A Comparison of Semi-Solid, Liquid, and Temporary Immersion Bioreactor Systems for Effective Plant Regeneration of Gerbera jamesonii “Shy Pink”

Abstract

Temporary immersion system (TIS) cultures are reported to be superior when compared to semi-solid (SS) and liquid (LQ) cultures for the in vitro plant regeneration of many plant species. In the present study, we used a TIS for plant regeneration of Gerbera jemesonii “Shy Pink” and compared the results with that of SS and LQ cultures. The number of shoots regenerated in the SS, LQ, and TIS cultures was 6.93, 3.13, and 3.03, respectively. The shoots regenerated with the SS and LQ cultures demonstrated 3.33 and 4.22% hyperhydricity, whereas the shoots regenerated with the TIS were healthy even though the number of shoots regenerated was less. The plantlets regenerated with the TIS demonstrated higher values for the number of roots, root length, biomass of the plantlets, leaf length/width, and area compared to the SS and LQ cultures. When the G. jemesonii “Shy Pink” plants were regenerated using the TIS, their levels of photosynthetic pigments were highest. The number of stomata on the abaxial surface of their leaves was 11.40, and the frequency of closed stomata was 59% for the plants regenerated with the TIS. The number of stomata was 21.4 and 14.5 for the plants regenerated with the SS and LQ cultures, respectively. Meanwhile, the frequency of closed stomata was 13% and 15% for the plants regenerated with SS and LQ cultures. Furthermore, G. jemesonii “Shy Pink” showed the highest survival of plants when regenerated in the TIS compared to SS and LQ cultures. The TIS was found to be the most suitable culture system for plant regeneration of G. jemesonii “Shy Pink” compared to SS and LQ cultures.

1. Introduction

Gerberas are popular commercial flowers that are grown for their colorful blooms and are popular as potted plants or cut flowers in flower markets worldwide. They are known for their good durability, resilience to transportation, and wide range of colors [1]. Gerbera cultivars are normally propagated using tissue cultures since seed propagation has shown a high level of heterozygosity [2,3]. However, conventional micropropagation is a labor-intensive and expensive process because it involves the maintenance of a large number of culture vessels, semi-solid media, and the periodic transfer of plant material into fresh media due to the exhaustion of nutrients in the medium [4]. Furthermore, Garcia-Ramirez [5], Paek et al. [6], Watt [7], and Murthy et al. [8] reported that the cost of the gelling agent is an important element contributing to an increase in production costs. As a result, liquid media micropropagation of plants has emerged as a desirable substitute for traditional micropropagation. Improved multiplication and plantlet growth, as well as the ability to generate a large number of plantlets in a shorter amount of time, are benefits of LQ cultures [6]. Furthermore, it is feasible to multiply commercially significant plants at a vast scale using a scale-up procedure [5,7,8]. However, because the propagules are continuously submerged in liquid cultures, there are significant drawbacks of micropropagation utilizing LQ cultures, such as morphological, anatomical, and physiological problems, known as “hyperhydricity” [5,6,7,8,9]. To address these issues, devices known as temporary immersion systems/bioreactors (TISs) have been developed. These systems enable the cultured tissue to be immersed in a liquid medium for a predetermined amount of time before being exposed to a sterile gaseous environment within the cultures [5,6,7,8,9]. The proper growth and development of the propagules is facilitated by the gaseous environment of temporary immersion bioreactor cultures [5,7,10]. Furthermore, to facilitate a smooth transition upon ex vitro transplantation, in the plants regenerated in TISs, physiological functions such as photosynthesis, respiration, chlorophyll synthesis, and stomata function are stimulated [11,12]. Several temporary immersion bioreactor systems have been developed by various researchers, such as the Recipient for Automated Temporary Immersion system (RITA^®), a twin-flask system, an ebb and flow system, and a rocker system [8,10]. Among various TIS bioreactors, particularly SETISTM [13], which operates on the ebb and flow principle, has several advantages in size and handling of culture vessels. This system has been used efficiently for the regeneration of varied horticulture and medicinal plants, such as Chrysanthemum morifilium, Cinnidium officinale and Fragaria x ananassa [12], Colocasia esculenta [14], Malus domestica [15], Musa [16], and Prunus avium [17].

Gerbera jamesonii “Shy Pink” is one popular cultivar developed and released in the Republic of Korea, and it has long vase life of 12–14 days and a bloom yield of 50–54 stems per plant per year. This cultivar has pink-colored petals and brown core disk florets, and it is a well-liked cultivar with considerable market demand (Figure 1) [3]. A method of micropropagation using various explants in several cultivars of G. jemesonii has been established [2], although micropropagation systems using temporary immersion cultures have been tested for few cultivars. However, a comparison of SS, LQ, and TIS cultures has not been carried out. Because of this, the main goal of the current study was to micropropagate G. jemesonii “Shy Pink” in temporary immersion bioreactors using the SETIS^® system. Following this, the results of this approach were compared with those of conventional methods that entail the use of LQ and SS cultures for plantlet regeneration.

2. Materials and Methods

2.1. Plant Material and Culture Conditions

In the present study, in vitro regenerated plantlets of Gerbera jamesonii Bolus ex Hooker f. “Shy Pink” was used as the initial research material, and tissue cultures were established as per the protocol of Lim et al. [3]. For shoot multiplication, five shoot tips (30–40 mm in height with two to three leaf primordia) were used as explants, and they were cultured on 50 mL of Murashige and Skoog semi-solid medium [18] supplemented with 0.1 mg L⁻¹ benzyladenine (BA), 30 g L⁻¹ sucrose, and 0.8% (w/v) agar in 400 mL capacity magenta boxes (Magenta GA-7-3 Plant Culture Box, 350 mL, 75 × 75 × 100 mm, Magenta Corp., Chicago, IL, USA, containing 50 mL of medium). All the plant tissue culture chemicals were procured from Duchefa Biochemie, Haarlem, The Netherlands. Similarly, in another set of experiments, five shoot tips were cultured in 50 mL of MS LQ medium containing 0.1 mg L⁻¹ benzyladenine (BA) and 30 g L⁻¹ sucrose in magenta boxes. Shoot tip explants were cultured on a plastic net so that the explants were imbibed in the liquid medium with the LQ cultures. Cultures were also established using a TIS (SETISTM bioreactor, VERVIT, Zelzate, Belgium), and the TIS consisted of an upper container (5.5 L capacity) capable of cultivating the explants and a lower container (4 L capacity) which contained the liquid medium. The lower and upper containers were connected by a silicone hose, and filters (0.22 µm pore size) were attached to each area where air entered and exited to maintain sterilization. The TIS was supplied with an air volume of 0.1 vvm (air volume/medium volume/minute), and the time was set so that the explants were immersed in the MS liquid (500 mL) medium containing 0.1 mg L⁻¹ benzyladenine for 10 min for every 3 h. Fifty shoot tip explants were used for shoot multiplication in the TIS. The cultures were maintained for four weeks, and data on shoot regeneration were collected at the end of four weeks.

For root induction from the shoots, shoot cultures were established as above by using five shoot tips (2–4 cm in height with two or three leaves) in the semi-solid cultures and LQ cultures and using MS medium supplemented with 0.1 mg L⁻¹ indole butyric acid (IBA) and 30 g L⁻¹ sucrose. Meanwhile, for the TIS cultures, fifty shoot tips were cultured for the induction of rooting. The cultures were maintained for four weeks, and data on the root regeneration were collected at the end of four weeks. The medium pH was adjusted to 5.8 before filter sterilization or autoclaving at 121 °C for 15 min.

All the cultures were maintained under white light-emitting diodes (LEDs, 400–700 nm, PSLED-1203) with a light intensity of a 40 µmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) and under a 16 h/8 h (d/n) photoperiod. The cultures were maintained for 4 weeks at a temperature of 25 °C in growth chambers and 50% relative humidity.

2.2. Collection of Data on the Morphological and Growth Parameters

The total number of shoots regenerated per explant, the length of the shoot (cm), fresh weight (mg), leaf length (mm), leaf width (mm), and leaf area (mm²) were measured at the shoot multiplication stage. Similarly, total fresh weight (mg), dry weight (mg), the length of the shoots (cm), the length of the roots (cm), number of roots (per plantlet), number of leaves (per plantlet), plantlet height (cm), petiole length (cm), leaf length (mm), leaf width (mm), leaf index (length/width), and leaf area (mm²) were measured at the end of the rooting stage. The data were recorded after each of the 4-week cycles. The length of the shoots, leaf length, leaf width, plantlet height, and petiole length were measured using a ruler. The dry weight of the shoots and plantlets was taken after drying the material in an oven until it reached constant weight.

2.3. Estimation of the Chlorophyll and Carotenoid Contents

The content of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in the in vitro regenerated plants after acclimatization was analyzed. In brief, 200 mg fresh-weight tissue samples were collected from the third leaf from the top of the plantlets and were subjected to extraction using 80% acetone [19]. The absorbance was measured using a spectrophotometer (Libra S22, Biochrome Ltd., Cambridge, UK) at the following wavelength maxima (Amax): chlorophyll a at 663.2 nm, chlorophyll b at 646.8 nm, and total carotenoids at 470 nm. The amount of chlorophyll a, chlorophyll b, and carotenoids was calculated using the following equations [19].

Chl a (mg g⁻¹) = (12.72 × OD663 − 2.5 × OD645) V/1000 W

Chl b (mg g⁻¹) = (22.88 × OD646 − 4.67 × OD663) V/1000 W

Car (mg g⁻¹) = [(1000 × OD470 − 3.27 × Chl a − 104 × Chl b)/229] V/1000 W

where V is the total volume of acetone extract (mL), and W is the FW (G) of the sample.

2.4. Measurement of Chlorophyll Fluorescence

The chlorophyll fluorescence parameters were measured on the third leaves of plants using a potable FluorPen FP100 (Photon Systems Instruments, Drasov, Czech Republic). The ground-state fluorescence (F₀) was measured in 30 dark-adapted leaves of plants moved into a dark room. The maximum fluorescence level in the dark-adapted state was triggered by a 1 s saturating light pulse of a 3000 µmol m⁻² s⁻¹ PPFD (Fm). The maximum quantum efficiency of PSII photochemistry was calculated as Fv/Fm, where Fv = Fm − F₀.

2.5. The Stomatal Index

Five randomly selected plants were used to count the number of stomata for the plants regenerated with each culture system. The excised samples were placed on glass slides and observed with an optical microscope (ECLIPSE Ci-L, Nikon Corporation, Tokyo, Japan). The stomatal density was calculated as the number of stomata divided by the area where the number of stomata was recorded.

2.6. Acclimatization

Plants grown under different culture treatments were harvested, and their roots were carefully washed with distilled water to remove the adhering medium; then, the plants were transplanted into plastic trays containing potting medium (cocopeat 51%, peat moss 10%, vermiculite 13%, humic acid 0.1%, perlite 15%, zeolite 10%, fertilizer 0.4%; Shinsung Mineral Co., Ltd., Dunchon-aero, Republic of Korea). Fifteen plants were transplanted to 16 well-plastic trays, and three replicates were maintained for each culture treatment. The plants were maintained in a growth chamber with 80% humidity, with a 300 µmol m⁻² s⁻¹ PPFD, with a 16 h/8 h (d/n) photoperiod, and at a temperature of 25 ± 2 °C. Four weeks after transplantation, data on the percentage survival of the plants were evaluated.

2.7. Statistical Analysis

Experiments were set up according to a completely randomized design. During shoot proliferation and rooting of the shoots in the SS and LQ media, there were ten replicates, and for the TIS, there were three replicates. The data obtained were subjected to a one-way analysis of variance (ANOVA). The statistical significance of the differences between the mean values was assessed using Duncan’s multiple range test at p < 0.05. All the statistical analyses were performed using SAS 9.4 software (SAS Institute Inc., Cary, NC, USA).

3. Results

3.1. Morphological and Growth Parameters of Shoots and Plantlets Regenerated with Different Culture Systems

The results for different culture systems in terms of the shoot regeneration of G. jemesonii “Shy Pink” are presented in Figure 2. The number of shoots regenerated in the SS cultures was 6.93, whereas 3.13 and 3.03 shoots were regenerated from the shoot tip explants in the LQ and TIS cultures, respectively (Figure 2A). These results were statistically significant according to Duncan’s multiple range test (DMRT) at p < 0.05. The fresh weight of the shoots was 0.93, 1.52, and 0.59 g and the dry weight of shoots was 131.53, 1.64, and 0.55 mg with the SS, LQ, and TIS cultures, respectively (Figure 2B). Both the fresh and dry mass of the shoots in the LQ cultures was significantly higher when compared to the shoots regenerated in the SS and TIS cultures. In addition, the shoot and petiole lengths were highest with the LQ cultures (64.97 mm and 28.01 mm) as compared to the shoots regenerated in SS cultures (40.53 mm and 20.25 mm), which was significant as per DMRT (Figure 2D). On the other hand, the shoots that were regenerated in the TIS cultures were healthy and did not exhibit hyperhydricity, while 3.33 and 4.22% of the shoots that were regenerated in the SS and LQ cultures showed hyperhydricity (Figure 2C). The percentage of hyperhydricity of the shoots regenerated with the SS and LQ cultures was statistically significant. The stems and leaves of the hyperhydric shoots were thick, rigid, water-soaked, and easily breakable. The morphology of the shoots regenerated with different culture systems is depicted in Figure 3.

The average length and width of the leaves, as well as the number of leaves that formed on the axillary shoots that were regenerated using various culture systems, were measured. The results are shown in Figure 4. With the SS, LQ, and TIS cultures, the corresponding numbers of leaves with shoots were 26.80, 18.60, and 16.33 (Figure 4A). With the SS, LQ, and TIS cultures, the length of the leaves with regenerated shoots was 11.33, 19.32, and 13.60 mm, while the breadth of the leaves was 6.33, 13.6, and 9.44 mm, respectively (Figure 4B–D). According to DMRT, these values were statistically significant.

Figure 5 displays the outcomes of rooting the G. jemesonii “Shy Pink” shoots on rooting media using various approaches, and

Table 1. Effect of culture system on rooting of G. jamesonii “Shy Pink” shoots after four weeks of culture.

Culture System	Total FW (g)	Total DW (mg)	Plantlet Height (mm)	Shoot Height (mm)	No of Roots	Root Length (mm)	Petiole Length (mm)	Leaf Length (mm)	Leaf Width (mm)	Leaf Area (mm2)	Leaf Index
SS	0.62 ± 0.0 c	60.90 ± 1.0 c	71.74 ± 0.8 c	54.92 ± 0 c	6.60 ± 0.4 b	16.40 ± 0.7 b	28.92 ± 1.2 c	14.81 ± 0.4 c	9.32 ± 0.2 c	91.30 ± 3.9 c	1.59 ± 0.0 a
LQ	0.71 ± 0.0 b	70.45 ± 1.1 b	80.13 ± 1.5 b	65.85 ± 1.3 b	7.80 ± 0.2 a	14.12 ± 0.8 c	34.56 ± 1.5 b	17.84 ± 0.5 b	11.42 ± 0.4 b	136.78 ± 8.7 b	1.59 ± 0.0 a
TIS	0.87 ± 0.0 a	75.80 ± 2.3 a	88.87 ± 1.5 a	74.88 ± 0.8 a	7.85 ± 0.2 a	18.80 ± 0.6 a	42.85 ± 0.9 a	22.78 ± 0.3 a	14.21 ± 0.3 a	206.94 ± 6.8 a	1.62 ± 0.0 a

Leaf index: length/width. SS: semi-solid culture, LQ: liquid culture, TIS: temporary immersion system. Different letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test (n = 10 and n = 3 for SS, LQ, and TIS cultures, respectively).

presents the data. When compared to the SS and LQ cultures, the growth characteristics of the plantlets grown under the TIS cultures were all optimal. These included total fresh weight (0.87 g), dry weight (75.80 mg), plantlet height (88.87 mm), shoot height (74.88 mm), number of roots (7.85 per shoot), root length (18.80 mm), petiole length (42.85 mm), leaf length (22.78 mm), leaf width (14.21 mm), leaf area (206.94 mm²), and leaf index (1.62). The total fresh weight, plantlet height, shoot height, root length, and leaf area values of the TIS-regenerated plants were statistically significant at p < 0.05, according to DMRT. The leaf index values, however, did not reach statistical significance.

3.2. Chlorophyll and Carotenoid Contents of the Regenerated Plants Using Different Culture Systems

The G. jemesonii “Shy Pink” plantlets’ chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid concentrations were tested for each treatment; the results are shown in Figure 6. The plantlets that were regenerated using SS cultures had concentrations of 0.89, 0.37, 1.25, and 0.24 mg g⁻¹ DW for chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid. With the plantlets that were regenerated using LQ cultures, the pigment concentration was 0.86, 0.36, 1.21, and 0.23 mg g⁻¹ DW. These were, however, 0.96, 0.39, 1.34, and 0.26 mg g⁻¹ DW when the plantlets that were grown using the TIS cultures were used. There was no statistically significant difference found between the values of carotenoid and chlorophyll in the regenerated plants in the various systems.

3.3. Chlorophyll Fluorescence Parameters

Following the conclusion of the culturing, measurements were made of the chlorophyll fluorescence parameters of the plantlets regenerated using the various culture systems, including minimal fluorescence (F₀), maximal fluorescence (Fm), maximal variable fluorescence (Fv), and the photosynthetic efficiency of PSII (Fv/Fm). The results are displayed in

Table 2. Effect of different culture systems on chlorophyll fluorescence of G. jamesonii Shy Pink plantlets.

Culture System	F0	Fm	Fv	Fv/Fm
SS	8070.88 ± 71.9 b	44,926.13 ± 54.5 b	36,855.25 ± 487.0 b	0.82 ± 0.0 a
LQ	8095.00 ± 190.2 b	46,620.75 ± 1073.8 ab	38,525.75 ± 904.3 ab	0.83 ± 0.0 a
TIS	8627.80 ± 147.2 a	48,778.63 ± 1494.5 a	40,150.88 ± 1386.4 a	0.82 ± 0.0 a

F₀: minimum fluorescence, Fm: maximum fluorescence, Fv: maximal variable fluorescence, Fv/Fm: maximum quantum efficiency of photosystem Ⅱ. SS: semi-solid culture, LQ: liquid culture, TIS: temporary immersion system. Different letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test (n = 10 and n = 3 for SS, LQ, and TIS cultures, respectively).

. Higher F₀, Fm, and Fv values were shown by the plants that were regenerated utilizing the TIS cultures; these values were statistically significant based on DMRT at p < 0.05. The Fv/Fm values, on the other hand, did not show statistical significance when the plants were regenerated using various culture systems.

3.4. Analysis of the Stomatal Index

The culture techniques used to regenerate the plants had an impact on the stomatal characteristics of the leaves of G. jemesonii “Shy Pink” (Figure 7A,B). In the leaves of plants that were regenerated using SS cultures, there were 18.70, 2.70, and 21.40 open, closed, and total stomata. These values for the plant leaves that were regenerated using LQ cultures were 12.40, 2.10, and 14.50. On the other hand, the leaves of the plants that were regenerated utilizing the TIS had 4.40, 7.00, and 11.40 open, closed, and total stomata, respectively. There was a statistically significant difference in the number of stomata and the frequency of closed stomata in the plants that were regenerated using the TIS. In Figure 7A, stomatal micrographs are displayed.

After being successfully transplanted into the potting media, the regenerated plants in the SS, LQ, and TIS cultures demonstrated 85%, 80%, and 100% survival four weeks following transplanting (Figure 8A–C). There was statistical significance in the survival rate of the plants that were regenerated using various culture techniques.

4. Discussion

While semi-solid cultures are typically used for micropropagation, it is well recognized that mass plant propagation through tissue culture in semi-solid media is an expensive and labor-intensive procedure. According to Garcia-Ramirez [5] and Murthy et al. [8], gelling agents like agarose, gelrite, and agar greatly raise the cost of plantlet regeneration in vitro and restrict the potential for automated commercial micropropagation. An excellent method for lowering plant production costs and facilitating automation is the use of LQ media in plant regeneration systems [6,7]. Hyperhydricity, on the other hand, frequently counteracts the use of LQ media, resulting in morphological, anatomical, and physiological abnormalities in regenerated plants [9]. To address these problems, several researchers have developed temporary immersion systems. These systems are intended to periodically submerge cultivated propagules in the LQ medium, drain the plant tissue, and then expose them to a sterile gaseous environment [7,20]. Over time, scientists have developed several TISs, most of which consist of two or more compartments in a container or separate vessel. The medium is moved from the reservoir compartment to the compartment or vessel that is used to cultivate the explants or plants. These systems aid in providing a sufficient amount of nutrients and also help to solve the hyperhydricity issues by providing proper aeration to the propagules, and TISs have been used efficiently for the micropropagation of many horticultural species [12,21,22,23,24,25]. Moreover, according to Georgiev et al. [10], Garcia-Ramirez [5], and Murthy et al. [8], TISs enhance the gaseous environment and offer the most natural setting for in vitro plant regeneration in terms of both shoot proliferation and the rooting of shoots compared to SS and liquid cultures. In the present study, we utilized semi-solid, LQ, and TIS cultures for the plant regeneration of G. jemesonii “Shy Pink” and analyzed the growth parameters during the shoot proliferation stage and the rooting of the shoots. We also analyzed the biochemical characteristics of the regenerated plantlets, such as estimation of the chlorophyll and carotenoid contents and the stomatal index in the regenerated plants.

The evaluation of different culture systems, including SS, LQ, and TIS cultures, demonstrated that the number of shoots, fresh and dry weight, and the length of the shoots were higher in the SS and LQ cultures when compared to the TIS. However, 3.33% and 4.22% of the shoots regenerated in the SS and LQ cultures showed hyperhydricity. An optimum 3.03 shoots were regenerated from the shoot tip explants of G. jemesonii “Shy Pink” in the TIS, and they were healthy and did not show any hyperhydricity. The number of leaves, the leaf length/breadth, and the leaf area were better with the LQ and TIS cultures compared to the SS cultures. Overall, the TIS was found to be superior even though the number of shoots regenerated was less when compared to the SS and LQ cultures. The TIS culture combines the advantages of immersion and a dry period, which maximizes gas exchange and facilitates overcoming abnormalities like hyperhydricity in the cultures [15,23]. In addition, the exposure of the explants to proper aeration during growth and differentiation in vitro will help in the improvement of the morphology and physiology of their shoots [5,10]. Additionally, Frometa et al. [21] tested the immersion frequency (6, 8, 12 h), additional ventilation (1 min every 2 h), and the length of the duration of the shoot cultures (14, 21, 28 and 35 days), and they reported immersion every 8 h with additional ventilation was beneficial in the regeneration of shoots of G. jemesonii. However, in this study, variation in the immersion frequency (10 min every 4/8/12 h) and variation in the number of explants per culture (culture density of 25, 50, 75, and 100 shoots/culture) did not yield better results.

The results of root induction of shoots of G. jemesonii “Shy Pink” in three different culture systems reveal that the TIS was superior for the regeneration of roots from the shoots, and the number of roots, the root length, the fresh and dry weight of the plantlets, plantlet height, petiole length, leaf length/width, and leaf area were all optimum with the TIS cultures. Rooting of the shoots is a critical step during micropropagation which favors proper survival and the involvement of in vitro regenerated plants upon transplantation into soil. The TIS’s ability to easily manipulate culture conditions helps enhance the physiological quality of the propagated plants for horticulture species like Curcuma longa [22], Anthurium andreanum [23], Vaccinium vitis-idea ssp. minus [24], Chrysanthemum morifolium [12], and Epipactis flava [25] and medicinal plants like Cnidium officinale [12].

The two groups of pigments used in photosynthetic processes in plants are chlorophylls and carotenoids [26]. The present investigation found no statistically significant differences in the levels of carotenoid, chlorophyll a, or chlorophyll b among the plants that were regenerated in various systems. However, the levels of carotenoid, chlorophyll a, and chlorophyll b in the leaves of the G. jemesonii “Shy Pink” plants were at their peak when the plants were regenerated using the TIS method. This could be because the TIS culture conditions enhanced the biochemical capacity of the regenerated plants.

Since the stomata are an essential component of photosynthesis, their size and density are thought to be markers of a plant’s ability to adapt and thrive in a variety of environments. Some morphological and physiological alterations that are frequent in conventional micropropagation can be avoided by employing ventilation, according to Zobayed [27]. One such change is the production of dysfunctional stomata, which remain wide open continuously. One of the most common causes of plant death during acclimatization is dehydration, which can be prevented by having closed stomata and a low stomatal index, which results in a low transpiration rate [11,28]. In contrast to the plants regenerated with the SS and LQ cultures, the G. jemesonii “Shy Pink” plants that were regenerated in the TIS in this study favored a low stomatal index and a high percentage of closed stomata. This suggests the functionality of stomata, thereby promoting a lower transpiration rate and a higher photosynthetic rate.

According to Martre et al. [29], the quality and vigor of the regenerated plants are equally important to the effectiveness of a micropropagation technique as the number of shoots produced by the explants. Acclimatization is thus a critical step that impacts the outcome of micropropagation. Comparing the plants regenerated with the SS and LQ cultures to those regenerated with the TIS technique, the latter showed a one hundred percent survival rate four weeks following transplantation into garden soil. Similar studies have shown in reports [30,31] that the TIS environment helps the plantlets cope with the stress of acclimatization. In comparison to SS cultures, Ahmadian et al. [32] and Martinez-Estrada [23] found that a TIS has an environment that is favorable for plantlet growth, particularly for the root development of Dianthus caryophyllus and Anthurium andreanum plantlets during plant regeneration. Numerous other earlier studies have shown that TIS plantlets outpaced SS and LQ-derived plants in terms of development and rapid adaptation to ex vitro environments upon transplantation [25,33].

5. Conclusions

The current experiments indicated that SS cultures were appropriate for the shoot proliferation stage, while TIS cultures were more effective during the rooting stage when compared to standard SS and LQ cultures. Shoot regeneration, biomass accumulation, and growth were all at their peak in the SS cultures, whereas the root regeneration and biomass accumulation by the plantlets were optimal with the TIS cultures. Thus, G. jemesonii “Shy Pink” plants can be mass-propagated and regenerated effectively using both SS and TIS cultures. The plantlets obtained from the TIS exhibited the highest rate of survival and good adaption at the acclimatization stage.