore mother cells or during meiosis in the male flowers, whereas anther development was aberrant in the bisexual flowers. Morphologically bisexual flowers of X. sorbifolium do not have male function and are functionally female. Comparison of floral developmental characters did not support the separation of the Chinese monotypic genus Xanthoceras from the family Sapindaceae.

Shoots of the aquatic eudicot family, Podostemaceae, exhibit unusual organogenesis with mixed leaf and stem identities. New shoots arise at the base of the older shoot with shoot apical meristem (SAM) identity but the entire SAM differentiates into a "leaf" as it develops in the Podostemoideae subfamily. The "leaves" are tightly arranged in a zigzag manner to form an apparent distichous shoot as a whole. Although previous studies have suggested that Podostemoideae shoots have evolved by modifying the ancestral sympodial branching system in the basal Tristichoideae subfamily, this evolutionary scenario requires elucidation at the molecular level. To confirm that the shoots arise as axillary shoots, in the present study, we examined gene expression patterns in plumular shoots of Zeylanidium tailichenoides using CUP-SHAPED COTYLEDON 3 (CUC3) and SHOOT MERISTEMLESS (STM) orthologs, which are involved in the determination of axils and meristem formation in model plants. Expression of the CUC3 ortholog was detected at the adaxial base of cotyledons and parental shoots where the new shoots are initiated, while STM ortholog was expressed at the initiation site and in the young shoot primordia throughout early shoot development. The results demonstrate that each Z. tailichenoides shoot arises as an axillary bud in a manner similar to axillary meristem formation in model plants involving CUC3 and STM genes. Considering that each of the two cotyledons produces an axillary bud that in turn continues to form its own axillary bud independently, the apparent distichous shoot in Z.tailichenoides is not a single shoot, but a composite of two sympodially branched shoots.

Morphogenesis of a population of the marine euplotid ciliate, Uronychia binucleata, which was found in Yellow Sea coastal waters next to a sewage outfall at a beach near Zhanqiao Pier, Qingdao, China, was investigated using protargol staining. The main pattern of morphogenesis is typical for the genus and can be summarized as follows: 1) the oral primordium in both proter and opisthe develops de novo in a subcortical pouch. In each daughter cell, the developing adoral zone of membranelles divides into two parts. The new membranelles formed in the proter’s oral primordium will replace the leftmost five parental ones; six parental membranelles are retained by the proter; 2) the undulating membranes anlage is formed and develops independently from the oral primordium within the same subcortical pouch; 3) five primary front oventral-transverse cirral anlagen appear de novo on the cell surface; 4) the marginal cirral anlagen are formed de novo; 5) the leftmost frontal cirrus develops de novo on the cell surface; 6) two caudal cirri are formed at the posterior end of the rightmost anlage while the second primordium from the right gives rise to the third caudal cirrus. In contrast to its congeners, the anlage of the leftmost frontal cirrus is formed to the right of the undulating membranes anlage and before the formation of the latter.

We developed an efficient plant regeneration technique for turmeric (Curcuma longa L.) via somatic embryogenesis derived from embryogenic cell suspensions cultured from petioles. We initiated in vitro callus cultures from the plantlets of turmeric cultured on Murashige and Skoog (MS) medium. Then leaf, petiole, stem, and root explants of in vitro-grown plantlets were cultured on Schenk and Hildebrandt (SH). The stem and root explants formed white nodular calluses at a frequency of 86.7% and 85.8%, respectively, when cultured on SH medium; however, we used the calluses derived from the root explants for initiating a cell-suspension culture. In this study, the actively growing cell suspension culture comprised small, highly cytoplasmic cell aggregates and large vacuolated cells. On being transferred to a basal SH medium (without growth regulators), a few of these calluses differentiated into somatic embryos, which indicated that the initial calluses were embryogenic cells. We stimulated additional shoot differentiation by adding 0.5 mg L−1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) and various concentrations of N6-benzyladenine (BA) to the embryogenic cell suspension. The highest frequency at which shoot primordia developed from embryogenic cell clusters was 70% (when cultured in the dark on the supplemented MS medium), with more than 83.3% of regenerated shoot primordia producing roots. We successfully transplanted rooted plantlets into a soil mixture of sterile vermiculite and potting soil (1:1) and grew them to maturity in a growth chamber, achieving a survival rate of > 95%. To our knowledge, this is the first report of complete plant regeneration of turmeric from embryogenic cell-suspension cultures. The embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and gene manipulation for improving turmeric quality.

Detailed information on viroid invasion of the shoot apical meristem is important for the efficient production of viroid-free plants by meristem tip culture. Additionally, knowledge on viroid distribution in plant reproductive tissues, especially the embryo, is crucial to assess seed transmission of viroids. In this study, we analyzed histochemically the distribution of chrysanthemum chlorotic mottle viroid (CChMVd) in the shoot meristems and flower buds of CChMVd-infected chrysanthemum plants (cv. ‘Okayama-heiwa’). In situ hybridization revealed that CChMVd invaded the shoot apical meristems and leaf primordia in the shoot meristem at a high frequency (ca. 70%). In flower buds, CChMVd was found in the bract, receptacle vascular, petals, ovules, and immature stamens and pistils. Furthermore, we showed that seed transmission of CChMVd occurred at a high frequency when CChMVd-infected plants were used as female parents (♀). Overall, our findings suggest that CChMVd infection of ovules in flower buds is involved in seed transmission of the viroid.

Flavonoids are widely distributed secondary metabolic products with many biological functions in plants. Further elucidation of the accumulation and localization patterns of its biosynthesis enzymes will broaden our understanding of flavonoids biosynthesis and regulation. Chalcone isomerase (CHI, EC 5.5.1.6) is an early-step enzyme in the flavonoids biosynthesis pathway. In this study, using an antibody specifically developed against grapevine CHI enzyme, we found that the accumulation of CHI protein exhibited temporal and spatial specificity. In grape berries, CHI was investigated mainly in the outer hypodermis cells of exocarp tissues, in the vascular bundles of mesocarp; and in the integument and the cells around the raphe of seeds. At the subcellular level, CHI was visualized in the cytoplasm, nucleus, and plastids (chloroplasts) of the exocarp cells, while only in the cytoplasm of mesocarp vascular bundle cells. In grapevine vegetable organs, the leaf mesophyll and phloem of leaf veins, the pith ray and primary phloem of stems, the primary phloem and endoderm of roots, and the young leaves, leaf primordium, and the growth point of leaf buds were CHI signal-positive. In these tissue cells, CHI was primarily observed in the cytoplasm, cell wall, and nucleus. The distinct localization patterns of CHI suggested the complexity of flavonoids biosynthesis in grapevine. The distribution of CHI protein exhibited temporal and spatial specificity. Different subcellular localization patterns were observed in grape berries and grapevine vegetable tissues.

Lateral Organ Boundaries Domain 13 (LBD13), which is expressed in emerged lateral roots and encodes a transcriptional activator, plays an important role in lateral root formation in Arabidopsis. Lateral roots (LRs) are major determinants of root system architecture, contributing to the survival strategies of plants. Members of the LBD gene family encode plant-specific transcription factors that play key roles in plant organ development. Several LBD genes, such as LBD14, 16, 18, 29, and 33, have been shown to play important roles in regulating LR development in Arabidopsis. In the present study, we show that LBD13 is expressed in emerged LRs and LR meristems of elongated LRs and regulates LR formation in Arabidopsis. Transient gene expression assays with Arabidopsis protoplasts showed that LBD13 is localized to the nucleus and harbors transcription-activating potential. Knock-down of LBD13 expression by RNA interference resulted in reduced LR formation, whereas overexpression of LBD13 enhanced LR formation in transgenic Arabidopsis. Analysis of β-glucuronidase (GUS) expression under the control of the LBD13 promoter showed that GUS staining was detected in LRs emerged from the primary root, but not in LR primordia. Moreover, both the distribution of LR primordium number and developmental kinetics of LR primordia were not affected either by knock-down or by overexpression of LBD13. Taken together, these results suggest that LBD13 is a nuclear-localized transcriptional activator and controls LR formation during or after LR emergence.

Adventitious roots (ARs) induced are essential for apple rootstock vegetative propagation, however, a transcriptomic analysis of long non-coding RNAs (lncRNAs) that mediate AR formation under exogenous indole-3-butyric acid (IBA) treated in the apple dwarf rootstock ‘M26’ (Malus pumila Mill.) rarely been explored. To explore this, we examined ‘M26’ stem cuttings treated with IBA (1 mg L−1) and control (without IBA treatment), where anatomical analysis showed IBA-treated stems began to develop AR primordia within third day and ARs occurred after 16 days, while the control had no ARs developed. An assessment of hormone content levels in basal stem cuttings showed that, after IBA treatment, indole-3-acetic acid, zeatin riboside, and jasmonic acid were increased at third day but subsequently decreased later, however gibberellin-3 was decreased at third day. The control and IBA-treated third day basal stem cuttings were sent for whole-genome, high-throughput RNA sequencing. The results showed a total of 855 reliable lncRNAs were identified as novel lncRNAs and 63 novel lncRNAs were defined as IBA-responsive lncRNAs. Fifteen IBA-responsive lncRNAs were predicted to be putative targets for 94 miRNAs and three IBA-responsive lncRNA (CUFF.16781, CUFF.2143 and CUFF.41325) was predicted to be the target mimic of mdm-miR156 and mdm-miR396. Functional annotations of these potential target genes suggest that they are involved in many different biological processes, including plant hormone and sucrose signaling, which suggests that IBA-responsive lncRNAs may participate in AR formation in apple rootstocks. This study sheds light on IBA-mediated AR formation in apple rootstock cuttings and provides a foundation for further research.

A highly efficient regeneration system of Anthurium andraeanum Linden was established using root segments as explants of four genotypes, Arizona, SWD, OYH, and Mississippi. The root explants were firstly incubated on callus induction media containing NH4NO3 level of 206 mg L−1 or 825 mg L−1, and the calluses were formed from both ends of the root segments after 1 mo of culture. Root-derived calluses were then transferred to shoot regeneration media with NH4NO3 level of 412 mg L−1 or 1650 mg L−1, and numbers of shoot were successfully produced from the surface of calluses after 2 mo of culture. Medium containing 206 mg L−1 NH4NO3 induced more callus compared to 825 mg L−1. In contrast, the number of shoots per callus was the same between the above two shoot regeneration media indicating that NH4NO3 level had little effect on shoot regeneration. Long-termin vitro maintenance of the callus-bearing shoot initials was possible without compromising shoot regeneration and subsequent rooting. Histological analysis showed that early cell division was initiated from the inner cortical parenchyma cells near the vascular tissue and from the vascular parenchyma cells adjacent to the xylem vessel. The primary callus mainly comprised inner parenchyma cells with scattered procambial-like cells and xylem elements and two different meristematic zones beneath epidermal layers. Transfer of callus to the regenerative medium caused meristemoid formation and subsequent shoot primordia production. Calcium oxalate crystals and tannin-like deposits were visible on the outer callus layer or within the cotyledon-like structure of shoot primordium throughout callus development. Starch grains were abundant in root explants, but disappeared in the profusely growing primary callus, and were mobilized in the shoot primordium, indicating energy requirements of these two developmental stages. Flow cytometry analysis demonstrated that ploidy levels were maintained during in vitro cultures, including long-term regenerated plants. The inter-simple sequence repeat analysis indicated genomic DNA stability of Arizona and OYH but occasional genetic variation in primary regenerates of SWD and Mississippi.

The organogenesis and development of reproductive organs, i.e., stamen and gynoecium, are important floral characteristics that are closely related to pollinators and reproductive fitness. As a genus from Magnoliaceae, Liriodendron has only two relict species: L. chinense and L. tulipifera. Despite the similar flower shapes of these species, their natural seed-setting rates differ significantly, implying interspecies difference in floral organogenesis and development. MADS-box genes, which participate in floral organogenesis and development, remain unexplored in Liriodendron. Here, to explore the interspecies difference in floral organogenesis and development and identify MADS-box genes in Liriodendron, we examined the stamen and gynoecium primordia of the two Liriodendron species by scanning electron microscopy combined with paraffin sectioning, and then collected two types of primordia for RNA-seq. A total of 12 libraries were constructed and 42,268 genes were identified, including 35,269 reference genes and 6,999 new genes. Monoterpenoid biosynthesis was enriched in L. tulipifera. Genome-wide analysis of 32 MADS-box genes was conducted, including phylogenetic trees, exon/intron structures, and conserved motif distributions. Twenty-six genes were anchored on 17 scaffolds, and six new genes had no location information. The expression profiles of MIKC-type genes via RT-qPCR acrossing six stamen and gynoecium developmental stages indicates that the PI-like, AG/STK-like, SEP-like, and SVP-like genes may contribute to the species-specific differentiation of the organogenesis and development of reproductive organs in Liriodendron. Our findings laid the groundwork for the future exploration of the mechanism underlying on the interspecific differences in reproductive organ development and fitness in Liriodendron.

Background Chrysanthemum morifolium is one of the most popular ornamental crops. The capitulum, which is the main ornamental part of chrysanthemum plants, consists of ligulate marginal ray florets, an attractive corolla (petals), and radially hermaphroditic disc florets, but no stamens. In Asteraceae species, the zygomorphic ray florets evolved from the actinomorphic disc florets. During this process, the zygomorphic ligulate corolla arose and the stamens were aborted. Although molecular genetic research has clarified ray floret development to some extent, the precise molecular mechanism underlying ray floret development in chrysanthemum remained unclear. Results A CYC2-like gene, Cyc2CL, was cloned from C. morifolium ‘Fenditan’. Subsequent analyses revealed that the alternative splicing of Cyc2CL, which occurred in the flower differentiation stage, resulted in the production of Cyc2CL-1 and Cyc2CL-2 in the apical buds. Prior to this stage, only Cyc2CL-1 was produced in the apical buds. A fluorescence in situ hybridization analysis of labeled Cyc2CL-1 and Cyc2CL-2 RNA indicated that Cyc2CL-2 was first expressed in the involucre tissue during the final involucre differentiation stage, but was subsequently expressed in the receptacle and floret primordia as the floral bud differentiation stage progressed. Moreover, Cyc2CL-2 was highly expressed in the inflorescence tissue during the corolla formation stage, and the expression remained high until the end of the floral bud differentiation stage. Furthermore, the overexpression of Cyc2CL-1 and Cyc2CL-2 in transgenic Arabidopsis inhibited stamen and petal development. Therefore, both Cyc2CL-1 and Cyc2CL-2 encode candidate regulators of petal development and stamen abortion and are important for the ray floret development in chrysanthemum. Conclusion In this study, we characterized the alternatively spliced transcripts of the CYC2-like gene that differ subtly regarding expression and function. The data presented herein will be useful for clarifying the regulatory mechanisms associated with the CYC2-like gene and may also be important for identifying the key genes and molecular mechanisms controlling the development of ray florets in chrysanthemum.

Made available in DSpace on 2021-06-25T10:15:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Mapanioideae (besides Cyperoideae the other subfamily of Cyperaceae) is divided in two tribes (Hypolytreae and Chrysitricheae) in which the reproductive units (spicoids) are interpreted either as flowers or as inflorescences. Little is known about the ontogenetic factors operating during the spicoid development and how they may contribute to the spicoid final morphology. In this context, we studied the spicoid development in the monotypic genus Diplasia and compared it with other developmental studies in Mapanioideae. Our results show that the main developmental variations are related to the sequence of organ formation, the size and shape of the spicoid primordium and the influence of the subtending bract constraints in different sides of such primordium. Our hypotheses to explain the spicoid’s morphological variation in Mapanioideae are: that size and shape of the spicoid primordium contribute to changes in the number and position of organs to be formed, and spatial constraints of the subtending bracts influence the reduction of adaxial parts. Developmental features of the spicoids showed to be stable within the tribe. Our observations of the nature of spicoids in Diplasia give arguments for both, floral and inflorescence interpretations, sustaining the uncertainty towards a satisfactory definition. The difficulty in distinguishing the boundaries of flower and inflorescence nature in the spicoids raised a third hypothesis where the spicoid could be interpreted as a de novo structure, due to a reorganisation of regulatory genes resulting in a single unit with flower and inflorescence features combined, considered a synapomorphy of Mapanioideae. Departamento de Botânica Instituto de Biociências Universidade de São Paulo Departamento de Botânica Instituto de Biociências Universidade Estadual Paulista (UNESP), Caixa Postal 199 Jodrell Laboratory Royal Botanic Gardens Departamento de Botânica Instituto de Biociências Universidade Estadual Paulista (UNESP), Caixa Postal 199

Background Propagation of cuttings is frequently used in various plant species, including blueberry, which shows special root characteristics that may hinder adventitious root (AR) formation. AR formation is influenced by various factors, and auxin is considered to play a central role; however, little is known of the related regulatory mechanisms. In this study, a comparative transcriptome analysis of green cuttings treated with or without indole-butyric acid (IBA) was performed via RNA_seq to identify candidate genes associated with IBA-induced AR formation. Results Rooting phenotypes, especially the rooting rate, were significantly promoted by exogenous auxin in the IBA application. Blueberry AR formation was an auxin-induced process, during which adventitious root primordium initiation (rpi) began at 14 days after cutting (DAC), root primordium (rp) was developed at 21 DAC, mature AR was observed at 28 DAC and finally outgrowth from the stem occurred at 35 DAC. Higher IAA levels and lower ABA and zeatin contents might facilitate AR formation and development. A time series transcriptome analysis identified 14,970 differentially expressed genes (DEGs) during AR formation, of which there were 7467 upregulated and 7503 downregulated genes. Of these, approximately 35 candidate DEGs involved in the auxin-induced pathway and AR formation were further identified, including 10 auxin respective genes (ARFs and SAURs), 13 transcription factors (LOB domain-containing protein (LBDs)), 6 auxin transporters (AUX22, LAX3/5 and PIN-like 6 (PIL6s)) and 6 rooting-associated genes (root meristem growth factor 9 (RGF9), lateral root primordium 1 (LRP1s), and dormancy-associated protein homologue 3 (DRMH3)). All these identified DEGs were highly upregulated in certain stages during AR formation, indicating their potential roles in blueberry AR formation. Conclusions The transcriptome profiling results indicated candidate genes or major regulatory factors that influence adventitious root formation in blueberry and provided a comprehensive understanding of the rooting mechanism underlying the auxin-induced AR formation from blueberry green cuttings.

Blue light is an important environmental factor that induces mushroom primordium differentiation and fruiting body development. Although blue-light treatment has been applied for the production of oyster mushroom (Pleurotus ostreatus), the blue-light response mechanisms of P. ostreatus still remain unclear. In the present study, we exposed the primordium of P. ostreatus to blue-light, red-light, and dark conditions for 7 days. Subsequently, comparative transcriptomics analysis of the stipe, pileus, and gill under the three light conditions was performed to reveal the gene expression response mechanism of P. ostreatus to blue light and red light. The results showed that blue light enhanced the growth and development of all the three organs of P. ostreatus, especially the pileus. In contrast, red light slightly (non-significantly) inhibited pileus growth. When compared with red-light and dark treatments, blue-light treatment significantly upregulated gene expression involved in glycolysis/gluconeogenesis, the pentose phosphate pathway and the peroxisome in the pileus, but not in the gill or stipe. Most of the glycolysis and pentose phosphate pathway genes were upregulated in the pileus by blue light. When compared with dark treatment, red-light treatment downregulated the expression of many respiration metabolism genes in the pileus. These results revealed that blue light enhanced the activation of glycolysis and the pentose phosphate pathway, whereas red light weakened glycolysis and pentose phosphate pathway activation. The conclusion can be drawn that blue light improved P. ostreatus fruiting body (particularly, the pileus) growth rate via enhancement of glycolysis and the pentose phosphate pathway.

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.

Morphogenesis of pycnidium of Truncatella angustata BPF5 has been studied under in vitro culture on l-arabinoze medium. A single stalked, lemon shaped and papillate initial cell seems to take part in the formation of pycnidium. The apical papilla of initial cell elongates so as to give the initial a central position flanked by hyphae. This single initial by successive divisions becomes an oval pycnidial primordium (trichomeristogenous). Further enlargement of pycnidium seems to take place due to mechanical force exerted by successive conidial formation. At maturity, the pycnidium is melanized. Some primordia abort leaving behind ghost pycnidia in the central disc. The minimal supplemented media like l-arabinoze medium reduce vegetative growth but induce early pycnidial formation thus requiring less teasing operations during microscopy. This in turn avoids damage of fragile young structures and facilitates study of developmental stages. This is the first report of early stages of pycnidial development under in vitro condition in any of the Coelomycetous fungus.