Immersion Systems Research Articles

A temporary immersion system for mass micropropagation of pitahaya (Hylocereus undatus)

Scaling-up techniques in temporary immersion systems are an alternative for commercial micropropagation. In vitro propagation of pitahaya (Hylocereus undatus) using temporary immersion with liquid culture medium improves micropropagation efficiency compared to the conventional method in semisolid medium. The objective of this work was to evaluate the efficiency of traditional culture and temporary immersion during micropropagation of pitahaya to facilitate the rapid establishment of new commercial plantations of high genetic and phytosanitary quality. Semisolid culture, liquid media in partial immersion and temporary immersion in an Ebb-and-Flow bioreactor were evaluated. Also, in temporary immersion, different immersion frequencies (every 4, 8, 12, and 16h) and culture densities (5, 10, 15 and 20 explants per bioreactor) were evaluated. For the multiplication stage, new shoots and length were recorded per explant at 45 d of in vitro culture and in the acclimatization stage, the survival percentage was determined at 30 d of greenhouse cultivation. A temporary immersion of 2min every 4h and 15 explants per bioreactor was the best culture system, obtaining on average 10.7 shoots per explant with a length of 1.9cm. No significant differences were observed among treatments during acclimatization, obtaining survival percentages of 98%-100%. This study reports for the first time a protocol for scaling-up techniques in temporary immersion for commercial micropropagation of pitahaya (and for any species of the Cactaceae family) and its establishment in a productive plantation.

REGENERACIÓN DE PLANTAS DE Agave marmorata Roelz EN SISTEMAS DE INMERSIÓN TEMPORAL, MEDIANTE ORGANOGÉNESIS Y EMBRIOGÉNESIS SOMÁTICA

<p><strong>Background.</strong> The genus Agave has social, cultural, environmental and economic importance in Mexico. Approximately 15 species of agave are used as raw material for the production of mezcal, among them is the <em>Agave marmorata</em> Roelz, which is considered the longest-lived agave for the preparation of this drink because its maturation period ranges from 20 to 35 years. Agave plant tissue culture is a technique that allows obtaining plants by organogenesis (O) and / or somatic embryogenesis (ES) in a short period of time. The quality and quantity of plants propagated by these methods can be favored with the use of Temporary Immersion Systems (SIT). <strong>Objective.</strong> Establish a protocol for the regeneration of <em>A. marmorata</em> seedlings in SIT, with the methods of direct organogenesis and indirect somatic embryogenesis. <strong>Methodology</strong>. Two regeneration methods, direct organogenesis and indirect somatic embryogenesis, were evaluated. For the first, the effect of the frequency and number of explants grown in SIT in the sprouting and growth stages of the explant was determined. For the second, the effect of the number of explants grown in SIT on the percentage of germination and the growth rate of somatic embryos was evaluated. <strong>Results</strong>. The best treatment in the sprouting stage was obtained with 80 explants grown in SIT with an immersion frequency of 8 h and an immersion time of 2 min, with 84% sprouting and 17.5 sprouts / explant of 1 cm in height. The best growth of the explants originated from direct organogenesis was obtained with 180 cultured explants per flask with an immersion frequency of 8 hours and an immersion time of 2 min. On the other hand, in the somatic embryogenesis method, the highest percentage of embryo germination (62.2%) was achieved with 80 somatic embryos cultured in SIT with an immersion frequency of 8 hours. <strong>Implications.</strong> The results of this research contribute to a better understanding of the regeneration process of direct organogenesis and somatic embryogenesis in temporary immersion systems. This can help to scale the system commercially and to conserve germplasm. <strong>Conclusions</strong>. It was possible to establish an effective protocol for the regeneration of agave seedlings.</p>

Xanthones Production in Gentiana dinarica Beck Hairy Root Cultures Grown in Simple Bioreactors.

The hairy root clones of Gentiana dinarica cl-B, cl-D, cl-3, and cl-14 were cultivated in parallel in diverse simple bioreactors, including temporary immersion systems RITA® (TIS RITA®), bubble column bioreactors (BCB), and Erlenmeyer flasks (EF), and evaluated for biomass production and xanthone content. The obtained results showed that TIS RITA® and BCB containing ½ MS medium with 4% sucrose provided equally good growth conditions in which the majority of the clones displayed the higher percentage of dry matter (DM%), and xanthones norswertianin-1-O-primeveroside (nor-1-O-prim) and norswertianin production than those cultivated in EF. Thin and well branched hairy root clone cl-B grown in BCB for 7 weeks was superior regarding all growth parameters tested, including growth index (19.97), dry weight (2.88 g), and DM% (25.70%) compared to all other clones. Cl-B cultured in TIS RITA® contained the highest amount of nor-1-O-prim (56.82 mg per vessel). In BCB with constant aeration, cl-B accumulated the highest norswertianin content reaching 18.08 mg/vessel. The optimized conditions for cultivation of selected G. dinarica hairy root clones in highly aerated TIS RITA® and BCB systems contribute to the development of bioreactor technology designed for the large scale commercial production of xanthones nor-1-O-prim and norswertianin.

SETIS™ bioreactor increases in vitro multiplication and shoot length in vanilla (Vanilla planifolia Jacks. Ex Andrews)

Temporary immersion systems (TIS) are extensively used for the in-vitro regeneration of plants. These systems have been proposed as a suitable technique for propagating vanilla (Vanilla planifolia Jacks) for commercial purposes. This study compares three different culture systems for the micropropagation of vanilla: semi-solid (SS), liquid medium under partial immersion (PI), and temporary immersion (TI) using the SETIS™ bioreactor. In all cases, the culture systems included the Murashige and Skoog culture medium (MS) added with 30 g L−1 sucrose and 9.55 μM benzyladenine (BA). The number of shoots per explant, as well as shoot length, number of leaves per shoot, fresh weight, dry weight, stomatal index, and chlorophyll content were recorded after four weeks of culture. Also, the ex-vitro survival rate was estimated for all the culture systems investigated. The highest values of shoot proliferation and development were observed in TI (11.41 shoots/explant), followed by PI (6.53), and SS (3.76); the highest fresh and dry weights were obtained in TI, followed by PI, and SS. A higher synthesis of chlorophyll was observed in PI relative to TI and SS. Stomata showed a similar distribution and morphology in leaves of plants under the different culture systems. T1 showed peak values of the percentage of closed stomata and stomatal index. The highest percent survival during acclimatization was observed in TI (98%), followed by PI (85%), and SS (75%). At the end of the micropropagation process, 227 plants of V. planifolia were obtained in SS, 500 plants in PI and 3026 in TI. In conclusion, the SETIS ™ bioreactor improves the physiological conditions of in-vitro development and is an alternative for the propagation of V. planifolia at large scale.

Sucrose concentration and blueberry plant density in temporary immersion systems (TIS)

Abstract The aim was to determine the ideal concentration of sucrose along with the number of plants per container in the face of micropropagation of blueberry under TIS. In the experiment conducted with sucrose concentration (0, 10, 20, e 30 g L-1), we utilized a WPM medium, taking into account 30 explants per container. For the experiment dealing with blueberry plant density, the same medium with 20 g L-1 of sucrose in conjunction with 30, 40, 50 and 60 explants per container was used. After 90 days from the installation of the trial, the following response variables were assessed herein: total length of the plant, number of shoots, length of the largest shoot, number of hyperhydric shoots, length of hyperhydric shoots, and number of healthy leaves. Under a sucrose concentration corresponding to 20 g L-1 the best possible results were obtained. Survival rates of 22 and 80% under the influence of both 10 and 20 g L-1 treatments were reported, pointing out that plants exposed to 20 g L-1 treatment were more amenable to acclimatization. Thus, for the micropropagation of blueberry under TIS the use of 20 g L-1 sucrose and 60 plants/container comes to being highly recommended to increase yield.

Temporary immersion systems to improve alder micropropagation

A protocol has been developed for the propagation of Alnus glutinosa axillary shoots in liquid medium. The explants were cultured in Woody Plant Medium supplemented with 0.1 mg l− 1 benzyladenine and 0.5 mg l− 1 indole acetic acid. The effect of the bioreactor type (RITA® and Plantform™), frequency of the immersions, regulator concentrations, and volume of medium per explant was investigated. All the treatments with the temporal immersion systems (TIS) increased the proliferation rates. The best results were obtained on using the Plantform™ vessels and, unlike the shoots cultured in RITA® vessels, the culture in Plantform™ had very low hyperhydricity percentages. Shoots originating from the culture in semi-solid medium and in Plantform™ rooted in semi-solid medium with 0.1 mg/l indole butyric acid for 7 days. No significant differences were observed in the rooting or acclimatization percentages, with survival percentages greater than 85% being achieved. This is the first work on the use of TIS systems in alder propagation, with the results of this study providing new perspectives for its mass propagation. A new protocol is described in order to reduce the propagation costs of alder.

Effects of meta–topolin derivatives and temporary immersion on hyperhydricity and in vitro shoot proliferation in Pyrus communis

Although micropropagation in temporary immersion systems might increase plant growth and multiplication, it can also cause specific problems such as hyperhydricity and losses by contamination. A new commercial temporary immersion bioreactor, SETIS™, was used to micropropagate two Tunisian pear cultivars, ‘Arbi’ and ‘Mahdia 6’. The latter cultivar was endogenously contaminated by Sphingomonas. Hyperhydricity was inevitable when 5 µM benzyladenine was applied. However, the symptoms could be reduced by lowering the immersion frequency to 3 times per day. Applying 5 µM meta–Methoxy topolin riboside (MemTR) or meta–Topolin riboside (mTR) completely inhibited hyperhydric shoot formation. Moreover, the addition of Plant Preservative Mixture was effective to control Sphingomonas and allowed the plants to proliferate. For both pear cultivars, the highest number of shoots per explant was induced by 5 µM MemTR, whereas the highest leaf area was obtained with 5 µM mTR. The longest shoots were obtained with 5 µM mTR for ‘Arbi’ and 5 µM MemTR for ‘Mahdia 6’. Methoxy topoline-riboside (MemTR) and meta-topoline-riboside (mTR) were used as alternatives to benzyladenine to prevent hyperhydricity during the micropropagation of pears in a new temporary immersion system. These cytokinins also increased the number of good quality shoots, characterized by large leaves and longer shoots.

Direct contact ultrasound assisted freezing of chicken breast samples

Nowadays, rapid freezing is sought to favor the formation of small ice crystals. Several studies have shown that the application of ultrasounds (US) accelerates the processes of energy and mass transfer when they are applied through immersion systems. However, there are hardly any studies on its application in direct systems without the use of a liquid medium for its transmission. Therefore, the objective of this work was to evaluate the potential of the application of US for improving the freezing process of chicken breast samples. First, the application of intermittent US treatments at different net sonication times of 7, 17, 37, 50 and 67% during the freezing of distilled water samples in a conventional freezer was evaluated. It was observed that net sonication times of 37, 50 and 67% reduced the phase change period by 30.0, 21.4, 27.0%, respectively. The effective freezing time was also reduced by 12.4 and 12.8% by applying net sonication times of 37 and 50%. Considering these results, an intermittent US treatment with a net sonication time of 37% was chosen for chicken breast freezing in an air-forced cooling tunnel at ambient temperatures from −13 to −22 °C. The length of all the freezing phases was reduced upon application of US, leading to an overall process time reduction of approx. 11%. On the other hand, no significant differences were found either in the Water Holding Capacity (WHC) or Cooking Loss (CL) values between control and US assisted frozen chicken breast samples. Furthermore, in vitro experiments showed that US-assisted freezing did not influence protein digestibility of chicken meat samples.This study demonstrates the potential of the application of US by direct contact to favor energy transfer processes during freezing of water and chicken breasts samples. However, its effect on the quality of the frozen products should be further studied.

Batch and Flow Ultrasound-Assisted Extraction of Grape Stalks: Process Intensification Design up to a Multi-Kilo Scale.

Nowadays, approximately 1 billion kg/y of grape stalks, with a remarkable polyphenols content, are produced worldwide. In this paper, the extraction process intensification of polyphenols in water was achieved under ultrasound-assisted recovery, focusing on kinetics and scaling-up factors. Immersion and cup-horn systems were exploited as acoustic cavitation sources, and the total phenolic content (TPC) was chosen to assess the process efficiency. The kinetics were evaluated by Peleg’s hyperbolic model, and the effect of both the initial feedstock granulometry and ultrasound size-reduction were determined. The results were compared with conventional extraction methods (data analysis by ANOVA). The best polyphenols yield was obtained after 45 min of sonication, giving between 29.71 and 31.89 mg/g (gallic acid equivalents over the dry matter). The extracts were characterized using HPLC-DAD, UPLC-ESI-MS/MS, DPPH• assay (2,2-diphenyl-1-picrylhydrazyl), TEAC assay (Trolox equivalent antioxidant capacity), and proanthocyanidin content determination. The flow-mode extraction procedure of grape stalks (2 kg) was carried out in a 15 L reactor. A semi-industrial decanter unit and a bag-filter were the keys units of the downstream operations. The resulting particle-free solution underwent nanofiltration on a membrane pilot skid, providing a final polyphenols-enriched stream concentrated up to 355.91%, as shown by the antioxidant activity and TPC.

USO DE INSUMOS ALTERNATIVOS PARA LA MICROPROPAGACIÓN EN MEDIO SEMISÓLIDO Y SISTEMA DE INMERSIÓN TEMPORAL DE Solanum tuberosum, Ullucus tuberosus Y Oxalis tuberosa

<p><strong>Background. </strong>Micropropagation allows high rates of clonal multiplication of plants in short periods of time. However, despite being a simple technique, its implementation can be costly for regions and producers with limited resources. One of the main limiting factors is the lack of access to the inputs (QP) for the basic media that is widely published and disseminated in the literature. Other limiting factors include high costs, logistical difficulties and timely access to such inputs. <strong>Objective. </strong>In order to address these problems, the present work proposes the use of alternative inputs in the composition of the culture media for micropropagation of potatoes (<em>Solanum tuberosum</em>), ulluco (<em>Ullucus tuberosus</em> Caldas), and oca (<em>Oxalis tuberosa</em> Molina) in semi-solid and liquid media, as well as in temporary immersion systems (SIT). <strong>Methodology.</strong> All the macro and micronutrients of the basic media were replaced with inputs available at low cost in any local market. <strong>Results.</strong> The best results for micropropagation rate, plant vigor and rooting were obtained in the medium with substituted and accesible inputs (EP medium). These results were validated for micropropagation by nodal cutting in liquid and semi-solid médium and SIT. <strong>Implications.</strong> The basic medium (EP), formulated herein may be used by any laboratory and resource-poor farmers to apply micropropagation and improve revenues. <strong>Conclusion.</strong>The production cost of the EP medium, with easily accessible inputs in low-cost local markets, is significantly cheaper than MS medium used as a control, without differences in micropropagation.</p>

Influence of Vitrofural® on sugarcane micropropagation using temporary immersion system

Sugarcane cultivation is of great economic and social importance worldwide. In vitro cloning using temporary immersion systems is an option to produce vitroplants of high genetic and phytosanitary quality to establish stock seedbeds. However, the sterilization of culture media through autoclaving increases production costs and limits the efficiency of temporary immersion systems. For that reason, the objective of this study was to evaluate the morphological and physiological changes caused by Vitrofural® in sugarcane plants under temporary immersion. We compared two types of culture media sterilization: chemical sterilization using Vitrofural® and autoclaving using Temporary Immersion Bioreactors. After four weeks of cultivation, we evaluated morphological (number of shoots, shoot length, and number of leaves per shoot) and physiological indicators (soluble and bound phenolics, and phenolics excreted to the culture medium). As well as chlorophyll-a, -b and total chlorophyll content. The results show that when Vitrofural® was added to culture medium, a higher number of shoots per explant, length of the shoots and number of leaves were observed in comparison with the shoots obtained in autoclaved sterilized medium. Furthermore, a reduced the amount of phenolics compounds were found in Vitrofural® treatent .In addition, the use of the Vitrofural® increased the synthesis of photosynthetic pigments respect to autoclaving. In conclusion, Vitrofural® has a positive effect in morphological and physiological of sugarcane cultured in vitro and is a low-cost alternative for the commercial micropropagation of sugarcane. Vitrofural® produced a positive effect on the growth, content of photosynthetic pigments and compounds phenols during in vitro culture of sugar cane.

INDUKSI PERKECAMBAHAN PADA BIJI KARET (Hevea brasiliensis Muell) DENGAN SISTEM PERENDAMAN DAN TANPA PERENDAMAN AIR

This study aims to see the differences in the speed of sprout growth with immersion systems and without water immersion. The results showed that there were differences percentage of seeds germinated between seeds that were given water immersion for 12 hours (85%) and were not given water immersion (65%). For the other parameter average days of sprouting, where the seeds that are given water immersion are more quickly induced by sprouts as much as 60% age 7 days after seedling, compared not given water immersion can only be induced by 40%. Likewise, the 17th day after the seedlings were induced by 85% for soaking seeds and 65% for seeds that were not soaking. Keywords: Rubber, Sprout, Water immersion

Micropropagation of valuable walnut genotypes for timber production: new advances and insights

The intensive production of timber from walnuts is mainly hampered by the scarcity of varieties for this purpose. While the hybrid progeny Mj209xRa is considered suitable for timber production in Europe, problems associated with its recalcitrance, the low ability for rooting and the high mortality of acclimated vitroplants, limit the reproduction of elite trees. This research was aimed to assess the influence of two methods for the in vitro introduction of several walnut genotypes, to determine the effects of temporary immersion systems (TIS) on proliferation and rooting, and to reduce the random losses of acclimated vitroplants. Hence, trees from Mj209xRa progeny as well as some common walnuts were used. As an outstanding result, the in vitro establishment of 6 out of 7 trees was obtained. Also important was the improving of quality of microshoots, the multiplication ratios and the rooting using TIS through the management of the kind of bioreactor, the volume of culture media, and the kind of explant inoculated. The direct transplant to field nursery of acclimated vitroplants considerably increased their quality, while mortality was highly reduced. Certainly, these results represent a great contribution to the current micropropagation protocol, especially with the potential introduction of TIS for massive plant production

Ultrasonic fingerprint sensor in underglass prototype using impedance mismatching

Fingerprint imaging is a powerful tool in biometric identification systems. There are several methods for measuring fingerprints, such as optics, capacitive, and ultrasonic methods. The previous ultrasonic imaging techniques have been available in immersion systems which require the hands to be put into water. Also, pulse-echo system in dry condition has coupling layer which is similar acoustic impedance to human tissue or high impedance materials based on waveguide. For fingerprint imaging in the dry state, we propose an ultrasonic imaging technique in underglass by using the impedance mismatch between the fingertip and the solid plate. We present the image results according to the plate material such as acrylic, glass and PDMS. In addition, simulation results based on small 2-D array transducers in a near field suggest the feasibility of compact and fast fingerprint scanning system. Therefore, this work presents feasibility of ultrasonic pulse-echo fingerprint sensing method for dry hand.

Micropropagation of Guarianthe skinneri (Bateman) Dressler et W. E. Higging in Temporary Immersion Systems.

This study's aim was to establish a protocol for the micropropagation of G. skinneri using temporary immersion system (TIS). Different concentrations of 6-Benzylaminopurine (0, 1, 2, and 3mgL-1), three different systems of cultivate semi-solid (SS) and liquid media under partial (PI) and temporary immersion systems (TIS), different compositions of the inorganic salts, and the number of subcultures were evaluated. The results showed a maximum of 16.56 shoots per explant obtained through TIS, adjusting all the parameters evaluated in our study. One higher number of shoots per explant was observed in the micropropagation of G. skinneri TIS compared to SS and PI. While the use of 3mgL-1 of BAP + MS (Murashige and Skoog) media was better than 3mgL-1 of BAP VW (Vacint and Went) for the generation of a greater number of shoots per explant, 6.33 and 2.72, respectively. The immersion frequency of 2min every 4h allowed the production to be scaled to 8.54 shoots per explant. While it was necessary to perform three subcultures every 30days, to obtain 16.56 shoots per explant, a rooting phase was not required due to the generation of adventitious roots during the different subcultures. However, a phase of elongation of the regenerated plants with ½ MS + GA3 (gibberellic acid) was needed to guarantee 100% survival in the process of acclimatization. In conclusion, this plant production system can be applied for the commercial micropropagation of this species for ornamental purposes, as well as for its reintroduction in protected natural areas.

An efficient protocol for commercial micropropagation of malanga (Colocasia esculenta L. Schott) using temporary immersion

Temporary immersion systems allow semi-automatization and reduce production costs during micropropagation on a commercial scale. The objective of this study was to evaluate temporary immersion (TI) as a new technique for commercial micropropagation of malanga (Colocasia esculenta). The effect of the semi-solid and liquid medium using the Temporary Immersion Bioreactor (TIB) was compared. For the TIB culture system, the immersion frequency and volume of culture medium per explant on shoot development were evaluated. At 30 d of culture, the number of shoots per explant, shoot length and number of leaves per shoot were scored. In addition, the chlorophyll content during multiplication and survival during acclimatization were evaluated in the different culture systems. The results showed that the highest shoot production, shoot length and number of leaves per shoot were obtained in TI with an immersion frequency of 2 min every 4 h and with a volume of culture medium of 25 and 50 mL per explant. An increase in chlorophyll content in TI was observed with an immersion frequency every 4 h and with a culture medium volume of 50 and 100 mL per explant. Regarding the percentage of survival during acclimatization, no significant differences were observed between culture systems, immersion frequencies and volume of culture medium per explant. In conclusion, the evaluated immersion system is an alternative for commercial micropropagation of C. esculenta.

INDUKSI PERKECAMBAHAN PADA BIJI KARET (Hevea brasiliensis Muell) DENGAN SISTEM PERENDAMAN DAN TANPA PERENDAMAN AIR

This study aims to see the differences in the speed of sprout growth with immersion systems and without water immersion. The results showed that there were differences percentage of seeds germinated between seeds that were given water immersion for 12 hours (85%) and were not given water immersion (65%). For the other parameter average days of sprouting, where the seeds that are given water immersion are more quickly induced by sprouts as much as 60% age 7 days after seedling, compared not given water immersion can only be induced by 40%. Likewise, the 17th day after the seedlings were induced by 85% for soaking seeds and 65% for seeds that were not soaking. Keywords: Rubber, Sprout, Water immersion

Use of bioreactor systems in the propagation of forest trees.

Plant biotechnology can be used to conserve the germplasm of natural forests, and to increase the productivity and sustainability of plantations. Both goals imply working with mature trees, which are often recalcitrant to micropropagation. Conventional in vitro culture uses closed containers and gelled medium with sugar supplementation. Bioreactor culture uses liquid medium and usually incorporates aeration. The increased absorption of nutrients via the liquid medium together with the renewal of the air inside the bioreactors may improve the physiological state of the explants. In this review, we will explore the feasibility of using bioreactors to overcome the recalcitrance of many trees to micropropagation and/or to decrease the cost of large‐scale propagation. We will focus on the recent use of bioreactors during the multiplication, rooting (plant conversion in the case of somatic embryos), and acclimation stages of the micropropagation of axillary shoots and somatic embryos of forest trees (including some shrubs of commercial interest), in both temporary and continuous immersion systems. We will discuss the advantages and the main obstacles limiting the widespread implementation of bioreactor systems in woody plant culture, considering published scientific reports and contributions from the business sector.

In Vitro Propagation of Rheophytic Orchid, Epipactis flava Seidenf.-A Comparison of Semi-Solid, Continuous Immersion and Temporary Immersion Systems.

Epipactis flava Seidenf. is an endangered Thai rheophytic orchid that has recently shown a rapid decrease in its natural habitat, prompting an urgent need for conservation using ex situ reintroduction methods. Temporary immersion system (TIS) has been successfully applied for large-scale propagation in various plants species. Propagation efficiency of E. flava using TIS was investigated and compared with conventional semi-solid system (SSS) and liquid continuous immersion system (CIS). The highest percentage of new shoot and shoot bud formation was obtained from TIS, followed by CIS and SSS, respectively. Growth parameters as indicated by number of new shoots, shoot buds, shoot height and leaves per explant were significantly higher using TIS than with SSS and CIS. Moreover, the maximum number of new shoots and shoot buds per replication were reliably obtained from TIS higher than SSS and CIS. After acclimatization, the highest survival percentage of plantlets was observed in TIS (76.7%), with 60% surviving after eight weeks of transplantation in artificial stream. TIS was determined as the most suitable culture system for in vitro mass propagation of E. flava compared to CIS and SSS.

Micropropagation of ‘MSXJ’ hybrid of papaya in temporary immersion systems

Micropropagation of ‘MSXJ’ hybrid of papaya in temporary immersion systems