Next Article in Journal
Using HPLC–MS/MS to Assess the Quality of Beet, Mizuna, Lettuce and Corn Salad after Juglone and Walnut Leaf Extract Treatments
Next Article in Special Issue
In Vitro Organogenesis of Critically Endangered Lachenalia viridiflora
Previous Article in Journal
Characterization of Berry Aromatic Profile of cv. Trebbiano Romagnolo Grapes and Effects of Intercropping with Salvia officinalis L.
Previous Article in Special Issue
In Vitro Propagation of the Dendrobium anosmum Lindl. Collected in Vietnam
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 2
10.3390/agronomy12020346
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Temporary Immersion Bioreactor System as an Efficient Method for Mass Production of In Vitro Plants in Horticulture and Medicinal Plants

by
Ho-Dong Hwang
1,2,
Suk-Hyun Kwon
1,
Hosakatte Niranjana Murthy
1,3,
Seung-Won Yun
2,
Sung-Soo Pyo
2 and
So-Young Park
1,*
1
Department of Horticulture, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28466, Korea
2
Oriental Medicine Cenetr, Chungbuk Technopark, Yeongudangi-ro 76, Ochang 28116, Korea
3
Department of Botany, Karnatak University, Dharwad 580003, India
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(2), 346; https://doi.org/10.3390/agronomy12020346
Submission received: 19 December 2021 / Revised: 27 January 2022 / Accepted: 28 January 2022 / Published: 29 January 2022
(This article belongs to the Special Issue In Vitro-Based Systems for Horticultural and Floricultural Crop Improvement)
Browse Figures
Versions Notes

Abstract

:
A temporary immersion system (TIS) bioreactor has been used as an efficient and cost-effective method for the in vitro propagation of many plant species. In the current study, the applicability of a TIS bioreactor for plantlet regeneration Chrysanthemum morifolium Ramat., Fragaria × ananassa Duch., and Cnidium officinale Makino was studied. Shoot length, a number of leaves per regenerated shoot, fresh, and dry biomass of plantlets were optimal with the TIS compared to semi-solid and liquid immersion cultures. The leaf area in cryshanthmum, strawberry, and C. afficinale were 2.87 cm2, 3.51 cm2, and 1.43 cm2, respectively, in the plants regenerated by TIS. The photosynthetic pigments were highest in strawberry plants grown in TIS bioreactor culture, and there was no significant difference between semi-solid and liquid culture while the highest values were obtained in C. officinale maintained in semi-solid culture. The chrysanthemum and strawberry plants showed a 100% acclimatization rate in all culture systems. C. officinale plants showed the highest survival rate at 96.9%, which were regenerated in the TIS. TIS bioreactor culture, thus, provides a convenient method that could be adopted for commercial in vitro propagation of chrysanthemum, strawberry and C. officinale plants.

1. Introduction

In vitro culture of the plant is an effective technique for producing genetically homogeneous plants in horticulturally important species [1,2,3]. By optimizing in vitro culture conditions, millions of elite clones can theoretically be produced from the mother plant within a year [1]. It is well known that the use of a semi-solid medium using conventional agar increases production costs by the gelling agent during commercial mass propagation and limits the possibility of automation; thus, it is the main reason to increase the cost of in vitro culture products [4,5]. To overcome these bottlenecks of the conventional method of in vitro culture, a bioreactor culture method using a liquid medium was introduced. Since the bioreactor uses a liquid medium, undesirable physiological phenomena such as hyperhydricity may occur in the cultured plants depending on the species and culture methods [5]. Temporary Immersion System (TIS) was introduced to the bioreactor culture to supplement the immersion culture method of bioreactor culture. This system is a method of reducing hyperhydricity of plants and producing physiologically healthy plants by repeating the period in which the liquid medium ebb and flood the bioreactor for a certain period of time by a timer and solenoid valve [5]. In this system, plantlets are immersed in the medium only temporarily to provide nutrients for growth, and the immersion period is followed by a drying period, thereby reducing hyperhydricity.
Since Alvard et al. [6] used TIS for the first time on banana (Musa AAA cv. Grand Naine) micropropagation, many studies using TIS have been reported to overcome the limitations of semi-solid culture and liquid culture [7,8,9,10,11]. TIS is a system in which cells, tissues, and organs of plants are immersed in a liquid medium for a specific time period, semi-automatically using a bioreactor [12]. This system increases the growth rate of plants in many species by improving the ventilation of the culture vessel [13]. In addition, because of these advantages, plants produced in TIS promote physiological processes such as photosynthesis, respiration, chlorophyll development, and stomata function so that they can adapt well to the ex vitro environment during the acclimatization [14].
TIS developed to date are Temporary Immersion (RITA®), Temporary Immersion Bioreactor (TIB®), Ebb-and-Flow bioreactor [15,16], Monobloc Advance Temporary Immersion System (MATIS® [17]), and SETIS™ [12]. Among various TIS bioreactors, SETIS TM has the advantage that the size of the culture vessel is relatively large, about 6 L, and easy to handle. Kim et al. [5] successfully produced virus-free apple plantlets (Malus domestica) in TIS, and it was reported that sweet cherry (Prunus ovatum) and mlalanga (Colocasia esculenta) also showed excellent growth of plantlets in this system [18,19].
Chrysanthemum (Chrysanthemum morifolium) is one of the most popular potted and herbaceous landscape plants worldwide. In vitro culture of chrysanthemum is necessary for virus-free plant production and mass propagation of new varieties [20]. Strawberry (Fragaria × ananassa Duch.) is also a crop that has vegetative propagation, such as chrysanthemums, and it is necessary to mass produce excellent individuals by using in vitro culture [21]. In addition, Cnidium officinale (synonym: Ligusticum officinale (Makino) Kitag.) is a medicinal plant that grows with rhizomes (Cinidii Rhizoma). The plants produce a flower, but if the plant does not commonly produce seeds because of the incompatibility with chromosomes, it is necessary to produce healthy plantlets by using in vitro culture [22,23]. Therefore, the production of healthy plantlets of these crops is of great commercial importance. In addition to the importance of these three kinds of species in the horticultural industry, each of these plants has different growth characteristics (i.e., chrysanthemums that grow stems in nodes, strawberries that grow as runners on root leaves, and C. officinale that has rhizomes on root leaves); thus, these plants were suitable for the application of plant production in the TIS bioreactor. Strawberries have been studied by Hanhineva et al. [24] in the RITA® system to regenerate a shoot from leaf explants, and chrysanthemums have been studied by Paek et al. [25] for micropropagation with TIS. However, most of the research focuses only on plant growth while studies related to acclimatization are insufficient. Therefore, in this study, the efficiency of the TIS bioreactor was compared by using semi-solid and liquid cultures using chrysanthemum, strawberry, and C. officinale. In addition, through histological analysis and acclimatization of the plants produced from this system, the optimal system was investigated for the production of plantlets.

2. Materials and Methods

2.1. Plant Material and Culture Conditions

In the present experiment, in vitro plantlets of chrysanthemum ‘Golden bel,’ strawberry ‘Seolhyang,’ and C. officinale were subcultured at 4-week intervals in MS medium [26] containing 30 g·L−1 sucrose and maintained for one year before the use in the present study. Chrysanthemum was prepared as a 2 cm long explant including one node with two leaves, and strawberries and C. officinale were prepared as a 2 cm long plantlet including two leaves and used in the experiment. According to the in vitro culture method, explants were cultured using a semi-solid culture, a liquid stasis culture, and a temporary immersion system bioreactor, respectively. The TIS (SETIS TM bioreactor, VERVIT, Zelzate, Belgium) was used for temporary immersion bioreactor culture.
The TIS consisted of an upper container (5.5 L) capable of cultivating plantlets and a lower container (4 L) with a liquid medium. The lower and upper containers are connected by a silicone hose, and the filter (0.22 μm pore size, VERVIT, Zelzate, Belgium) is attached to each area where air enters and exits to maintain sterilization. At this time, the air injection volume is 0.1 vvm (aeration volume/medium volume/minute), and the timer is set so that the plantlet is immersed by the liquid medium for 10 min every 3 h.
In the current experiment, all plants were cultured in MS medium to which no plant growth regulator was added, and 0.8% (w/v) of agar was added for semi-solid culture. TIS added 1 L of the medium, and semi-solid and liquid cultures were added at 100 mL medium to 500 mL in volume plastic containers (SPL, W 120 mm × H 80 mm, Pocheon, Korea). In liquid culture, a plastic net was used to fix the explants. Culture density was set to culture plantlets of 70 individuals per 1 L of the medium in all experiments. Medium pH was adjusted to 5.8 before sterilization and autoclaved at 121 °C for 15 min.
All plants were cultured under white light-emitting diodes (LED, 400–700 nm, PSLED-1203) with a light intensity of 80 μmol m−2 s−1 photosynthetic photon flux density (PPFD) and under the 16 h photoperiod. The cultures were maintained for 6 weeks in a culture room at 25 ± 1 °C, and then the plants harvested in each culture method were examined for growth such as plant height, number of leaves, and root length and sampled for analysis.

2.2. Cytological and Histological Analysis

Cytologic analysis of the leaves of harvested plantlets was peeled 3 min after manicure was applied in the middle area between the central vein and the edge of the leaf at the front and back of the leaf. The peeled manicure section (hardened film strip) was observed with an optical microscope (Olympus BX40, Tokyo, Japan) to measure the size of the epidermal cells (version 4.5, Leica Application Suite, Wetzlar, Germany).
For histological analysis of chrysanthemum, the stem segments were embedded in 3% agar solution, then solidified, and cut thinly with a razor blade. Agar pieces containing stems were stained with Toluidine blue O (0.05% in water, pH 4.4 buffered with 10 mM Na-acetate) for 10 min. The cut surface of the dyed stem was observed with an optical microscope (Olympus BX40, Tokyo, Japan), and the diameters of cortex, pith, and secondary xylem tissue were examined according to Yeung’s method [27].

2.3. Chlorophyll and Carotenoid Content Measurement

The content of chlorophyll a, b, and carotenoids of in vitro cultivated plant leaves of the three species was analyzed. Around 25 mg of the third leaf cut from the plantlet, and then immersed in 6 mL of 80% (v/v) acetone, and maintained for 48 h in a cool and dark state. The absorbance of the supernatant was measured at 645 and 663 nm with a spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). Total chlorophyll content and carotenoid content were calculated according to the method described in Lichtenthaler et al. [28].

2.4. Acclimatization of Harvested Plants from TIS

In order to evaluate the effect of the culture system on the acclimatization of chrysanthemum, strawberry, and C. officinale plants, 30 plantlets were collected from each culture system and then planted in 42 pore floral foams in plastic trays. The plants were watered once every 3 days during the 30 days of the acclimatization process. Acclimatization was maintained with a 300 μmol m−2 s−1 PPFD in an external environment of 25 ± 2 °C. After 4 weeks, the plant length, number of leaves, and the number and length of roots (≥0.5 cm) were evaluated.

2.5. Statistical Analysis

All experiments were repeated 3 times in a completely randomized. Data are expressed as mean ± standard error. Statistically significant differences were determined using Duncan’s multiple range test with SAS (SAS10, Cary, NC, USA).

3. Results and Discussion

3.1. Plant Growth in Different Culture Systems

The comparison of plant growth showed significant differences among the three culture systems in all plant species (Figure 1, Figure 2 and Figure 3). Chrysanthemum plants had the highest fresh weight of 2.75 g and 0.44 g of dry weight per explant in TIS culture, 2.9 times of fresh weight compared to semi-solid culture, and 2.2 times of liquid culture. Strawberry plants also showed the best growth in TIS culture with 1.10 g (semi-solid four times, liquid three times) of the fresh weight per explant and 0.17 g of the dry weight per explant, and C. officinale also showed the best growth with 0.85 g (semi-solid 8.1 times and liquid 2.3 times) of the fresh weight per explant. In the shoot growth, the highest shoot length was obtained in the plants grown in the TIS in all three species, chrysanthemum (10.6 cm), strawberry (10.8 cm), and C. officinale (8.6 cm), and the number of leaves was also the highest in TIS in all three species. In root length, the plants grown in TIS and liquid culture showed longer roots than semi-solid culture ones in chrysanthemum and C. officinale, but the root of strawberry plants showed the best development in TIS treatment, and the highest number of roots was obtained in TIS in all three species.
The TIS culture combines the advantages of immersion and dry periods to maximize gas exchange and increase the absorption of nutrients [5,29]. Plants in the in vitro culture absorb nutrients not only through roots but also through leaves [30]. Under TIS, when plants are immersed in the medium, they are exposed to the medium from the entire epidermis, allowing them to absorb nutrients [5,29]. Therefore, it is thought that the growth and proliferation rate of plantlets grown in TIS was superior to that of plants grown in semi-solid or liquid culture. In addition, one of the main advantages of TIS is that volatile compounds such as ethylene are removed by promoting ventilation in the culture vessel by forcing aeration. Through this, carbon dioxide required for photosynthesis can be resupplied [29]. Compared to TIS, semi-solid and liquid cultures are thought to have poor growth because there is no exchange of gas inside or outside the culture vessel. When pineapple (Ananas comosus L. Merr) was cultured in the semi-solid, liquid, and TIS bioreactor, the highest growth of plants was obtained from TIS [8]. According to studies of Thi et al. [30], when carnations (Dianthus caryophyllus cv. Dreambyul) were cultured in the TIS (RITA®), their fresh weight was nine times higher than in semi-solid cultures. RITA® bioreactor used in a carnation study by Thi et al. [31] consists of 1-L cylindrical vessels and two compartments: an explant culturing container and medium conservation container; thus, the volume of the container for explant culturing is about 0.5 L, which is relatively small compared to the SETIS (6 L, explant culturing container) used in this study. Therefore, the forcing aeration of the TIS bioreactor and widening the contact with the liquid medium were considered as important factors to promote the growth of the shoot and root of many horticultural crops.

3.2. Cytological and Histological Analysis

As a result of examining the area of three species of plant leaves cultured in a three-types of culture system, chrysanthemum (2.87 cm2), strawberry (3.51 cm2), and C. officinale (1.43 cm2) all had the largest leaf area in TIS, and the leaf area of plants grown in this TIS increased by 1.4 times, 2.8 times, and 3.6 times, respectively, compared to plants grown in semi-solid culture in each plant species (Figure 4B,C). As a result of examining the leaf cell size through a microscope, the area of epidermal cells was the largest in TIS in chrysanthemum (2986.8 µm2) and C. officinale (1610.8 µm2), which were 2.2 times and 2.0 times larger, respectively, compared to plants grown in semi-solid medium. However, while the leaf area of strawberry plants increased 2.8 times in TIS compared to semi-solid culture, interestingly, the area of epidermal cells was not significantly different from that of plants grown in semi-solid medium (Figure 4A,C).
Microscopic observation of in vitro grown plants showed that the reason why the size of the leaves in chrysanthemum and C. officinale increased is due to the increase in epidermal cell area. In contrast, strawberry plants confirmed that the number of cells increased and not the size of epidermal cells. Histological observations were performed on the stems of chrysanthemum plants cultured in three different culture systems (Figure 5A). The lengths of the chrysanthemum stem pith (0.69 mm), secondary xylem (0.27 mm), and cortex (0.28 mm) were the longest plants regenerated in TIS (Figure 5B). The stem diameter was one of the most important features showing a significant difference in the growth of chrysanthemum plants cultured in TIS when compared to the control. The development of the secondary xylem in TIS was the best, which was responsible for the diameter of the stem being thicker than the control. Plants grown in TIS culture have a good development of secondary xylem tissue, which can withstand acclimatization stress better than those produced in semi-solid or liquid culture and, thus, increase the plant’s survival rate when acclimatization occurred [32,33].

3.3. Chlorophyll and Carotenoid Contents

The contents of chlorophyll and carotenoid content in the leaves of the plantlets harvested in the three culture systems showed significant differences between the culture methods for strawberries and C. officinale, but there was no difference between treatments for chrysanthemums (Figure 6). The photosynthetic pigments, Chl a (18.2 mg∙g−1 FW), Chl b (6.7 mg∙g−1 FW), and carotenoid (4.5 mg∙g−1 FW), were the highest in strawberries grown in TIS culture, and there was no significant difference between semi-solid and liquid culture while the highest values were obtained in C. officinale maintained in semi-solid culture (Figure 6). It was interesting that strawberries had the highest chlorophyll content per unit weight in the TIS system, which had the largest leaf area, while C. officinale had the highest chlorophyll content in the plants grown on semi-solid culture, which had the smallest leaf area.
The opposite results obtained from strawberry and C. officinale in the relationship between chlorophyll content and leaf size could be interpreted by the differences in the cell size between the two species. In the case of strawberries, plants had a similar cell size in all cultures systems regardless of the leaf area, whereas C. officinale had a positive correlation in cell size and leaf area and small cell size in the semi-solid culture with a small leaf area. Therefore, in the case of C. officinale, the cell size and area of the leaf were the smallest cultured in semi-solid medium; thus, more mesophyll cells might be contained than other two treatments when the same fresh weight of leaf samples was taken for chlorophyll content analysis. It might have resulted in high chlorophyll content as single mesophyll cells containing a similar number of chloroplasts.
The increase in the photosynthetic pigment of strawberry leaves grown in TIS culture can be seen as an increase in photosynthesis efficiency due to gas exchange in the bioreactor [13]. A recent study of Matuszkiewicz et al. [34] using Arabidopsis showed that the plants cultured in ventilated culture sealed with permeable tape enhanced efficiency of photosynthesis and decreased ethylene production by decreasing the stress marker compared to the ones cultured in air-tight sealed plates. The results clearly indicate that stress is greater in conventional culture.

3.4. Acclimatization of Three Horticultural Species

As a result of investigating growth after 4 weeks of transplanting three types of plants cultured in three different systems into floral foam, chrysanthemum and strawberry plants showed 100% acclimatization rate in all culture systems. C. officinale plants showed the highest survival rate at 96.9%, which were regenerated in the TIS, whereas 89% and 86% of plants survived, which were regenerated in semi-solid and in liquid cultures, respectively (Table 1). In all three plant species, above-ground growth, such as fresh weight, dry weight, length of the above-ground part, and a number of leaves, was the best in the plants produced in TIS (Table 1). Zobayed et al. [35] reported that the histological and physiological changes in leaves, including stomata, are closer to ex vitro plants by ventilating the culture container during in vitro cultured plantlets. In this study, the TIS is ventilated culture condition; thus, the plants cultured in TIS might have received relatively less stress during the acclimatization process compared to other cultures, which is interpreted as affecting improved growth.
Unlike strawberry and C. officinale plants, which are radical leaf plants, chrysanthemum plants were grown by elongating nodes, and the plants were cut into 3 cm lengths (2~3 nodes) and acclimatized. Therefore, 30 individuals produced in semi-solid, liquid, and TIS increased to 44, 48, and 59, respectively, during the acclimatization process (Figure 7). These results reveal that TIS culture is more advantageous not only in the growth and development of the plants but also in their multiplication during the process of acclimatization. In particular, great effects on the mass propagation of plant species such as chrysanthemums, which grow faster and increase in their nodal length. Successful micropropagation depends not only on the number of shoots propagated from the explants but also on the morphological quality and vitality of the plants produced [36]. Therefore, acclimatization is an important step in determining the success of microreproduction [37]. According to a study by Martínez-Estrada et al. [29], Anthurium andreanum plants cultivated in TIS showed more accumulation of photosynthetic products and a higher percentage of closed pores than plants cultivated in a semi-solid medium so that acclimatization could be improved. In this regard, Yang and Yeh [38] reported that the Calathea orbifolia plants produced in TIS had higher photosynthetic rates during the ex vitro acclimatization process and that the leaf area and fresh weight were higher than those cultured in a semi-solid medium. Similarly, Aragón et al. [14] reported that TIS-derived plants showed better growth and control of pore function in plantain (Musa AAB) plants. In addition, in this study, it is said that plants grown in a ventilated TIS have less moisture loss and can accumulate more starch in the leaves upon acclimatization. Since the accumulated starch can be used as an energy source during the first few days of ex vitro adaptation, it can be seen that plants produced in TIS are advantageous for acclimatization.

4. Conclusions

The results of current experiments have demonstrated that TIS is an efficient system for mass propagation of plantlets as compared to the conventional in vitro regeneration systems such as semi-solid culture and liquid stationary cultures. Regeneration, growth, and accumulation of biomass by the plantlets were optimal in TIS. Histological observations have revealed that the excellent growth of regenerated plants in TIS was a result of an increase in cell size. In particular, chrysanthemum showed that the development of the secondary xylem from the stem is essential for the survival and growth of plants upon ex vitro transplantation. The results of this study showed that the TIS can be an optimal system for mass production of regeneration of ornamentals, crops, and medicinal plants.

Author Contributions

H.-D.H. and S.-H.K. contributed to data acquisition, experiment, and wrote the manuscript. S.-W.Y. and S.-S.P. participated in data interpretation and H.N.M. participated in editing of the manuscript. S.-Y.P. made substantial contributions to data interpretation, revised the manuscript, and the conception and design of this study. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Industrial Strategic Technology Development Program (Grant number P0018148) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare that they have no conflict of interest.

References

  1. Diengngan, S.; Mahadevamma, M.; Murthy, B.N. Efficacy of in vitro propagation and crown sizes on the performance of strawberry (Fragaria × ananassa Duch) cv. Festival under field condition. J. Agric. Sci. Technol. 2016, 18, 255–264. [Google Scholar]
  2. Ghaderi, N.; Normohammadi, S.; Javadi, T. Morpho-physiological responses of strawberry (Fragaria × ananassa) to exogenous salicylic acid application under drought stress. J. Agric. Sci. Technol. 2015, 17, 167–178. [Google Scholar]
  3. Sowik, I.; Borkowska, B.; Markiewicz, M. The activity of mycorrhizal symbiosis in suppressing Verticillium wilt in susceptible and tolerant strawberry (Fragaria × ananassa Duch.) genotypes. Appl. Soil. Ecol. 2016, 101, 152–164. [Google Scholar] [CrossRef]
  4. Zhang, B.; Song, L.; Bekele, L.D.; Shi, J.; Jia, Q.; Zhang, B.; Chen, J. Optimizing factors affecting development and propagation of Bletilla striata in a temporary immersion bioreactor system. Sci. Hortic. 2018, 232, 121–126. [Google Scholar] [CrossRef]
  5. Kim, N.Y.; Hwang, H.D.; Kim, J.H.; Kwon, B.M.; Kim, D.; Park, S.Y. Efficient production of virus-free apple plantlets using the temporary immersion bioreactor system. Hortic. Environ. Biotechnol. 2020, 61, 779–785. [Google Scholar] [CrossRef]
  6. Alvard, D.; Cote, F.; Teisson, C. Comparison of methods of liquid medium culture for banana micropropagation: Effects of temporary immersion of explants. Plant Cell Tissue Organ Cult. 1993, 32, 55–60. [Google Scholar] [CrossRef]
  7. Businge, E.; Trifonova, A.; Schneider, C.; Rödel, P.; Egertsdotter, U. Evaluation of a new temporary immersion bioreactor system for micropropagation of cultivars of eucalyptus, birch and fir. Forests 2017, 8, 196. [Google Scholar] [CrossRef]
  8. Escalona, M.; Lorenzo, J.C.; González, B.; Daquinta, M.; González, J.L.; Desjardins, Y.; Borroto, C.G. Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep. 1999, 18, 743–748. [Google Scholar] [CrossRef]
  9. Jang, H.R.; Lee, H.J.; Shohael, A.M.; Park, B.J.; Paek, K.Y.; Park, S.Y. Production of biomass and bioactive compounds from soot cultures of Rosa rugosa using a bioreactor culture system. Hortic. Environ. Biotechnol. 2016, 57, 79–87. [Google Scholar] [CrossRef]
  10. Gianguzzi, V.; Inglese, P.; Barone, E.; Sottile, F. In vitro regeneration of Capparis spinosa L. by using a temporary immersion system. Plants 2019, 8, 177. [Google Scholar] [CrossRef] [Green Version]
  11. Posada-Pérez, L.; Montesinos, Y.P.; Guerra, D.G.; Daniels, D.; Gómez-Kosky, R. Complete germination of papaya (Carica papaya L. cv. MaradolRoja) somatic embryos using temporary immersion system type RITA® and phloroglucinol in semi-solid culture medium. In Vitro Cell Dev. Biol. Plant 2017, 53, 505–513. [Google Scholar] [CrossRef]
  12. Vervit SETIS™. Bioreactor Temporary Immersion Systems in Plant Micropropagation. 2021. Available online: http://www.setis-systems.be (accessed on 28 March 2021).
  13. Bello-Bello, J.J.; Cruz-Cruz, C.A.; Pérez-Guerra, J.C. A new temporary immersion system for commercial micropropagation of banana (Musa AAA cv. Grand Naine). In Vitro Cell Dev. Biol. Plant 2019, 55, 313–320. [Google Scholar] [CrossRef]
  14. Aragón, C.E.; Sánchez, C.; Gonzalez-Olmedo, J.; Escalona, M.; Carvalho, L.; Amâncio, S. Comparison of plantain plantlets propagated in temporary immersion bioreactors y gelled medium during in vitro growth y acclimatization. Biol. Plant 2014, 58, 29–38. [Google Scholar] [CrossRef]
  15. Tisserat, B.; Vandercook, C.E. Development of an automated plant culture system. Plant Cell Tissue Organ Cult. 1985, 5, 107–117. [Google Scholar] [CrossRef]
  16. Ducos, J.P.; Labbe, G.; Lambot, C.; Pétiard, V. Pilot scale process for the production of pre-germinated somatic embryos of selected robusta (Coffea canephora) clones. In Vitro Cell Dev. Biol. Plant 2007, 43, 652–659. [Google Scholar] [CrossRef]
  17. Etienne, H.; Bertrand, B.; Georget, F.; Lartaud, M.; Montes, F.; Dechamp, E.; Verdeil, J.L.; Barry-Etienne, D. Development of coffee somatic and zygotic embryos to plants differs in the morphological, histochemical and hydration aspects. Tree Physiol. 2013, 33, 640–653. [Google Scholar] [CrossRef] [Green Version]
  18. Godoy, S.; Tapia, E.; Seit, P.; Andrade, D.; Sánchez, E.; Andrade, P.; Prieto, H. Temporary immersion systems for the mass propagation of sweet cherry cultivars and cherry rootstocks: Development of a micropropagation procedure and effect of culture conditions on plant quality. In Vitro Cell Dev. Biol. Plant 2017, 53, 494–504. [Google Scholar] [CrossRef]
  19. Arano-Avalos, S.; Gómez-Merino, F.C.; Mancilla-Álvarez, E.; Sánchez-Páez, R.; Bello-Bello, J.J. An efficient protocol for commercial micropropagation of malanga (Colocasia esculenta L. Schott) using temporary immersion. Sci. Hortic. 2020, 261, 108998. [Google Scholar] [CrossRef]
  20. Song, J.Y.; Mattson, N.S.; Jeong, B.R. Efficiency of shoot regeneration from leaf, stem, petiole and petal explants of six cultivars of Chrysanthemum morifolium. Plant Cell Tissue Organ Cult. 2011, 107, 295. [Google Scholar] [CrossRef]
  21. Akbar Mozafari, A.; Havas, F.; Ghaderi, N. Application of iron nanoparticles and salicylic acid in in vitro culture of strawberries (Fragaria × ananassa Duch.) to cope with drought stress. Plant Cell Tissue Organ Cult. 2018, 132, 511–523. [Google Scholar] [CrossRef]
  22. Lee, C.Y.; Kim, Y.K.; Kim, Y.S.; Suh, S.Y.; Lee, S.Y.; Park, S.U. Somatic embryogenesis and plant regeneration in Cnidium officinale Makino. J. Med. Plants Res. 2009, 3, 96–100. [Google Scholar]
  23. Kim, D.H.; Park, J.M.; Kang, S.M.; Lee, S.M.; Seo, C.W.; Lee, I.Y.; Lee, I.J. Distribution characteristics of weeds and vegetation types in Cnidium officinale field. Weed Turf. Sci. 2015, 4, 279–287. [Google Scholar] [CrossRef] [Green Version]
  24. Hanhineva, K.; Kokko, H.; Kärenlampi, S. Shoot regeneration from leaf explants of five strawberry (Fragaria × ananassa) cultivars in temporary immersion bioreactor system. In Vitro Cell Dev. Biol. Plant 2005, 41, 826–831. [Google Scholar] [CrossRef]
  25. Paek, K.Y.; Chakrabarty, D.; Hahn, E.J. Application of bioreactor systems for large scale production of horticultural and medicinal plants. Plant Cell Tissue Organ Cult. 2005, 81, 287–300. [Google Scholar] [CrossRef]
  26. Murashige, T.; Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 1962, 15, 473–497. [Google Scholar] [CrossRef]
  27. Yeung, E.C. The use of histology in the study of plant tissue culture systems-some practical comments. In Vitro Cell Dev. Biol. Plant 1999, 35, 137–143. [Google Scholar] [CrossRef]
  28. Lichtenthaler, H.K. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol. 1987, 148, 350–382. [Google Scholar]
  29. Martínez-Estrada, E.; Islas-Luna, B.; Pérez-Sato, J.A.; Bello-Bello, J.J. Temporary immersion improves in vitro multiplication and acclimatization of Anthurium andreanum Lind. Sci. Hortic. 2019, 249, 185–191. [Google Scholar] [CrossRef]
  30. Etienne, H.; Berthouly, M. Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult. 2002, 69, 215–231. [Google Scholar] [CrossRef]
  31. Thi, L.T.; Park, Y.G.; Jeong, B.R. Growth and development of carnation ‘Dream Yul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods. In Vitro Cell Dev. Biol. Plant 2019, 55, 539–548. [Google Scholar] [CrossRef]
  32. Debergh, P.; Aitken-Christie, J.; Cohen, D.; Grout, B.; Von Arnold, S.; Zimmerman, R.; Ziv, M. Reconsideration of the term ‘vitrification’ as used in micropropagation. Plant Cell Tissue Organ Cult. 1992, 30, 135–140. [Google Scholar] [CrossRef]
  33. Picoli, E.A.; Otoni, W.C.; Figueira, M.L.; Carolino, S.M.; Almeida, R.S.; Silva, E.A.; Fontes, E.P. Hyperhydricity in in vitro eggplant regenerated plants: Structural characteristics and involvement of BiP (binding protein). Plant Sci. 2001, 160, 857–868. [Google Scholar] [CrossRef]
  34. Matuszkiewicz, M.; Koter, M.D.; Filipecki, M. Limited ventilation causes stress and changes in Arabidopsis morphological, physiological and molecular phenotype during in vitro growth. Plant Physiol. Biochem. 2019, 135, 554–562. [Google Scholar] [CrossRef] [PubMed]
  35. Zobayed, S.M.A. Ventilation in micropropagation. In Photoautotrophic (Sugar-Free Medium) Micropropagation as a New Micropropagation and Transplant Production System; Springer: Dordrecht, The Netherlands, 2005; pp. 147–186. [Google Scholar]
  36. Martínez, M.T.; Corredoira, E.; Vieitez, A.M.; Cernadas, M.J.; Montenegro, R.; Ballester, A.; San José, M.C. Micropropagation of mature Quercus ilex L. trees by axillary budding. Plant Cell Tissue Organ. Cult. 2017, 131, 499–512. [Google Scholar] [CrossRef]
  37. Gonçalves, S.; Martins, N.; Romano, A. Physiological traits and oxidative stress markers during acclimatization of micropropagated plants from two endangered Plantago species: P. algarbiensis Samp. and P. almogravensis Franco. In Vitro Cell Dev. Biol. Plant 2017, 53, 249–255. [Google Scholar] [CrossRef]
  38. Yang, S.H.; Yeh, D.M. In vitro leaf anatomy, ex vitro photosynthetic behaviors and growth of Calathea orbifolia (Linden) Kennedy plants obtained from semi-solid medium and temporary immersion systems. Plant Cell Tissue Organ Cult. 2008, 93, 201–207. [Google Scholar] [CrossRef]
Agronomy 12 00346 g001 550
Figure 1. Fresh weigh, shoot length, and leaf number of chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants after 4 weeks in semi-solid, liquid, and TIS cultures. Bars with different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Figure 1. Fresh weigh, shoot length, and leaf number of chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants after 4 weeks in semi-solid, liquid, and TIS cultures. Bars with different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Agronomy 12 00346 g001
Agronomy 12 00346 g002 550
Figure 2. Chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants after 4 weeks in semi-solid liquid and TIS cultures. Scale bars = 5 cm.
Figure 2. Chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants after 4 weeks in semi-solid liquid and TIS cultures. Scale bars = 5 cm.
Agronomy 12 00346 g002
Agronomy 12 00346 g003 550
Figure 3. Harvested plants of chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants from semi-solid, liquid, and TIS cultures. Scale bars = 3 cm.
Figure 3. Harvested plants of chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants from semi-solid, liquid, and TIS cultures. Scale bars = 3 cm.
Agronomy 12 00346 g003
Agronomy 12 00346 g004 550
Figure 4. Cytological analysis of chrysanthemum (CM); strawberry (SB); and Cnidium officinale (CO) plants grown in semi-solid, liquid, and TIS cultures. (A) Cytological observation of adaxial leaf surface of CM, SB, and CO plants grown in semi-solid, liquid, and TIS cultures (scale bar = 100 µm). (B) Morphological observation of leaves of CM, SB, and CO plants grown in different culture systems (scale bar = 1 cm). (C) Leaf area and epidermal cell area of CM, SB, and CO plants grown in semi-solid (S-solid), liquid, and TIS cultures. Bars with different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Figure 4. Cytological analysis of chrysanthemum (CM); strawberry (SB); and Cnidium officinale (CO) plants grown in semi-solid, liquid, and TIS cultures. (A) Cytological observation of adaxial leaf surface of CM, SB, and CO plants grown in semi-solid, liquid, and TIS cultures (scale bar = 100 µm). (B) Morphological observation of leaves of CM, SB, and CO plants grown in different culture systems (scale bar = 1 cm). (C) Leaf area and epidermal cell area of CM, SB, and CO plants grown in semi-solid (S-solid), liquid, and TIS cultures. Bars with different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Agronomy 12 00346 g004
Agronomy 12 00346 g005 550
Figure 5. Histological analysis of chrysanthemum plants grown using semi-solid, liquid, and TIS cultures. (A) Histological observation of chrysanthemum stems in semi-solid, liquid and TIS cultures (p: pith, sx: secondary xylem, ct: cortex). (B) Stem diameter of chrysanthemum plants in semi-solid, liquid and TIS cultures. Different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Figure 5. Histological analysis of chrysanthemum plants grown using semi-solid, liquid, and TIS cultures. (A) Histological observation of chrysanthemum stems in semi-solid, liquid and TIS cultures (p: pith, sx: secondary xylem, ct: cortex). (B) Stem diameter of chrysanthemum plants in semi-solid, liquid and TIS cultures. Different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Agronomy 12 00346 g005
Agronomy 12 00346 g006 550
Figure 6. Chlorophyll a, Chlorophyll b, and Carotenoid content of in vitro grown chrysanthemum (CM), strawberry (SB) and Cnidium officinale (CO) plants grown in semi-solid (S-solid), liquid, and TIS cultures. Bars with different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Figure 6. Chlorophyll a, Chlorophyll b, and Carotenoid content of in vitro grown chrysanthemum (CM), strawberry (SB) and Cnidium officinale (CO) plants grown in semi-solid (S-solid), liquid, and TIS cultures. Bars with different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Agronomy 12 00346 g006
Agronomy 12 00346 g007 550
Figure 7. Acclimatization of in vitro grown chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants in TIS and semi-solid and liquid cultures. (A) Acclimatized chrysanthemum plants after 4 weeks harvested from semi-solid, liquid, and TIS cultures. (B) Number of acclimatized plants after acclimatization of semi-solid, liquid, and TIS cultures. NS: no significance. Different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Figure 7. Acclimatization of in vitro grown chrysanthemum (CM), strawberry (SB), and Cnidium officinale (CO) plants in TIS and semi-solid and liquid cultures. (A) Acclimatized chrysanthemum plants after 4 weeks harvested from semi-solid, liquid, and TIS cultures. (B) Number of acclimatized plants after acclimatization of semi-solid, liquid, and TIS cultures. NS: no significance. Different letters differ significantly from each other by Duncan’s multiple-range test (DMRT; p ≤ 0.05).
Agronomy 12 00346 g007
Table
Table 1. Comparison of different culture systems on growth of chrysanthemum (CM), strawberry (SB) and Cnidium officinale (CO) plantlets after 4 weeks of acclimatization.
Table 1. Comparison of different culture systems on growth of chrysanthemum (CM), strawberry (SB) and Cnidium officinale (CO) plantlets after 4 weeks of acclimatization.
SpeciesCultureFresh Weight
(g)		Dry Weight
(g)		Shoot Length
(cm)		No. of Leaves/PlantletRoot Length
(cm)		No. of Roots/PlantletSurvival Rate
(%)
Semi-solid6.82 b z0.69 b5.03 b10.80 b5.30 a19.70 a100 a
CMLiquid6.11 c0.62 c4.99 b10.00 b4.53 b14.30 b100 a
TIS7.84 a0.91 a5.79 a14.20 a5.13 a14.10 b100 a
Semi-solid3.01 c0.67 c4.87 c8.80 a4.62 a8.20 b100 a
SBLiquid4.32 b0.79 b6.83 b7.50 b3.77 a8.60 b100 a
TIS7.11 a0.14 a8.65 a8.30 a4.36 a10.50 a100 a
Semi-solid1.75 b0.27 b5.32 b5.20 b2.33 a6.80 a89.0 b
COLiquid1.92 b0.30 b6.32 b5.60 b2.52 a6.20 a86.0 b
TIS4.42 a0.74 a8.14 a7.70 a2.56 a8.60 a97.0 a
z Different letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Hwang, H.-D.; Kwon, S.-H.; Murthy, H.N.; Yun, S.-W.; Pyo, S.-S.; Park, S.-Y. Temporary Immersion Bioreactor System as an Efficient Method for Mass Production of In Vitro Plants in Horticulture and Medicinal Plants. Agronomy 2022, 12, 346. https://doi.org/10.3390/agronomy12020346

AMA Style

Hwang H-D, Kwon S-H, Murthy HN, Yun S-W, Pyo S-S, Park S-Y. Temporary Immersion Bioreactor System as an Efficient Method for Mass Production of In Vitro Plants in Horticulture and Medicinal Plants. Agronomy. 2022; 12(2):346. https://doi.org/10.3390/agronomy12020346

Chicago/Turabian Style

Hwang, Ho-Dong, Suk-Hyun Kwon, Hosakatte Niranjana Murthy, Seung-Won Yun, Sung-Soo Pyo, and So-Young Park. 2022. "Temporary Immersion Bioreactor System as an Efficient Method for Mass Production of In Vitro Plants in Horticulture and Medicinal Plants" Agronomy 12, no. 2: 346. https://doi.org/10.3390/agronomy12020346

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop