Scanning electron microscopy
Significant differences in stomata morphology were observed between hyperhydric and normal shoots. SEM showed low stomata density and closed stomata of larger size, with larger stomata pore on the leaf surface of HS compared to normal shoots (Fig. 1). This could explain that the presence of stomata with larger area and size on HS of B. vulgaris could indicate low development of the stomatal complex compared to normal shoots.
Dutta and Prasad (2010) demonstrated a positive correlation between normal leaf development and a higher rate of stomata occurrence. Picoli et al. (2001) also reported, based on studies (SEM), that stomatal differentiation is impaired in hyperhydric plants. These changes are consistent with the characteristics exhibited by hyperhydric plantlets of other species, such as Tectona grandis L. (Quiala et al.2012) and Vaccinium spp. (Gao et al. 2018).
In general, normal shoots had elliptically shaped and closed stomata (Fig. 1). Therefore, we conclude that normal shoots had normal development of the stomata complex compared to HS. According to Tian et al. (2015) in hyperhidric plants leaves are one of the most affected and sensitive organ during the ex vitro survival in the greenhouse. Thus, stomata morphology is a valuable tool for predicting ex vitro survival in plants. Deformation stomatal complex development in hyperhydric plants negatively affects physiological processes such as photosynthesis, tissue drying, and plant death during the acclimatization phase (Palma et al. 2011).
Therefore, stomata structure and density, as well as stomata movement, play a very important role in regulating the water content of a plant (Apóstolo and Llorente 2000; Picoli et al. 2001). These changes can cause more stomata to malfunction and stomata to movement abnormally. Abnormal stomata can affect the overall physiology of the plant, especially the relationship between cell and water. Therefore, changes in stomata structure and stomata movement may be critical for the induction, promotion and development of HS in B. vulgaris.
Changes in chlorophyll content
The highest values for total chlorophyll content were obtained in shoots of B. vulgaris cultured in TIS (Fig. 2). HS, however, those cultured in static liquid medium showed a significant decrease in the content of this photosynthetic pigment. Saher et al. (2004) suggested that hyperhydricity causes inhibition of electron transport, increasing the likelihood of generation ROS, which can damage the membrane components of PSII, leading to causing oxidative damage in the photosynthetic apparatus. These results may suggest that the HS may have damage to photosynthetic pigments due to hyperhydricity. According to Tian et al. (2015), HS showed lower total chlorophyll content compared to shoots without hyperhydricity, which might be due to the harmful effects of oxygen species (ROS). In addition, they suggested that these conditions could cause an alteration in cellular redox homeostasis and damage to chloroplasts.
Similarly, Chakrabarty et al. (2006) reported that hyperhydric leaves had significantly lower chloroplast number per cell and chloroplasts showed reduced thylakoid stacking compared to healthy leaves. Also, the chlorophyll content in Allium sativum L., decreased which affected the physiological response of the plants (Wu et al. 2009). Petruş‑Vancea et al. (2018) indicated that hyperhydricity leads to a decrease in chlorophyll content in Beta vulgaris var. Conditiva. These authors shared that low chlorophyll content in shoots is one of the most common physiological responses induced by hyperhydricity. In this study Ρathares et al. (2018) found similar results in hyperhydric shoots of Lippia grata Schauer, which reduced chlorophyll content.
Comparison of hyperhydric shoots with normal plants showed a significant increase in H2O2 and MDA content in HS (Fig. 3A and B). The lowest values were obtained in normal shoots with significant differences compared with HS. The highest values for MDA content were obtained in HS, suggesting that lipid peroxidation might be increased in these shoots, (ROS). Oxidative stress could be induced in these shoots and affect their morphology. According to Hamed et al. (2013), lipid peroxidation is a consequence of oxidative damage to cell membranes and leads to the formation of MDA. MDA is an indicator oxidative stress response in plants (Perveen et al. 2013; Sen and Alikamanoglu, 2013; Mittler, 2017).
The activity APX and CAT was significantly lower in normal shoots compared with hyperhydric tissue (Figs. 3C). According to Malik et al. (2014), oxidative stress is defined by an imbalance between oxidants and reductants in the cell. This imbalance is due to excessive production of ROS and the cell is unable to counteract the harmful effects of ROS. This means that the increase in H2O2 accumulation normally occurs at a low level, due to the presence in the plant of several antioxidant systems are present that allow eliminating the excessive production of H2O2 and maintaining the optimal levels of ROS to achieve a normal dynamic balance.
The morpho-physiological, anatomical, and biochemical changes differ among plants species and depend on the specific responses of each species and the culture conditions used. Shoots of B. vulgaris cultured in static liquid culture responded strongly to hyperhydricity. Knowledge of the changes associated with hyperhydricity could prevent this anomaly in bamboo shoots cultured in vitro.
Several studies have been conducted to investigate the relationship between hyperhydricity and oxidative stress (Gupta and Prasad 2010; Balen et al. 2011; Dewir et al. 2014; Tian et al. 2017). The time course of H2O2 generation in hyperhydric tissues of Dianthus caryophyllus confirmed the close relationship between hyperhydricity and oxidative stress in this species (Saher et al. 2004). In this context, these authors described that HS could cause greater oxidative stress, due to the accumulation of ROS in cells. They also reported that ROS is one of the first cellular responses that stimulate defense mechanisms against oxidative stress in several plant species. However, high concentrations of ROS can cause uncontrolled oxidation of various cellular components and degradation of chlorophyll proteins. To counteract these effects, the cell must activate various antioxidant defense mechanisms to eliminate the harmful effects of these ROS.
According to Dewir et al. (2006) HS, a typical stress-induced change in physiological state is evident. They note an oxidative stress characterized by markedly increased MDA content in HS of Euphorbia millii Des Moul.. At the same time, oxidative stress was show to be reduced in normal shoots by increased activities of SOD, POX and CAT and enzymes of the ascorbate-glutathione cycle (APX, GR, MDHAR and DHAR). These enzymes play a crucial role in the elimination of H2O2 from plant cells.
In this context, Balen et al. (2009) showed higher oxidative stress in Mammillaria gracilis Pfeiffdue to an increase in MDA and ROS content compared to shoots without hyperhydricity. They also suggested that the increased activities of the antioxidant enzymes SOD, APX, CAT in shoots indicate activation of a defense mechanism against the increased production of ROS in these tissues. Therefore, oxidative stress could be responsible for the deficient morpho-physiology and biochemistry of HS in this species.
Tian et al. (2015) indicate that under stressful conditions in an in vitro environment, the increased production and decreased degradation of ROS lead to oxidative damage. The antioxidant capacity may be the crucial factor in the production of excessive ROS. Therefore, the remarkable change in antioxidant enzyme activity and antioxidant molecule content at the subcellular level may be a hallmark for the induction of hyperhydricity. In this work, the results confirm the aspects previously reported.
Isah (2019) share that plants have developed an antioxidant defense machinery against ROS that could cause membrane damage to cellular structures; it consists of antioxidant compounds and enzymes that include CAT and APX. If the enzymes did not detoxify the accumulation of ROS and H2O2 in the cells or if they were absent this could lead to hyperhydricity in the in vitro cultures. They reported that the activity of CAT and APX regulates the accumulation of ROS that result from cellular oxidative stress.
Ex vitro acclimatization
After 7 days of acclimatization, a decreased ex vitro survival rate of B. vulgaris hiperhidric plantlets was observed. The highest survival rate 94.23% was observed in the normal plantlets after transfer to the greenhouse (Fig. 4).
The lowest percentage of survival was obtained in hyperhydric plants, which coincided with the highest number of HS and the highest water content in the shoots. In addition, these shoots had the highest number of open and probably non-functioning stomata and the highest content of antioxidant. The highest percentage of surviving plants from TIS without symptoms of hyperhydricity could be due to the fact that they showed a better morpho-physiological response in vitro. This response was related to the highest values of total chlorophyll content and better development of the stomatal complex. On the other hand, these plants showed less oxidative stress. All this indicates that they were better prepared to prevent water loss by transpiration and to adapt to environmental changes in vitro and ex vitro; this confirms the description of Zobayed et al. (2000).
This could lead to better morpho-physiological, anatomical and biochemical development of plants. These characteristics could facilitate the adaptation response during acclimatization of B. vulgaris plants in the culture house, as reported by Kozai (2010). In agreement with Hazarika (2006), the results obtained show that defining the morpho-anatomical and physiological characteristics of plants propagated in vitro allows early selection of those plants that can survive during acclimatization.
The results obtained in the present study show that the plants from TIS have a higher anatomical and biochemical response ex vitro compared to the SLM plants. In addition, Vidal and Sánchez (2019) confirmed the positive effect of TIS on morphological quality and ex vitro adaptation in different woody species. These authors indicated that these conditions reduced stress during ex vitro acclimatization and increased the growth of these plants. These results were consistent with those obtained in B. vulgaris plants in the present study. However, other authors described a different response to this variable for this species only when they used a different culture system. For example, Gajjar et al. (2017) achieved a lower survival percentage (80%) in B. vulgaris than in this study when they used a semisolid culture medium. Similarly, Desai et al. (2019) achieved a survival rate of 85%. dos Santos Ribeiro et al. (2020) determined an ex vitro survival rate of 88.88% in SLM in this species. However, in this work the percentage of survival (94.23%) increased by using TIS.