Temporary Immersion Bioreactors Research Articles

Cattleya walkeriana growth in different micropropagation systems

The aim of the present research was to verify the in vitro growth of orchids in different systems of micropropagation, being cultivated in a bioreactor, with natural ventilation and conventional systems. Cattleya walkeriana plants were obtained from the germination of seeds in culture medium. After 8 months, seedlings with 1 cm of length were placed in a culture vessel according to the treatments, which counted with two micropropagation systems (conventional and natural ventilation) in three media of culture (liquid, solid with 5 or 6g L-1 of agar). Two additional treatments in bioreactor of temporary and continuous immersion were performed. The design was entirely randomized (ERD), consisting of a 2x3 factorial with two additional treatments, totaling 8 treatments with three repetitions. The temporary immersion bioreactor promoted a bigger growth of the aerial part and of the root system, bigger accumulation of dry mass and better control of water loss by the plants. The temporary immersion bioreactor is the best micropropagation system for the C. walkeriana growth in vitro.

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.

Integrated criteria to identify the best treatment in plant biotechnology experiments

Plant scientists usually record many indicators in their experiments. The common statistical management involves univariate analyses. Such analyses generally create a split picture of the effects of experimental treatments since each indicator is addressed independently. The Euclidean distance combined with the expert’s criteria seems to show potential as an integrating indicator. The Euclidean distance has been widely used in many scientific fields nevertheless, as far as we know, it has not been frequently employed in plant science experiments. To exemplify the use of the Euclidean distance in this field, we performed an experiment focused on the effects of gibberellic acid on protease excretion during pineapple micropropagation in temporary immersion bioreactors. Five gibberellic acid concentrations were compared: 0.0, 1.4, 2.8, 4.2 and 5.6 μM. Four dependent variables were recorded: increase in fresh shoot mass, protein concentration, proteolytic activity and specific activity in the culture media. The statistical protocol carried out integrated these four dependent variables to easily identify the best gibberellic acid treatment. Based on expert’s criteria the integral analysis provided by the Euclidean distance indicated that 4.2 μM gibberellic acid is the best treatment to produce proteases in the experimental conditions described here. This treatment showed the shortest (statistically significant) Euclidean distance to expert’s criteria (0.61).

The physiology of ex vitro pineapple (Ananas comosus L. Merr. var MD-2) as CAM or C3 is regulated by the environmental conditions: proteomic and transcriptomic profiles

Proteomic and transcriptomic profiles of key enzymes were monitored in pineapple plants propagated under C3 and CAM-inducing metabolisms to obtain insight into the CAM-facultative metabolism and the relationship of CAM plants with oxidative stress. Pineapple is one of the most important tropical crops worldwide. The use of temporary immersion bioreactors for the first stages of pineapple propagation enables precise control of plant growth, increases the rate of plant multiplication, decreases space, energy and labor requirements for pineapple plants in commercial micropropagation. Once the plantlets are ready to be taken from the reactors, they are carefully acclimatized to natural environmental conditions, and a facultative C3/CAM metabolism in the first 2 months of growth is the characteristic of pineapple plants, depending on environmental conditions. We subjected two sets of micropropagated pineapple plants to C3 and CAM-inducing environmental conditions, determined by light intensity/relative humidity (respectively 40 μmol m−2 s−1/85 % and 260 μmol m−2 s−1/50 %). Leaves of pineapple plants grown under CAM-inducing conditions showed higher leaf thickness and more developed cuticles and hypodermic tissue. Proteomic profiles of several proteins, isoenzyme patterns and transcriptomic profiles were also measured. Five major spots were isolated and identified, two of them for the first time in Ananas comosus (OEE 1; OEE 2) and the other three corresponding to small fragments of the large subunit of Rubisco (LSU). PEPC and PEPCK were also detected by immunobloting of 2DE at the end of both ex vitro treatments (C3/CAM) during the dark period. Isoenzymes of SOD and CAT were identified by electrophoresis and the transcript levels of OEE 1 and CAT were associated with CAM metabolism in pineapple plants.

Secoiridoid glycosides production by Centaurium maritimum (L.) Fritch hairy root cultures in temporary immersion bioreactor

Secoiridoid glycosides are naturally occurring phytochemicals of great importance for the food and pharmaceutical industry because of their various biological activities. Certain Gentiana and Centaurium species, which are recognized as the most important sources of these compounds, have become critically endangered due to overexploitation. In this study we describe a laboratory-scale approach for further implementation in large-scale production of secoiridoid glycosides, using a hairy root culture system of Centaurium maritimum L. Fritch, an underutilized and phytochemically unexplored species. Hairy roots were induced by Agrobacterium rhizogenes strain A40M70GUS and grown in Erlenmeyer flasks, as well as in RITA® temporary immersion bioreactors (TIBs). About 2–4 times higher biomass production rate and up to 8 times higher total secoiridoid glycosides production rate were achieved in RITA® bioreactors. Among the selected hairy root lines, line HR3 cultured in RITA® TIBs proved to be the most efficient considering both biomass and secoiridoid glycosides production rate.

Establishment of photomixotrophic cultures for raspberry micropropagation in Temporary Immersion Bioreactors (TIBs)

A novel protocol for raspberry (Rubus spp.) micropropagation in photomixotrophic conditions was optimized for the commercial genotypes Heritage, Meeker and Amity. Plant cultures were established in Temporary Immersion Bioreactors (TIBs) under a controlled environment: 550ppmv CO2; light intensity 80μMm−2s−1; sucrose concentrations 15gr/L and 30gr/L. Results showed that both CO2 flux and chlorophyll fluorescence were increased in plants cultured in TIBs with sucrose-reduced medium (15gr/L) in comparison with those plants micropropagated in TIBs+30gr/L sucrose, confirming a consistent photomixotrophic stage since the 5th day of culture. Raspberry plants multiplied in TIBs+15gr/L sucrose demonstrated the highest values of plant size, total number of internodes, and percent of acclimatization to greenhouse conditions. Studied variables could be related to an increase of the in vitro photosynthetic activity which might prime plants for the ex vitro environment.

Development of coffee somatic and zygotic embryos to plants differs in the morphological, histochemical and hydration aspects

In Coffea arabica L., the development of direct sowing of somatic embryos (SE) in planting substrate, with subsequent nursery production of plants, has promoted the industrialization of somatic embryogenesis. However, plant conversion rates are still low and require improvements to enhance the cost-effectiveness of commercial micropropagation. With the aim of improving plant regeneration from SE, we studied the morphological and histological criteria and water characteristics during germination and plant conversion of zygotic embryos (ZE) and SE. At the cotyledonary stage, SE produced in a 1 l RITA(®) temporary immersion bioreactor (area 55.8 cm(2)) were morphologically similar in size (2-3 mm) but abnormal as compared with mature ZE. Protein and starch reserve levels were extremely low throughout germination and conversion to plantlets, while the water status remained steady [water content (WC) from 76 to 87%, Ψ from -0.37 to -0.47 MPa, pressure potential from 0.69 to 0.24 MPa]. In ZE, spectacular hydration occurred during the first 3 weeks (WC from 37 to 75%; Ψ from -6.24 to -1.0 MPa). Cotyledons remained undifferentiated for 10 weeks after sowing. Conversely, after only 3 weeks under germination conditions in a RITA(®) bioreactor, spongy and palisade parenchyma and stomata formed in SE cotyledons. The ZE plant conversion was faster than that of SE (14 vs. 22 weeks) and more efficient (rates 96 vs. 55%), with much more substantial hypocotyl and cotyledon development. The use of a new 5 l MATIS(®) bioreactor (area 355 cm(2)), designed especially to favor embryo dispersion and light transmittance to SE, markedly improved the embryo-to-plantlet conversion rate (91%). These results highlight the morphological heterogeneity and lack of protein reserves in SE at the beginning of the germination phase and marked differences in water characteristics. However, they also reveal high phenotypic plasticity, leading to a highly efficient plantlet conversion rate due to better embryo dispersion and light transmittance in more horizontal bioreactors.

Nodule cluster cultures and temporary immersion bioreactors as a high performance micropropagation strategy in pineapple (Ananas comosus var. comosus)

Biotechnology tools based on tissue culture techniques allow mass clonal propagation of plants with desired genetic and sanitary features. In the present work a pineapple micropropagation protocol was established based on nodule cluster cultures (NC) associated with different culture systems. Leaf segments from in vitro-grown plantlets cultivated in culture medium supplemented with 2μM NAA and 8μM 2-iP or BAP resulted in the highest rates of NC induction. NC fresh weight increased ratio was more efficient in a culture medium supplemented with 2μM NAA and 2μM BAP in twin-flasks temporary immersion system (TIS-TF). This culture medium was also efficient for the microshoots development in RITA® temporary immersion system (TIS-RITA®). Microshoots elongation in permanent immersion system (PIS) was more efficient in a culture medium supplemented with 10μM GA3. Shoots longer than 2cm showed 92% survival rate when acclimatized. Free polyamine endogenous levels was measured and showed higher contents for the TIS-TF, followed by TIS-RITA®, and PIS, respectively. Putrescine levels also followed this pattern, being absent in PIS, which, on the other hand, showed higher levels of spermine and spermidine. The results indicate the establishment of efficient protocol for pineapple micropropagation, with a high multiplication and regenerative rates and great potential for agriculture application. The biochemical analysis suggests that besides culture medium composition, flask design and culture system also drastically affects the plant's metabolism toward multiplication and plant regeneration.

An Approach for Micropropagation of Blueberry (<i>Vaccinium corymbosum</i> L.) Plants Mediated by Temporary Immersion Bioreactors (TIBs)

A new procedure for blueberry (Vaccinium corymbosum L.) micropropagation in programmed Temporary Immersion Bioreactors (TIBs based on two separate bottles) was developed for the commercial genotypes Biloxi, Sharp Blue and Brillita. Plant cultures were developed in a controlled environment with 0.4 MPa CO2 enrichment, sucrose-reduced medium, and light intensity of 60 mM m-2·s-1. Principal component analysis showed that component 1 (C1) grouped 64.08% of the total variability, while the first two components accounted for 86.97%. Representation of the principal components demonstrated three clusters corresponding with the blueberry genotypes, and within each cluster plants micropropagated in agar-base medium grouped separately from those plants multiplied in TIBs. Both plant number and total internodes traits (related to the productive efficiency) were demonstrated superior in blueberries propagated in TIBs. Additionally, when transferred to greenhouse conditions, blueberries propagated in TIBs showed higher adaptability and growing rates than those cultured by the conventional approach, altogether evidencing the occurrence of a photomixotrophic stage in the vitroplantlets cultured in TIBs.

Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

Chloroplast transformation technology is a promising approach for the production of foreign proteins in plants with expression levels of up to 70 % of total soluble protein (TSP) achieved in tobacco. However, expression of foreign protein in the chloroplast can lead to drastic or even lethal effects in transplastomic plants grown in soil, thereby potentially limiting the applicability of this technology. For instance, previous attempts to express the outer surface protein A (OspA) from Borrelia burgdorferi in tobacco chloroplasts led to plant death when expressed at 10 % TSP. We show here that this earlier transplastomic line, as well as a new plant line, OspA:YFP, expressing OspA fused to the yellow fluorescent protein, can be propagated in temporary immersion bioreactors (TIBs) using AlkaBurst™ technology to produce leafy biomass that expressed OspA at levels of up to 7.6 % TSP, to give a maximum yield of OspA of about 108 mg/L. Our results show that TIBs provide an alternative method for the production of transplastomic biomass expressing proteins toxic for plants and is a particularly useful approach when ‘absolute’ containment is required.

Hydrogen peroxide induced phenylpropanoids pathway eliciting a defensive response in plants micropropagated in Temporary Immersion Bioreactors (TIBs)

The relation between the oxidative burst and phenylpropanoid pathways has been studied using the sugarcane cultivar C86-56, which does not release phenolics in agar-base micropropagation systems. In stationary liquid culture, a significant production of phenolic compounds and plant survival were determined in sugarcane plants treated with 5mM H2O2. The spectrophotometer determinations and the gene expression analysis corroborated that releasing of phenolics and soluble θ-quinones was induced during the first 24h of treatment. In comparison with the control treatments, sugarcane plants treated with H2O2 demonstrated differences in the micropropagation-related variables when multiplied in Temporary Immersion Bioreactors (TIBs) supplemented with polyethyleneglycol (PEG 20%). Expression of selected genes related to photosynthesis, ethylene, auxins, oxidative burst, and defense pathways were confirmed during the entire PEG 20% stress in the plants coming from the 5mM H2O2 treatment; whereas, much more heterogeneous expression patterns were evidenced in plants stressed with PEG but not previously treated with H2O2. RT-PCR expression analysis supports the hypothesis that while H2O2 induces the oxidative burst, the phenylpropanoids pathways elicit and maintain the defensive response mechanism in micropropagated sugarcane plants.

Effect of different carbon sources on the accumulation of carbohydrate, nutrient absorption and the survival rate of Chinese Ash (Fraxinus mandshurica) explants in vitro

In this study, we analyzed the effect of different carbon sources on: (i) carbohydrate and nutrient absorption in Chinese Ash (Fraxinus mandshurica) explants in vitro, and (ii) explant survival rate during ex vitroacclimatization. The explants were cultured under one of five conditions: (1) in a solid medium supplemented with 30 g l-1 sucrose (SM); (2) in a modified temporary immersion bioreactor (TIB) supplemented with sucrose at 0 (TIB0) or (3) 30 g l-1 (TIBS) concentration; (4) in TIB flushed with CO2 at 700 to 900 µl l-1 (TIBC) with no sucrose; or in TIB flushed with CO2 at 700 to 900 µl/l while simultaneously adding sucrose 30 g l-1 (TIBCS). The explants cultured under TIBCS exhibited the highest level of root induction and acclimatization survival rate. In addition, these explants exhibited maximized fresh weights (FW), dry weights (DW), and sucrose contents. Explants cultured in SM showed the highest rate of carbohydrate accumulation. At the end of the culture incubation period, absorption of nitrogen, phosphorus, potassium, magnesium, iron, zinc and manganese was also higher in explants cultured in SM than in TIB. This study revealed that the use of TIBCS provided optimum explant survival. Key words: Fraxinus mandshurica, temporary immersion bioreactor, CO2 enrichment, nutrient absorption.

Comparison of liquid culture methods and effect of temporary immersion bioreactor on growth and multiplication of banana (Musa, cv. Dwarf Cavendish)

Four different liquids, as well as solid culture methods used in shoot propagation of banana were compared. Treatments studied were solid medium (A), liquid medium with immersion of plants (B), liquid medium with cotton culture support (C), liquid medium aerated by bubbling (D), and liquid medium with a temporary immersion bioreactor system (TIB) for 20 min every 1 h (E). After 4 weeks of culture, shoots in liquid medium with immersion and liquid medium aerated by bubbling showed none too little proliferation. Shoots in the solid medium and those cultured in liquid medium containing cotton culture supported played multiplication rates of 2.7 to 3.5 with the highest multiplication rate (> 7.00) observed in the explants that were subjected to the TIB in the medium. Three treated groups differed in the accumulation of dry matter; the lowest weight (around 0.6 g) was observed in treatments B and D, while 2 to 4 times greater accumulation was observed in the explants in the solid medium and those cultured in the liquid medium with a cotton culture support. The highest multiplication rates and weight gains were observed in the liquid medium with a TIB (E). Shoots in liquid medium continuously aerated by bubbling, displayed hyperhydricity of the outer leaf sheaths. However, this was not observed with temporary immersion of explants. Keywords: Banana, micropropagation, dwarf cavendish, temporary immersion bioreactor (TIB)

The Potential of Temporary Immersion Bioreactors (TIBs) in Meeting Crop Production Demand in Nigeria

Micropropagation, popularly known for large-scale clonal propagation, is the first major and widely accepted practical application of plant biotechnology. The commercial utility of conventional micropropagation of important crop species is limited as a result of the large numbers needed annually to start up new farms in addition to high production costs. These result primarily from high labour cost, low multiplication rate or long duration of multiplication before plantlets are taken to the field, and poor survival rates resulting from contamination risks and during acclimatization. All of these constitute a major setback in the use of Micropropagation for scaling up of the several economic species for commercialization. Temporary Immersion Bioreactor system (TIBs) is a relatively recent micropropagation procedure that employs the use of automated gadgets to control rapid multiplication of plant cultures under adequate conditions. TIBs provide a more precise control of the adequate conditions (gaseous exchange, illumination etc.) required by plants for growth, development and survival than the conventional culture vessels. This bioreactor system incorporates a number of features specifically designed to simplify its operation and reduce production costs. The set-up consists of two vessels, one for the plantlets and the other one for the liquid culture media coupled together through a perforated rubber tubing that permits the flow of the liquid media from one vessel to the other. TIBs consist of three main phases: Multiplication, Elongation and Rooting phase. Plantlets propagated in TIBs have better performance than those propagated by conventional methods of micropropagation. This is as a result of a better handling of the in vitro atmosphere and the nutrition. TIBs provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. In addition to diminishing production costs regarding labour force, Temporary Immersion Bioreactors save energy, augment micropropagation productivity and efficiency.

Improved Mass Multiplication of Rhodiola crenulata Shoots Using Temporary Immersion Bioreactor with Forced Ventilation

A temporary immersion bioreactor system was found to be suitable for mass shoot proliferation of Rhodiola crenulata. The shoot multiplication ratio and hyperhydration rate reached 46.8 and 35.4%, respectively, at a temporary immersion cycle of 3-min immersion every 300 min. Forced ventilation was employed in the temporary immersion bioreactor culture in order to decrease the hyperhydration rate, improve shoot quality and enhance the multiplication ratio. The highest multiplication ratio of 55.7 was obtained under a temporary immersion cycle of 3-min immersion every 180 min with the forced ventilation at an air flow rate of 40 l/h, and the hyperhydration rate was reduced to 26.1%. Forced ventilation also improved the subsequent elongation and rooting rate of these proliferated shoots, and the shoot cultures from the temporary immersion bioreactor formed complete plantlets when subcultured onto a rooting medium containing 5 μmol/l indole-3-acetic acid.

Efeitos do meio de cultura e da relação BAP/ANA na multiplicação in vitro de clones de Eucalyptus grandis x E. urophylla em biorreator de imersão temporária

Foram realizados três experimentos individuais com o objetivo de testar diferentes meios de cultura e combinações entre os fitorreguladores BAP e ANA na multiplicação de clones de Eucalyptus grandis x E. urophylla, utilizando o biorreator de imersão temporária RITA®. O meio de cultura MS e a frequência de imersão a cada 2 h promoveram maior massa fresca e número de brotos por explantes. No entanto, houve diferença quanto ao crescimento das culturas entre os dois clones avaliados. A combinação 1,0 µM de BAP com 0,5 µM de ANA foi a que resultou maiores médias em relação à massa fresca e ao número de brotos. As culturas apresentaram alto percentual de hiper-hidricidade, sendo essa desordem fator limitante nas condições deste estudo para o cultivo de Eucalyptus em biorreatores.

Production of laccase and decolouration of the textile dye Remazol Brilliant Blue R in temporary immersion bioreactors

Laccase production by Trametes pubescens grown on sunflower-seed shells (SS) under solid-state fermentation (SFF) conditions in temporary immersion bioreactors was studied. Three immersion cycles were considered: 1 min immersed and 9 min non-immersed, 1 min immersed and 30 min non-immersed and 1 min immersed and 60 min non-immersed. The latter led to the highest laccase activities (4000–6000 U l −1). Also, the in vitro and in vivo decolouration of the recalcitrant textile dye Remazol Brilliant Blue R (RBBR) was assessed. It was found that RBBR (133.33 mg l −1) was efficiently decolourised by T. pubencens grown on SS under SSF conditions in temporary immersion bioreactors in five successive batches. The percentage of RBBR decolouration was higher than 55% in 4 h and around 70% in 24 h in all the batches. However, it was found that RBBR decolouration by the crude culture filtrates was more advantageous. Thus, an RBBR decolouration percentage of nearly 80% in 2 h was obtained.

Biotechnology of Cannabis sativa L.

Plant-based medicines play an important role in the lives of human beings. Identification of active principles and their molecular targets from traditional medicine provides an enormous opportunity for modern drug development. Using modern biotechnology, plants of specific chemical composition can be mass propagated for the extraction of bulk active pharmaceuticals. In the present study, we have used Cannabis sativa, as a model to assess the potential of the applications of biotechnology for the multiplication of elite medicinal plants. Cannabis contains Δ9-tetrahydrocannabinol (THC), a unique terpeno-phenolic compound which accumulates mainly in the glandular trichomes of the plant. Besides its psychoactivity, THC possesses analgesic, anti-inflammatory, appetite stimulant and anti-emetic properties making it a very promising therapeutic drug especially for cancer and AIDS patients. Because of the allogamous (cross fertilization) nature of this species it is difficult to maintain the reproducibility of the plants with unique chemical profile, if grown from seeds. Therefore, screening of elite clones and their propagation using biotechnological tools is the most suitable way to maintain the quality and the efficacy of the product. This report describes our recent biotechnological advancements in Cannabis propagation using tissue culture, synthetic seeds and temporary immersion bioreactors and, in-vitro conservation of elite propagules using slow growth conditions [1–5]. Acknowledgement: This work was supported in part by the National Institute on Drug Abuse (NIDA), National Institute of Health (NIH), Department of Health and Human Services, USA, Contract No. N01DA-10–7773.

Optimización del protocolo de propagación del plátano cv. CEMSA ¾ en Biorreactores de Inmersión Temporal

La automatización de la producción de plantas con el empleo de Biorreactores de Inmersión Temporal (BIT) constituye una herramienta eficaz para la propagación in vitro con la cual se elevan los coeficientes de multiplicación y se aumenta la calidad del material propagado. Algunos factores que influyen en este proceso son las características de los explantes que se emplean para inocular los BIT, la manipulación de los mismos y su estado fisiológico. Estos, aparejados a los indicadores de eficiencia del sistema, son elementos a tener en cuenta en el estudio de la morfogénesis de cultivos como el plátano, que presenta índices de proliferación bajos en medio semisólido. Con el objetivo de optimizar el protocolo descrito por Roels, et al. (2005), se evaluó el efecto del manejo del explante (con división y sin división), el volumen de medio de cultivo durante tres ciclos de proliferación de brotes de plátano cv. CEMSA ¾ sobre el coeficiente de multiplicación y la calidad de los mismos. El mayor coeficiente de multiplicación se logró con el tratamiento en el que se incrementó el volumen del medio de cultivo en función de la biomasa sin seccionar el explante durante los tres ciclos de propagación. Estas modificaciones al protocolo de Roels, et al. (2005), al reducir la manipulación de los explantes durante cada ciclo de cultivo y aumentar las tasas de multiplicación, lo hacen más eficiente y simple, y de esta manera más aplicable para la propagación de plátano a gran escala.

IN VITRO CULTURE OF CHESTNUT (CASTANEA SATIVA MILL.) USING TEMPORARY IMMERSION BIOREACTORS

Chestnut (Castanea sativa Mill.) is difficult to propagate by grafting and cuttings. However, as an alternative to conventional vegetative propagation methods, efforts have been made to establish reliable in vitro regeneration systems that allow clonal propagation. The two major systems are based on embryogenesis and on micropropagation of axillary shoots. The use of temporary immersion bioreactors may provide an efficient and economic tool for mass clonal propagation of chestnut. Seedlings of Castanea sativa were established in vitro and used as the starting material for the experiments. Apical and axillary explants were grown in liquid multiplication media with a composition based on the semi-solid substrates using the temporary immersion bioreactors (TIB), twin flasks system. Apical explants were qualitatively better (longer shoots, higher number of leaves) compared to axillary explants. Duration and frequency of an immersion affected the multiplication rate and quality of plantlets of Castanea sativa.