Effect of activated charcoal on multiplication of African yam (Dioscorea cayenensis-rotundata) nodal segments using a temporary immersion bioreactor (RITA®)

In this study, we compared the growth of Dioscorea cayenensis-rotundata (African yam) nodal segments, using semisolid medium in test tubes and liquid medium in 1-L Recipient for Automated Temporary Immersion (RITA^®) temporary immersion bioreactors (TIB), and the application of various culture parameters. The addition of activated charcoal (AC) had a positive effect on the growth of nodal segments, both in semisolid medium and in liquid medium in RITA^® bioreactors. After 2 mo culture in the presence of AC, plantlets were 6.4–6.6 cm long compared to 3.2–3.8 cm in absence of AC, with no significant difference observed between the culture systems. In the range of inoculation densities tested (5–20 nodal segments per RITA^® bioreactor), there was no effect on the number of buds produced per nodal segment, the moisture content of plantlets (fresh weight basis), or on net fresh weight gain. By contrast, the individual leaf surface area of plantlets decreased in line with increasing inoculation density. Among the range of benzylaminopurine (BAP) concentrations tested (0–17.6 μM), 0.44 μM induced the highest number of buds (3.8 buds per nodal segment) in the TIB. However, comparable numbers of buds could be produced with media devoid of BAP, either by increasing the frequency of 1-min daily immersion cycles in RITA^® bioreactors from one every 12 h to one every 4 h or by using semisolid medium containing AC.     

High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR

An efficient protocol for genetic transformation of somatic embryos of Quercus robur by selection in a temporary immersion system is reported. The transformation frequency was 5 times higher than achieved by conventional culture on semi-solid medium, ranging between 6 and 26 % for the four genotypes evaluated. Clumps of globular or torpedo somatic embryos were precultured for 7–10 days, inoculated with Agrobacterium tumefaciens strain EHA105:p35SGUSINT and cocultivated for 4 days before being cultured for 4 weeks on semi-solid selection medium supplemented with 25 mg L^?1 kanamycin. Explants were transferred to RITA^® bioreactors and subjected to a two-step selection protocol involving immersion in liquid medium supplemented with 25 mg L^?1 kanamycin, for 18 weeks, and then with 75 mg L^?1 kanamycin. Putatively transformed explants appeared after serial transfer to selection medium over 12–16 weeks. The presence of neomycin phosphotransferase II and β-glucuronidase genes in the plant genome was confirmed by histochemical and molecular analysis, and the copy number was determined by Southern blotting and real-time quantitative polymerase chain reaction. Transformed somatic embryos were germinated and transferred to soil for acclimatization, approximately 8 months after inoculation of the original tissue with bacteria. As the limiting factor for recovery of plants from oak embryogenic lines is the low embryo conversion rate, axillary shoot lines were established from transformed germinated embryos. Transformed embryos and shoots were cultured in medium with or without kanamycin and the responses to several morphogenetic processes (recovery after cryopreservation, germination, shoot proliferation, and rooting) were evaluated.     

Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems

Airlift bioreactors were programmed for continuous and temporary immersion culture to investigate factors that affect the rhizome proliferation, shoot formation, and plantlet regeneration of Cymbidium sinense. During rhizome proliferation, the continuous immersion bioreactor system was used to explore the effects of activated charcoal (AC) in the culture medium, inoculation density, and air volume on rhizome differentiation and growth. The optimum conditions for obtaining massive health rhizomes were 0.3 g l^?1 AC in the culture medium, 7.5 g l^?1 inoculation density, and 150 ml min^?1 air. In addition, the temporary immersion bioreactor system was used for both shoot formation and plantlet regeneration. Supplementing 4 mg l^?1 6-benzylaminopurine and 0.2 mg l^?1 naphthalene acetic acid (NAA) to the culture medium promoted shoot induction from the rhizome. Cutting the rhizome explants into 1 cm segments was better for massive shoot formation than cutting into 0.25 and 0.5 cm explant segments. NAA promoted plantlet regeneration and the rooting rate (94.7 %), with whole plantlets growing well in culture medium containing 1.0 mg l^?1 NAA. Therefore, applying bioreactors in C. sinense micropropagation is an efficient way for scaling up the production of propagules and whole plantlets for the industrial production of high-quality seedlings.     

Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system

Here, we evaluated the efficiency of shoot multiplication of Vanilla planifolia Jacks. ex Andrews using solid medium, partial immersion, and a temporary immersion system (TIS) to improve micropropagation in this species. Clusters of shoots were cultivated in vitro using Murashige and Skoog (MS) medium supplemented with 9.55 μM benzyladenine (BA) and 100 mL L^?1 coconut water. For the TIS, a RITA^® system was used and three immersion frequencies were evaluated (every 4, 8, and 12 h) with an immersion time of 2 min. After 30-d culture, the TIS produced the maximum multiplication rate (14.27 shoots per explant) when using an immersion frequency of 2 min every 4 h, followed by the partial immersion system (8.64 shoots per explant), and solid medium (5.80 shoots per explant). Next, the effect of the volume of culture medium per explant was also evaluated for TIS. The most suitable volume of culture medium for shoot formation was 25 mL per explant, which increased the rate of multiplication to 17.54 shoots per explant. Root initiation was 90% successful in TIS using half-strength MS medium supplemented with 0.44 μM naphthaleneacetic acid (NAA) and an immersion frequency of 2 min every 4 h. With this system, the shoot multiplication rate increased threefold compared to that obtained with solid medium. In addition, this system produced good results for the transplantation and acclimation (90% of survival) of in vitro-derived plants. These results offer new options for large-scale micropropagation of vanilla.     

Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system

The efficiency of micropropagation of double-node shoots of hop (Humulus lupulus L. cv. Tettnanger) was evaluated using semi-solid and liquid culture medium in RITA® temporary immersion bioreactors. The highest fresh and dry weight of shoots, average number of shoots, and multiplication rate were obtained using the RITA® system, whereas the longest shoots were obtained on semi-solid medium. Moreover, shoot length was affected significantly by the inoculum density of double-node shoots in RITA® vessels. In addition, the RITA® bioreactors were suitable for shoot induction from organogenic calli. The percentage of shoot induction and the shoot fresh and dry weights were significantly higher in the RITA® system than in semi-solid medium. The age of organogenic calli and inoculum density significantly affected the induction of shoots from organogenic calli. The optimum conditions for DNA delivery into hop organogenic calli using the biolistic particle delivery system were also determined. Organogenic calli were bombarded with the plasmid pSR5-2 (gusA and nptII) varying helium pressure (900, 1,100, or 1,350 psi) and target distance (6, 9, or 12 cm). The highest gusA transient activity was obtained using a pressure of 900 psi and a target distance of 6 cm. For stable genetic transformation, 3-wk-old organogenic calli were bombarded with the plasmid pCAMBIA1303 (gusA, mgfp5, and hpt) using these optimum conditions. Stable gusA expression was observed in organogenic calli and shoots after 4 wk of culture on selection medium containing 2.5 mg l^?1 hygromycin. The presence of the mgfp5 gene in the hop genome was confirmed by PCR.     

Development of a temporary immersion system (RITA®) for mass production of sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. interspecific hybrids)

Commercial micropropagation of sugarcane is largely determined by the clonal fidelity and the cost of plants produced. Rapid production of plants in vitro reduces the frequency of offtypes in many species. By exploiting the concept of transverse thin cell layer culture, we have developed a rapid, high frequency direct plant regeneration system, called SmartSett®, for commercial sugarcane cultivars grown in Australia. Similar to conventional micropropagation, labour remains the major cost of this plant production system. Hence, to reduce the labour component, we have integrated the SmartSett® system with the RITA® temporary immersion bioreactor. Thin transverse leaf sections or fragmented leaves cultured on agar-based SmartSett® shoot induction medium were used as the starting material for RITA®. Shoot initiation on semi-solid medium prior to transferring to RITA®, culture immersion frequency, explant size and genotype determined the productivity (number of plants produced per unit culture) of the system. Results obtained with cultivar Q165 indicate that explants cultured for 45 d on SmartSett® shoot induction medium were the most prolific, producing on average 275 shoots per vessel after 45 d of culture in RITA with 1 min immersion every 12 or 24 h. Using the fragmented tissue, 14-d-old explants and 3-mm leaf tissue fragments were the most productive. Experiments with three cultivars (Q117, Q165 and Q205) showed that RITA® culture conditions need to be optimised for each cultivar for maximum plant production.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

Temporary immersion systems (RITA®) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production

Somatic embryogenesis in cork oak (Quercus suber L.) is an efficient tool that allows the production of large number of embryos from selected quality and productive trees. Temporary immersion systems (TIS) are an alternative to semi-solid or liquid culture that combine the advantages of liquid culture and avoid the associated problems. Parameters that affect the TIS multiplication efficiency of Q. suber L. embryogenic cultures were evaluated. Immersion frequencies of 1 min every 6 or 4 h increased the fresh weight 3.7 or 7.5-fold compared with an immersion frequency of 1 min every 12 h or cultures on semi-solid medium, respectively. The cellular fate of embryogenic cultures was also affected by the immersion frequency, 1 min every 6 h was the best for mass propagation of proliferative developmental stages (embryogenic calli and embryo clusters) while 1 min every 4 h promoted the formation of single, fully developed cotyledonary embryos. An initial amount of 1.5 g fresh weight of proliferative tissues produced the best results in RITA^® containers while 0.5 g of embryogenic callus was the best for semi-solid cultures.     

Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.     

Micropropagation of <Emphasis Type="Italic">Asparagus macrorrhizus</Emphasis>, a Spanish endemic species in extreme extinction risk

Asparagus macrorrhizus: is a new species, which has been recently described. It is limited to the area surrounding the “Mar Menor” lagoon, in Murcia (Spain), and is the only “Critically Endangered” species of the genus Asparagus. Despite being protected, the number of plants has decreased in the last years due to the urbanization of its natural habitat. This species is a valuable genetic resource for asparagus breeding because of its special characteristics. So, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The micropropagation protocol from asparagus rhizome buds previously developed by our research group has been adapted for A. macrorrhizus. Rhizome buds of A. macrorrhizus were extracted, disinfected, and then cultured on Asparagus Rhizome Bud Medium (ARBM) consisting of MS medium supplemented with 0.3 mg l^??1 NAA, 0.1 mg l^??1 KIN, 2 mg l^??1 ancymidol and 6% sucrose. A percentage of 69.7?±?8.0% of the rhizome buds developed shoots, but only 17.4?±?7.9% of them rooted. To increase this low rooting rate, the shoots were cultured on Macrorrhizus Rooting Media (MRM) supplemented with three different concentrations of IBA. The highest rooting rate (55.0?±?7.9%) was reached when shoots were incubated in MRM-2 consisting of MS medium supplemented with 2 mg l^??1 IBA and 4% sucrose. The acclimatization rate of the micropropagated plantlets was 90%. The method developed in this study allows the micropropagation of A. macrorrhizus, offering a new option to preserve this almost extinct species.     

In vitro propagation of the neotropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation

Guadua angustifolia Kunth was successfully propagated in vitro from axillary buds. Culture initiation, bud sprouting, shoot and plant multiplication, rooting and acclimatization, were evaluated. Best results were obtained using explants from greenhouse-cultivated plants, following a disinfection procedure that comprised the sequential use of an alkaline detergent, a mixture of the fungicide Benomyl and the bactericide Agri-mycin, followed by immersion in sodium hypochlorite (1.5% w/v) for 10 min, and culturing on Murashige and Skoog medium containing 2 ml l^?1 of Plant Preservative Mixture^®. Highest bud sprouting in original explants was observed when 3 mg l^?1 N⁶-benzylaminopurine (BAP) was incorporated into the culture medium. Production of lateral shoots in in vitro growing plants increased with BAP concentration in culture medium, up to 5 mg l^?1, the highest concentration assessed. After six subcultures, clumps of 8–12 axes were obtained, and their division in groups of 3–5 axes allowed multiplication of the plants. Rooting occurred in vitro spontaneously in 100% of the explants that produced lateral shoots. Successful acclimatization of well-rooted clumps of 5–6 axes was achieved in the greenhouse under mist watering in a mixture of soil, sand and rice hulls (1:1:1).     

Improving secondary embryogenesis in <Emphasis Type="Italic">Quercus robur</Emphasis>: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA^® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.     

Micropropagation of apple — A review

Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed.     

Antimicrobial and hormetic effects of silver nanoparticles on in vitro regeneration of vanilla (<Emphasis Type="Italic">Vanilla planifolia</Emphasis> Jacks. ex Andrews) using a temporary immersion system

Microbial contamination is a serious problem in temporary immersion systems (TIS) during commercial micropropagation. The use of adequate doses of silver nanoparticles (AgNPs), formulated as Argovit?, is an alternative to reduce the contamination indices and promote development in plants. The aim of this study was to evaluate the antimicrobial and hormetic effects of Argovit on in vitro regeneration of vanilla (Vanilla planifolia) using a TIS. In vitro regenerated shoots were grown in Murashige and Skoog (MS) liquid medium with Argovit at five different concentrations (0, 25, 50, 100 and 200 mg/l) using a temporary immersion bioreactor system (RITA^®). At 30 days of culture, contamination percentage was evaluated and shoot regeneration and length were used to determine the hormetic response. Analysis of macro and micronutrient contents was performed. In addition, the effect of Argovit on total phenolic content (TPC), reactive oxygen species (ROS) production, antioxidant capacity (ORAC) and lipid peroxidation (LP-MDA) was determined. Results showed that bacterial contamination was reduced at 50, 100 and 200 mg/l of Argovit. Growth stimulation was observed at 25 and 50 mg/l of Argovit, while significant inhibition was detected at 100 and 200 mg/l of Argovit. Mineral nutrient analysis revealed changes in macro and micronutrient concentrations exerted by Argovit. Moreover, the presence of Argovit induced the production of ROS and increased total phenolic content, antioxidant capacity and lipid peroxidation with a dose-dependent effect. Results suggested that the production of ROS and mineral nutrition are key mechanisms of AgNPs-induced hormesis for vanilla. Therefore, the addition of 50 mg/l of Argovit in the culture media had an antimicrobial and hormetic effect. Use of Argovit could be an efficient strategy for commercial micropropagation of vanilla and other species.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

Comparative analysis of direct plant regeneration from immature leaf whorl and floral explants for three elite US sugarcane (Saccharum spp. hybrids) genotypes

Sugarcane (Saccharum spp. hybrids) is an important commodity field crop in tropical and subtropical countries providing sugar and biofuel feedstock and occupying a critical and strategic position in the global economy. This study was conducted to evaluate, compare, and optimize a rapid direct regeneration tissue culture system from immature leaf whorl and pre-emergent floral explants for three elite US sugarcane genotypes: CP84-1198, CP88-1762, and CP89-2143. Direct regeneration of adventitious shoot buds from the immature leaf roll explants and subsequent elongation and rooting of shoot buds was successfully obtained on modified Murashige and Skoog salt medium supplemented with 5 mg l^–1 α-naphthaleneacetic acid and 0.5 mg l^?1 kinetin. Significant genotype-specific differences in the morphogenetic potential of leaf roll explants were discernible with the explant developmental stage (explant position along the leaf roll axis) and orientation during in vitro culture. The highest number of shoots was regenerated from CP88-1762, followed by CP89-2143 and CP84-1198 from explants closest to the meristem that were oriented horizontally (CP88-1762) or vertically (CP89-2143 and CP84-1198) on the culture medium. Immature inflorescence-derived explants from all three genotypes when cultured on the above medium for 2 wk rapidly produced shoots, followed by rooting on medium supplemented with 4 mg l^?1 indole-3-butyric acid. The regeneration protocols yielded robust rooted plantlets from immature leaf roll explants within 4 to 6 wk, which were readily acclimatized under greenhouse conditions.     

Shoot regeneration from leaf explants of five strawberry (<Emphasis Type="Italic">Fragaria × Ananassa</Emphasis>) cultivars in temporary immersion bioreactor system

Summary A temporary immersion bioreactor system (TIB system) provides a convenient and efficient way to propagate plant material in vitro while requiring significantly lower labor input than conventional methods. The applicability of a TIB system for adventitious shoot regeneration from strawberry leaf explants was studied. Five commercial cultivars, i.e. Bounty, Jonsok, Korona, Polka, and Zephyr, were propagated in regeneration medium in commercially available TIB bioreactors (RITA^?) and, for comparison, on the same medium solidified with agar. The TIB system proved to be well suited for shoot propagation and for subsequent subculture of the developing plantlets. Regeneration frequencies were 70±8 to 94±2% and 83±5 to 92±3% in the TIB system and on semi-solid medium, respectively. The labor time taken by the TIB system was less than half of the time required for handling plant material for cultivation on semi-solid medium. This system thus provides a convenient method that could be adopted for commercial in vitro propagation or for regeneration of transgenic strawberry cultivars.     

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l^?1 6-benzyladenine (BA) and 0.1 mg l^?1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l^?1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.     

Bioreactor shoot cultures of <Emphasis Type="Italic">Rhododendron tomentosum</Emphasis> (<Emphasis Type="Italic">Ledum palustre</Emphasis>) for a large-scale production of bioactive volatile compounds

Rhododendron tomentosum Harmaja (Ledum palustre), a peat bog plant from Ericaceae family, has been used in traditional medicine as the anti-arthritis agent. Although modern researches confirm its anti-inflammatory properties, it remains threatened by habitat degradation and possibilities to collect this endangered species from its natural environment for further biological activity studies are limited. Therefore, R. tomentosum liquid in vitro cultures were established as the alternative source of that valuable plant material. Schenk–Hildebrandt medium with 24.60 μM 2-isopentenyladenine and 592.02 μM adenine provides intensive growth and proper morphology of the obtained microshoots. The R. tomentosum biomass was scaled up using the various bioreactors (immersion, temporary immersion and spraying systems) for better growth and improved volatile oil production. The largest biomass accumulation (fresh weight?=?250 g l^?1, growth index?=?280, dry weight?=?20 g l^?1) and essential oil content (0.5% v/m) were achieved with application of commercially available RITA^® bioreactor. GC/MS analysis revealed the high content of p-cymene (6.9%), alloaromadendrene (5.5%), shyobunone (8.2%) and ledene oxide (II) (13.0%) in the volatile fraction obtained from RITA^® system. The biomass growth parameters and production profile in terms of essential oil and selected terpenoid compounds were determined during the 2 month period. The influence of culture conditions and bioreactor construction on the growth and volatile oil production in R. tomentosum biomasses was discussed.