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Qualification of sugars by Thin-layer Chromatography during the induction and development of PLBs in C. tigrina. Fructose, glucose, xylose, maltose and sucrose were used as markers.
Source publication
Tissue culture techniques have been employed for orchid mass propagation by means of the morphogenetic route of protocorm like-bodies (PLBs). This study aimed to analyze and compare Indole-acetic acid (IAA) and sugar endogenous levels in protocorm-like bodies (PLBs) induction and development in Cattleya tigrina, in order to better understand this p...
Context in source publication
Context 1
... analysis of soluble sugars in the different cultivation times, during PLB induction and development, showed the presence of sugar alcohols, especially glucose, fructose and sucrose (Figure 3), in all culture times. Regeneration from organogenesis or somatic embryogenesis is well known, and it is known that regeneration is strongly correlated with the concentration of endogenous hormones or hormones externally applied to the culture medium (Grzyb, Kalandyk, Waligórski, & Mikuła, 2017). IAA, the main natural representative of the auxin group, is responsible for regulating plant growth and development, besides being related to a wide variety of plant responses (Sairanen et al., ...
Citations
... The increased carbohydrate content in explants facilitated improved propagation, as evidenced by enhanced fresh and dry weight, length, volume, number of plantlets, and leaf area, with a positive correlation observed between carbohydrate content and growth parameters across all systems in these two media (Fig. 9). Similarly, de Conti et al. (2018) identified a correlation between increased soluble sugars in PLBs of Cattleya tigrina and the presence of sucrose in the culture medium [51]. Similar findings were reported during PLBs proliferation of Phalaenopsis amabilis [18], germination of Dactylorihza umbrosa [52], somatic embryogenesis of walnut [53,54] and organogenesis of Cedrela fissilis [37], where high sugar content in samples was associated with elevated carbon concentrations in the culture media. ...
Background
Micro-propagation is the primary technique for the mass propagation of greenhouse orchids. However, various factors, including culture media and cultivation systems, influence the scaling-up and efficient commercialization of in vitro techniques. The utilization of liquid cultivation systems and bioreactors are relatively cost-effective and has attracted significant attention for mass production. In this study, we evaluated the effects of eight culture media, in both semi-solid and liquid forms, on the growth of in vitro mini-plantlets of Phalaenopsis orchids. We subsequently assessed the performance of four selected media: half-strength modified Murashige and Skoog (1/2 MMS), modified FAST (MFAST), and two simplified media (SM1 and SM2), across four types of cultivation systems, including semi-solid media in glass jars, liquid media in a permanent immersion system using Erlenmeyer flasks on a shaker (PIS), and a temporary immersion system (TIS) in two forms: FA-Bio bioreactor (TIS-FA-Bio) and RITA® bioreactor (TIS-RITA®).
Results
The results indicated that the optimal culture medium for orchid growth depends on the cultivation system used. Among the eight evaluated semi-solid and liquid media, the highest plant growth was observed in the SM2 medium, attributed to the presence of additives (banana powder and activated charcoal), two types of sugar (with high carbon content), and small amounts of nickel and ascorbic acid. The high carbon content in the SM2 liquid medium, combined with the temporary immersion explants in a large volume of medium in the TIS-RITA®, resulted in the highest total carbohydrate content and enhanced plant growth. The maximum quantum efficiency of photosystem II (Fv/Fm) and the number of plantlets per liter in SM2 semi-solid medium were higher than those in the 1/2 MMS semi-solid medium (control), leading to a 61.6% reduction in production costs. Furthermore, the number of plantlets per liter in the TIS-FA-Bio containing SM2 (10.8 USD) was higher than in TIS-RITA® with 1/2 MMS (39.3 USD), resulting in a 72.5% decrease in production costs due to a lower volume of medium and the use of inexpensive equipment.
Conclusions
We recommend the use of SM2 in TIS-FA-Bio and TIS-RITA® for economical and efficient mass propagation of Phalaenopsis orchid.
... The appearance of starch grains in the cells of the L. loeselii protocorms was also evident (Figure 2). The deposition of starch grains also applied to the PLBs, as was previously described for leaf-induced PLBs of Cattleya tigrina, for example [61]. In the case of the PLBs of L. loeselii, we also noticed the deposition of starch grains that resembled those that were found in the protocorms ( Figure 6). ...
Liparis loeselii (L.) Rich, an endangered member of the Orchidaceae family, is found in alkaline fens. With the declining populations of L. loeselii, there is a pressing need to reintroduce this species in Central Europe. As in vitro germination is a crucial tool for obtaining plants for introduction into the environment, we looked at the morphological changes occurring during the early stages of L. loeselii development in vitro. As the early stages of orchid development, especially the protocorm stage, are thought to be responsible for SAM formation and the initiation of symbiotic association, we focused on cell wall elements whose epitopes have been found in similar processes in other species: the extensin and pectin rhamnogalacturonan I (RG-I) side chain epitopes. We addressed the following questions: Does the cell wall of L. loeselii change its composition during the early stages of development, as noted in other species? Are there noticeable similarities in the cell wall to organs of different species whose function is to contact microorganisms? Are there regularities that allow the recognition of individual structures on this basis? Immunolocalization revealed changes in the distribution of certain extensins (JIM11 and JIM20) and RG-I (LM5 and LM6) side chain epitopes. Extensins, a type of cell wall protein, were observed during the initial stages of the formation of PLB and the shoot apical meristem of protocorms and PLBs. RG-I, on the other hand, was found to play a significant role in the development of the protocorm and PLB. In pseudobulbs, which appeared on the protocorms, extensins occurred in their storage part. However, RG-I side chains (1→4)-β-galactans (LM5), and (1→5)-α-L-arabinans (LM6) were not found in pseudobulbs. We revealed that a common feature of protocorms and PLBs was an increased amount of extensins, which were detected with the JIM11 antibody, and pectins, which were detected with the LM5 antibody, that were present together, which may prove helpful in determining the identity of the induced structures and distinguishing them from pseudobulbs. Thus, our study unveiled the role of extensins and RG-I during the growth of protocorms and PLBs. We suggest that PLBs may mimic the wall remodelling that occurs in protocorms, which indicates that using cell wall components is an invitation to be colonised by a fungal partner. However, this needs to be tested in future research. The findings of this research can help interpret future studies on the propagation, acclimatisation, and introduction of L. loeselii into the natural environment.
... The determination of endogenous hormones during spontaneous morphogenesis of centaury revealed that the IAA concentration started to decrease after only two days of the experiment and decreased continuously until the day 21, when a significant increase was detected. The similar results were observed during the induction and development of protocorm-like bodies of Cattleya tigrina, when the authors linked the decrease in endogenous IAA content to intense cell division and differentiation (De Conti et al., 2018). As previously mentioned, the significantly increased IAA content in centaury root explants was detected on day 21, when the first regenerated adventitious shoots were noticed. ...
... In centaury root explants, total soluble sugars (TSS) began to increase after two days and continued to increase after four days. The strong increase was also observed after two days during organogenesis of Cedrela fissilis (Aragão et al., 2016) and two days after induction of protocorm-like bodies from leaf explants of C. tigrina (De Conti et al., 2018). This high endogenous TSS content indicates that the first days of culture are an important period for the uptake of exogenous sucrose from the culture medium. ...
Common centaury (Centaurium eryhtraea Rafn) is a medicinal plant species with vigorous morphogenic potential in vitro. The process of spontaneous shoot regeneration in a solid root culture is characteristic for this plant species. In this context, the aim of this work was to investigate the dynamic changes of endogenous phytohormones and carbohydrates content in root explants at different time points (0, 2, 4, 7, 14, 21, 28, and 60 days) during spontaneous centaury morphogenesis in vitro. Detailed analysis of cytokinins (CKs) showed that trans-zeatin (tZ) was the major bioactive CK at all time points. The corresponding riboside, tZ9R, was also determined in the majority of the identified transport forms, at all time-points. Further analysis of endogenous auxin revealed a significant increase in endogenous indole-3-acetic acid (IAA) after 21 days, when a huge jump in the ratio of IAA/bioactive CKs was also observed. The maximum total soluble sugar content was measured after 14 days, while a significant decrease was determined after 21 days, when the first regenerated adventitious shoots appeared. This undoubtedly indicates an increased energy requirement prior to the actual regeneration of the shoots. The obtained results indicate that the period from day 14 to day 21 involves the most dramatic disturbances in endogenous bioactive CKs, IAA and carbohydrate balance, which are very important and valuable factors for the onset of shoot regeneration.
... A positive correlation between soluble, storage, and total carbohydrate content and the fresh and dry weight, volume, and number of PLBs was also found (Fig. 4). These findings were consistent with those of de Conti et al. (2018), who observed a positive correlation between the increased soluble and storage carbohydrate content of PLBs and growth and development in Cattleya tigrina orchids. ...
... This, in turn, promoted PLBs proliferation across various culture systems ( Fig. 2a to d; Table 2). These findings align with previous studies, such as de Conti et al. (2018), which observed a correlation between increased soluble sugar content and the presence of sucrose in the culture medium in Cattleya tigrina orchids. Similar patterns were observed in studies on somatic embryogenesis of Medicago sativa (Horbowicz et al. 1995), somatic embryogenesis of walnut (Vahdati et al. 2006), and organogenesis of Cedrela fissilis (Aragão et al. 2016) where high sugar content in the samples was associated with elevated carbon concentration in the culture medium. ...
Plant growth regulators play a crucial role in the proliferation of protocorm-like bodies through somatic embryogenesis in Phalaenopsis orchids. Additionally, media components and cultural systems can impact their proliferation and reduce costs. This research aimed to enhance proliferation rates of orchid protocorm–like bodies and decrease costs of both media providing and culture systems. To achieve this, protocorm explants were cultivated in four different systems: solid, liquid permanent immersion (Erlenmeyer flasks on a shaker), and temporary immersion (RITA® and FA-Bio bioreactor, a new laboratory mini-bioreactor). The media used included half-strength Murashige and Skoog (1/2 MS), modified FAST medium (MFAST), and low-cost medium (LC). The solid medium contained thidiazuron at concentrations of 0, 1.5, or 3.0 mg L−1. Solid 1/2 MS without thidiazuron was the control. Protocorm-like bodies in solid LC and MFAST exhibited high chlorophyll and anthocyanin content. Their proliferation rates increased by 32.6% and 29.6%, respectively, while culture costs decreased by 45.2% and 43.1%, respectively, compared to solid 1/2 MS with 3.0 mg L−1 thidiazuron. The production of protocorm-like bodies and their carbohydrate content were significantly higher in MFAST and LC media, compared to 1/2 MS media across all culture systems. The RITA® bioreactor with liquid LC showed higher proliferation rates than the other treatments. Liquid LC reduced costs by 57.6% compared to solid LC, making it a cost-effective option for mass propagation. The FA-Bio bioreactor reduced costs by 70.5% compared to RITA® for laboratory research evaluations in liquid culture. MFAST media in FA-Bio bioreactors can be used commercially for cost-effective propagation of Phalaenopsis orchids.
... In Cattleya tigrine, a similarly significant rise of endogenous IAA content occurred when PLBs differentiated into shoots [53]. Besides the absolute contents, the transportation and distribution of auxin are crucial for PLB formation and shoot development [25]. ...
Background
Paphiopedilum, commonly known as slipper orchid, is an important genus of orchid family with prominent horticultural value. Compared with conventional methods such as tillers and in vitro shoots multiplication, induction and regeneration of protocorm-like bodies (PLBs) is an effective micropropagation method in Paphiopedilum. The PLB initiation efficiency varies among species, hybrids and varieties, which leads to only a few Paphiopedilum species can be large-scale propagated through PLBs. So far, little is known about the mechanisms behind the initiation and maintenance of PLB in Paphiopedilum.
Results
A protocol to induce PLB development from seed-derived protocorms of Paphiopedilum SCBG Huihuang90 (P. SCBG Prince × P. SCBG Miracle) was established. The morphological characterization of four key PLB developmental stages showed that significant polarity and cell size gradients were observed within each PLB. The endogenous hormone level was evaluated. The increase in the levels of indoleacetic acid (IAA) and jasmonic acid (JA) accompanying the PLBs differentiation, suggesting auxin and JA levels were correlated with PLB development. Gibberellic acid (GA) decreased to a very low level, indicated that GA inactivation may be necessary for shoot apical meristem (SAM) development.
Comparative transcriptomic profiles of four different developmental stages of P. SCBG Huihuang90 PLBs explore key genes involved in PLB development. The numbers of differentially expressed genes (DEGs) in three pairwise comparisons (A vs B, B vs C, C vs D) were 1455, 349, and 3529, respectively. KEGG enrichment analysis revealed that DEGs were implicated in secondary metabolite metabolism and photosynthesis. DEGs related to hormone metabolism and signaling, somatic embryogenesis, shoot development and photosynthesis were discussed in detail.
Conclusion
This study is the first report on PLB development in Paphiopedilum using transcriptome sequencing, which provides useful information to understand the mechanisms of PLB development.
... Leaves are the most common target tissue used for SEs induction in orchid (Jainol and Gansau, 2017). Different protocols have been developed for a number of orchid species through somatic embryogenesis using leaf explants (De Conti et al. 2018;Shen et al., 2018, Zanello andCardoso, 2019). However, orchid leaves are known to be recalcitrant to regeneration and possessed high oxidation rate (Temjensangba and Deb, 2005;Kaur and Bhutani, 2009). ...
Phalaenopsis amabilis is an Indonesian native orchid often used as parent to produce various orchid hybrids. However, this natural orchid is increasingly difficult to find growing naturally in the forest due to over-harvesting and destruction of its natural habitat. The objectives of this study were to investigate the effect of plant growth regulators (PGRs), light regime, and organic substances on the induction and regeneration of somatic embryos (SEs) of P. amabilis orchid. Root, stem, leaf, and protocorm explants were cultured on New Phalaenopsis (NP) medium supplemented with thidiazuron (TDZ) (0.0, 1.0, 2.0, 3.0 mg L-1) in combination with α-napthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole acetic acid (IAA) with concentrations of 1.0, 2.0, and 3.0 mg L-1. Light and dark conditions were tested for their effectiveness to induce the formation of SEs, and the resulting SEs were cultured on NP medium supplemented with various organic substances (banana, bean sprout, tomato, and potato extracts) with concentrations of 50, 100, 150, 200, and 250 g L-1. Results showed that the highest number of SEs (36.45 ±0.26 embryos) was found in stem explants cultured in NP medium supplemented with 3.0 mg L-1 TDZ and 1 mg L-1 NAA. When explants were cultured in dark conditions, the number of SEs significantly increased with the highest number of SEs achieved in stem explants culture in 3.0 mg L-1 TDZ and 1.0 mg L-1 NAA. NP medium supplemented with 150 g L-1 tomato extract was the most effective medium for growth of SEs-derived plants. Seedlings of this treatment produced an average of 4.20 ±0.17 leaves and 3.20 ±0.11 roots after 12 weeks of culture. In conclusion, SEs can be produced effectively from stem explants with a combination of 3.0 mg L-1 TDZ and 1.0 mg L-1 NAA, one month early in dark conditions, and regenerated on NP medium with addition of tomato extract.
... For histological analysis, a procedure based on the work described by De Conti et al. (2018) was applied. Samples ~ 5 mm in length were fixed in 2.5% paraformaldehyde in 0.2 M (pH 7.2) phosphate buffer overnight. ...
Cattleya tigrina is an endangered Brazilian native orchid with ornamental potential. Biotechnological tools based on tissue culture techniques are effective for its mass propagation and conservation. Protocorm-like bodies (PLBs) were induced from leaves in culture medium supplemented with plant growth regulators, and generated complete in vitro plants. To deepen on the mechanism underlying this morphogenetic route, this study is aimed to identify and characterize differentially expressed proteins during C. tigrina PLBs development. Leaves were inoculated in in culture medium Murashige and Skoog (MS) supplemented with 9 μM Thidiazuron, and PLBs t were collected after 30, 60 and 100 days in culture. Proteomic analysis performed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry detected 122, 132 and 447 protein after 30, 60 and 100 days, respectively. During PLBs development, 36 proteins were differentially expressed. Proteins related to energy and carbohydrate metabolism, cell proliferation, protein processing, and secondary metabolism and phytohormones were assessed showing their relevance in the process of development of PLBs. Many proteins related to metabolic and energy processes were identified after 60 and 100 days in culture, suggesting their role in which is cell division and differentiation. Enolase was only present after 100 days in culture, thus being a candidate as a molecular marker in this developmental phase.
... The supplementation of sucrose or sugar is essential to stimulate axillary bud growth in vitro [47]. Sucrose in the cultivation media may increase the endogenous levels of carbohydrates, such as sucrose, glucose, fructose, and starch [48,49], and plant hormones, such as IAA, isopentenyl adenine riboside 5 -monophosphate, isopentenyl adenine riboside, isopentenyl adenine, zeatin riboside 5 -monophosphate, and zeatin riboside [50], that are important for various phases of plant growth. Starch accumulation is a prerequisite for shoot initiation in numerous plants [51]. ...
This study aimed to investigate the impact of plant growth regulators, sucrose concentration, and the number of subcultures on axillary shoot multiplication, in vitro flowering, and somaclonal variation and to assess the phytochemical composition, antioxidant capacity, and enzyme inhibitory potential of in vitro-established callus, somaclonal variant, and normal green shoots of Catharanthus roseus. The highest shoot induction rate (95.8%) and highest number of shoots (23.6), with a mean length of 4.5 cm, were attained when the C. roseus nodal explants (0.6-1 cm in length) were cultivated in Murashige and Skoog (MS) medium with 2 µM thidiazuron, 1 µM 2-(1-naphthyl) acetic acid (NAA), and 4% sucrose. The in vitro flowering of C. roseus was affected by sucrose, and the number of subcultures had a significant effect on shoot multiplication and somaclonal variation. The highest levels of phenolics and flavonoids were found in normal green shoots, followed by those in somaclonal variant shoots and callus. The phytochemicals in C. roseus extracts were qualified using liquid chromatography-tandem mass spectrometry. A total of 39, 55, and 59 compounds were identified in the callus, somaclonal variant shoot, and normal green shoot tissues, respectively. The normal green shoot extracts exhibited the best free radical scavenging ability and reducing power activity. The strongest acetylcholinesterase inhibitory effects were found in the callus, with an IC50 of 0.65 mg/mL.
... Thanks and appreciation to my daughter, Ms. Coerulea Hegde, for her voluntary help extended in coordinating with M/S Seair Exim Solutions and analysing the enormous data of import and export of orchids from various ports of India. Almeida et al. (2017), Caovila et al. (2016), Cardoso and Ono (2011), Cavalcante Martini et al. (2001), Corrêa Pereira et al. (2011, Da Silva et al. (2014), Da Silva Júnior et al. (2012), 2013, De Carvalho et al. (2018), De Conti et al. (2018), De Melo Ferreira et al. (2017, De Menezes Gonçalves et al. (2012, Dezan et al. (2012), Dignart et al. (2009), Dorneles and Trevelin (2011), Endres Júnior et al. (2014), Favetta et al. (2017), Galdiano Júnior et al. (2011,b, 2013 Acknowledgements We are grateful to our colleagues at the Yeomiji Botanic Garden, Jeju, Korea, for providing plant material. ...
The history of the creation and development of the orchid collection of the Stock greenhouse of the Tsitsin Main Botanical Garden of the Russian Academy of Sciences is shown. The first orchids were obtained from the botanical gardens and flower firms of Germany in 1948; the number of natural species was 140. A complete list of preserved German orchid species, including 65 species from the 29 genera, is given. Further stages forming of the collection of orchids are described. Now the collection of tropical orchids of the Stock greenhouse includes 1328 species, subspecies, and varieties of orchids, belonging to 250 genera, and 69 species that are included in various categories of IUCN. The main areas of research with living collection plants are listed. These include the inventory and maintenance of an electronic database, the study of orchid adaptation to cultivation in greenhouses (morphology, phenology, symbiotrophism), reproductive biology issues (embryology, artificial pollination, seed morphometry), cryopreservation, and in vitro cultivation.
Orchids are an important part of plant biodiversity on this planet due to their high variability among species and their habitats. South America represents more than thirty percent of all known orchid species, Colombia, Ecuador, Brazil, Peru, and Bolivia being among the richest countries in the world in terms of orchid biodiversity. Nevertheless, concerning the orchid conservation status, in Colombia precisely orchids occupy the unlucky first place as the plant family with the highest number of threatened species. There is a similar situation in the rest of the South American countries. The two main threats to orchid survival are both anthropogenic: the first one is deforestation, and the second largest threat to orchids is collection from the wild. One desirable action to safeguard these endangered species is to develop procedures that make possible their massive propagation, which would provide material for both environmental restoration and commercial purposes avoiding extractions from nature. Likewise, the development of systems that allow the ex situ conservation of orchid germplasm is imperative. This chapter reviews the progresses of different in vitro approaches for orchid propagation and germplasm conservation, safeguarding the genetic biodiversity of these species. Several study cases are presented and described to exemplify the protocols developed in the Botanical Institute of Northeast (UNNE-CONICET) for propagating and long-term storing the germplasm of wild orchids from Argentina (Cattleya lundii, Cohniella cepula, C. jonesiana, Gomesa bifolia, Aa achalensis, Cyrtopodium brandonianum, C. hatschbachii, Habenaria bractescens). Moreover, it has been attempted to put together most of the available literature on in vitro propagation and germplasm conservation for South American orchids using different explants and procedures. There are researches of good scientific quality that even cover critical insights into the physiology and factors affecting growth and development as well as storage of several orchid materials. Moreover, studies are still necessary to cover a major number of South American species as well as the use of selected material (clonal) for both propagation and conservation approaches.
