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Induction of in vitro organogenesis in the root culture of Centaurium erythraea. a Detail of the centaury root with longitudinal rupture in the epidermis, b the centaury root with regenerated adventitious buds with a well-developed first leaf (arrows) and second leaf, c detail of the centaury root with fully developed adventitious buds, and d four-week-old centaury shoots in vitro on MS hormonefree medium
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Phytohormones are important regulators of numerous developmental and physiological processes in plants. Spontaneous morphogenesis of the common centaury (Centaurium erythraea Rafn.) is possible on nutrition medium without addition of any plant growth regulator depending solely on endogenous phytohormone levels. Thus, this plant species represents a...
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... CK nucleobases are considered the bioactive CK forms in plants [153]. They are the least abundant of all CK groups [154,155], with kohlrabi seedlings being no exception [60,63], and with transZ mostly contributing throughout all developmental stages studied [64]. The strong increase of these bioactive CK forms argues for their specific role in controlling important processes in young developing kohlrabi seedlings, together with shaping the shoot apical meristem [156]. ...
Kohlrabi (Brassica oleracea var. gongylodes) is a vegetable cultivated for its edible stem tuber. Although valued for its nutritional properties and tolerance to abiotic stress, kohlrabi is one of the least studied brassicas. In this review, we summarize the results of our decade-long research on in vitro morphogenesis of kohlrabi, starting from 2013. Protocols for efficient in vitro regeneration with minimal requirements for external application of plant growth regulators (PGRs) have been developed, both by somatic embryogenesis (SE) and by de novo shoot organogenesis (DNSO). Efficient regeneration by direct SE was achieved using immature zygotic embryos as explants incubated on PGR-free media, and the production process was maintained in culture thanks to highly efficient secondary SE. Conversely, efficient regeneration by indirect DNSO was achieved using entire seedlings as explants and adding only cytokinin (CK) without the need for exogenous auxin. Comprehensive phytohormone analyses revealed that different exogenously applied CKs differentially affected the composition of endogenous phytohormones and induced changes in the expression of cell cycle-related genes and other genes involved in the organogenic response. The addition of high sucrose concentrations to the nutrient media failed to induce the formation of stem tubers in in vitro culture, but revealed a complex interaction with exogenously applied CKs, interfering with both the endogenous phytohormonome and the expression of organogenesis-related genes. Our work has provided substantial biotechnological advances in the field of in vitro regeneration of kohlrabi, as well as in understanding the underlying phytohormonal regulation. The review aims to introduce kohlrabi to the scientific community as a model system for both basic and applied research, while we continue seeking answers to the outstanding questions and trying to pave the way for the development of more resistant varieties.
... CK free bases are generally considered to be bioactive CK forms [57] and usually account for only a small portion of total CKs in various plant species [58,59], including kohlrabi seedlings grown in vitro [36,39]. The most abundant CK free base in this study was transZ, which is usually considered the most active of all [60][61][62]. ...
The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1–T4), additionally including the effects of cytokinins (CKs)—trans-zeatin (transZ) and thidiazuron (TDZ)—and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA20 increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses.
... Existing research data show the types and concentrations and combinations of endogenous PGRs influence the callus induction frequency and callus morphogenesis [28]. In the process of culturing the roots and stem segments of Centaurium erythraea, IAA content is higher than that of cytokinins in the roots, and it is also twice the IAA contents in the stem [29]. These PGRs may significantly promote the expansion of dividing cells in explants and callus initiation. ...
Agapanthus praecox has become a burgeoning variety in the flower market due to its high ornamental value with unique large blue-purple inflorescence. For rapid entering into the market, tissue culture technology or organogenesis has an attractive application over the conventional reproduction approach. In this study, a highly efficient protocol based on indirect organogenesis has been successfully established for A. praecox subsp. orientalis ‘Big Blue’. Two types of explants, root tips versus root segments, were compared for callus induction frequency in response to the induction culture media. The induction media contain Murashige and Skoog’s (MS) Basal Salt supplemented with various concentrations of picloram (PIC), 2,4-Dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), kinetin (KT) and naphthalene acetic acid (NAA). Of the two types of explants, root tips were found to be more effective for callus induction than root segments. Among the induction media tested, the highest callus induction rate (100.00%) was achieved when cultured on MS supplemented with 2.0 mg/L PIC, 1.5 mg/L KT and 0.1 mg/L NAA, which was probably accredited to higher endogenous phytohormone contents, especially of 3-indoleacetic (IAA). The optimal medium for callus proliferation was MS + 1.0 mg/L PIC + 1.0 mg/L 6-BA + 0.4 mg/L NAA, and the fresh weight increased by 72.74%. After being transferred onto the adventitious bud induction medium for 25 days, shoots were dedifferentiated from the surface of the flourishing callus, which then developed to the plantlet with roots in 90 days. The plantlets were transplanted in a greenhouse with a survival rate of 92.86%. This study innovatively established an indirect organogenesis tissue culture system of A. praecox with roots as explants, which provided a practical reference in its application.
... The appropriate CKs type for MNs induction and differentiation was genotype dependent, for example, BA was recommended for MNs induction of Radiata pine (Aitken-Christie et al.1988) and Eucalyptus globulus (Trindade and Pais 2003), TDZ for Pelargonium × hortorum, Pelargonium × domesticum (Haensch 2004) and Acacia mangium (Xie and Hong 2001), CPPU for Eucalyptus botryoides (Ito et al.1996). According to previous studies, high endogenous CKs promoted cell fate transition from callus cells to de novo shoot organogenesis by mediating up-regulated expression of WUS gene (Trifunović-Momčilov et al. 2016;Gordon et al. 2007;Meng et al. 2017;Xiao et al. 2018). On the contrary, some studies had emphasized that a proper auxin/CK ratio was key factor for effective shoot regeneration, instead of sole CK or auxin level (Cheng et al. 2010;Zhang et al. 2008). ...
Tree peony ( Paeonia sect. Moutan ) is an economically important multipurpose woody plant in terms of its medical, ornamental and oil values, but its breeding and industrial development are severely limited due to inefficient traditional propagation methods and existing in vitro regeneration systems. Meristematic nodules (MNs) are an attractive alternative to solve this problem. This study first presented a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ via MN culture with four consecutive steps, including embryogenic callus (EC) formation, MN induction and leaf cluster differentiation, shoot elongation, rooting and acclimatization. The highest EC induction rate (81.25%) was achieved when cotyledons were cultured on modified Murashige and Skoog (mMS) medium with 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 5.37 µM α-naphthylacetic acid (NAA) for 30 days. The optimal MN induction rate (100%) and leaf cluster differentiation rate (45.83%) were obtained when ECs were cultured on modified woody plant medium (mWPM) supplemented with 2.02 µM CPPU + 2.27 µM thidiazuron (TDZ) for a subculture time of 10 days. The combination of 1.29 µM 6-benzyladenine (BA) + 0.58 µM gibberellin (GA 3 ) yielded the best shoot elongation (13.40 shoots per nodule), rooting rate (43.33%) and consequently survival rate (45.83%). The study will be beneficial to the mass propagation, breeding and genetic improvement of tree peony.
... The process of ISE from the centaury leaf explants is illustrated in Figure 2 and the Supplementary video. Since the two main systems for the SE induction in centaury, DSE from root culture [35] and ISE from leaf culture [28], differ in their requirements regarding the addition of PGRs for the SE induction, the endogenous contents of different CKs, IAA, salicylic acid (SA) and abscisic acid (ABA) were analyzed in the roots and shoots of the in vitro grown plants as the sources of explants [71]. It was found that the total amount of endogenous CKs was 1.4 times higher in shoots as compared to shoots, but inactive or weakly active N-glucosides were the predominate CK forms in both organs, whereas free bases and O-glucosides represented only a small portion of the total CK pool. ...
... Planned investigation considering the determination of endogenous levels of phytohormones at different stages of somatic embryo development aims to relate these levels to the embryogenic capacity of centaury root and shoot explants. The processes of SE from centaury root and leaf cultures are presented in Figure 3. Since the two main systems for the SE induction in centaury, DSE from root culture [35] and ISE from leaf culture [28], differ in their requirements regarding the addition of PGRs for the SE induction, the endogenous contents of different CKs, IAA, salicylic acid (SA) and abscisic acid (ABA) were analyzed in the roots and shoots of the in vitro grown plants as the sources of explants [71]. It was found that the total amount of endogenous CKs was 1.4 times higher in shoots as compared to shoots, but inactive or weakly active N-glucosides were the predominate CK forms in both organs, whereas free bases and O-glucosides represented only a small portion of the total CK pool. ...
... The roots were characterized with higher IAA content but lower IAA/free CK bases ratio and lower ABA content in comparison to roots. The most significant difference, however, was a 44-fold higher SA content in the roots as compared to shoots [71]. It is not clear which of these differences allows spontaneous SE from roots but not from shoots; for example, Quiroz-Figueroa et al. [2] demonstrated that very low concentrations of salicylates could induce cellular growth and enhance somatic embryogenesis in Coffea arabica. ...
Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum of secondary metabolites with confirmed healing properties. Centaury is an emerging model in plant developmental biology due to its vigorous regenerative potential and great developmental plasticity when cultured in vitro. Hereby, we review nearly two decades of research on somatic embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic embryos. When SE is initiated from centaury root explants, the process occurs spontaneously (on hormone-free medium), directly (without the callusing phase), and the somatic embryos are of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is indirect (preceded by callusing). Histological observations and culture conditions are compared in these two systems. The changes in antioxidative enzymes were followed during SE from the leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were analyzed using a variety of approaches. The newest and preliminary results, including centaury transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed as perspectives of centaury research.
... Fortunately, even almost twenty years ago centaury showed vigorous regenerative potential in vitro [93][94][95][96][97]. The most interesting and essential information is that spontaneous morphogenesis of centaury is possible on nutrition medium without addition of any plant growth regulator [98]. In addition, during the years, centaury has also been used for studies of numerous developmental processes [99][100][101]. ...
The production and breeding of plants with desired properties are possible by a fundamental biotechnological technique, genetic engineering. Applying and developing of genetic engineering procedures also enable preservation and improvement of plant species endangered in nature, including medicinal plant common centaury (Centaurium erythraea Rafn.). Numerous developmental processes in plants are controlled by cytokinins (CKs). The only so far known enzyme involved in CK catabolism is cytokinin oxidase/dehydrogenase (CKX). Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were successfully introduced into centaury root explants. Subsequently, the contents of endogenous CKs in AtCKX-overexpressing centaury plants grown in vitro were investigated. Simultaneous secondary metabolite analyses showed antibacterial and antifungal activity of transgenic centaury plants and suggested their use as potential producers of anti-cancer compounds. Considering that centaury can inhabit saline soils in natural habitats, following investigations included evaluation of salinity tolerance in vitro. All obtained and summarized results indicated that transgenic AtCKX centaury plants could serve as a suitable model for studies of numerous physiological and developmental processes under endogenous phytohormonal control.
... In the study carried out to analyze the endogenous hormones in the plant, the amount of total endogenous cytokinin on the shoots was determined 1.4 times more than the roots. Roots were reported to have higher IAA content than shoots [41]. Free cytokinins in the plant are carried from root to shoots [42]. ...
Tissue culture techniques in tomato, pepper and eggplant are important for the development of disease-resistant and high yielding varieties, which require a suitable regeneration protocol. Although shoot regeneration has been achieved by using different explants and cytokinin doses in Solanaceae species, very few studies have reported in vitro regeneration using root tissues. The current study is the first report to compare direct shoot regeneration capabilities using root node explants in three Solanaceae species (tomato, pepper, and eggplant) under three cytokinins (BAP, TDZ, and GA3) hormone. Plantlets were regenerated from the root node explants of tomato, pepper and eggplant in the media containing 0, 0.5, 1, 1.5 and 2mg/L of BAP, TDZ and GA3. Results revealed that the shoot regeneration of root node explants varied according to the species, cytokinins (BAP, TDZ and GA3) and doses of hormones. Among the species, the best shoot regeneration was observed in tomato followed by eggplant and pepper plants. While the shoot length was statistically significant in tomato, it was observed to be insignificant in pepper and eggplant. The highest number of root regeneration and root length was observed in tomatoes. The results obtained from the study will contribute to the development of successful/reproducible tissue culture protocols from roots node explants.
... N-glucosylation thus seems to be an evolutionarily recent mechanism to inactivate biologically active CKs. In vascular plants however, the abundance of CK N-glucosides is not proportional to the evolutionary age of the species and neither is their distribution with respect to monocotyledonous or dicotyledonous plants since the prevalence of N-glucosides occurs in a wide variety of evolutionarily distinct species such as Petunia hybrida, Nicotiana tabacum, Musa acuminata, Lilium elodie, Anthurium andreanum (Gajdošová et al., 2011), Arabidopsis thalina (Jiskrová et al., 2016;Šmehilová et al., 2016), Centaurium erythraea (Trifunović-Momčilov et al., 2016), Hordeum vulgare (Jiskrová et al., 2016), Raphanus sativus (Blagoeva et al., 2004), Solanum lycopersicum (Žižková et al., 2015) and others. The opposite is the case in e.g., Manihot esculenta, Zea mays, Triticum aestivum, Phragmites australis, Avena sativa (Gajdošová et al., 2011) with only a minor portion of CK N-glucosides in whole CK spectrum. ...
Cytokinin (CK) N-glucosides are the most abundant group of CK metabolites in many species; however, their physiological role in planta was for a long time perceived as irreversible storage CK forms only. Recently, a comprehensive screen showed that only vascular plants form CK N-glucosides in contrast to mosses, algae, and fungi. The formation of CK N-glucosides as biologically inactive CK conjugates thus represents an evolutionarily young mechanism for deactivation of CK bases. Even though CK N-glucosides are not biologically active themselves due to their inability to activate the CK perception system, new data on CK N-glucoside metabolism show that trans-zeatin (tZ) N7- and N9-glucosides are metabolized in vivo, efficiently releasing free CK bases that are most probably responsible for the biological activities observed in a number of bioassays. Moreover, CK N-glucosides’ subcellular localization as well as their abundance in xylem both point to their possible plasma membrane transport and indicate a role also as CK transport forms. Identification of the enzyme(s) responsible for the hydrolysis of tZ N7- and N9-glucosides, as well as the discovery of putative CK N-glucoside plasma membrane transporter, would unveil important parts of the overall picture of CK metabolic interconversions and their physiological importance.
... Values that are even considerably lower than those presented here, have been reported for seedlings of basal-media grown kohlrabi (Brassica oleracea var. gongylodes) 40 and for in vitro grown centaury (Centaurium erythraea Rafn.) 41 . Also, higher values for total endogenous CKs in the roots of potato compared to the shoots, are consistent with the observation that roots are generally more abundant in CKs, being their primary site of synthesis 42 . ...
... Cytokinin nucleobases comprised a low proportion of total CK levels in both shoots (1.8%) and roots (0.6%) of potato, while N-glucosides, which are widely considered to be irreversibly inactive, accounted for 94% of the total CK pool in the shoots and 82% in the roots (Fig. 2). A similar pattern was found in CK composition of C. erythraea 41 , indicating that the utilisation of only a small portion of total CKs is a common phenomenon at least among asterids, if not in broader groups of plants. ...
... Unlike nucleobases, CK ribosides were remarkably more abundant in potato roots than in shoots (Fig. 4). The same has been observed in C. erythraea 41 . The most abundant CK riboside in potato shoots was tZR, and it was also remarkably abundant in the roots. ...
A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.
... In this study, five C. erythraea genotypes of 2x and five of 4x ploidy level were tested for their in vitro regeneration ability. The use of root explants for in vitro regeneration in centaury, as well as high regeneration potential of the centaury roots was described earlier (Subotić et al., 2004;2006;Subotić and Grubišić, 2007;Trifunović-Momčilov et al., 2016). The authors showed that morphogenesis in vitro in centaury root culture is direct, without callus phase, and spontaneous, on medium without addition of any plant growth regulators (Subotić et al., 2004;Trifunović-Momčilov et al., 2016). ...
... The use of root explants for in vitro regeneration in centaury, as well as high regeneration potential of the centaury roots was described earlier (Subotić et al., 2004;2006;Subotić and Grubišić, 2007;Trifunović-Momčilov et al., 2016). The authors showed that morphogenesis in vitro in centaury root culture is direct, without callus phase, and spontaneous, on medium without addition of any plant growth regulators (Subotić et al., 2004;Trifunović-Momčilov et al., 2016). Roots provide an excellent source of explants for mass clonal propagation because of their efficiency in the multiplication of a large number of genetically identical plants, as well as due to their easy maintenance and manipulation in vitro (Pant et al., 2010;Vinocur et al., 2000). ...
The impact of ploidy level on both the regenerative potential under in vitro conditions and the production of major bioactive specialized metabolites, such as iridoids and xanthones, was examined in Centaurium erythraea Rafn. Shoot regeneration frequency was genotype dependent, but not affected by explant ploidy level. In most cases, the regenerated shoots of autotetraploid (4x) genotypes were more robust than diploid (2x) ones. Regeneration efficiency of root explants declined from the apical to the basal root segment. Shoot and root biomass production of two month-old plants was not significantly different between 2x and 4x genotypes. Both 4x and 2x genotypes were characterized by the predominance of secoiridoid glucoside gentiopicrin in shoots and roots, which is followed by sweroside and swertiamarin. Loganic acid, loganin and secologanin were much less abundant. Methylbellidifolin was the major xanthone in both shoots and roots. Diploid plants showed higher biosynthetic capacity for the production of secoiridoids and xanthones in both shoots and roots. Results highlight a higher potential of diploid C. erythraea genotypes for biotechnology-based sustainable production of secoiridoids in comparison to tetraploid genotypes.
