Efficient production of virus-free apple plantlets using the temporary immersion bioreactor system


Viral pathogens reduce the quality and yield of apple (Malus domestica) fruits by 30–50%. A mass production system of virus-free apple plantlets is needed to meet the demand of the domestic fruit tree industry. In this study, we compared the production of virus-free plants in different in vitro culture systems, including a temporary immersion bioreactor (TIB), a continuous immersion bioreactor, and conventional solid and liquid culture systems (controls). Apple plantlets were immersed in the TIB once every 3 h (TIB-3) or 6 h (TIB-6). The fresh weight of apple plants was the highest in the TIB-3 system and lowest in the liquid culture. Shoots were the longest in the TIB-3 system, approximately twofold longer than those in solid culture and liquid culture. Roots of apple plants were the longest in the TIB-3 system compared with solid and liquid cultures. Root number in the TIB-3 system was also higher than that in solid and liquid cultures. Moreover, leaf area was the highest in plants grown in the TIB-3 treatment. The total stem area of TIB-3 plants was the largest at 1.46 mm2. This study suggests that the airlift bioreactor is capable of producing a large number of virus-free plants in a short time compared with conventional culture systems. Additionally, secondary xylem was well developed in the stems of plants grown in the TIB-3 system. Therefore, this system shows a high potential for producing healthy plants suitable for acclimatization.


Apple (Malus domestica) is a temperate fruit tree of the Rosaceae family and one of the most cultivated fruit trees in the world. Apple trees are usually propagated using vegetative methods such as grafting and cuttings; however, vegetative propagation increases the risk of virus infection (Chen et al. 2019; Li et al. 2016). The four major viral pathogens of apple include Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV), and Apple scar skin viroid (ASSVd) (Li et al. 2016). Virus infections are reported to reduce the quality and quantity of apple fruits by 30–50% (Cieślińska and Rutkowski 2008; Hadidi and Barba 2011). Therefore, the production of virus-free plants via in vitro culture has been a major focus of recent studies (Chen et al. 2017, 2019; Hu et al. 2017; Ko et al. 2018).

In vitro culture is a powerful method of rapid mass production of virus-free plantlets and is unaffected by seasonal changes and cultivation area (George et al. 2008; Kovalchuk et al. 2009). Conventional culture uses semi-solid medium containing agar and gelrite (Quiala et al. 2012). However, it is difficult to increase the scale of conventional culture, as the vessel volume is a limitation in this method. Moreover, since the medium is not completely removed from plantlet roots during acclimatization, bacterial and fungal infections are highly likely in conventional culture, which decreases the acclimatization rate. These factors increase the production costs associated with conventional culture (Gatti et al. 2017; Georgiev et al. 2014; Quiala et al. 2012).

To overcome these problems, studies were performed using liquid culture (Ascough and Fennel 2004; Cuenca et al. 2017; Sreedhar et al. 2009). In liquid culture, plantlets are in direct contact with the growth medium; therefore, all cells are able to absorb the nutrients uniformly, which promotes plant growth (Ascough and Fennel 2004). Moreover, the secreted waste products are diluted in the liquid medium. However, this method could cause hyperhydricity because of the high relative humidity in the culture vessel (Ascough and Fennel 2004; Cuenca et al. 2017; Sreedhar et al. 2009).

Recently, the bioreactor system has been reported as an ideal method for the mass production of plants, while overcoming the problems of semi-solid and liquid culture systems and reducing the cost of production (Martínez-Estrada et al. 2019). The bioreactor system can be easily automated and scaled up to increase production (Benelli and Carlo 2018; Georgiev et al. 2014; Le et al. 2019; Martínez-Estrada et al. 2019). It also prevents the accumulation of ethylene and other harmful gases by forced aeration, thus promoting the growth and quality of cultured plantlets (Martínez-Estrada et al. 2019).

Among various bioreactor systems, the temporary immersion bioreactor (TIB) system is ideal. In this system, plantlets are immersed in the medium only temporarily to provide nutrients for growth, and the immersion period is followed by the drying period, thereby reducing hyperhydricity (Frometa et al. 2017; Vives et al. 2017). Recently, mass production of plantlets using TIB has been reported in gerbera (Frometa et al. 2017), pistachio (Akdemir et al. 2014), stevia (Ramírez-Mosqueda et al. 2016), and olive (Benelli and Carlo 2018).

In this study, we evaluated the conventional culture systems (solid and liquid) and bioreactor systems (continuous immersion bioreactor [CIB] and TIB) for the efficient mass production of virus-free apple plantlets. Additionally, histological analysis was conducted to investigate the growth parameters of apple plantlets produced in various in vitro culture systems.

Materials and methods

Plant materials and culture conditions

The apple rootstock M9, cultured in vitro, was used in this study. Plantlets were cultured on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 2.2 µM 6-benzylaminopurine (BA), 30 g L− 1 sucrose, and 9.0 g L− 1 agar. Shoot cultures were maintained at 24 ± 1 °C under a 16-h-light/8-h-dark photoperiod using white light emitting diodes (LEDs) at an intensity of 88 µmol photons m− 2 s− 1. Subculturing was conducted at regular monthly intervals.

Bioreactor culture system

The efficiency of mass production of virus-free plants was compared among the different culture systems, including conventional (solid and liquid) cultures, CIB, and TIB (Fig. 1). Apple shoots were cut into 1.5-cm segments. To initiate immersion cultures using CIB and TIB, a 5-L bioreactor containing 2 L of medium was inoculated with 100 shoots. To initiate conventional (solid and liquid) cultures, an SPL culture vessel containing 100 ml medium was inoculated with five shoots. The TIB system comprised two flasks connected via a silicon tube. A net was placed in the bioreactor culture vessel and liquid culture vessel to prevent the plantlets from being submerged in the liquid medium. Apple plantlets were cultured on MS medium supplemented with 2.17 µM indole-3-butyric acid (IBA) and 30 g L− 1 sucrose. In the TIB system, growth medium was supplied in a controlled manner, i.e., every 3 h (TIB-3) or every 6 h (TIB-6) for 10 min, using a timer and a solenoid valve. In the CIB system, the growth medium was supplied for 24 h. All cultures were maintained at a temperature of 24 ± 1 °C under a 16-h-light/8-h-dark photoperiod and 88 µmol photons m− 2 s− 1 light intensity. After 6 weeks of culture, the pigment content and histology of plantlets were investigated.

Fig. 1

Schematic diagram of conventional culture and bioreactor systems used in this study. a Conventional (solid and liquid culture), b continuous immersion bioreactor (CIB), c temporary immersion bioreactor (TIB). (1) Air inlet; (2) air flow meter; (3) membrane filter; (4) medium supply; (5) glass sparger; (6) supporter (net); (7) air outlet; (8) sampling port; (9) valve control air supply

Determination of chlorophyll (Chl) and carotenoid contents

To determine the Chl content of apple plants, 0.05 g of fresh leaves was harvested from plantlets grown in each treatment and extracted in 6 mL of 80% acetone for 48 h in the dark. Contents of Chl a and Chl b were measured at 663.2 and 646.8 nm, respectively, using a spectrophotometer (Uvikon-930, Kontron Instruments, Zurich, Switzerland). The carotenoid content was measured at 470 nm, as described previously (Lichtenthaler 1987).

Histological analysis

Leaf and stem tissues were harvested from apple plantlets and fixed in formalin–alcohol–acetic acid (FAA) solution (formalin:glacial acetic acid:ethanol:distilled water = 10:5:50:35) for 24–48 h and then dehydrated in a graded ethanol series. Subsequently, the samples were embedded in Technovit 7100 (Kulzer, Wehrheim, Germany), as described previously (Yeung 1999), and 5-µm-thick sections were prepared using a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with toluidine blue O and observed under a light microscope (Olympus BX40, Tokyo, Japan).

Statistical analysis

All experiments were replicated at least three times using a completely randomized design. Data are expressed as mean ± standard error (SE). Statistically significant differences were determined using Duncan’s multiple range test in SAS (version 9.4; SAS, USA).

Results and discussion

The growth of apple plantlets varied among the different culture systems (Table 1, Fig. 1). Overall, plant growth was better in the bioreactor systems (CIB and TIB) than in conventional (solid and liquid) cultures (control; Table 2). Shoots were the longest in the TIB-3 system and approximately twofold longer than those in solid culture and liquid culture. The FW of apple plantlets was the highest in the TIB-3 system and lowest in liquid culture. Additionally, the accumulation of ethylene and other gases was less in the TIB culture vessel than in conventional culture because of forced aeration, which likely promoted shoot growth in the TIB system (Georgiev et al. 2014; Martínez-Estrada et al. 2019). Consistent with our results, Ramírez-Mosqueda et al. (2016) reported that the length and FW of stevia plants grown in the TIB system were higher than those grown in semisolid culture. Similarly, Zhang et al. (2018) showed that the TIB system enhanced the growth and leaf and stem development of Bletilla plants.

Table 1 Comparison of culture scale depending on the culture systems used in this study
Table 2 Effect of in vitro culture systems on the growth of apple (rootstock M9) plantlets

Like shoots, roots of apple plantlets were also the longest in the TIB-3 system (84.1 cm) compared with solid culture (5.2 cm) and liquid culture (5.1 cm) systems (Table 2). The root number was also higher in TIB-3 than in solid and liquid cultures (Table 2). In bioreactor culture systems (TIB and CIB), the medium is rich in dissolved oxygen because of continuous air supply. This facilitates breathing in roots, which promotes root growth (Gao et al. 2015; Ramos-Castellá et al. 2014; Valdez-Tapia et al. 2014).

The TIB-3 system enhanced all plant growth factors, whereas the TIB-6 system showed slightly lower shoot growth and FW (Table 2). This is probably because in the TIB-6 treatment, growth medium was supplied to plants once every 6 h, thus drying and delaying the initial plantlet growth.

A comparison between bioreactors and conventional culture systems showed that shoot development in both CIB and TIB was better than in solid and liquid cultures (Fig. 2). The lack of space and ventilation in conventional culture vessels may be responsible for poor shoot development in these systems (Fig. 2). Moreover, the number of leaves per plantlet was higher in the bioreactors, particularly TIB-3, than in the control (Fig. 3). Moreover, leaf area in TIB-3 (ca. 19 cm2) was approximately 2.1- and 3.2-fold higher than that in solid and liquid cultures, respectively (Fig. 3).

Fig. 2

Images of apple (rootstock M9) plantlets grown in different culture systems for 6 weeks. a Solid culture system, b liquid culture system, c and e continuous immersion system (CIB); d and f temporary immersion bioreactor (TIB) supplied with the growth medium once every 3 h (TIB-3), g TIB supplied with the growth medium once every 6 h (TIB-6)

Fig. 3

Leaf area and development in apple plants harvested from different culture systems after 6 weeks. Data represent mean ± SE of three biological replicates. Different lowercase letters indicate significant differences at p < 0.05 (Duncan’s multiple range test)

Previously, Ševčíková et al. (2018) showed that the leaf area of tobacco, strawberry, rapeseed, and potato plants grown in the air exchange treatment was higher than that of plants grown without air exchange. Additionally, in poppy, Ziska et al. (2008) showed that an increase in the level of carbon dioxide (CO2) following gas exchange is accompanied by a corresponding increase in leaf area. This suggests that the CO2 level in the bioreactor affects leaf development.

We also analyzed the contents of Chl a, Chl b, and carotenoids in plantlets grown in different culture systems. Total Chl content was the highest in solid culture (39.1 mg g− 1 FW) (Fig. 4). Among the bioreactor systems, total Chl content (36.2 mg g− 1 FW) and Chl a content (25.33 mg g− 1 FW) were higher in the TIB-6 system than in the TIB-3 system (Fig. 4). Chl b content was the lowest in liquid culture, although no significant differences were detected in Chl b content among the bioreactor treatments. Carotenoid content was the highest in the TIB-6 treatment (approximately 5.7 mg g− 1 FW) and lowest in the CIB treatment (approximately 4.9 mg g− 1 FW) (Fig. 4). Previous studies have shown that gas exchange increases the Chl and carotenoid contents of plants and subsequently plant growth (Alvarez et al. 2012; Larema et al. 2012; Park et al. 2011). However, Sáez et al. (2012) reported that the growth rate of Castanea shoots is not associated with the Chl content. In our study, the Chl content of plants in solid culture and TIB-6 was higher than that in liquid culture and TIB-3, possibly because plant shoots in solid culture and the TIB-6 system were exposed to air for a longer duration than in the other treatments, which probably dried the leaves. Therefore, Chl content in this study did not show a significant correlation with plant growth.

Fig. 4

Contents of chlorophyll (Chl) a, Chl b, and carotenoids in apple plantlets harvested from different culture systems. Data represent mean ± SE of three biological replicates. Different lowercase letters indicate significant differences at p < 0.05 (Duncan’s multiple range test)

Histological evaluation revealed that the total stem area was the highest in TIB-3 (1.46 mm2) (Fig. 5). The pith area was the highest in plants grown in TIB-3 (0.23 mm2) and lowest in plants grown in TIB-6 (0.16 mm2) (Fig. 5). The xylem area showed no significant difference among the various treatments (Fig. 5). The area of the cortex layer was the highest in plants grown in TIB-3 (Fig. 5). Previously, Kwon et al. (2015) showed a correlation between stem diameter and secondary xylem formation in Populus euramericana cultured in vitro; however, these results are inconsistent with the results of our study. In apple, cortex and pith contribute to the thickness of the stem. Generally, thick lignified stems exhibit low water loss and enhanced plant strength (Schuetz et al. 2012). Thus, our results regarding pith and cortex thickness in apple suggest that plantlets grown in the TIB-3 system will be highly adaptive to ex vitro conditions during acclimation.

Fig. 5

Development of leaf and stem tissues of apple plantlets harvested from various culture systems. a Areas of the cortex, xylem, and pith in the stem. Data represent mean ± SE of three biological replicates. Different lowercase letters indicate significant differences at p < 0.05 (Duncan’s multiple range test), b histological sections of stem and leaf. Scale bars = 100 µm


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This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant Number 315003-5).

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NYK contributed to the data acquisition and wrote the manuscript. HDH, JHK, and BMK participated in the experiment and sample analysis. DK participated in data interpretation and revising of the manuscript. S-YP made substantial contributions to data interpretation, revising of the manuscript, the conception, and design of this study.

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Correspondence to So-Young Park.

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Kim, N., Hwang, H., Kim, J. et al. Efficient production of virus-free apple plantlets using the temporary immersion bioreactor system. Hortic. Environ. Biotechnol. (2020). https://doi.org/10.1007/s13580-020-00257-3

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  • Acclimatization
  • Chlorophyll
  • Histology
  • Leaf area
  • Malus domestica