Immersion Frequency Research Articles

Optimization of cultivation conditions of Salvia viridis L. shoots in the Plantform bioreactor to increase polyphenol production

Salvia viridis is a rich source of pharmacologically-important polyphenolic compounds with anti-inflammatory, antioxidant and anticancer activities. The aim of the present study was to optimise the large-scale cultivation of sage shoots in the Plantform bioreactor. It evaluated the effect of culture duration (two or three weeks), the use of a semi-continuous system and the immersion frequency in a liquid medium (every 80 or 170 min) on the growth and accumulation of secondary metabolites. The content of bioactive phenolic acids and phenylethanoids in the obtained material was determined using HPLC. The most efficient growth parameters, expressed as fresh (26.99 g) and dry weight (3.51 g) as well as proliferation ratio (18.6), were observed for shoots grown in a fed-batch system, immersed every 80 min; this approach yielded a 33-fold biomass increase within four weeks. However, the most efficient production of bioactive compounds was achieved for shoots grown for three weeks in a batch system, immersed every 80 min: total phenolic acid, total phenylethanoid and total phenol contents of 18.3, 11.4 and 29.7 mg/g DW. In these conditions, within three weeks, total phenolic acid level was almost 10 times higher than that found in the aerial parts of four-month-old soil-grown plants with a similar phenylethanoid level. Hence, the described S. viridis shoot culture cultivated in the Plantform bioreactor may be an alternative efficient source of plant material rich in valuable compounds.

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>

Did the animal move? A cross-wavelet approach to geolocation data reveals year-round whereabouts of a resident seabird

Considerable progress in our understanding of long-distance migration has been achieved thanks to the use of small geolocator devices (GLS). The tracking of resident or short-distance migrant animals remains however challenging because geolocation errors are substantial and difficult to estimate. This study aims to examine the sex-specific marine space uses of a resident tropical seabird, the masked booby (Sula dactylatra), during its full annual life cycle at the Fernando de Noronha archipelago (Brazil). Masked boobies (n = 31) tagged with GLS recording light intensity, seawater immersion, and water temperature showed a resident behaviour over their entire annual cycle. A wavelet analysis of GLS data revealed oscillatory patterns of inferred longitude correlated with changes in immersion frequency. This synchronicity demonstrated that birds traveled away and back from the colony on consecutive trips of short length (\(\sim\) 2–4 days) and short range (\(\sim\) 100–300 km) eastward of the colony. Duration and range of trips depended on the sex of the individual and on the time of the year. Trip duration increased gradually from the end of the breeding period to the post-breeding period, probably due to the release of the central-place breeding constraints. During the pre-breeding period, females had farther ranges eastward and spent more time in water than males. Despite inherent limits of light-based geolocation, this study demonstrates the relevance of synchronicity analysis of GLS data for investigating year-round movements of resident or short-distance migrants.

The relative frequency of immersion of rye grains in liquid

The grain heap of rye, in addition to high-grade grain, contains weeds and harmful impurities. Poisonous ergot sclerotia are harmful impurities. Modern grain cleaning machines do not provide the allocation of ergot sclerotia in one technological process by air flow and on sieves. Ergot sclerotia have a specific gravity lower than that of rye grain. Purification of rye seeds from ergot sclerotia by specific gravity is possible in a salt solution. To create a machine for the insulation of toxic ergot from grain, practical experiments were carried out on throwing single grains of winter rye of the Falenskaya 4 variety into a liquid with a specific gravity of ρ zh = 1.0·103; 1.09·103; and 1.15·103 kg/m3. Sodium salt of hydrochloric acid (NaCl) was used to create the solution. The results obtained are presented in the form of the distribution of the relative frequency of grains when immersed in a liquid from the height h of fall. It was found that 100% immersion of grains in water occurs at a height h of not less than 43·10−3 m, and an aqueous solution of salt – 58·10−3 m. When developing a device for the isolation of ergot sclerotia by specific gravity in a salt solution, it is necessary to take into account that the grain will move in a flow and exclude the appearance of an undesirable trapping of an air bubble by a grain, which will worsen the results of separating impurities from the grain.

Drought stress and hurricane defoliation influence mountain clouds and moisture recycling in a tropical forest

Mountain ranges generate clouds, precipitation, and perennial streamflow for water supplies, but the role of forest cover in mountain hydrometeorology and cloud formation is not well understood. In the Luquillo Experimental Forest of Puerto Rico, mountains are immersed in clouds nightly, providing a steady precipitation source to support the tropical forest ecosystems and human uses. A severe drought in 2015 and the removal of forest canopy (defoliation) by Hurricane Maria in 2017 created natural experiments to examine interactions between the living forest and hydroclimatic processes. These unprecedented land-based observations over 4.5 y revealed that the orographic cloud system was highly responsive to local land-surface moisture and energy balances moderated by the forest. Cloud layer thickness and immersion frequency on the mountain slope correlated with antecedent rainfall, linking recycled terrestrial moisture to the formation of mountain clouds; and cloud-base altitude rose during drought stress and posthurricane defoliation. Changes in diurnal cycles of temperature and vapor-pressure deficit and an increase in sensible versus latent heat flux quantified local meteorological response to forest disturbances. Temperature and water vapor anomalies along the mountain slope persisted for at least 12 mo posthurricane, showing that understory recovery did not replace intact forest canopy function. In many similar settings around the world, prolonged drought, increasing temperatures, and deforestation could affect orographic cloud precipitation and the humans and ecosystems that depend on it.

USE OF RITA® TEMPORARY IMMERSION SYSTEM TO OBTAIN MICROTUBERS OF SEVERAL MASHUA (Tropaeolum tuberosum Ruiz & Pavón) MORPHOTYPES

<p><strong>Background. </strong>The Temporary Immersion System (TIS) is an effective strategy to obtain quality mashua microtubers through the <em>in vitro</em> propagation of seedlings in a small space and in a short period of time. <strong>Objective. </strong>Obtain microtubers of 27 mashua morphotypes (<em>Tropaeolum tuberosum</em>)<em> </em>using the TIS-RITA<sup>®</sup>. <strong>Methodology.</strong> The seedlings were previously propagated in MS (Murashige & Skoog) solid medium, supplemented with 3% sucrose, at 5.6 pH and 0.7% agar. After fifteen days of cultivation in this medium, the seedlings were transferred to a MS liquid. For the tuberization process, seedlings with an average size of 10 cm were transferred to TIS-RITA® systems for ten weeks, under conditions of complete darkness, at 19 ± 2 ° C, an immersion frequency of 2 minutes every 3 hours in a MS liquid medium supplemented with 8% sucrose and 2 mg.L<sup>-1</sup> benzylamino purine (BAP). <strong>Results. </strong>The best results of microtubers were obtained with different morphotypes, which are respect to i/size, MAC 06A (1.9 cm) and MAC 056 (1.8 cm); ii / fresh weight, MAC 06A (0.2g) and MAC 092 (0.18 g); iii / quantity, MAC 08A (51 units) and MAC 048 (56 units). <strong>Implications</strong>. TIS-RITA<sup>®</sup> can be used to obtain quality and virus-free seeds of a wide variety of commercially important crops. <strong>Conclusion</strong>. The SIT-RITA® based on the intermittent contact of the explants cultivating medium, is an efficient system to obtain mashua microtubers.</p>

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.

Distribution of the relative frequency of immersion of pea grains in the liquid depending from the height of transportation

One of the main ways to protect seed material from various diseases is treatment with chemicals. The most effective way to protect seeds from various diseases is wet etching with the simultaneous release of grain impurities. To develop a device for cleaning and etching seeds by wet density, studies were conducted to determine the minimum height of the loading hopper relative to the surface of the water solution of the etcher when transporting spherical grains to it in order to ensure that the surface tension forces are overcome. Practical experiments were conducted on throwing single grains of peas of the Vita variety (spherical grain) with a humidity of 14% into a liquid with a density of ρzh = 1000, 1090 and 1150 kg/m3. Sodium chloride salt (NaCl) was used to change the density liquid. To obtain more reliable information, the experiments were carried out in three-fold repetition. The temperature of the surrounding air and liquid of different densities ρzh was 20°C. It is established that when single pea grains fall 100%, the immersion in water (ρzh = 1000 kg/m3) occurs at a transport height h of at least 12.0·10−3 m. In the case of 100% immersion of pea grains in an aqueous solution of sodium chloride (NaCl) with a density of ρzh = 1090 and 1150 kg/m3, the minimum transport height h is 15.0·10−3 and 18.0·10−3 m, respectively.

Experimental Investigation of the Mechanical Failure Behavior of Coal Specimens With Water Intrusion

Mining fissures formed by coal mining can easily open bedrock and loose aquifers, and can thus cause water–rock interaction. The strength of coal and rock and their pore and fracture development characteristics are closely related to moisture content. Therefore, this paper studied the strength, porosity, fracture propagation, and failure characteristics of coal with different moisture contents. Six representative coal samples with different moisture contents of 0, 3.79, 6.10, 9.17, 11.01, and 11.68% were prepared by non-destructive water immersion experiment, and the samples were analyzed using. Nuclear Magnetic Resonance (NMR) and uniaxial compression acoustic emission experiments. Then, the temporal and spatial variation of the moisture contents of the coal samples and the characteristics of crack propagation and failure were studied before and after water immersion. Non-destructive water immersion and NMR analysis showed that the moisture content increases exponentially with increasing water immersion time and immersion frequency. Additionally, it was shown that with increasing water immersion time and frequency, the moisture distribution within the coal samples changes from uneven to uniform, and micro-pores and meso-pores develop into larger pores with increasing water content. A coupled uniaxial compression–acoustic emission experiment showed that acoustic emissions are closely related to the macroscopic failure mode of the coal sample. With increasing sample moisture content, the degree of sample destruction and the concentration of acoustic emissions both reduced. The increase of moisture content promotes the change of the macroscopic failure mode of the coal samples from tensile failure to tensile–shear composite failure. The results of this study have important reference value for analyzing the stability of coal pillars and surrounding rock under the action of water, and especially for the design of coal pillar dam bodies in coal mines with groundwater reservoirs.

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.

Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andreanum).

Anthurium has been micropropagated mainly through conventional techniques in semisolid culture medium. However, this culture system involves constraints due to the low number of shoots produced and the high costs of the gelling agent and labor. Temporary immersion systems (TIS) are an alternative for increasing biological performance, reducing costs, and facilitating a semi-automated micropropagation process. The objective of this study was to compare the efficiency of different types of TIS during the in vitro propagation of anthurium. We used 2-cm-long nodal segments from in vitro plants. Explants were cultured in different TIS: temporary immersion bioreactors (TIB®), Ebb-and-Flow bioreactor, and recipient for automated temporary immersion (RITA®), with a 2-min immersion frequency at 12-h intervals. We used Murashige and Skoog (MS) medium supplemented with 3% (w/v) of sucrose and 8.88µM benzylaminopurine. After 60days of culture, we evaluated various physiological variables and the percent survival in the different TIS. The largest numbers of shoots per explant were observed in TIB® and Ebb-and-Flow, with 50.83 and 43.16 shoots per explant, respectively; the lowest number of shoots per explant was observed in RITA®, with 30.66. TIB® yielded the highest content of photosynthetic pigments (chlorophyll a, b, and total chlorophyll), stomatal index, and percentage of closed stomata relative to both Ebb-and-Flow and RITA®. The TIB® and RITA® systems showed a 99% shoot survival, while Ebb-and-Flow yielded 86% survival. In conclusion, TIS design and type affect a number of physiological processes and in vitro development, with TIB® as a feasible option for the commercial micropropagation of anthurium.

Micropropagation of Stevia rebaudiana Bertoni Using RITA® Bioreactor

Stevia rebaudiana Bertoni is used for medicinal and food purposes. The effectiveness of different culture systems in Stevia micropropagation was evaluated. Node explants were cultured in semisolid or RITA® bioreactor containing woody plant medium (WPM) without plant growth regulators (PGRs). The effect of three medium volumes (100, 200, and 300 mL) and four immersion frequencies (10 seconds every 1, 4, 6, or 8 hours) were assessed. The shoots and roots developed simultaneously in the RITA® bioreactor treatment containing 100 mL of WPM at 10 seconds/4 hours immersion frequency and the healthiest plantlets were obtained from this RITA® treatment using 5 mL of medium per explant. The root induction rate was 100% in nonhyperhydrated shoots obtained from both semisolid and RITA® bioreactor, and all of them were successfully acclimatized. Thus, an economical and viable protocol was developed by performing the micropropagation in one step, eliminating the use of agar and PGRs, and decreasing the amount of medium used per explant.

Temporary immersion improves in vitro multiplication and acclimatization of Anthurium andreanum Lind.

A lack of automation and the high production costs entailed by the use of labor and a gelling agent limit micropropagation on a commercial scale. The aim of this study was to evaluate the temporary immersion (TI) technique in in vitro multiplication and acclimatization of Anthurium andreanum cv. Rosa. Nodal segments from in vitro-derived adventitious shoots were cultivated in different culture systems: semisolid medium, liquid medium with partial immersion and TI using an Ebb-and-Flow bioreactor. The effect of the culture system, immersion frequency, and culture medium volume per explant on shoot multiplication of A. andreanum was evaluated after 45 days of culture. In addition, chlorophyll content, stomatal index and survival rate during acclimatization were evaluated in different culture systems. A completely randomized experimental design was used for all experiments, with three replicates. An analysis of variance (ANOVA) and Tukey’s range test (p ≤ 0.05) were performed. The results showed significant differences in the variables evaluated among the different culture systems. The highest shoot production was obtained in TI with 31.50 ± 0.50 shoots per explant, followed by the partial immersion system and culture in semisolid medium, with 7.25 ± 0.16 and 4.50 ± 0.18 shoots per explant, respectively. Immersion frequency and the amount of culture medium per explant did not show significant differences, which allows us to recommend the immersion frequency of every 12 h and the culture medium volume of 25 mL per explant. TI favored an increase in chlorophyll content, a low stomatal index and a high percentage of closed stomata, suggesting an increase in the functionality of the stomata and probably a higher photosynthetic rate. The survival rate during acclimatization increased when using temporary immersion systems (TIS). This study shows for the first time an efficient TIS for commercial micropropagation of A. andreanum that produces plants with a high survival rate during acclimatization.

Simple Protocol for the Micropropagation of Teak (<i>Tectona grandis</i> Linn.) in Semi-Solid and Liquid Media in RITA<sup>® </sup> Bioreactors and <i>ex Vitro</i> Rooting

In Latin America the forestry of exotic species such as teak has been increasing in recent decades, due to their advantages in wood quality, rapid growth; and the relative ease of producing clones and their multiplication with respect to native species. Therefore, there is great interest in developing larger-scale propagation strategies that reduce costs and intensive manual labor. Culture in liquid media with temporary immersion and the semi-automation of the system has raised expectations for large-scale micropropagation. We report a protocol for teak, which reuses the primary explants in several culture cycles in semi-solid medium to produce nodal explants for the multiplication phase in temporary immersion bioreactors (RITA® ). The control of factors such as cytokinin concentration, explants density, immersion frequencies and culture duration was analyzed. The number of shoots increased with 0.5 mg·l-1 of BA (6-Benzyladenine), alone or in combination with 0.5 mg·l-1 of Kinetin, with 2 daily immersions of 1 minute each; however, these shoots showed a high degree of hyperhydricity. When 0.05 mg·l-1 of BA was used with 1 immersion of 1 minute every 2 days, the hyperhydricity decreased. Although the number of shoots was lower, they showed good length to be used during multiplication and rooting ex vitro. Our results suggest that teak micropropagation can be simplified in two phases in vitro, the establishment and multiplication; followed by rooting ex vitro and acclimatization. This would imply a reduction in production costs, since most of the multiplication would take place in RITA® containers.

Temporary Immersion System for Micropropagation of Tree Species: a Bibliographic and Systematic Review

This paper was characterized by a bibliometric and systematic review on the database ISI Web of Science, aiming to provide an update of the main points addressed regarding the Temporary Immersion Systems (TIS) for micropropagation of tree species. It was pointed out that the frequency and time of immersion were one of the main parameters studied in the papers and 35% these papers worked with eucalyptus species. The main problem reported in the papers was the hyperhydricity, but it was overcome via procedures such as: a) air injection into the system, b) increasing the immersion intervals and decreasing the immersion time and c) decreasing concentration of cytokinin. Most papers reported that TIS produced plants that were more successful in surviving the ex-vitro acclimation stage than those produced on semi-solid media or continuous immersion systems. Few studies compared different types of temporary immersion bioreactors on micropropagation of plants and within the established criteria, papers with TIS tree species represented only 15% of the total. This system has presented promising results for most of the tree species, and although some gaps have been identified and few are the works with tree species, this process has been taking an increasingly larger space in the propagation of plants.

High-frequency protocorm-like bodies and shoot regeneration through a combination of thin cell layer and RITA® temporary immersion bioreactor in Cattleya forbesii Lindl.

An efficient in vitro mass propagation through protocorm-like bodies (PLBs) was established in Cattleya forbesii Lindl., a commercially important orchid. Whole PLBs (W-PLB) and transverse thin cell layers of PLB (tTCL-PLB) explants were cultured in RITA® bioreactors based on temporary immersion system. Explants were transferred in semi-solid or RITA® bioreactor for protocorm production or shoot regeneration. The effect of different immersion frequencies, medium volumes and inoculum densities were studied and optimized. RITA® bioreactor cultures were found to be superior compared with semi-solid cultures regarding PLB production and shoot regeneration. tTCL-PLB explant types cultured in the RITA® bioreactor with immersion for 1 min/4 h, 250 mL of medium and 20 explants showed the highest number of PLBs per RITA® (2237 PLBs) and per explant (111.9 PLBs). The highest number of PLBs per explant was 21 times higher than those from semi-solid culture. The highest number of shoots per RITA® (3998 shoots) and per explant (199.9 shoots) were observed on tTCL-PLB cultured in RITA® bioreactor (1 min/4 h; 150 mL of medium and 20 explants). The highest number of shoots per explant was 95 times higher than those grown on semi-solid culture. Mass propagation of PLBs and shoots of C. forbesii Lindl. using combined thin cell layer and RITA® temporary immersion has been adapted in commercial practice.

Production of biomass and flavonoid of Gynura procumbens (Lour.) Merr shoots culture in temporary immersion system.

Gynura procumbens (Lour.) Merris one of medicinal plant which was carried out used as antioxidant, anticancer, anti-inflammatory, hepatoprotective, and antimicrobial. Many strategies were used to increase the production of biomass and valuable compounds. This study was to investigate the variation effect of growth regulators and immersion frequency on production of biomass and flavonoid contained of G. procumbens shoots culture in temporary immersion bioreactor. Stem nodes were used as an explants and induction of shoots were done in solid MS medium supplemented with many kinds of growth regulator. The best treatments were used to produce biomass and flavonoid compounds in temporary immersion bioreactor; there are combination of IAA 2 mg/L and BA 4, 6, 8 mg/L and immersion frequency (5 min each 3 h; 15 min each 12 h). Results showed that the growths of G. procumbens shoots in solid MS medium were influenced by supplementation of growth regulators. MS medium supplemented with single cytokinine (6 mg/L kinetin) and combination of auxin (IAA) and cytokinine (BA) caused increasing of shoots growth. Production of biomass of G. procumbens in temporary immersion bioreactor was achieved in long immersion interval (12 h) and highest flavonoid production was obtained in combination treatment of immersion frequency 15 min each 12 h and MS medium supplemented with IAA 2 mg/L, BA 8 mg/L.

Optimizing factors affecting development and propagation of Bletilla striata in a temporary immersion bioreactor system

Culturing factors regarding to B. striata plantlets growth and pseudobulb development through temporary immersion bioreactor system (TIBS) were studied. Immersion frequency, sucrose concentration and inoculant amount were analyzed for their influence on seedling development. An immersion frequency of 3 min per 2 h was found to substantially promote the development of pseudobulbs. Seedling growth and development were also affected by the immersion frequency. The best seedling development was observed among those cultures that were immersed at a rate of 3 min per 6 h. Similarly, sucrose concentration was identified to affect the development of seedlings and pseudobulb formation obviously, with the best development recorded at 40 g/L weight ratio. A prominent seedling development, in terms of stem diameter, plant height and leaf width, were observed when 300 explants was loaded in the TIBS tank. The development of pseudobulbs was also found to be obviously enhanced at this explants loadings. The current findings verified the optimal immersion frequency, sucrose concentration and explant loads for improved seedling growth and pseudobulb development strategy for B. striata.

Liquid Culture Systems Affect Morphological and Biochemical Parameters during Rosa canina Plantlets In Vitro Production

Wild roses are an important group of plants used as decorative shrubs and cut flowers. They are also a row material for pharmaceutical, cosmetic and food industries. For rose in vitro propagation, solid media are commonly used. Up till now there is a few reports confirming the beneficial effect of liquid media on Rosa shoot growth and multiplication. The aim of the study was to investigate different culture systems, temporary immersion system (TIS) (immersion frequencies of 15 min every 6, 8 and 12 h), rotary shaker (RS) and stationary liquid (SL) for propagation of R. canina and compare with solid medium culture. Shoot tips and stem explants were grown on basic Murashige and Skoog medium with 20 mg dm-3 Fe EDDHA, 1 µM BA, 1.5 µM GA3 and 3% sucrose for six weeks. Liquid cultures stimulated biomass growth. The highest biomass growth in RS cultures were observed however, RS reduced the shoot dry mass content. TIS cultures immersed every 6 and 8 h increased dry mass content. In TIS and on solid medium shoot multiplication was 1.5-2 times better than in other systems and stem explants were more efficient. Solid medium improved the content of chlorophyll a, b, a+b and carotenoids. Higher contents of photosynthetic pigments were determined in shoot tips than stem explants. TIS-derived plantlets accumulated the largest amount of phenolic compounds. As the frequency of immersion increases the concentration of these compounds were reduced. In turn, SL cultures favored the accumulation of soluble sugars.