Cytokinins induce hyperhydricity in leaves of in vitro grown Arabidopsis thaliana

Transcription

1 Cytokinins induce hyperhydricity in leaves of in vitro grown Arabidopsis thaliana

2 Cytokinins induce hyperhydricity in leaves of in vitro grown Arabidopsis thaliana Ziqi Zeng Reg. Num: Msc thesis Plant Breeding Department of Plant Breeding, Wageningen UR PBR Supervisors: Dr. Frans Krens Nurashikin binti Kemat Examiner: Dr. Frans Krens Nurashikin binti Kemat Wageningen UR Plant Breeding, Droevendaalsesteeg 1, 6700 AA Wageningen, The Netherlands August 2016 I

3 Abstract Tissue culture is a useful and powerful tool for plant breeding and vegetative propagation. However, physiological disorder hyperhydricity (HH) that leads to morphological abnormalities such as translucent, thick and brittle leaves in tissue culture seriously affect the quality and multiplication rate of micropropagation. It is known that hyperhydricity is resulted from flooding of apoplast and might be due to deficiency in lignin synthesis. Cytokinins as a growth regular are vital hormone for multiplication rates in tissue culture that we cannot do without. In order to study th effect of different types of cytokinins on HH development of Arabidopsis, we examined different types of cytokinins. We have found that all cytokinins induced HH. The highest amount of apoplastic water in the apoplast was found on TDZ at concentration of 0.5µM on Arabidopsis thaliana (col-0). Besides, there were significantly different between the cytokinins types and their concentration on stomata aperture, but there were no difference in stomatal density. Using Zeatin or BAP at low concentration of 0.1µM resulted less HH symptoms in all treatments. These results indicate that adenine type of cytokinin produce less HH than phenylurea type. Keywords: Apoplast, Arabidopsis thaliana, hyperhydricity, cytokinins, lignin, stomatal, water accumulation. II

4 TABLE OF CONTENT INTRODUCTION... 1 MATERIALS AND METHODS... 3 PLANT GROWTH AND TREATMENTS... 3 ESTIMATION OF APOPLASTIC WATER AND APOPLASTIC AIR VOLUMES IN LEAVES... 3 MEASUREMENT OF STOMATAL APERTURE... 4 RESULTS... 5 CULTURING ON GELRITE AND/OR CYTOKININS INDUCED HH IN ARABIDOPSIS THALIANA (COL-0) SEEDLINGS... 5 APOPLASTIC WATER AND APOPLASTIC AIR VOLUMES IN ARABIDOPSIS THALIANA (COL-0) SEEDLINGS... 6 STOMATAL APERTURE AND DENSITY IN ARABIDOPSIS THALIANA (COL-0) SEEDLINGS... 7 APOPLASTIC WATER VOLUMES IN ARABIDOPSIS THALIANA MUTANTS (REF1-4, REF3-3 AND FLP) SEEDLINGS... 8 DISCUSSION... 9 CAUSES OF HH IN ARABIDOPSIS THALIANA SEEDLINGS... 9 CONSEQUENCE OF HH IN ARABIDOPSIS THALIANA SEEDLINGS CONCLUSION REFERENCE APPENDIX III

5 Introduction Tissue culture is a useful and powerful tool for plant breeding and vegetative propagation both in horticulture and agriculture (Van Den Dries et al. 2013). However, during tissue culture, plants are undergoing unnatural and extreme conditions, which may result in physiological disorder, for example hyperhydricity (HH) (Van Den Dries et al. 2013; Debergh et al. 1981; Rojas-Martinez et al. 2010). HH causes translucent, thick and brittle leaves of the explants that are visible to naked eyes, seriously reducing the quality and multiplication rate of commercially vegetative propagation in tissue culture (Van Den Dries et al. 2013; Gaspar et al. 1995; Sadhy Saher et al. 2005; Kevers et al. 1987). It was reported that HH occurs in many plant species (Van Den Dries et al. 2013; Wu et al. 2009; Chakrabarty et al. 2005). In Arabidopsis, Delarue et al. (1997) found that a mutant (cril) severely formed HH in vitro cultured with 0.7% (w/v) agar. The water content in hyperhydric plant is usually high. This is because plants that are affected by HH are not capable of keeping water balance correctly (Rojas-Martinez et al. 2010). It was observed that hyperhydric plants have excess water in apoplast (Gribble et al. 1996; Gribble et al. 1998), which is the intercellular space. Water accumulation in apoplast may result in physiological disorder by reducing gas exchange (Gribble et al. 2003), because diffusion rate of gases is much more lower in water than in air in plant tissues as it would be expected (Jackson 1985). There are several factors that will result in HH. First of all, as plants are cultured in a sealed vessel, the gas exchange is affected and the humidity is relative high, which are the major contribution to the formation of HH (Ivanova & van Staden 2010; Debergh et al. 1992). Furthermore, other factors such as the type and concentration of gelling agent (Debergh et al. 1992; Ivanova & Van Staden 2011), cytokinins (Ivanova & Van Staden 2011; Kadota & Niimi 2003) may also play a significant role in the development of HH. Besides, it was reported that deficiency of cellulose and lignin may also result in HH since symplast may take up more water by reducing cell wall pressure in hyperhydric plants (Kevers et al. 1987; Van Den Dries et al. 2013). In addition, malformed stomata was also found in leaves of hyperhydric plants (Apóstolo & Llorente 2000), and stomatal closure in hyperhydric leaves may impair transpiration and thereby aggregate HH (Van Den Dries et al. 2013). 1

6 Many researches have been done on HH, however, the phenomenon and underlying mechanism of HH remain poorly understood (Van Den Dries et al. 2013; Ziv 1991; Gribble et al. 2003). The present research examined on how the apoplast is flooded. One such condition may due to plant hormone effect which are necessary for multiplication, cytokinins. The objectives of this study were: (1) To evaluate the effect of different cytokinins on morphological and physiological characteristics in leaves of in vitro grown Arabidopsis plants. (2) To study on the effect of lignin and stomata on development of HH. 2

7 Materials and methods Plant growth and treatments Arabidopsis thaliana (col-0) seeds were sterilized with 70% (v/v) ethanol for 1 min and 2% (w/v) sodium hypochlorite for 15 min. After then, the seeds were rinsed three times with sterilized distilled water for 10 min. Subsequently, sterile seeds were transferred to a Petri dish with half-strength Murashige and Skoog (MS) basal salt mixture including vitamins (Murashige & Skoog 1962) with a supplement of 1.5% (w/v) sucrose and solidified with 0.7% (w/v) Micro-agar (all of them are from Duchefa Biochemie, Haarlem, The Netherlands). Seeds were first stratified in dark for 3 d at 4 0 C and subsequently germinated in a growth chamber with 16h light/8h dark period (30µmol m -2 s -1, Philips TL33) at 21 0 C. 7-days-old wild type Arabidopsis thaliana (col-0) seedlings were transferred and cultured in high-sided Petri dish with ten seedlings per dish solidified with 0.2% (w/v) Gelrite and/or 0.7% (w/v) Micro-agar. Four types of cytokinins were supplied (zeatine, 6-Benzylaminopurine (BAP), 6-(3-hydroxybenzylamino) purine (meta-topolin) and Thidiazuron (TDZ)) with three levels of concentration (0µM, 0.1µM and 0.5µM respectively). Each treatment replicated three times. 7-days-old lignin mutant (ref1-4 and ref3-3) and stomata mutant (flp) Arabidopsis thaliana seedlings were transferred and cultured in high-sided Petri dish with five seedlings per dish solidified with 0.2% (w/v) Gelrite and/or 0.7% (w/v) Micro-agar. Each treatment replicated three times. Estimation of apoplastic water and apoplastic air volumes in leaves The apoplastic water was extracted from leaves tissues with mild centrifugation (Terry & Bonner 1980; Van Den Dries et al. 2013). Leaves were excised from plants, weighed and subsequently put into a spin mini filter microcentrifuge tube (Starlab, Ahrensburg, Germany), centrifuging at 3000g for 20 min at 4 0 C. After centrifugation, leaves were weighted again. The formula: V water = [(FW - W ac ) x ρh 2 O] / FW was used to calculate the apoplastic water volume (V water ) in µl g -1 fresh weight (FW). FW represented the fresh weight of leaves in mg, while W ac represented the weight of leaves after centrifugation and ρh 2 O represented water density that was taken as equal to 1 g ml -1. The apoplastic air volumes in leaves was assessed with the help of a pycnometer with 3

8 a stopper (Raskin 1983; Van Den Dries et al. 2013). Leaves were excised, weight and subsequently put into the pycnometer, which was then filled with distilled water and stoppered. Filter paper was used to remove any excess water on the exterior of the pycnometer. The combined weight of the full pycnometer and leaves was measured. The pycnometer, with both water and leaves, underwent a vacuum (about 500 mm Hg) for 5 min in order to remove air from the apoplast and replace it by water. If needed, this vacuum treatment was re-did till all air was supposed to be removed out of the apoplast and the leaves had sunk to the bottom of the pycnometer. After vacuum infiltration, the pycnometer was refilled, dried and then weighted again. The formular: V air = [(W bv W av ) x ρh 2 O] / FW was used to calculated rge apoplastic air volume (V air ) in µl g -1 FW. W bv represented the weight in mg of the pycnometer including both leaves and water before vacuum infiltration, while W av represented the weight of the pycnometer including both leaves and water after vacuum infiltration. FW represented the fresh weight of the leaves, and ρh 2 O represented the water density that was taken as equal to 1 g ml -1. Measurement of stomatal aperture The stomatal aperture was explored by making epidermal impressions of adaxial leaf. Leaves were excised from plants, and then adaxial surface were placed onto impression material and then solidified. The impression material was polyvinylsiloxane-based high precision President Light Body impression material (Coletene/Whaledent AG, Altstatten, Switzerland) (Geisler et al. 2000). Subsequently, leaves were removed. Transparent nail polish was applied to the epidermal imprints leaving over. After drying, the nail polish were gently and carefully peeled and placed on a microscope slide. The leaf stomata impressions were then shown under an Axiophot light microscope (Zeiss, Oberkochen, Germany). Images were subsequently captured using AxioCam ERc5S digital camera (Zeiss), and AxioVision software (Zeiss) was used to measure the stomatal aperture. Guard cell lengths (µm) were measured to the nearest micrometer viewed at 40x magnification. In addition, stomatal density was measure by counting the number stomata per field (200x200µm) of view at 40x magnification. Sample size was three leaves per plant and three fields per leaf, replicated twice. 4

9 Results Culturing on Gelrite and/or cytokinins induced HH in Arabidopsis thaliana (col-0) seedlings It is shown in figure 1 that on days 14 after transfers, the control seedlings (non-hyperhydric) has no symptoms of HH (fig.1a). The seedlings grown on Gelrite, gelrithe plus cytokinins, and agar plus cytokinins showed HH symptoms with thick, wrinkle and translucent leaves, as well as elongated petioles (fig.1). Overall, Gelrite-grown (Gelrite and Gelrite plus cytokinins) seedlings apparently developed severer HH than agar-grown (agar and agar plus cytokinins) seedlings, comparing figure 1B to figure 1A. Besides, either grown on Gelrite or agar, it is shown that TDZ induced more HH symptoms while meta-topolin and BAP were moderate, and zeatine induced the least severe HH (fig.1). Increasing cytokinins concentration induced more HH, as it can be seen that seedlings from 0.5µM cytokinins had thicker, curler leaves and anthocyanin production, compared to those from 0.1µM for all four types of cytokinins (fig.1). It is noticed in figure 2A that on days 7 after transfer, the control seedlings were healthy, whereas those on agar plus cytokinins (0.5µM BAP, 0.5µM TDZ) started to show HH symptoms with anthocyanin production (fig 2A). The seedlings grown on Gelrite, and Gelrite plus cytokinins (0.5µM BAP, 0.5µM TDZ) developed symptoms of HH with curled leaves, anthocyanin production and elongated petioles, and it was apparently severer on Gelrite (without cytokinins) (fig. 2A). After 12 days, it is shown that Gelrite-grown seedlings grew faster than agar-grown seedlings (fig.2b). The control seedlings were healthy, whereas seedlings grown on agar plus cytokinins started to show HH symptoms (fig. 2B). The seedlings grown on Gelrite and Gelrite plus cytokinins apparently developed severer HH than those on days 7 (fig. 2A). After 19 days, the control seedlings were healthy (fig. 2D), whereas the HH symptoms of seedlings grown on agar plus cytokinins were severer than those on days 15 (fig. 2C). The HH symptoms was even more severer in seedlings grown on Gelrite and Gelrite plus cytokinins, and the seedlings already gave signs of highly stress with anthocyanin production and leaves necrosis (fig. 2D). After 25 days, the control seedlings were still healthy (fig. 2E). The seedlings grown on agar plus cytokinins developed severer HH than those on days 19 (fig.2d) but they were still alive (fig. 2E). The seedlings grown on Gelrite and Gelrite plus cytokinins had mostly necrosis, 5

10 which indicated the later stage of HH (fig. 2E). Apoplastic water and Apoplastic air volumes in Arabidopsis thaliana (col-0) seedlings After 14 days, it is shown in figure 3A that overall Gelrite-grown (Gelrite and Gelrite plus cytokinins) seedlings accumulated remarkably more water in apoplast than agar-grown (agar and agar plus cytokinins) seedlings. Either grown on agar or Gelrite, the apoplastic water was significantly different (p-value <0.05) for both cytokinins types (zeatine, meta-topolin, BAP and TDZ) and cytokinins concentration (0µM, 0.1µM and 0.5µM) (fig. 3A). When grown on agar, seedlings from agar (the control, without cytokinins) accumulated the lowest amount of apoplastic water, comparing to agar plus cytokinins. Regarding the cytokinins types added, seedlings from agar plus TDZ had the largest amount of apoplatic water, whereas seedling from agar plus zeatine had the less effect. Regarding concentration, seedlings from 0.5µM cytokinins accumulated significant larger apoplastic water than those from 0.1µM for all four types of cytokinins. When grown on Gelrite, seedlings from Gelrite (without cytokinins) accumulated the significantly largest amount of apoplastic water, comparing to those from Gelrite plus cytokinins. Regarding cytokinins types, seedling from Gelrite plus TDZ showed the significantly largest amount of apoplastic water, whereas seedlings from Gelrite plus zeatines showed the least. Generally, seedlings from 0.5µM cytokinins accumulated significant larger apoplastic water than those from 0.1µM for all four types of cytokinins. On the apoplastic air, it is shown in figure 3B that overall agar-grown seedlings had substantially more volume of apoplastic air in apoplast than Gelrite-grown seedlings. Either grown on agar or Gelrite, the apoplastic air was significantly different (p-value <0.05) for both cytokinins types and cytokinins concentration (fig. 3B). When grown on agar, the control seedlings had significantly larger volume of apoplastic air compared to seedlings from agar plus cytokinins. As for cytokinins types added, seedlings from agar plus zeatine had the significantly largest volume apoplastic air, whereas seedlings from agar plus TDZ had the lowest. The seedlings from 0.5µM cytokinins showed significantly larger volume of apoplastic air than those from 0.1µM for all four types of cytokinins. A similar pattern of apoplastic air was also observed for seedlings grown on Gelrite and Gelrite plus cytokinins (fig. 3B). 6

11 Adding the apoplastic water and apoplastic air volumes, the total apoplast was calculated. According to figure 4, it is shown that when seedlings were grown on Gelrite and Gelrite plus cytokinins, the apoplastic water accounted for more than 90% (fig.4a) whereas apoplastic air account for less than 10% (fig.4b), indicating that the apoplast was saturated by water. However, when seedlings were grown on agar and agar plus cytokinins, it is shown that the control seedlings had the lowest percentage of apoplastic water (fig.4a) and highest percentage of apoplastic air (fig.4b) (11.6% and 88.4 %, respectively), followed by agar plus zeatine. On the other hand, agar plus TDZ showed the highest percentage of apoplastic water and lowest percentage of apoplastic air (fig. 4). Overall, seedlings from 0.5µM cytokinins accounted for larger percentage of apoplastic water and smaller percentage of apoplastic air than those from 0.1µM for all four types of cytokinins (fig.4). These results indicate that the increase of apoplastic water in hyperhydric seedlings were at the expense of decreased apoplastic air volume. Figure 5 shows that from days 7 to days 15 the apoplastic water in hyperhydric seedlings grown from Gelrite, Gelrite plus cytokinins (0.5 BAPµM and 0.5µM TDZ) and agar plus cytikinins (0.5µM BAP and 0.5µM TDZ) increased along with the development of HH, and Gelrite-grown seedlings accumulated remarkably more water in apoplast than agar-grown seedlings. From days 15 to days 19, the increasing of apoplastic water in hyperhydric seedlings from Gelrite and Gelrite plus cytokinins slowed down, indicating that the apoplast was close to saturated. After days 19, the apoplastic water in hyperhydric seedlings from agar plus cytokinins continually and slowly increased, whereas those from Gelrite and Gelrite plus cytokinins started to decline because of necrosis of leave tissues. On the other hand, the apolastic water in the control seedlings remained more or less constant during the whole period, and it was at least three times less than that of hyperhydric seedlings on days 25. Stomatal aperture and density in Arabidopsis thaliana (col-0) seedlings It is shown in figure 6 that after 14 days of culture the stomata in the leaves were partially or fully closed in hyperhydric seedlings compared to the control seedlings, and the opening of stomata decreased along with the development of HH. It is shown in figure 7 that either grown on agar or Gelrite, the stomatal aperture in seedlings was 7

12 significantly different (p-value <0.05) for cytokinins types (BAP and TDZ) and cytokinins concentration (0µM, 0.1µM and 0.5µM). Overall, the control seedlings had the significantly largest value of stomatal aperture than that of hyperhydric seedlings. Hyperhydric seedlings grown on Gelrite (without cytokinins) had the significantly lowest value of stomatal aperture than those grown on Gelrite plus cytokinins and agar plus cytokinins. In addition, hyperhydric seedlings from 0.5µM cytokinins had significantly smaller stomatal aperture than those from 0.1µM for both BAP and TDZ. However, no significant difference (p-value >0.05) in stomatal density between hyperhydric and non-hyperhydric (the control) seedlings were observed (fig. 7). Apoplastic water volumes in Arabidopsis thaliana mutants (ref1-4, ref3-3 and flp) seedlings It is shown in figure 8 that on days 14 after transfers, wild type seedlings grown on agar were healthy, whereas those on Gelrite developed HH. The mutants (ref1-4, ref3-3 and flp) seedlings grown on agar and Gelrite showed HH symptoms with thick, wrinkle and translucent leaves, as well as elongated petioles (fig.8). Overall, Gelrite-grown seedlings apparently developed severer HH than agar-grown seedlings (fig.8). After 14 days, it is shown in figure 9 that overall Gelrite-grown seedlings accumulated remarkably more water in apoplast than agar-grown seedlings. Apoplastic water was significantly different (p-value <0.05) for wild type and mutants. Either grown on agar or Gelrite, wild type (col-0) seedlings accumulated significantly less apoplastic water than mutants (ref1-4, ref3-3 and flp), but the apoplastic volumes within mutants were not significantly different (fig.9). 8

13 Discussion In many previous researches on HH (Debergh et al. 1981; Rojas-Martinez et al. 2010; Gaspar et al. 1995; Sadhy Saher et al. 2005; Gribble et al. 2003; Ziv 1991), the extent of HH was visually accessed and evaluated based on the malformation and morphological abnormality of hyperhydric plants. However, seldom of them gave research on the underlying mechanism of HH. Gribble et al. (1996; 1998; 2003) assumed that HH resulted from accumulation of apoplastic water that interrupt and reduce gas exchange in apoplast of plant tissues, but the volume of apoplastic water and air were not adequately quantified. The studies of Van den Dries et al. (2013), for the first time, assessed the severity of HH by measurement of apoplastic water and apoplastic air volume in hyperhydric plant leaves tissues. In the present study, we followed the methods of Van den Dries et al. (2013) and quantified the apopalstic water and air volume in both hyperhydric and non-hyperhydric Arabidopsis thaliana seedlings. We found that in hyperhydric seeldings, the volume of apoplastic water were significantly larger than that of non-hyperhydric ones (fig.3), which accumulated over time (fig.5) and was at the cost of the volume of apoplastic air (fig.4). It was calculated that in hyperhydirc Arabidopsis thaliana seedlings, the percentage of apoplastic water accounted for more than 90% in total apoplast (fig.4). These findings are in lines with the study of Van den dries et al. (2013). Causes of HH in Arabidopsis thaliana seedlings Obviously, the main cause of HH is the flooding of apoplast that affects the water balance, reduces gas exchange in plant tissues and eventually leads to physiological disorders (Rojas-Martinez et al. 2010; Gribble et al. 1996; Gribble et al. 1998; Gribble et al. 2003; Van Den Dries et al. 2013). According to figure 3 and figure 4, it is shown that generally the seedlings grown on (0.2%) Gelrite accumulated significantly more apopalstic water than those grown on (0.7%) agar. This can be expected because Gelrite are supposed to release much more water than agar over time, increase the water uptake of plants and generate HH in many plant species such as Calophyllum inophyllum,prunus avium and Aloe polyphylla other than Arabidopsis thaliana (Turner & Singha 1990; Franck et al. 2004; Ivanova & Van Staden 2011; Van Den Dries et al. 2013). On the other hand, the less severe of HH on medium with agar compared with Gelrite may be due to the sulphated galactan in agar that has the ability to control HH, according to Nairn et al. (1995). 9

14 It was reported that cytokinins could induce HH in vitro propagation (Kadota & Niimi 2003; Bairu et al. 2007; Debnath 2009). However, cytokinins-induced HH may be a difficult problem to overcome because cytokinins as a growth regular are vital hormone for multiplication rates in tissue culture that we cannot do without (Huang et al. 1998). Therefore, we need to establish how exactly cytokinins induce HH and to maintain the multiplication factor. In the present study, four types of cytokinins were used to induce HH, which are zeatine, meta-topolin as well as BAP from adenine derivatives, and TDZ from phenylurea derivatives. Three level of concentration were applied, which were 0µM, 0.1µM and 0.5µM. It is found that TDZ generated the severest HH among others, whereas zeatine has the least effect (Fig. 1 and 3). Generally, phenylurea derivative has higher activities in terms of growth promotion, shoot proliferation and regeneration than adenine derivative (Kadota & Niimi 2003). Although TDZ has powerful cytokinins activities regarding yield and multiplication (Ružić & Vujović 2008; Pawlicki & Welander 1994; Briggs et al. 1988), its shoots quality is usually lesser because of compact hyperhydric growth (Briggs et al. 1988). It is reported that TDZ has a stronger effect on inducing HH compared with zeatine, meta-topolin and BAP. This is exemplified in the work undertaken by Kadota & Niimi (2003) in which HH in explants of Pyrus pyrifolia was found to be more affected by TDZ than by BAP. Another example of this is the study carried out by Debnath (2009) in which Rhodiola rosea L showed rapid proliferation with HH symptoms at more than 0.5µM TDZ but produced normal shoots within four weeks of culture after transfer to 1-2µM zeatine. Furthermore, it is observed in figure 1 and figure 3 that zeatine generate less HH than meta-toploin and BAP. The study of Bairu et al. (2007) found that when culturing Aloe polyphylla in vitro, there were more hyperhydric shoots generated on medium with BAP than with meta-topolin or zeatine. Besides, Ivanova & Van Staden (2011) also discovered in their studies on Aloe polyphylla that zeatine induced less hyperhydric shoots than BAP. These findings are in consistent with our results. It is shown in our results that overall increasing the concentration of cytokinins aggravated the severity of HH. This is supported by the study of Bairu et al. (2007), which found that the occurrence of HH increased with an increasing concentration (0.5µM - 15µM) of zeatine, meta-topolin and BAP in Aloe polyphylla s tissue culture. Debnath (2009) found that in roseroot (Rhodiola rosea L) tissue culture HH was induced in high concentration (4µM) of TDZ, but the hyperhydric symptom was 10

15 almost absent in lower concentration (1µM) of TDZ. In addition, it was also reported that the higher concentration of cytokinins, the easier to induce HH in many plant species such as Olearia microdisca (Williams & Taji 1991), Lavandura vera (Andrade et al. 1999), Malus sylvestris (Phan 1991) etc. All these findings support our results. It was suggested that due to its physical structure, Gelrite enhance the absorption of cytokinins (Williams & Taji 1991; Franck et al. 2004; Ivanova & Van Staden 2011), which is expected to generate sever HH than Gelrite only. According to our result (figure 1 and 3), it shows that Arabidopsis thaliana seedlings grown on Gelrite develop severer HH and accumulated significantly more apoplastic water than seedlings grown on Gelrite plus cytokinins, which seems to be contradictive with what we expect. However, it is shown in figure 4A that the apoplast water (%) of all the seedlings grown on either Gelrite and Gelrite plus cytokinins account for more than 90% out of total apoplast. This implies that the fact seedlings on Gelrite appeared to develop severer HH than those on Gelrite plus cytokinins does not indicate Gelrite was a stronger competitor. As a matter of fact, the apoplast of the seedlings grown on Gelrite plus cytokinins were also saturated by water. Less lignin and reduced lignification were constantly considered to be one of the causes of HH (Piqueras et al. 2002; Shady Saher et al. 2005). Our results show that both lignin mutants (ref1-4 and ref3-3) developed severer HH than wild type (fig. 8 and 9). Lignin quality and quantity is influenced by certain ref Arabidopsis mutants (Ruegger & Chapple 2001). It was reported that ref1-4 and ref3-3 Arabidopsis mutants accumulated less sinapate esters than wild type Arabidopsis (Ruegger & Chapple 2001; Nair et al. 2004). Mutation that affected sinapate ester biosynthesis may negatively impact lignin biosynthesis and thereby reduce lignification of plants (Ruegger & Chapple 2001). This was exemplified in the work undertaken by Schimiller et al. (2009) and Ruegger & Chapple (2001) who found that there was less lignin content in ref1 and ref3 Arabidopsis mutants compared to wild type. Kevers & Gaspar (1986) and Kevers et al. (1987) suggested that due to the deficiency in lignin, hyperhydric plants may have reduced cell wall pressure, which consequently make the symplast uptake more water and aggravate HH. Furthermore, Kevers et al. (1987) confirmed that lignin accumulated over time in normal tissues in carnation, but there was a deficiency in lignin content in hyperhydric tissues. Therefore in our study, the development of HH in ref1-4 and ref3-3 Arabidopsis mutant may be resulted from 11

16 deficiency in lignin synthesis. Figure 8 and 9 showed that the stomata mutant developed HH in both agar and gelrite. This result complies with Yang & Sackr (1995) which found that flp Arabidopsis mutant displays abnormal stomata including paired stomata and a small amount of unpaired of guard cells in cotyledons and the stomata were partially close compared to wild type. The closure of the stomata evidently reduces transpiration and thereby contributes to the flooding of the apoplast which hyperhydricity (Yang & Sackr 1995). Consequence of HH in Arabidopsis thaliana seedlings HH of Arabidopsis thaliana seedlings mainly results from the flooding of the apoplast within plant tissues, and the consequence is that it impairs gas exchange and leads to physiological disorders in plants (Van Den Dries et al. 2013). Visually, hyperhydric plants exhibited morphological abnormalities such as thick, curled and translucent (watery and glassy appearance) shoots as well as anthocyanin production (fig. 1 and 2) (Gaspar et al. 1995; Kadota & Niimi 2003; Sadhy Saher et al. 2005; Van Den Dries et al. 2013). The reduced gas exchange in hyperhydric plant tissues may issue in hypoxic stress that may consequently lead to oxidative damage (Olmos et al. 1997; Saher et al. 2004; Chakrabarty et al. 2005; Vergara et al. 2012; Van Den Dries et al. 2013), or issue in accumulation of gaseous compounds like ethylene in plant cells (Voesenek et al. 1993; Van Den Dries et al. 2013). It is shown in figure 1 and figure 2 that hyperhydic Arabidopsis seedlings also exhibited elongated petioles, which was likely caused by accumulated ethylene, according to Millenaar et al. (2005). It was reported that hyperhydric plants also exhibited anatomical defects such as epidermal discontinuity (Olmos & Hellin 1998; Apóstolo & Llorente 2000), chlorophyll deficiency (Franck et al. 1998), low lignification (Kevers & Gaspar 1986; Kevers et al. 1987), malformed non-functional stomata (Werker & Leshem 1987; Apóstolo & Llorente 2000; Picoli et al. 2001; Olmos & Hellin 1998) etc. In the present study, it is observed that there was a reduction of stomatal aperture in hyperhydric Arabidopsis thaliana shoots (fig. 6 and 7A), which is consistent with the study of Van Den Dries et al. (2013). Besides, Ziv & Ariel (1994) also observed stomatal closure in his research on HH in carnation. The reasons for stomatal closure may be due to the increasing stress triggered by, for example flooding of (shoots) apoplast (Van Den Dries et al. 2013) or flooding of plants roots (Atkinson et al. 2008; 12

17 Else et al. 2009), in hyperhydric plants. Furthermore, it is shown that the stomatal densities were not significantly different for agar, agar plus cytokinins, Gelrite and Gelrite plus cytokinins (fig. 7B). This result is contradictive with the study of Apóstolo & Llorente (2000) that found a decreasing stomatal density in hyperhydric Simmondsia chinensis, and the stomatal density is expected to be lower since the size of epidermal cells is larger in hyperhydric leaves (Olmos & Hellin 1998; Mossad et al. 2013). However, the study of Van Den Dries et al. (2013) also did not find significant change in stomatal density between hyperhydic and non-hyperhydric Arabidopsis thaliana seedlings, which is in lines with our results. 13

18 Conclusion In the present study, the effect of cytokinins types and concentration on HH in wild type Arabidopsis thaliana (col-0) in vitro was investigated. Applying cytokinins induced HH. Hyperhydric plants displayed malformation such as thick, brittle, curled, glassy leaves as well as petioles elongation. In hyperhydric seedlings, apopalstic water volume was significantly large and accumulated over time, which was at the expense of apoplastic air volume. Beside, stomatal closure was also observed. In our research, cytokinins concentration of 0.1µM resulted less HH symptoms than 0.5 µm in all treatments. TDZ generate significantly severer HH than BAP, meta-topolin and zeatine, which makes it unsuitable for multiplication of Arabidopsis thaliana. Although zeatine induces the least sever HH among others, it was rather expensive. Furthermore, it shows that meta-topolin has a similar effect with BAP on inducing HH. Therefore giving the fact that BAP is more common in tissue culture, it is likely that BAP would be the most suitable cytokinins for Arabidopsis thaliana, which guarantees adequate proliferation of seedlings and keeps the damage of HH under critical level. In addition, the effect of lignin and stomata on development of HH in Arabidopsis thaliana mutants was also studied. It was found that both lignin mutants (ref1-4 and ref3-3) and stomata mutant (flp) developed HH that maybe due to lignin deficiency and reduced transpiration, and their apoplastic water volume was significantly large than wild type (col-0). Future research can be carried out to further investigate the effect of HH on lignification. Also, molecular study on HH can provide more details into the disorder and help to create new treatment to prevent it. 14

19 Reference Andrade, L. et al., The effect of growth regulators on shoot propagation and rooting of common lavendar (Lavandura vera DC). Plant Cell Tiss, 56, pp Apóstolo, N.M. & Llorente, B.E., Anatomy of normal and hyperhydric leaves and shoots of in vitro grown Simmondsia chinesis (link) schn. In Vitro Cellular & Developmental Biology - Plant, 36(4), pp Atkinson, C.J., Harrison-Murray, R.S. & Taylor, J.M., Rapid flood-induced stomatal closure accompanies xylem sap transportation of root-derived acetaldehyde and ethanol in Forsythia. Environmental and Experimental Botany, 64(2), pp Bairu, M.W. et al., Optimizing the micropropagation protocol for the endangered Aloe polyphylla: Can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell, Tissue and Organ Culture, 90(1), pp Briggs, B., McCulloch, S. & Edick, L., Micropropagation of azaleas using thidazuron. Acta Hort, 226, pp Chakrabarty, D. et al., Hyperhydricity in apple: ultrastructural and physiological aspects. Tree physiology, 26(3), pp Debergh, P. et al., Reconsideration of the term vitrification as used in micropropagation. Plant Cell, Tissue and Organ Culture, 30(2), pp Debergh, P., Harbaoui, Y. & Lemeur, R., Mass propagation of globe artichoke (Cynara scolymus): Evaluation of different hypotheses to overcome vitrification with special reference to water potential. Physiologia Plantarum, 53, pp Debnath, S.C., Zeatin and TDZ-induced Shoot Proliferation and Use of Bioreactor in Clonal Propagation of Medicinal Herb, Roseroot ( Rhodiola rosea L ). Journal of Plant Biochemistry & Biotechnology, 18(2), pp Delarue, M. et al., Cristal mutations in Arabidopsis confer a genetically heritable, recessive, hyperhydric phenotype. Planta, 202(1), pp

20 Van Den Dries, N. et al., Flooding of the apoplast is a key factor in the development of hyperhydricity. Journal of Experimental Botany, 64(16), pp Else, M. a. et al., Root signals and stomatal closure in relation to photosynthesis, chlorophyll a fluorescence and adventitious rooting of flooded tomato plants. Annals of Botany, 103(2), pp Franck, T. et al., Cytological comparison of leaves and stems of Prunus avium L. shoots cultured on a solid medium with agar or gelrite. Biotechnic & histochemistry : official publication of the Biological Stain Commission, 73(August), pp Franck, T. et al., Hyperhydricity of Prunus avium shoots cultured on gelrite: A controlled stress response. Plant Physiology and Biochemistry, 42(6), pp Gaspar, T. et al., Paradoxical results in the analysis of hyperhydric tissues considered as being under stress: questions for a debate. Bulg. J. Plant Physiol, 21, pp Gribble, K. et al., Environmental scanning electron microscopy of the surface of normal and vitrified leaves of Gypsophila paniculata (Babies Breath) cultured in vitro. Plant Cell Reports, 15(10), pp Gribble, K. et al., Position of water in vitrified plants visualised by NMR imaging. Protoplasma, 201(1-2), pp Gribble, K. et al., Vitrified plants: towards an understanding of their nature. Phytomorphology, 53, pp Huang, L.-C., Huang, B.-L. & Murashige, T., A micropropagation protocol for Cinnamomum camphora. In Vitro Cell Dev Biol Plant, 34, pp Ivanova, M. & Van Staden, J., Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. Plant Cell, Tissue and Organ Culture, 104(1), pp Ivanova, M. & van Staden, J., Natural ventilation effectively reduces hyperhydricity in shoot cultures of Aloe polyphylla Sch??nland ex Pillans. Plant Growth Regulation, 60(2), pp Jackson, M.B., Waterlogging and Submergence. 16

21 Kadota, M. & Niimi, Y., Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots. Plant Cell, Tissue and Organ Culture, 72(3), pp Kevers, C. & Gaspar, T., Vitrification of carnation in vitro: changes in water cintent extracellular space, air volume, and ion levels. Physiologie Vegetale, 24, pp Kevers, C., Prat, R. & Gaspar, T., Vitrification of carnation in vitro: Changes in cell wall mechanical properties, cellulose and lignin content. Plant Growth Regulation, 5(1), pp Millenaar, F.F. et al., Ethylene-Induced Differential Growth of Petioles in Arabidopsis. Analyzing Natural Variation, Response Kinetics, and Regulation. Plant Physiology, 137(3), pp Mossad, A. et al., Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content., 25(4), pp Murashige, T. & Skoog, F., A revised medium for rapid growth and bioassays with tabacco cultres. Physiology Plantarum, 15, pp Nair, R.B. et al., The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis. The Plant cell, 16(2), pp Available at: Nairn, B., Furneaux, R. & Stevenson, T., Identification of an agar constituent responsible for hydric control in micropropagation of radiata pine. Plant Cell Tissue Organ Cult, 43, pp Olmos, E. et al., The subcellular localization of peroxidase and the implication of oxidative stress in hyperhydrated leaves of regenerated carnation plants. Plant Science, 130(1), pp Olmos, E. & Hellin, E., Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants. Scientia Horticulturae, 75(1-2), pp Pawlicki, N. & Welander, M., Adventitious shoot regeneration from leaf segments of in vitro cultured shoots of the apple rootstock. Hortscience, 9, pp

22 Phan, C., Vitreous state in vitro culture: ethylene versus cytokinins. Plant Cell Rep9, ( ). Picoli, E. a T. et al., Hyperhydricity in in vitro eggplant regenerated plants: Structural characteristics and involvement of BiP (Binding Protein). Plant Science, 160(5), pp Piqueras, a. et al., Polyamines and hyperhydricity in micropropagated carnation plants. Plant Science, 162(5), pp Raskin, I., A method for measuring leaf volume, density, thickness, and internal gas volume. Hortscience, 18, pp Rojas-Martinez, L., G.F.Visser, R. & De Klerk, G.-J., The hyperhydricity syndrome: waterlogging of plant tissues as a major cause. Propagation of Ornamental Plants, 10, pp Ruegger, M. & Chapple, C., Mutations that reduce sinapoylmalate accumulation in Arabidopsis thaliana define loci with diverse roles in phenylpropanoid metabolism. Genetics, 159(4), pp Ružić, D. V. & Vujović, T.I., The effects of cytokinin types and their concentration on in vitro multiplication of sweet cherry cv. Lapins (Prunus avium L.). Horticultural Science, 35(1), pp Saher, S. et al., Hyperhydricity in micropropagated carnation shoots: The role of oxidative stress. Physiologia Plantarum, 120(1), pp Saher, S. et al., Pectin methyl esterases and pectins in normal and hyperhydric shoots of carnation cultured in vitro. Plant Physiology and Biochemistry, 43(2), pp Saher, S. et al., Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling: Implications of oxidative stress. Plant Cell, Tissue and Organ Culture, 81(2), pp Schilmiller, A.L. et al., Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis. Plant Journal, 60(5), pp Terry, M. & Bonner, B., An examination of centrifugation as a method of extracting an extracellualr solution from peas, and its use for the study of indoleacetic acid-induced growth. Plant Physiology, 66, pp

23 Turner, S. & Singha, S., Vitrification of crabapple, pear and geum on gellan gum-solidified culture-medium. Hortscience, 25, pp Vergara, R. et al., Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene. Journal of Experimental Botany, 63, pp Voesenek, L.A.C.J. et al., Submergence-induced ethylene synthesis, entrapment, and growth in two plant species with contrasting flooding resistances., 103(3), pp Werker, E. & Leshem, B., Structural changes during vitrification of carnation plantlets. Annals of Botany, 59, pp Williams, R.R. & Taji, A.M., Effect of temperature, gel concentration and cytokinins on vitrification of Olearia microdisca (J.M. Black) in vitro shoot cultures. Plant Cell, Tissue and Organ Culture, 26(1), pp.1 6. Wu, Z., Chen, L. & Long, Y., Analysis of ultrastructure and reactive oxygen species of hyperhydici garlic (Allium sativum L.) shoots. In Vitro Cellular and Development Biology, 45, pp Yang, M. & Sackr, F.D., The Too Many Mouths and Four Lips Mutations Affect Stomatal Production in Arabidopsis., 7(12), pp Ziv, M., Vitrification: morphological and physiological disorders of in vitro plants. In: Debergh PC, Zimmerman RH, eds. Micropropagation: technology and application. Dordrecht: Kluwer Academic Publishers, pp Ziv, M. & Ariel, T., Vitrification in relation to stomatal deformation and malfunction in carnation leaves in vitro. In:Lumsden PJ, Nicholas JR, Davies WJ, eds. Physiology, growth, and development of plants in culture, Dordrecht:, pp