Hyperhydricity (HH) is a significant obstacle in enhancing the efficacy of plant in vitro cultivation due to its adverse effects on plant quality and survival rate, resulting in significant economic losses in the commercial sector. Previous studies have shown the presence of abnormalities in HH leaves, including reduced lignin (Kemat et al. 2021; Kevers et al. 1987), chlorophyll deficit, and deformed stomata (Apostolo and Llorente 2000). It was observed that the alterations in the morphological characteristics of hyperhydric leaves, such as a deficient formation of the epicuticular wax layer, a decreased number of palisade cells and increased intercellular spaces in the mesophyll (Jausoro et al. 2010; Olmos and Hellin 1998). Currently, over 200 species have been identified as being susceptible to HH, with approximately 150 of these species classified as severely hyperhydric (Abdoli et al. 2007; Bakir et al. 2016; Chakrabarty et al. 2006; De Carvalho et al. 2013; Ivanova and Van Staden 2010; Kadota and Niimi 2003; Liu et al. 2017; Mayor et al. 2003; Wu et al. 2009). Van den Dries et al. (2013) mentioned that the apoplast of leaves in hyperhydric Arabidopsis seedlings becomes saturated, resulting in the near-complete flooding of apoplastic air space. Waterlogging in the apoplast can lead to physiological disorders due to its negative impact on gas exchange (Gribble et al. 2003). Moreover, it has been shown that elevated levels of sucrose can lead to the development of HH and inhibited growth of plants cultured in vitro (Ševčíková et al. 2019).

The presence of media components substantially influenced the development of HH, including phytohormones and solidifying agents, as well as the high relative humidity maintained within the culture containers. The cytokinin, a significant category of plant growth hormones, are known for their pivotal involvement in the shoot induction and multiplication in vitro (Krikorian 1995). Therefore, cytokinin are frequently altered in micropropagation protocols with the aim of augmenting shoot output (Aremu et al. 2014). The ability of exogenous cytokinin to induce HH is often influenced by its concentration, as demonstrated in studies by Debergh (1983), Kataeva et al. (1991), and Ivanova and Van Staden (2008). According to Buah et al. (1999), the type and concentration of gelling agent present in the culture medium affected many in vitro shoot multiplications. Furthermore, the gelling agent has an impact on the physical characteristics of the medium, including water potential and nutrient availability (Kusumoto 1980; Singha et al. 1985). On the other hand, the use of liquid culture systems also contributed to HH development in some plants.

HH influenced by many related factors. The manifestation of symptoms varies according on the species or even cultivar, and HH is not always present. Gaining a comprehensive understanding of the elements that contribute to the reduction of HH can significantly improve developments for the plant cell in vitro. Some researchers reported the symptoms of HH by looking on the phenotypic appearance (Casanova et al. 2008; Dewir et al. 2006). The scoring methods by looking at phenotypical appearance is more subjective because it is influenced by individual perception. This subjectivity arises due to the varying degrees of symptom exhibited by different shoots. In order to have more understanding of the impact of medium components on HH, we examined the phenomenon of HH on Arabidopsis thaliana Col-0 seedlings comprehensively and quantifiably on different types of cytokinin and gelling agents. In addition, a study was carried out to examine the impact of the excessive buildup of water in the apoplast. This investigation focused on the characteristics of stomata, water retention capacity, transpiration rate and leaf cuticle abnormalities.

Plant materials

Arabidopsis thaliana Col-0 seeds were sterilized using 70% (v/v) ethanol for a duration of 1 min, followed by treatment with 2% (w/v) sodium hypochlorite for 15 min. Then, the seeds were rinsed three times for 10 min using sterilized distilled water. Next, the seeds were cultured on half-strength Murashige and Skoog (MS) (Murashige and Skoog 1962) medium with vitamins supplemented and 1.5% (w/v) sucrose. The media solidified with 0.7% (w/v) Micro-agar, pH 5.8. The seeds were stratified in the dark for a duration of 3 days at a temperature of 4°C. The seeds were then transferred to a controlled climate room at 21°C with 16 h light/ 8 h dark (30 µmol m^-2 s^-1, Philips TL33) for germination. In all treatment, seedlings cultured on half-strength MS on 0.7% (w/v) Micro-agar without any cytokinin act as control.

Effect of media components

i) Different gelling agents

Arabidopsis thaliana Col-0 seedlings (7-days-old) were placed on half-strength MS with 4 different concentrations of solidifying agent at 0.7% Micro-agar, 0.35% Micro-agar, 0.2% gelrite, and 0.4% gelrite (all w/v) and liquid culture media.

ii) Different types and concentrations of cytokinin

7-days-old Arabidopsis thaliana Col-0 seedlings were cultured on half-strength MS with 5 different cytokinin types; 6-benxylaminopurine (BAP), thidiazuron (TDZ), Zeatin (Z), meta-Topolin (MT) or Forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea) (CPPU) (Sigma, St Louis, MO, USA) at different concentrations of 0, 0.3, 1 and 3 µM solidified with two different gelling agents; 0.7% (w/v) Micro-agar and 0.2% (w/v) gelrite.

Measurement of apoplastic water and air

The measurement of apoplastic water was examined based on Terry and Bonner (1980) by using mild centrifugation. The leaves were excised, weighed and were centrifuged in Eppendorf 5418R Refrigerated microcentrifuge (Hamburg, Germany) at speed of 3,000 g at a temperature of 4°C for a duration of 20 min. Following centrifugation, the leaves were later reweighed. For apoplastic air volume, the leaves were removed, weighed then placed into the pycnometer following the methods of Raskin (1983). Distilled water was filled in the pycnometer until full and put a stopper. Next, the pycnometer was placed in a vacuum for 5 min until the leaves had sunk to the bottom. The apoplastic water volume and air were calculated using the formula (Van den Dries et al. 2013). Then, the mean percentages of apoplastic water and apoplastic air related to the total apoplast volume were counted using formula of %tmvw or %tmva = 100 × tmvw or tmva / Tmap. Where, tmvw = mean volume apoplastic water, tmva = mean volume apoplastic air, Tmap = mean total volume of the apoplast (water + air).

Stomatal traits

Stomatal aperture was examined by creating epidermal impressions of the abaxial surface of the leaf. Leaf impressions were prepared by using dental resin. When the impression material solidified, quickly applied transparent nail and then carefully stripped. Then the specimens were mounted on a microscope slide and observed under an Axiophot light microscope manufactured by Zeiss in Oberkochen, Germany with a 400× magnification. The pore aperture and pore length were measured by using AxioVision software release 4.8.2 (Zeiss). Stomata density was assessed by counting the number of stomata per field (200 × 200 µm) of view at 400× magnification. Stomata density calculated using the formula: The number of stomata in the field of view / the area (mm²).

Quantification of water loss

Water loss quantification was conducted according to Zhang et al. (2012) with minor modifications by using an analytical microbalance. The roots of Arabidopsis thaliana Col-0 seedlings were incised and the fresh weight (FW) of the leaves were measured at the starting point (0 min) was measured (FW₀). The leaves were then allowed to desiccate at ambient temperature at the bench within an open Petri dish and then were weighed at different intervals after 30, 60, 120 and 240 min (W_t). Subsequently, the leaves were put in an oven at 70°C overnight to get the dry weight (DW). The percentage of water content (WC) was calculated by using the following formula: %WC = (W_t-DW)/ (FW₀-DW) × 100.

Determination of transpiration rate

The measurement of the transpiration rate was examined using an analytical balance. Arabidopsis thaliana Col-0 seedlings (7-days old) were placed individually into test tube caps on the treatment of 0 and 1 µM cytokinin (BAP, TDZ, Z or MT). Seedlings on Micro-agar without cytokinin act as control and gelrite without cytokinin as HH control. Subsequently, the test tube caps were placed in the round closed plastic container to mimic the tissue culture environment condition. Then, a layer of 2 ml Paraffin oil (PO) (Sigma, St Louis, MO, USA) was added to each test tube to mitigate the evaporation of water from the media. The test tubes caps + media + PO and seedlings, was weighed separately at two different days and two-time intervals. The leaf areas of the samples were measured using ImageJ software. The transpiration rate was calculated according to the decreased weight (water loss) / total leaf area (cm²). For this experiment, treatment of 0 and 1 µM cytokinin has been chosen as concentration based on the optimal concentration in both Micro-agar and gelrite.

Anatomical structure analysis

14-days old Arabidopsis thaliana Col-0 seedlings were immersed in a 5% (v/v) solution of glutaraldehyde in 0.1 M phosphate-buffer with a pH of 7.2 for 2 hours at 4°C. Then, the specimens were washed with 0.1M phosphate-buffer (pH 7.2) for 15 min four times followed by distilled water. The samples were dehydrated using a gradient series of ethanol and subsequently embedded in Technovit 7100 (Heraeus-Kulzer Technik, Germany). The specimens were cross-sectioned using a rotary microtome and mounted to a glass slide subsequently stained with 0.05% (w/v) solution of toluidine Blue O in phosphate buffer with a pH of 6.8. The images were captured using an Axiophot light microscope (AxioVision software release 4.8.2, Zeiss, Jena, Germany).

Cuticle pattern of HH

The Toluidine Blue O (TB) test was performed according to Tanaka et al. (2004) to investigate the cuticle pattern of hyperhydric leaves in comparison to control leaves. Phenotypic observation was then conducted to assess the staining and distribution patterns according to Tanaka et al. (2004). The study was then identified five distinct staining patterns: (1) Class I, characterised by a uniform staining pattern across the entire sample; (2) Class II, characterised by a patchy staining pattern; (3) Class III, characterised by a proximal staining pattern; (4) Class IV, characterised by a distal staining pattern; and (5) Class V, characterised by a staining pattern of trichomes.

Statistical data analysis

Three replicates of each treatment with 15 plants were analysed for media components, apoplastic water and air and water loss except for stomatal, transpiration and anatomical structure analysis. The data were analyzed using one-way analysis of variance (ANOVA), followed by post-hoc mean separation was performed by Duncan’s Multiple Range Test (DMRT) at P ≤ 0.05. All experiments were carried out with a minimum of two repetitions.

HH characterization

Fig. 1a-d illustrated the morphological characteristics of Arabidopsis on Micro-agar and gelrite medium. No hyperhydric symptoms was observed on seedling grown Micro-agar medium (control) (Fig. 1a). Addition of cytokinin had a significant impact on the seedlings quality such as glassiness, wrinkled, long petiole, larger leaves and showed signs of anthocyanin formation on 14 days of culture (Fig. 1c-e). Likewise, the manifestations of HH were observed on gelrite alone medium (Fig. 1b).

Figure 1. Phenotypic characteristics of Arabidopsis on micro-agar (control) (a) and HH conditions of gelrite (0.2%) (b), 1 µM 6-benzylaminopurine (BAP) micro-agar (c), 1 µM BAP on gelrite (d) and 1 µM thidiazuron (TDZ) on micro-agar (e). Bar = 5 mm

HH development of different gelling agent concentration

A slight difference in media water potential resulted in a more pronounced impact on the prevalence of HH. The reduction in Micro-agar concentration resulted in a corresponding rise in the proportion of apoplastic water and a decline in the proportion of apoplastic air, with decreasing by 50% compared to the control. Besides, when doubled the gelrite concentration, the seedlings exhibited a mitigating effect on hyperhydric symptoms, leading to a consistent decline in the proportion of apoplastic water (Fig. 2a). Fig. 2b illustrates the typical characteristics observed in Arabidopsis seedlings grown in a liquid medium, which manifestation of HH symptoms. A notable of HH symptoms was seen when lowering the level of Micro-agar at 0.35% (w/v), increasing gelrite at 0.4% (w/v) and liquid culture (Fig. 2c-e). The results indicated that 90% of the apoplast being filled with water, which is comparable to the condition of 0.4% gelrite. The results align with the studies conducted by Deberg et al. (1981) and Kusumoto (1980), which observed that minor alterations in medium water potential led to significant variations in leaf water potential. According to the study conducted by Ghashghaie et al. (1991), it was observed that the leaf water potential and absolute water content were high when the concentration of the solidifying agent (agar) was low.

Figure 2. Proportion of water and air in relation to the total apoplast volume at different levels and types of gelling solidifying agents (a-b). The phenotypic manifestation of Arabidopsis on different types of gelling agents and concentrations; 0.35% (w/v) Micro-agar (c), 0.4% (w/v) gelrite (d) and liquid media (e). Different letters indicate statistically significant differences at P ≤ 0.05. Bar = 5 mm

Volume of apoplastic water and air in hyperhydric seedlings

The study revealed that the apoplastic volume in the Micro-agar (control) treatment consisted of approximately 13% water and 87% air. In contrast, hyperhydric seedlings exhibited a significant increase in water content, accounting for almost 90% of the total apoplastic volume (Fig. 3a-b). Irrespective of the specific cytokinin type and concentration used on the gelrite medium, the apoplastic water volume percentage consistently above 90%, mirroring the percentage observed on the gelrite alone. Among the four types of cytokinin examined on Micro-agar medium, it was observed that TDZ exhibited comparatively higher levels of water content at concentrations of 0.3, 1, and 3 µM, with percentages of 45.5%, 69.6%, and 80.8% respectively. Conversely, TDZ displayed lower levels of air content at these concentrations, with percentages of 54.5%, 30.4%, and 19.2% respectively, when compared to BAP, MT, and Z. This suggests that TDZ, a phenylurea-type, has more potent ability to induce HH compared to the adenine types (Z, MT, and BAP). The efficacy of a different phenylurea-type cytokinin, namely 1-(2-Chloro-4-pyridyl)-3-phenylurea (CPPU), was validated to ascertain the discernible variations among various types of cytokinin (Fig. 4a-b). However, Fig. 4a-b illustrates that the proportions of apoplast water in all the seedlings cultivated on both gelrite and gelrite with cytokinin exceed 90% of the total apoplast. This suggests that the observation of seedlings on gelrite exhibiting more pronounced hypocotyl hooking than those on gelrite supplemented with cytokinin does not necessarily imply that gelrite was a more potent competitor. This observation is consistent with the findings of Kadota and Niimi (2003), who reported that synthetic phenylurea derivatives resulted in a higher occurrence of hyperhydric shoots compared to adenine derivatives in Pyrus pyrifolia. Based on one hypothesis, an elevated concentration of cytokinin has been proposed to stimulate the production of ethylene, leading to the development of hyperhydricity, compromised shoot growth, and apical necrosis (Liu et al. 2017; Žd’árská et al. 2013). Additionally, it was shown that the application of cytokinin to hyperhydric leaves resulted in the formation of anthocyanins, indicating the presence of stress.

Figure 3. The relative amounts of apoplastic water (a) and air (b) after treatment with different cytokinin concentrations: (0, 0.3, 1, and 3) µM of 6-benzylaminopurine (BAP), thidiazuron (TDZ), Zeatin (Z) or meta–Topolin (MT). Different letters indicate statistically significant differences at P ≤ 0.05

Figure 4. The proportion of water and air in relation to the overall volume of apoplasts under different cytokinin concentrations (0, 0.3, 1, and 3 µM) and types (6-benzylaminopurine (BAP), 1-(2-Chloro-4-pyridyl)-3-phenylurea (CPPU), and thidiazuron (TDZ)). Different letters indicate statistically significant difference P ≤ 0.05

Stomata traits in hyperhydric seedlings

It was observed that the stomata were opened on control medium (Fig. 5a), partially open stomata on cytokinin 1 µM BAP on Micro-agar (Fig. 5c) and the stomata were closed on gelrite alone medium (Fig. 5b) and 1 µM BAP on (Fig. 5d). Table 1 showed the average of pore aperture and pore length measurements of the stomata on the treatment of control, 0.3 µM and 1 µM cytokinin on Micro-agar. In contrast, the stomata were closed in all gelrite cytokinin treatment (data not presented). The average width of apertures in control seedlings was approximately 6 µm and the average length was approximately 13 µm while the seedlings treated with cytokinin (Table 1). The pore diameters of hyperhydric seedlings exhibited alterations, accompanied by a reduction in stomatal density as compared to the control seedlings (Fig. 6). A reduction in stomata density of hyperhydric seedlings was seen when subjected to increasing concentrations of cytokinin in both Micro-agar and gelrite. The reductions were measured at 25%, 50%, and 65% for the corresponding concentrations. The study revealed that a decrease in stomatal density resulted in the buildup of water in the apoplast, primarily due to a reduction in transpiration. It was found that number of stomata per unit area were decreased in several hyperhydric leaves due to the increased size of the epidermal cells, which is around 2-3 times larger, resulting in a decreased stomatal density per unit area (Louro et al. 1999; Olmos and Hellin 1998). In our work, a notable reduction in stomatal opening size was seen following the introduction of cytokinin and when the medium consisted solely of gelrite without cytokinin. The guard cells in HH exhibited abnormalities characterised by deformations resulting from the disruption of the cell wall that delineates the stomatal pore. Consequently, this led to the partial opening of the stomata. Abnormalities in guard cell shape were seen in hyperhydric leaves of various plant species, as reported by Picoli et al. (2008) and Gupta and Prasad (2010). In addition, Yu et al. (2011) documented that the diminished survival rate of Brassica oleracea var. italica plantlets was attributed to the adverse alterations observed in leaf tissue, structure, density, stomatal size, guard cell shape, and chloroplast ultrastructure in hyperhydric plantlets.

Table 1 . Stomatal traits (pore aperture and pore length) at different cytokinin concentrations on micro-agar in Arabidopsis leaves.

Treatments (Micro-agar)	Pore aperture (µm)	Pore length (µm)
Control	6.3 ± 0.9a	12.7 ± 1.4a
0.3 µM TDZ	3.94 ± 1.2c	6.9 ± 0.8d
0.3 µM Z	4.83 ± 0.7b	8.5 ± 1.7b
0.3 µM MT	4.78 ± 1.6bc	8.05 ± 2.3bc
0.3 µM BAP	4.35 ± 0.7c	7.96 ± 1.9c
1 µM TDZ	2.07 ± 0.9e	4.01 ± 0.8f
1 µM Z	3.83 ± 0.5c	4.55 ± 0.7e
1 µM MT	2.67 ± 1.8d	4.24 ± 1.3e
1 µM BAP	2.63 ± 1.1d	4.37 ± 0.9e

The means of 9 leaves ± SE are presented; a, b, c, d, e, and f letters indicate significant differences between means at α = 0.05 level..

Figure 5. Abaxial leaf epidermal impressions of Arabidopsis leaves on micro-agar (control) (a), gelrite 0.2% (b), 1 µM 6-benzylaminopurine (BAP) micro-agar (c), and 1 µM BAP on 0.2% gelrite (d). Bar = 5 µm

Figure 6. Stomatal density of Arabidopsis seedlings on different cytokinin concentrations. Different letters indicate statistically significant differences at P ≤ 0.05

Water status and transpiration rates of hyperhydric seedlings

Closure of stomata in hyperhydric seedlings appears to enhance their ability to retain water, potentially leading to the buildup of water in the apoplast. To assess this phenomenon, we conducted an experiment to measure the amount of water lost by detached leaves following their transfer from a controlled environment with high relative humidity to the surrounding ambient relative humidity. During the first 30 minutes of being exposed to ambient relative humidity, the seedling on control medium experienced a water loss of approximately 37% in the leaves (Fig. 7a), in comparison to the initial point. In contrast, the water loss at about 15-25% on gelrite and cytokinin treatment (Fig. 7b). 50% of water loss was found at 48 minutes on control treatment whereas the range of 90-128 minutes for other treatments (Fig. 7c). The data presented in this study indicate that hyperhydric leaves had a much greater water retention capacity compared to non-hyperhydric leaves, as evidenced by the sustained presence of higher levels of water even after a four hours desiccation period.

Figure 7. The relative water content in Arabidopsis seedlings after treatment with various cytokinin concentrations (a-b) and at time of 50% of water loss (c). Different letters indicate statistically significant difference P ≤ 0.05

On both time intervals, seedlings on Micro-agar (control) resulted about ten times higher transpiration rate than seedlings on gelrite alone (Table 2). Additionally, it has been observed that the average transpiration rate (g/cm²) in the presence of cytokinin on both on gelling agents (except TDZ), showed slightly inclined as compared to gelrite alone. This finding demonstrates that the observed stomatal behaviour aligns with the measured transpiration rates in hyperhydric seedlings. The observed phenomenon may be attributed to a decrease in stomatal density and an alteration in stomatal opening size, which serves to regulate the exchange of gases and the process of transpiration. The average transpiration rate exhibited a noticeable rise during the second time interval (day 23-27) in comparison to the initial time interval (day 19-23). Theoretically, the transpiration rate decreased when the level of water retention increased. Our findings align with this hypothesis that seedlings with high water retention capabilities (referred to HH seedlings) had a slower water loss compared to the control group of seedlings.

Table 2 . Transpiration rate (g/cm²) at the 19–23 day interval and 23–27 day interval of different gelling agents and cytokinin concentrations.

Time interval	Treatments	Mean transpiration rate (g/cm2)
Days 19 - 23	Micro-agar (Control)	0.285a
	1 µM TDZ Micro-agar	0.070cd
	1 µM BAP Micro-agar	0.091c
	1 µM Z Micro-agar	0.122bc
	1 µM MT Micro-agar	0.098c
	Gelrite (HH Control)	0.025e
	1 µM TDZ gelrite	0.026e
	1 µM BAP gelrite	0.055d
	1 µM Z gelrite	0.050d
	1 µM MT gelrite	0.051d
Days 23 - 27	Micro-agar (Control)	0.380a
	1 µM TDZ Micro-agar	0.098d
	1 µM BAP Micro-agar	0.130c
	1 µM Z Micro-agar	0.154b
	1 µM MT Micro-agar	0.130c
	Gelrite (HH Control)	0.030f
	1 µM TDZ gelrite	0.035f
	1 µM BAP gelrite	0.071de
	1 µM Z gelrite	0.057e
	1 µM MT gelrite	0.055e

The mean of 9 leaves; a, b, c, d, e, and f indicate significance of the difference between means at an level of α 0.05..

Anatomical and cuticle abnormalities of hyperhydric leaves

Normal (control) leaves were shown to have a distinct dorsiventral homogenous mesophyll, characterised by a single layer of epidermis and palisade, leaf epidermis with a thin cuticle, and the presence of collateral vascular bundles was also noted (Fig. 8a). In contrast, hyperhydric plants exhibited disorganized mesophyll characterised by globular cells and the absence of a distinct palisade parenchyma. Furthermore, the presence of substantial intercellular gaps and the inadequate organization of vascular bundles can be observed (Fig. 8b-c). The findings align with the observations made by Barbosa et al. (2013), indicating that an increase of 6-benzyladenine level led to the gradual disappearance of the distinction between spongy and palisade parenchyma.

Figure 8. Transverse sections and microscopic views of Arabidopsis leaves on micro-agar (control) (a) and gelrite (0.2%) (b) and 1 µM 6-benzylaminopurine (BAP) on micro-agar (c). Bar = 50 µm

Hyperhydric leaves revealed irregular, often discontinuous growth of the epidermis and cuticle. Among the 24 seedlings that were examined, it was observed that the control seedlings displayed a proportion of 50% of seedlings that exhibited no staining and were devoid of any visible contaminants. This observation indicates that the leaves exhibit a fully developed cuticle. 20% of the control seedlings exhibited a patchy pattern (Class II), while 30% displayed a trichome staining pattern (Class V). In contrast, it was shown that all 24 seedlings exhibited discontinuous and poor cuticle development on gelrite medium. The abnormal cuticles facilitated the infiltration of TB into the epidermal surface, leading to distinct staining patterns. These patterns included a total absence of cuticle in 20% of complete loss of cuticle (Class I), 20% of incomplete cuticle (Class II), 30% of distal pattern (Class IV), and 20% of trichomes pattern (Class V) (Table 3). The results of this study confirm our previous findings regarding the disorganised ultrastructure of hyperhydric seedlings. These findings are consistent with the observations made by Jausoro et al. (2010) and Louro et al. (1999), who also noted a decrease in cuticle thickness and wax deposition in hyperhydric in vitro plants of Handroanthus impetiginosus and Eucalyptus urophylla.

Table 3 . Classification and percentages of TB-staining patterns of control and HH seedlings according to Tanaka et al. (2004).

The present study provided useful information regarding the physiological and anatomical alterations on HH manifestation. The physiological and anatomical alterations of HH in Arabidopsis were quantified using measurable techniques. The presence of water obtained from the medium is a crucial determinant in the manifestation of HH. The manipulation of solidifying agents concentrations had an impact on the availability of water and HH development. Our hypothesis is that water in the gelrite media is more easily accessible to the roots due to its physical structure as compare to Micro-agar. Additionally, high relative humidity in the headspace also contributed to the occurrence of HH. These findings suggested that the concentration of gelling agents was directly proportional to the water potential of the medium. Moreover, the level of plant growth regulators, as well as the specific hormone types also lead significantly influence the extent of HH produced. Exogenously applied cytokinin in the medium resulted in an increase of HH development. Addition of phenylurea-type exhibiting a greater negative impact compared to the adenine-type cytokinin in both media. This study becomes a benchmark for other plants that are sensitive to HH by providing a quantitative data on HH development through the percentage of water in the apoplast, an abnormal stomata trait, higher water retention capacity, lower transpiration rates, epidermal defects and impaired cuticle.

We would like to express our gratitude to Dr. G.J.M (Geert-Jan) de Klerk for his valuable and constructive recommendations during the design and development stages of this research project. We also want to express our heartfelt gratitude to Luo Rong and Ziqi Zeng for their dedicated efforts and valuable contributions on this research topic. This manuscript is a reworked version of chapter 2 and 6 from the first authors PhD thesis (Kemat 2020). This research was funded in part by the Dutch Ministry of Economic Affairs, TKI-TU grant KV1310-067 and by Ministry of Higher Education Malaysia and Universiti Putra Malaysia.