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Article

Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.)

by
Bruno Reyes-Beristain
1,
Eucario Mancilla-Álvarez
1,
José Abel López-Buenfil
2 and
Jericó Jabín Bello-Bello
3,*
1
Postgraduate College-Campus Cordoba, Amatlan de los Reyes, Veracruz 94953, Mexico
2
Postgraduate College-Campus Montecillo, Texcoco 56264, State of Mexico, Mexico
3
SECIHTI-Postgraduate College-Campus Cordoba, Amatlan de los Reyes, Veracruz 94953, Mexico
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 842; https://doi.org/10.3390/horticulturae11070842
Submission received: 20 June 2025 / Revised: 9 July 2025 / Accepted: 16 July 2025 / Published: 17 July 2025
(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)
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Abstract

Raspberry (Rubus idaeus L.) micropropagation is an alternative for obtaining plantlets with high genetic and phytosanitary quality. The objective of this study was to establish a protocol for the micropropagation of raspberry (Rubus idaeus L.) using the temporary immersion bioreactor, under intermittent immersion periods and different culture medium volumes. The effect of the liquid medium using the TIB and semisolid was evaluated. Different immersion frequencies and culture medium volumes per explant were evaluated in the TIB. In all treatments, the number of shoots per explant, shoot length, number of leaves per explant, percentage of hyperhydricity, and chlorophyll and β-carotene content at multiplication stage were evaluated. The generated shoots, without a root system, were transferred to the acclimatization stage. The results show that the TIB with an immersion frequency of 2 min every 8 h and a volume of 25 mL of culture medium per explant had the best developmental parameters, with 5.75 shoots per explant, a shoot length of 3.44 cm, and 2% hyperhydricity. The highest chlorophyll and β-carotene content was observed in the TIB at different immersion frequencies of 4, 8 and 12 h, with 25 and 50 mL per explant. Survival percentages higher than 96% were observed in all methods evaluated. In conclusion, the evaluated immersion system is an efficient alternative for R. idaeus micropropagation, without using a rooting stage.

1. Introduction

Raspberry (Rubus idaeus L.), of the Rosaceae family, belongs to the berry group and is cultivated because its fruits have a high nutritional value and are used to make flavorings and syrups [1,2]. In addition, this species has secondary metabolites with antitumor, antioxidant, antimicrobial and antimetastatic activity [3,4]. Despite its importance, this crop does not reach its maximum productive potential when propagation by root cuttings, stakes and plants division is used [5]. Micropropagation through tissue culture is a biotechnological technique used to produce a large number of plantlets under aseptic and controlled conditions [6].
Micropropagation in semisolid culture medium has disadvantages such as high production costs due to labor, use of gelling agents, low multiplication rate and lack of automation [7]. Temporary immersion systems (TISs) are a useful alternative to increase the multiplication rate and reduce operating costs through semi-automation compared to conventional micropropagation techniques in semisolid culture medium. TISs employ semi-automated bioreactors designed for large-scale production of cells, tissues or organs cultured in a liquid medium for a given time and frequency [7,8]. TISs improve efficiency during micropropagation by enhancing the availability and absorption of nutrients, growth regulators, vitamins and other organic compounds. In addition, with adequate immersion time and frequency, they can reduce hyperhydration, which allows increasing the multiplication rate in a shorter culture time [9]. Furthermore, TISs that work through an air flow can stimulate photosynthesis and respiration, processes associated with increased chlorophyll synthesis and improved stomatal functionality [10].
The TISs that have been used for micropropagation of R. idaeus are the LifeReactor™ [11], Temporary Immersion Bioreactor (TIB) [12], Recipient for Automated Temporary Immersion (RITA®) [13,14,15], SETISTM Bioreactor [16], and PlantFormTM Bioreactor [17,18]. However, neither immersion frequency nor explant density per bioreactor was assessed in these protocols. These variables are important for the scaling up of micropropagation using a TIS [7]. In addition, some of these TISs are trademarked, expensive, and sometimes difficult to acquire due to customs procedures in some countries. A TIB consists of twin flasks, one for growing plants and the other for liquid media. Both flasks are connected by platinum-cured silicone tubes and use hydrophobic filters, as described by Escalona et al. [19]. TIBs are designed with a minimum number of components and can be constructed from different materials with varying capacities, simplifying their assembly and maintenance. The design of TIBs not only facilitates quick and easy assembly but also improves operational efficiency. In addition, TIBs allow the volume of the culture medium to be adjusted according to production capacity or vessel size, making it ideal for both research and commercial applications. These features make the TIB a valuable tool for plant micropropagation [20]. The objective of this study was to establish a protocol for the micropropagation of raspberry (Rubus idaeus L.) using the temporary immersion bioreactor, under intermittent immersion periods and different culture medium volumes.

2. Materials and Methods

2.1. Plant Material, Establishment and In Vitro Culture Conditions

Rubus idaeus L. cultivar FRIDA plants were donated by the “Black Venture Farm” company in, Mich, MX (29°11′20.40″ N 102°56′42.83″ W). For in vitro establishment, new shoots of 10 cm in length were collected from the 1-year-old raspberry mother plant. The shoots were washed with running water and liquid soap (Axion Complete®, Mission Hills, S.A. de C.V., Guanajuato, Mexico) and rinsed with sterile water. In a laminar flow hood, the shoots were immersed in 70% (v/v) ethanol for 30 s, then immersed in a 0.25% (w/v) sodium hypochlorite solution with two drops of Tween 20® (Merck KGaA®, Darmstadt, Germany) added per 100 mL of water for five minutes, followed by five rinses with sterile distilled water. Finally, the shoots were reduced to 1 cm and individually cultured in test tubes (20 × 200 mm) containing 20 mL of semisolid MS (Murashige and Skoog) [21] multiplication medium supplemented with 30 g L−1 of sucrose, 0.6 mg L−1 of 6-benzylaminopurine (BAP, Merck KGaA®, Darmstadt, Germany) and 0.1 mg L−1 of naphthaleneacetic acid (NAA, Merck KGaA®, Darmstadt, Germany). This culture medium was prepared with stock solutions and was adjusted to a pH of 5.7 ± 0.1, 0.25% (w/v), Gelrite (Merck KGaA®, Darmstadt, Germany) was added as a gelling agent, and it was autoclaved at 121 °C and 1.03 bar for 20 min. The explants were incubated at 24 ± 2 °C and maintained under a photoperiod of 16/8 h (light:dark) with an irradiance of 40 ± 5 μmol m−2 s−1.

2.2. Evaluation of the Different Cultivation Methods

In vitro stabilized cultures were used for the evaluation of the different cultivation methods in the aforementioned multiplication medium, for which 2 cm long shoots were taken as a source of explants for the evaluation of the conventional system in semisolid medium and liquid medium in the TIB. For both cases, glass jars with a total capacity of 500 mL with 250 mL of culture medium was used at a rate of 50 mL per explant to maintain equal culture conditions. Four jars in semisolid medium and four bioreactors were used for each culture system.

2.3. Evaluation of Different Immersion Frequencies in Temporary Immersion

Three immersion frequencies were evaluated during the in vitro multiplication of R. idaeus. The frequencies were evaluated every 4, 8, and 12 h, with an immersion time of 2 min. The bioreactors and culture conditions were the same as those described above, with five explants placed in each bioreactor. Four TIBs were used for each immersion frequency.

2.4. Evaluation of Different Medium Volumes per Explant in Temporary Immersion

After determining the most appropriate immersion frequency for shoot multiplication (2 min every 8 h), a different number of explants per bioreactor were used (20, 10, 7 and 5 explants). In all cases, glass jars with a total capacity of 1000 mL with 500 mL of multiplication medium were used. The experiments under this design correspond to 25, 50, 71 and 100 mL per explant, respectively. The multiplication liquid culture medium and incubation conditions were the same as those previously described.

2.5. Chlorophyll and Carotenoid Determination

To determine the chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (chl t) content, the methodology proposed by Harborne [22] was used, where 0.25 g of fresh leaf tissue was used for all experiments. The tissue was macerated using a mortar and 80% acetone and left to stand at −4 °C for 24 h. Subsequently, the mixture was filtered with filter paper and adjusted to a final volume of 6.5 mL with 80% acetone. Finally, the absorbances were measured in a spectrophotometer (Genesys 10S, Thermo Scientific, Waltham, MA, USA) with absorbances of 663 and 645 nm.
On the other hand, β-carotene content was determined according to Biehler et al. [23]; 0.25 g of fresh tissue was used for all experiments and macerated with the help of a mortar and 80% acetone, and then allowed to stand at −4 °C for 24 h in 80% acetone to a final volume of 2.5 mL.

2.6. Determination of Hyperhydricity Rate

The shoots that were fragile and translucent leaves were identified as hyperhydric shoots. The hyperhydricity rate was calculated using the formula: hyperhydric shoots/normal shoots × 100.

2.7. Ex Vitro Acclimatization

Fifty shoots, approximately 2–3 cm in length, without hyperhydricity symptoms, were used from each culture system. The shoots used did not have a root system and were grown in 72-cavity polypropylene trays with a substrate composed of peat moss, compost and agrolite (1:1:1 v/v/v). The plantlets were maintained in greenhouse conditions with 50% shade at 35 ± 5 °C, 80 ± 10% relative humidity, light with an irradiance of 100 µmol m−2 s−1. The plantlets were irrigated with 40 mL of potable water thrice daily for four weeks. Subsequently, the plantlets were transplanted into 32-cavity trays exposed to light with an irradiance of 250 µmol m−2 s−1 and were irrigated with 200 mL of potable water twice daily for four weeks.

2.8. Data Analysis

All experiments were laid out in a completely randomized design with four replicates. After four weeks of culture, the number of shoots/explant, shoot length, number of leaves/explant, leaf hyperhydration (%), chlorophyll content, and β-carotene content were evaluated. Prior to statistical analysis, the assumptions of normality were verified using the Shapiro–Wilk test. An analysis of variance was performed with the transformed values using the formula: Y = arcsine (√ (x/100)), where “x” is the percentage value. Additionally, a comparison of means was performed using Tukey’s test (p < 0.05). The data were processed using the SPSS® v. 22 statistical packages for Windows.

3. Results

3.1. Effect of Cultivation Method on In Vitro Multiplication

When evaluating the effect of the different cultivation methods on the in vitro multiplication of R. idaeus, significant differences were observed for the variables number of shoots, shoot length, number of leaves, and percentage of hyperhydration (Table 1). For the first experiment (Table 1, I), the number of shoots per explant was better in temporary immersion cultivation method, with 5.76 shoots per explant, than in the semisolid cultivation method, with 2.45 shoots per explant. For the variable shoot length, the shoot length was longer in temporary immersion cultivation method, with 3.38 cm in length, than in the semisolid cultivation method, with 1.04 cm in length. Regarding the number of leaves per shoot, the number of leaves was higher in the temporary immersion cultivation method, with 7.47 leaves per shoot, than in the semisolid cultivation method, with 5.72 leaves per shoot. For the variable percentage of hyperhydration, the highest percentage was better in the temporary immersion cultivation method, with 17.20%, than in the semisolid cultivation method, with 1.00%.

3.2. Effect of Immersion Frequency on Shoot Regeneration

When evaluating the effect of the different immersion frequencies on the in vitro multiplication phase of R. idaeus, significant differences were observed for number of shoots, shoot length, number of leaves per explant, and percentage of hyperhydricity (Table 1, II). For the number of shoots, the highest multiplication rate was observed at the 4 and 8 h immersion frequencies with 5.81 and 5.76 shoots per explant, respectively, while the lowest number of shoots was observed at the 12 h immersion frequency with 4.15 shoots per explant. On the other hand, the greatest shoot length was obtained at the 8 h immersion frequency with 3.38 cm in length, while the shortest shoot length was observed at the 4 and 12 h immersion frequencies with 2.36 and 2.38 cm in length, respectively. The highest number of leaves was observed at the 12 h frequency with 7.69 leaves per shoot, while the lowest number of leaves was observed at the 4 and 8 h immersion frequencies with 6.54 and 7.47 leaves per shoot. The highest percentage of hyperhydration was observed in the 4 h temporary immersion treatment with 54% hyperhydration, while the lowest percentage of hyperhydrated shoots was observed at the 8 and 12 h immersion frequencies with 17.20 and 14% hyperhydrated shoots, respectively.

3.3. Effect of Medium Volume per Explant on Shoot Regeneration

When evaluating the effect of the different medium volumes per explant on the in vitro multiplication phase of R. idaeus, significant differences were observed for the number of shoots, shoot length, number of leaves and percentage of hyperhydration (Table 1, III). The highest number of shoots was obtained in the 25 and 50 mL medium volumes per explant with 5.76 and 5.75 shoots, respectively, while the lowest number of shoots was observed in the 71 and 100 mL medium volumes per explant with 3.81 and 4.17 shoots per explant, respectively. Regarding shoot length, the longest length was observed in the 100 mL medium volume per explant with 3.90 cm in shoot length, while the shortest shoots were observed in the 25 and 50 mL medium volumes per explant with 3.40 and 3.44 cm in length, respectively. For the number of leaves per shoot, the highest number of leaves was obtained in the 71 and 100 mL medium volumes per explant with 8.13 and 8.11 leaves per shoot, respectively, while the lowest number of leaves per shoot was obtained in the 25 mL medium volume with 6.46 leaves. Regarding the percentage of hyperhydration, the highest percentage was observed in the 71 and 100 mL volumes per explant, with 34 and 39% hyperhydration, respectively, while the lowest percentage of hyperhydration was observed in the 25 mL medium volume per explant, with 2% hyperhydration.
The effect of different cultivation methods during in vitro multiplication of raspberry is shown in Figure 1.

3.4. Chlorophyll and Carotenoid Determination During Different Cultivation Methods and Immersion Frequency

Chlorophyll (a, b and total) and carotenoid (β-carotene) contents showed significant differences between semisolid and temporary immersion cultivation methods (Figure 2a,b). The higher chlorophyll a, b and total contents were found in the temporary immersion cultivation method, with chlorophyll a, b and total 0.72, 0.62, and 1.35 mg g−1 FW, respectively, than in the semisolid cultivation method, with chlorophyll a, b and total 0.57, 0.27, and 0.85, respectively. Regarding carotenoid content, the higher β-carotene content was observed at the temporary immersion cultivation method, with 11.17 g−1 FW, than in the semisolid cultivation method, with 8.25 mg g−1 FW. For immersion frequency, no significant differences were observed in chlorophyll a contents at the immersion frequency of 4, 8 and 12 h, with 0.76, 0.72, and 0.78 mg g−1 fresh weight (FW), respectively. For total chlorophyll contents, no significant differences were observed at the immersion frequency of 4, 8 and 12 h, with 1.38, 1.35, and 1.22 mg g−1 fresh weight (FW), respectively, while the lowest chlorophyll b content was found at the immersion frequency of 12 h, with 0.44 mg g−1 FW. Regarding carotenoid content, no significant differences were observed among the different immersion frequencies.

3.5. Chlorophyll and Carotenoid Determination in Medium Volume

Chlorophyll (a, b and total) and carotenoid (β-carotene) contents showed significant differences among the different culture medium volumes per explant (Figure 3a,b). The highest chlorophyll a content was obtained in the 25 and 50 mL medium volumes per explant, with 0.74 and 0.76 mg g−1 FW, respectively, while the lowest chlorophyll a content was obtained in the 71 and 100 mL volumes per explant, with 0.64 and 0.65 mg g−1 FW, respectively. The highest chlorophyll b content was recorded in the 25 and 50 mL medium volumes per explant, with 0.64 and 0.59 mg g−1 FW, respectively, while the lowest chlorophyll b content was obtained in the 71 and 100 mL volumes per explant, with 0.56 and 0.48 mg g−1 FW, respectively. Regarding total chlorophyll content, the highest level was observed in the 25 and 50 mL medium volumes per explant, with 1.38 and 1.36 mg g−1 FW, respectively, while the lowest total content was observed in the 71 and 100 mL volumes per explant, with 1.22 and 1.23 mg g−1 FW, respectively. Regarding carotenoid content, the highest β-carotene content was obtained in the 25 and 50 mL medium volumes per explant, with 10.93 and 10.94 mg g−1 FW, respectively, while the lowest β-carotene content was found in the 71 and 100 mL volumes, with 8.77 and 9.08 mg g−1 FW, respectively.

3.6. Acclimatization

Shoots obtained in vitro (Figure 4a) showed no significant differences in the survival percentage among the evaluated culture systems, presenting a survival rate of between 96 and 99% at four weeks under greenhouse conditions (Figure 4b). Subsequently, the plantlets were transplanted to the field after eight weeks of greenhouse cultivation (Figure 4c,d).

4. Discussion

4.1. Effect of Cultivation Method on In Vitro Multiplication

The different cultivation methods had a significant effect on the number of shoots per explant, shoot length, number of leaves per shoot, percentage of hyperhydration, chlorophyll content, and β-carotene content. The temporary immersion culture system showed a higher in vitro multiplication rate compared to the semisolid culture system. This result could be attributed to the fact that the TIB allows explants to be exposed to the liquid culture medium for a scheduled time and frequency.
The efficiency of the TIS in increasing the number of shoots per explant under in vitro conditions in the genus Rubus spp. has been previously demonstrated in the Life Reactor™ [11], temporary immersion bioreactor (TIB) [12], PlantFormTM biorreactor [17], Recipient for Automated Temporary Immersion (RITA®) [13,14,15], SETISTM Bioreactor [16], and ElecTISTM bioreactor [24]. Debnath [11], in Rubus chamaemorus L., during in vitro multiplication in the Life Reactor™ obtained 6.1 shoots per explant, while in semisolid medium 3.2 shoots per explant were obtained. Arencibia et al. [12], in R. idaeus L. cv Meeker, developed an in vitro multiplication protocol in the TIB obtaining 3.9 shoots per explant, while, in semisolid medium, 1.8 shoots per explant were obtained. On the other hand, Welander et al. [17], in R. idaeus L. cv. Mormorshallon, observed no significant differences during in vitro multiplication in semisolid medium and the PlantFormTM bioreactor, obtaining 3.75 shoots per explant. Debnath [14], in R. idaeus L. cv. Latham, during in vitro propagation in RITA® obtained 4.1 shoots per explant, while in semisolid medium 1.9 shoots per explant were obtained. Geogieva et al. [15], in R. idaeus cv. Polka, developed an in vitro propagation protocol in RITA® obtaining 6.7 shoots per explant, while, in semisolid medium, 3.2 shoots per explant were obtained. Bošnjak et al. [16], in R. idaeus cv. HimboTop®, developed a protocol for in vitro propagation in the SETISTM Bioreactor obtaining 4.02 shoots per explant, while, in semisolid medium, 2.74 shoots per explant were obtained. Elazab et al. [24], in R. fruticosus L. cvs Thornfree and Chester, developed a protocol for in vitro multiplication using the ElecTISTM bioreactor obtaining 3.0 shoots per explant, while, in semisolid medium, 1 shoot per explant was obtained. The increase in the multiplication rate in temporary immersion could be explained by two factors: (1) a greater use of the components of the liquid culture medium such as nutrients, growth regulators, vitamins, sucrose and other organic compounds, and (2) the loss of apical dominance of the explants caused by the constant movement in the liquid medium during immersion [9]. In contrast, according to Mancilla-Álvarez et al. [7] a low multiplication rate in the semisolid cultivation method could be due to a lower availability of the culture medium components, since the contact of the explant base with the culture medium limits efficient absorption and there is no loss of explant apical dominance.
Regarding the number of leaves per shoot, Jones-Castro and Flores-Mora [13] reported that the shoots obtained in temporary immersion culture systems are more vigorous, have a greater number of leaves and possess a greater extension of the leaf area. However, one aspect to consider in temporary immersion culture systems is the increase in hyperhydration. This could be due to the fact that the liquid culture medium during temporary immersion facilitates greater hydration of the explants. This is because the liquid medium has the ability to be absorbed by the leaves, stems, stomata, lenticels and roots of the shoots. Hyperhydration is a severe physiological disorder involving apoplastic water accumulation, leading to a glassy appearance of tissues due to excessive contact between explants and liquid medium, in addition to wounds, high ionic strength and irradiance [25,26]. In contrast, the use of gelling agents in the semisolid medium decreases the availability of water and other compounds, which reduces the incidence of hyperhydration [27].

4.2. Effect of Immersion Frequency on Shoot Multiplication

Immersion frequency is a fundamental factor for in vitro multiplication. Immersion frequency controls nutrient availability in the culture medium, explant movement, gas exchange (in air-flow bioreactors) and relative humidity [9]. Proper control of immersion frequency regulates physiological processes in plants, including stomatal opening and functionality, synthesis of photosynthetic pigments, photosynthetic activity, respiration, nutrient uptake and hyperhydricity [28]. The frequency of immersion every 4 h presented a high number of shoots per explant, but a higher percentage of hyperhydricity. A higher immersion frequency can lead to hyperhydricity in plant tissues, which is detrimental to plantlet survival during acclimatization [29,30,31]. On the contrary, the immersion frequency of every 8 h for 2 min proved to be the most suitable for in vitro multiplication of R. idaeus, obtaining an adequate number of shoots per explant, with greater length and a lower percentage of hyperhydration. This behavior could be attributed to the balance between the adequate absorption of water and components of the culture medium [32,33,34]. Ayub et al. [35] used the TIB for in vitro multiplication of blackberry (R. ulmifulius) cv Tupy with an immersion frequency of every 4 h for 20 s, obtaining 5.4 shoots per explant and 1% hyperhydration.
In this study, the immersion frequency of every 12 h, being a prolonged period of time, limits the availability of the culture medium components. Immersion frequencies play an important role in nutrient utilization, as well as growth regulators and control of explant hyperhydration.

4.3. Effect of Medium Volumes on Shoot Multiplication

The volume of culture medium in temporary immersion plays an important role during explant development. In this study, the 71 and 100 mL medium volumes per explant presented the lowest number of shoots per explant, longest length per shoot and the highest number of leaves per shoot. In addition, these treatments showed an increase in the percentage of hyperhydration. According to Bello-Bello et al. [36], a low density of explants per bioreactor may not take advantage of the culture medium, causing a waste of resources. High concentrations of salts, such as nitrates (N), phosphates (P) and potassium (K), increase the absorption of water by osmosis, causing water-saturated tissues, thereby increasing the risk of hyperhydration. Etienne and Berthouly [37] indicated that the use of high medium volumes can be inefficient because some in vitro cultures produce compounds that stimulate the formation of shoots, which could unbalance the physiological processes of the explants. In contrast, the 25 and 50 mL medium volumes per explant did not affect the number of shoots per explant. However, a significant difference was observed in the percentage of hyperhydration, with the 25 mL volume per explant being the most efficient treatment to maintain the number of shoots without excessive hyperhydration. This could be due to the fact that the density of 20 explants per 500 mL bioreactor allowed a better use of the culture medium components, reducing the percentage of hyperhydration.
Debnath [38] developed a protocol for in vitro multiplication of lowbush blueberry (Vaccinium angustifolium Ait.), wild clone ‘NB1’ in the RITA® bioreactor, with a culture medium volume of 35 mL per explant, obtaining 8.5 shoots per explant, with no hyperhydration being observed. Similarly, Ayub et al. [39] used the TIB for in vitro multiplication of blueberry (Vaccinium ashei R.), with a volume of 90 mL of culture medium per explant, obtaining 1.73 shoots per explant, with 0.6% hyperhydration. On the other hand, Clapa et al. [18] used the PlantFormTM bioreactor for in vitro propagation of highbush blueberry (Vaccinium corymbosum L.), with a culture medium volume of 50 mL per explant, obtaining 12.65 shoots per explant, with no hyperhydration being observed. Kryukov et al. [40] used the RITA® bioreactor for in vitro propagation of strawberry (Fragaria × ananassa) cv. Murano, using a volume of 15 mL per explant, obtaining 33 shoots per explant, with no hyperhydration present.

4.4. Chlorophyll and Carotenoid Determination

Chlorophyll and carotenoid contents were greater in temporary immersion compared to semisolid medium. These results suggest that the increase in photosynthetic pigments during temporary immersion could promote in vitro shoot development. Also, chlorophyll and carotenoid contents are important for in vitro photosynthesis, also called photomixotrophism.
The highest chlorophyll content levels were obtained in the TIS at different frequencies. This could be due to the fact that the bioreactor allows the air exchange that occurs during temporary immersion, which may be the cause of the improvement in photosynthetic activity. The use of TI allows a greater flow of gases (O2, CO2 and N2) and improves the availability of nutrients such as nitrogen (N) and magnesium (Mg) in the liquid medium, important elements of the chlorophyll molecule.
The increase in chlorophyll is an indirect indicator of photosynthesis, which could promote in vitro photomixotrophism [41]. In vitro photomixotrophism refers to the capacity of explants to acquire energy from the culture medium and/or as a product of in vitro photosynthesis [42]. In contrast, the lowest chlorophyll content was observed in semisolid systems, possibly due to the lower availability of essential elements for chlorophyll synthesis and limited aeration in the culture vessels. Factors determining the photosynthetic rate include CO2 supply, irradiance, stomatal function and nutrient availability [7]. Elazab et al. [24], in blackberry (R. fruticosus L.) cv. Thornfree, reported an increase in chlorophyll using the ElecTIS bioreactor at a frequency of every 6 h for 8 min, compared to the semisolid medium. On the contrary, Clapa et al. [18], in blueberry (V. corymbosum L.), reported an increase in chlorophyll in the semisolid medium, compared to the PlantFormTM bioreactor. Regarding β-carotene content, the highest levels were obtained in the temporary immersion systems with different frequencies (4, 8 and 12 h). This effect was probably due to the availability of nutrients in the liquid culture medium. Furthermore, carotenoids are directly related to higher chloroplast density [43], which together could contribute to promoting photomixotrophism in vitro.
Regarding the chlorophyll content in the different culture medium volumes per explant, the highest chlorophyll content levels were obtained with the 25 and 50 mL medium volumes per explant. This fact could be due to an adequate proportion of essential elements available to the explants, which could favor chlorophyll synthesis and indirectly have an effect on photosynthesis in vitro. On the contrary, the lowest chlorophyll content was observed in the treatments with 71 and 100 mL of culture medium volume per explant. This effect is probably due to the excess of nutrients that alter the physiological balance. Excess N can promote disproportionate vegetative growth causing a relative reduction in photosynthetic pigments. Arigundam et al. [44], in ligonberry (Vaccinium vitis-idaea L.), reported an increase in chlorophyll using the RITA® bioreactor with 25 mL of culture medium per explant, compared to the semisolid medium. Similarly, Clapa et al. [18], in blueberry (Vaccinium corymbosum L.) cv. Duke, reported an increase in chlorophyll using the PlantFormTM bioreactor with 25 mL of culture medium per explant, compared to the semisolid medium. Regarding β-carotene content, it was observed that the lowest content of this pigment occurred in the treatments with 71 and 100 mL of culture medium volume per explant. This could be due to the higher percentage of hyperhydricity leading to poor photosynthesis.

5. Conclusions

Temporary immersion systems have a physiological effect on explant development, affecting the number of shoots per explant, shoot length, number of leaves per explant, hyperhydration, chlorophyll content, and β-carotene. Due to these effects, it is important to evaluate immersion frequencies and culture medium volumes per explant. The results show that the temporary immersion bioreactors with an immersion frequency of 2 min every 8 h and a volume of 25 mL of culture medium per explant had the best developmental parameters. In this study, a shoot elongation and rooting phase was not necessary, which represents advantages due to a shorter culture time and reduced labor and reagent costs. Temporary immersion bioreactors are an alternative for commercial raspberry micropropagation.

Author Contributions

Conceptualization, B.R.-B. and E.M.-Á.; methodology, E.M.-Á.; validation, E.M.-Á. and J.A.L.-B.; formal analysis, J.A.L.-B.; investigation, B.R.-B. and E.M.-Á.; resources, J.J.B.-B.; data curation, E.M.-Á.; contributed to the revising the manuscript, E.M.-Á. and J.A.L.-B.; validation, J.J.B.-B.; writing—original draft preparation, J.J.B.-B. and E.M.-Á.; writing—review and editing, J.J.B.-B.; visualization, J.J.B.-B.; supervision, J.J.B.-B. and E.M.-Á.; project administration, J.J.B.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data can be made available upon prior request.

Acknowledgments

This work was supported by Black Venture Farm, which generously donated the raspberry plant material used in this study.

Conflicts of Interest

The authors declare that they have no financial conflicts of interest that could have influenced the conduct of this study.

References

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Horticulturae 11 00842 g001
Figure 1. Effect of different culture methods during in vitro multiplication of raspberry (Rubus idaeus L.), obtained from semisolid medium and a temporary immersion bioreactor at 4 weeks. (a) Semisolid medium; (b–d) temporary immersion for 2 min every 4 h, 8 h, and 12 h, respectively; and (e–h) culture medium volume per explant at 25, 50, 71 and 100 mL, respectively (frequency of immersion for 2 min every 8 h). Bar = 10 cm.
Figure 1. Effect of different culture methods during in vitro multiplication of raspberry (Rubus idaeus L.), obtained from semisolid medium and a temporary immersion bioreactor at 4 weeks. (a) Semisolid medium; (b–d) temporary immersion for 2 min every 4 h, 8 h, and 12 h, respectively; and (e–h) culture medium volume per explant at 25, 50, 71 and 100 mL, respectively (frequency of immersion for 2 min every 8 h). Bar = 10 cm.
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Figure 2. Effect of different immersion frequencies during in vitro multiplication of Rubus idaeus L. after 4 weeks of culture. (a) Chlorophyll content, (b) β-carotene content. The values represent the means ± standard error. Different letters represent a significant difference (Tukey, p < 0.05). FW: fresh weight, Chl: Chlorophyll, TI: temporary immersion.
Figure 2. Effect of different immersion frequencies during in vitro multiplication of Rubus idaeus L. after 4 weeks of culture. (a) Chlorophyll content, (b) β-carotene content. The values represent the means ± standard error. Different letters represent a significant difference (Tukey, p < 0.05). FW: fresh weight, Chl: Chlorophyll, TI: temporary immersion.
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Horticulturae 11 00842 g003
Figure 3. Effect of different medium volumes per explant during in vitro multiplication of Rubus idaeus L. after 4 weeks of culture. (a) Chlorophyll content, (b) β-carotene content. The values represent the means ± standard error. Different letters represent a significant difference (Tukey, p < 0.05). FW: fresh weight, Chl: chlorophyll.
Figure 3. Effect of different medium volumes per explant during in vitro multiplication of Rubus idaeus L. after 4 weeks of culture. (a) Chlorophyll content, (b) β-carotene content. The values represent the means ± standard error. Different letters represent a significant difference (Tukey, p < 0.05). FW: fresh weight, Chl: chlorophyll.
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Figure 4. In vitro shoots and acclimatization of raspberry (Rubus idaeus L.) plantlets to ex vitro conditions. (a) Shoots after 4 weeks of in vitro culture in different culture systems, (b) acclimatized plantlets after 4 weeks in greenhouse, (c) plantlets after 8 weeks of cultivation, and (d) planting of R. idaeus after 12 weeks of culture.
Figure 4. In vitro shoots and acclimatization of raspberry (Rubus idaeus L.) plantlets to ex vitro conditions. (a) Shoots after 4 weeks of in vitro culture in different culture systems, (b) acclimatized plantlets after 4 weeks in greenhouse, (c) plantlets after 8 weeks of cultivation, and (d) planting of R. idaeus after 12 weeks of culture.
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Table 1. Effect of different immersion frequencies, culture medium volumes on in vitro multiplication and ex vitro survival rate during the acclimatization stage of raspberry (Rubus idaeus L.).
Table 1. Effect of different immersion frequencies, culture medium volumes on in vitro multiplication and ex vitro survival rate during the acclimatization stage of raspberry (Rubus idaeus L.).
TreatmentsNumber of Shoots Per ExplantShoot Length (cm)Number of Leaves Per ShootHyperhydricity (%)Survival
(%)
(I) Cultivation method
Semisolid2.45 ± 0.15 b1.04 ± 0.09 b5.72 ± 0.23 b1.00 ± 0.00 b96.33 ± 2.00 a
Temporary immersion5.76 ± 0.27 a3.38 ± 0.10 a7.47 ± 0.31 a17.20 ± 2.15 a98.33 ± 1.20 a
(II) Immersion frequency (2 min immersion)
Frequency every 4 h5.81 ± 0.16 a2.36 ± 0.08 b6.54 ± 0.19 bc54.00 ± 4.72 a98.00 ± 1.00 a
Frequency every 8 h5.76 ± 0.27 a3.38 ± 0.10 a7.47 ± 0.31 ab17.20 ± 2.15 b98.33 ± 1.20 a
Frequency every 12 h4.15 ± 0.22 b2.38 ± 0.12 b7.69 ± 0.36 a14.00 ± 1.73 b97.00 ± 1.52 a
(III) Culture medium volume per explant (mL) *
255.76 ± 0.18 a3.44 ± 0.07 b6.46 ± 0.19 c2.00 ± 0.64 c99.00 ± 1.00 a
505.75 ± 0.16 a3.40 ± 0.06 b7.34 ± 0.14 b17.55 ± 1.50 b99.00 ± 1.00 a
713.81 ± 0.15 b3.70 ± 0.19 ab8.13 ± 0.23 a34.00 ± 4.05 a97.00 ± 1.52 a
1004.17 ± 0.23 b3.90 ± 0.08 a8.11 ± 0.26 a39.60 ± 3.38 a97.00 ± 1.52 a
Average values of means ± standard error at 4 weeks of in vitro and ex vitro culture. Different letters indicate significant differences according to (I) Student t-tests at 95% confidence level. (II and III) analysis of variance (ANOVA, p < 0.05). * Immersion frequency of 2 min every 8 h.
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Reyes-Beristain, B.; Mancilla-Álvarez, E.; López-Buenfil, J.A.; Bello-Bello, J.J. Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.). Horticulturae 2025, 11, 842. https://doi.org/10.3390/horticulturae11070842

AMA Style

Reyes-Beristain B, Mancilla-Álvarez E, López-Buenfil JA, Bello-Bello JJ. Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.). Horticulturae. 2025; 11(7):842. https://doi.org/10.3390/horticulturae11070842

Chicago/Turabian Style

Reyes-Beristain, Bruno, Eucario Mancilla-Álvarez, José Abel López-Buenfil, and Jericó Jabín Bello-Bello. 2025. "Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.)" Horticulturae 11, no. 7: 842. https://doi.org/10.3390/horticulturae11070842

APA Style

Reyes-Beristain, B., Mancilla-Álvarez, E., López-Buenfil, J. A., & Bello-Bello, J. J. (2025). Temporary Immersion Bioreactor for In Vitro Multiplication of Raspberry (Rubus idaeus L.). Horticulturae, 11(7), 842. https://doi.org/10.3390/horticulturae11070842

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