Comparison of semi-solid, liquid, and temporary immersion bioreactor systems for efficient plant regeneration of Gerbera jamesonii cv. ‘Shy Pink’

doi:10.21203/rs.3.rs-4366227/v1

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Comparison of semi-solid, liquid, and temporary immersion bioreactor systems for efficient plant regeneration of Gerbera jamesonii cv. ‘Shy Pink’

https://doi.org/10.21203/rs.3.rs-4366227/v1

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Temporary immersion system (TIS) cultures are reported to be superior when compared to semi-solid (SS) and liquid cultures for in vitro plant regeneration of many plant species. In the present study, we used the TIS system for plant regeneration of Gerbera jemesonii cv. ‘Shy Pink’ and compared the results with that of SS and liquid cultures. The shoots regenerated with SS and liquid cultures demonstrated 3.33 and 4.22% hyperhydricity, whereas the shoots regenerated with TIS were healthy even though the number of shoots regenerated was less in number. The plantlets regenerated with TIS demonstrated higher values with the number of roots, root length, biomass of plantlets, leaf length/width, and area compared to SS and liquid cultures. The photosynthetic pigments were highest in Gerbera jemesonii cv. ‘Shy Pink’ plants were regenerated with TIS. The number of stomata on the abaxial surface of leaves was 11.40 and the frequency of closed stomata was 59% with plants regenerated with TIS. Furthermore, Gerbera jemesonii cv. ‘Shy Pink’ showed the highest survival of plants that were regenerated in TIS compared to SS and liquid cultures. TIS was found as the most suitable culture system for the micropropagation of Gerbera jemesonii cv. ‘Shy Pink’ compared to SS and liquid cultures.

Acclimatizationㆍ hyperhydricityㆍin vitro regenerationㆍliquid cultures

micropropagationㆍ stomatal index

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 (Zhou et al. 2022). Gerbera cultivars are normally propagated by using tissue cultures since seed propagation has shown a high level of heterozygosity (Cardoso and Teixeira da Silva 2013; Lim et al. 2023). 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 periodic transfer of plant material to fresh media due to exhaustion of nutrients in the medium (Maene and Debergh 1985). Furthermore, Garcia-Ramirez (2023) reported that the cost of the gelling agent is another element contributing to the 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 liquid cultures (Paek et al. 2005). Furthermore, it is feasible to multiply commercially significant plants on a vast scale using a scale-up procedure (Watt 2012; Garcia-Ramirez 2023; Murthy et al. 2023). However, because the propagules are continuously submerged in liquid cultures, there are significant drawbacks of micropropagation utilizing liquid cultures, such as morphological, anatomical, and physiological problems known as ‘hyperhydricity’ (Gao et al. 2018). To address these issues, devices known as temporary immersion systems/bioreactors (TIS) 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. The proper growth and development of propagules is facilitated by the gaseous environment of temporary immersion bioreactor cultures (Georgiev et al. 2014; Garcia-Ramirez 2023; Murthy et al. 2023). Furthermore, to facilitate a smooth transition upon ex vitro transplantation, the plants regenerated in TIS stimulates physiological functions such as photosynthesis, respiration, chlorophyll synthesis, and stomata function (Aragon et al. 2014; Hwang et al. 2022). Several temporary immersion bioreactor systems have been developed by various researchers such as the Recipient for Automated Temporary Immersion system (RITA®), Twin-flask system, Ebb and flow system, and rocker system (Georgiev 2014; Murthy et al. 2023). Among various TIS bioreactors namely SETIS™ (Vervit 2024) 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 (Hwang et al. 2022), Colocasia esculenta (Arano-Avalos et al. 2020), Malus domestica (Kim et al. 2020), Musa (Bello-Beloo et al. 2019) and Prunus avium (Godoy et al. 2017).

Gerbera jamesonii Bolus ex Hooker f. ‘Shy Pink” is one of the popular cultivars 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 (Lim et al. 2023). A method of micropropagation using various explants in several cultivars of Gerbera jemesonii has been established (Cardoso and Teixeira da Silva 2013), although micropropagation systems using temporary immersion cultures have not been tested. Because of this, the main goal of the current study was to micropropagate Gerbera jemesonii cv. ‘Shy Pink’ in temporary immersion bioreactors using the SETIS® system. Following that, the results of this approach are compared with those of conventional methods that entail liquid and semi-solid cultures for plantlet regeneration.

Plant material and culture conditions

In the present study, in vitro regenerated plantlets of Gerbera jemesonii cv. ‘Shy Pink’ was used as initial research material and tissue cultures were established as per the protocol of Lim et al. (2023). 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 MS semi-solid medium supplemented with 0.1 mg L^− 1 benzyl adenine (BA), 30 g L^− 1 sucrose, 0.8% (w/v) agar taken in 400 mL capacity magenta boxes (Magenta GA-7-3 Plant Culture Box, 350 mL, 75 x 75 x 100 mm, Magenta Corp., Chicago, IL, USA containing 50 mL of medium). Similarly, in another set of experiments, five shoot tips were cultured in 50 ml of MS liquid medium containing 1 mg L^− 1 benzyl adenine (BA), and 30 g L^− 1 sucrose taken in magenta boxes. Shoot tip explants were cultured on a plastic net so that explants were imbibed in the liquid medium with liquid cultures. Cultures were also established using TIS (SETIS™ bioreactor, VERVIT, Zelzate, Belgium) and TIS consisted of an upper container (5.5 L) capable of cultivating explants and a lower container (4 L) with a liquid medium. The lower and upper containers were connected by a silicone hose, and the filters (0.22 µm pore size) were attached to each area where air enters and exits to maintain sterilization. The TIS system 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 liquid medium for 10 min for every 3 h. Fifty shoot tip explants were used for shoot multiplication in TIS. The cultures were maintained for four weeks, and data on shoot regeneration was collected at the end of four weeks.

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

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

Collection of data on 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), length of the shoots (cm), length of root (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), leaf area (mm²), were measured at the rooting of shoots stage. The data were recorded after each of the 4-week cycles.

Estimation of chlorophyll and carotenoid content

The content of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids of in vitro regenerated plants after acclimatization was analyzed. 200 mg fresh-weight tissue samples were collected from the third leaf from the top of plantlets and were subjected to extraction by using 80% acetone (Lichtenthaler and Buschmann 2001). The absorbance was measured using a spectrophotometer (Libra S22, Biochrome Ltd., Cambridge, UK) at the following wavelengths maxima (A_max): Chlorophyll a at 663.2 nm, chlorophyll b at 646.8 nm, and total carotenoids at 470 nm.

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 (Fo) was measured on 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 3000 µmol m^− 2 s^− 1 PPFD (Fm). The maximum quantum efficiency of PSII photochemistry was calculated as Fv/Fm, where Fv = Fm – Fo.

Stomatal index

Five randomly selected plants were used for counting the number of stomata with 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.

Acclimatization

Plants grown under different culture treatments were harvested and roots were carefully washed with distilled water to remove the adhering medium, then plants were transplanted into plastic trays containing garden soil (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 of 80% humidity, 300 µmol m^− 2 s^− 1 PPFD, and at a temperature of 25 ± 2 ^oC. Four weeks after transplantation data on the percentage survival of plants were evaluated.

Statistical analysis

Experiments were set up in a completely randomized design. During shoot proliferation and rooting of the shoots in semisolid and liquid media, there were ten replicates and for 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 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).

Morphological and growth parameters of shoots and plantlets regenerated with different culture systems

The results of different culture systems on shoot regeneration of Gerbera jemesonii cv. ‘Shy Pink’ is presented in Fig. 1. The number shoots regenerated on semisolid (SS) cultures were 6.93, whereas 3.13 and 3.03 shoots regenerated from shoot tip explants in liquid and TIS cultures respectively (Fig. 1). The fresh weight of shoots was 0.93, 1.2 and 0.59 g and dry weight of shoots were 931.53, 1.64 and 0.55 mg with the SS, liquid and TIS cultures respectively. The shoot and petiole lengths were 40.53, 64.97 and37.17 mm and 20.25, 28.01, and 20.73 mm respectively with SS, liquid, and TIS cultures (Fig. 1). However, 3.33 and 4.22% of shoots demonstrated hyperhydricity regenerated in SS and liquid cultures, whereas shoots regenerated on TIS cultures were healthy without any hyperdyricity. The morphology of the shoots regenerated with different cultured systems is depicted in Fig. 2. The number of leaves with shoots was 26.80, 18.60, and 16.33 with SS, liquid, and TIS cultures. The length of leaves with regenerated shoots was 11.33, 19.32, and 13.60 mm, whereas the width of the leaves was 6.33, 1.36, and 9.44 mm respectively with SS, liquid, and TIS cultures (Fig. 3).

The results of rooting of shoots Gerbera jemesonii cv. ‘Shy Pink’ on rooting media by using different systems is shown in Fig. 4 and data is presented in Table 1. The growth characteristics such as 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) were all optimum with plantlets grown under TIS cultures when compared to SS and liquid cultures.

Table 1

Effect of culture system on rooting of *G. jamesonii* cv. 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^z
SS^y	0.62 c^x	60.90 c	71.74 c	54.92 c	6.60 b	16.40 b	28.92 c	14.81 c	9.32 c	91.30 c	1.59 a
Liquid	0.71 b	70.45 b	80.13 b	65.85 b	7.80 a	14.12 c	34.56 b	17.84 b	11.42 b	136.78 b	1.59 a
TIS	0.87 a	75.80 a	88.87 a	74.88 a	7.85 a	18.80 a	42.85 a	22.78 a	14.21 a	206.94 a	1.62 a

^zLeaf index: Length/width. ^ySS: semi-solid culture, Liquid: liquid culture, TIS: temporary immersion system. ^xDifferent letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test.

Chlorophyll and carotenoid contents of the regenerated plants using different culture systems

The chlorophyll a, chlorophyll b, and total chlorophyll and carotenoid contents of the Gerbera jemesonii cv. ‘Shy Pink’ plantlets for each treatment were measured and data has been presented in Fig. 5. The chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents were 0.89, 0.37, 1.25 and 0.24 mg g^− 1 DW with the plantlets regenerated with SS cultures. The pigment content was 0.86, 0.36, 1.21, and 0.23 mg g^− 1 DW with plantlets regenerated with liquid cultures. However, they were 0.96, 0.39, 1.34 and 0.26 mg g^− 1 DW with the plantlets regenerated with TIS cultures.

Chlorophyll fluorescence parameters

At the end of culture, the chlorophyll fluorescence parameters such as minimal fluorescence (Fo), maximal fluorescence (Fm), maximal variable fluorescence (Fv), and photosynthetic efficiency of PSII (Fv/Fm) values of plantlets regenerated with different culture systems were measured and data has been presented in Table 2. The highest value was recorded with the plants regenerated using the TIS system and the values Fo, Fm, Fv, and Fv/Fm were 8627.80, 48778.63, 40150.88, and 0.82 respectively.

Table 2

Effect of different culture systems on chlorophyll fluorescence of *G. jamesonii* cv. Shy Pink plantlets.
Culture system	Fo^z	Fm	Fv	Fv/Fm
SS^y	8070.88 b^x	44926.13 b	36855.25 b	0.82 a
Liquid	8095.00 b	46620.75 ab	38525.75 ab	0.83 a
TIS	8627.80 a	48778.63 a	40150.88 a	0.82 a
^zFo: minimum fluorescence, Fm: maximum fluorescence, Fv: maximal variable fluorescence, Fv/Fm: maximum quantum efficiency of photosystem Ⅱ,
^ySS: semi-solid culture, Liquid: liquid culture, TIS: temporary immersion system.
^xDifferent letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test.

Analysis of stomatal index

The stomatal properties of Gerbera jemesonii cv. ‘Shy Pink’ leaves were affected by culture systems in which the plants were regenerated (Fig. 6). The number of open, closed, and total number of stomata were 18.70, 2.70, and 21.40 with the leaves of plants regenerated with SS cultures. These values were 12.40, 2.10, and 14.50 with leaves of plants regenerated with liquid cultures. However, the number of open, closed, and total stomata were 4.40, 7.00, and 11.40 with the leaves of plants regenerated using TIS (Table 3). The stomatal micrographs are presented in Fig. 6.

The regenerated plants were transplanted to garden soil successfully and the plants regenerated in SS, liquid, and TIS showed 85, 80, and 100% survival after four weeks after transplantation (Fig. 7).

While semi-solid cultures are typically used for micropropagation, it is well-recognized that mass plant propagation through tissue culture in semisolid media is an expensive and labor-intensive procedure. According to Garcia-Ramirez (2023) and Murthy et al. (2023), gelling agents like agarose, gelrite, or 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 liquid media in plant regeneration systems (Peak et al. 2005; Watt 2012). Hyperhydricity, on the other hand, frequently counteracts the use of liquid media, resulting in morphological, anatomical, and physiological abnormalities in regenerated plants (Gao et al. 2018). To address these problems, several researchers have developed temporary immersion systems. These systems are intended to periodically submerge cultivated propagules in the liquid medium, drain the plant tissue, and then expose them to a sterile gaseous environment (Alvard et al. 1993; Watt 2012). Over time, scientists have developed several TIS systems, 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 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. Moreover, according to Georgiev et al. (2014), Garcia-Ramirez (2023), and Murthy et al. (2023), these systems enhance the gaseous environment and offer the most natural setting for the in vitro culture of plant regeneration at both shoot proliferation and rooting of shoots. In the present study, we utilized semisolid, liquid, and TIS systems for the plant regeneration of Gerbera jemesonii cv. ‘Shy Pink’ and analyzed the growth parameters during the shoot proliferation stage, and rooting of shoots. We also analyzed the biochemical characteristics of regenerated plantlets such as the estimation of chlorophyll and carotenoid content, and stomatal index in the regenerated plants.

The evaluation of different culture systems including SS, liquid, and TIS demonstrated that the number of shoots, fresh and dry weight, and length of shoots were higher in SS and liquid cultures when compared to the TIS system. However, the 3.33% and 4.22% shoots regenerated in SS and liquid cultures showed hyperhydricity. An optimum of 3.03 shoots was regenerated from the shoot tip explants Gerbera jemesonii cv. ‘Shy Pink’ in TIS and they were healthy and did not show any hyperhydricity. The number of leaves, leaf length/breadth, and leaf area were optimum with liquid and TIS cultures compared to SS cultures. Overall TIS system was found superior even though the number of shoots regenerated was less when compared to SS and liquid cultures. The TIS culture combines the advantages of immersion and a dry period which maximizes the gas exchange and facilitates overcoming abnormalities like hyperhydricity in the cultures (Kim et al. 2020; Martinez-Estrada et al. 2019). In addition, exposure of explants to proper aeration during growth and differentiation in vitro will help in the improvement of the morphology and physiology of shoots (Georgiev et al. 2014; Garcia-Ramirez 2023).

The results of root induction of shoots Gerbera jemesonii cv. ‘Shy Pink’ in three different culture systems reveals that TIS was superior for the regeneration of roots from the shoots, number of roots, root length, fresh and dry weight of plantlets, plantlet height, petiole length, leaf length/width and leaf area were all optimum with TIS cultures. Rooting of the shoot is a critical step during micropropagation which favors proper survival and involvement of in vitro regenerated plants upon transplantation to the soil. TIS's ability to easily manipulate culture conditions helps enhance the physiological quality of propagated plants in horticulture species like Curcuma longa (Marchant et al. 2021), Anthurium andreanum (Martinez-Estrada et al. 2019), Vaccinium vitis-idea ssp. minus (Arigundam et al. 2020), Chrysanthemum morifolium (Hwang et al. 2021), Epipactis flava (Kunakahonnuruk et al. 2019), and medicinal plants like Cnidium officinale (Hwang et al. 2021).

The two main pigments used in photosynthetic processes in plants are chlorophylls and carotenoids (Fraser et al., 2001). Plants that can produce larger amounts of these pigments in micropropagated plants would fare better when transplanted into an ex vitro environment. The present investigation found no statistically significant differences in the levels of carotenoid, chlorophyll a, or chlorophyll b between the plants that were regenerated in various systems. However, the levels of carotenoid, chlorophyll a, and chlorophyll b in the leaves of Gerbera jemesonii cv. "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 (2005). One such change is the production of dysfunctional stomata, which remain continuously widely open. 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 (Aragon et al. 2014; Vieira et al. 2015). In contrast to plants regenerated with SS and liquid cultures, the Gerbera jemesonii cv. "Shy Pink" plants that were regenerated in the TIS in this study favor 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. (2001), 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 plants regenerated with SS and liquid cultures to those regenerated with the TIS technique, the latter showed a hundred percent survival rate four weeks following transplantation to garden soil. Similar studies have shown in reports (Aragon et al. 2010; Regueira et al. 2018) that the TIS environment helps the plantlets cope with the stress of acclimatization. In comparison to SS cultures, Ahmadian et al. (2017) and Martinez-Estrada (2019) found that 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 liquid-derived plants in terms of development and rapid adaptation to ex vitro environments upon transplantation (Yang and Yeh 2008; Kunakhonnuruk et al. 2019).

The results of current experiments have demonstrated that TIS is an efficient system for mass propagation of Gerbera jemesonii cv. ‘Shy Pink’ plantlets as compared to the conventional semisolid and liquid cultures. Regeneration, growth, and accumulation of biomass by the plantlets were optimal in TIS. The plantlets obtained from TIS successfully adapted and had the highest survival rate during the acclimatization stage.

Data availability: Data will be made available on a reasonable request

Author Contributions: M.-J.L.; J.E.H.; S.-Y.L. and T.T. H. contributed to data acquisition and experiments. H.N.M. participated in the writing and editing of the manuscript. S.-Y.P. was involved in the conception and design of the study and made a substantial contribution to data interpretation, and revision of the manuscript. All authors have read and agreed to the final version of the manuscript.

Funding: This work was supported by the development of technology to maintain and manage the quality of superior and standard seedlings in floral crops (Project No. PJ017080), Rural Development Administration, Republic of Korea. "This work was conducted during the research year of Chungbuk National University in 2024."

Acknowledgment: Hosakatte Niranjana Murthy is thankful for the “Brain Pool” (BP) program, Grant No. 415 2022H1D3A2A02056665.

Informed Consent Statement: Not applicable

Conflict of Interest: The authors declare that they have no conflict of interest.

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Comparison of semi-solid, liquid, and temporary immersion bioreactor systems for efficient plant regeneration of Gerbera jamesonii cv. ‘Shy Pink’

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Abstract

Figures

Introduction

Materials and Methods

Plant material and culture conditions

Collection of data on morphological and growth parameters

Estimation of chlorophyll and carotenoid content

Measurement of chlorophyll fluorescence

Stomatal index

Acclimatization

Statistical analysis

Results

Morphological and growth parameters of shoots and plantlets regenerated with different culture systems

Chlorophyll and carotenoid contents of the regenerated plants using different culture systems

Chlorophyll fluorescence parameters

Analysis of stomatal index

Discussion

Conclusion

Declarations

References

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