Salinity tolerance improvement of in vitro propagated Paulownia tomentosa using proline

Paulownia tomentosa has been used as an ornamental plant and is still vastly used for this objective and can be utilized for the production of energy, wooden building materials, and paper pulp. The aim was to improve the in vitro propagation ability under salinity stress. This experiment was conducted on Paulownia tomentosa at Tissue Culture Technique Lab., Central Laboratories, Department of Ornamental Plants and Woody Trees, National Research Centre (NRC), Egypt, to examine the micropropagation behavior using different plant growth regulators (BA, Kin, and IBA) and improve the in vitro propagation ability of plant under the effect of salinity levels (500, 1000, 2000, and 4000 ppm) using two concentrations of proline (0.2 and 0.4 g/l). For in vitro propagation behavior, the best results for both shooting and rooting behaviors were obtained when MS culture medium was supplemented with BA at 0.2 mg/l, and Kin and IBA at 0.1 mg/l. For the improvement of the in vitro propagation ability under salinity stress, the survival was 100% when the shootlets of Paulownia grown on MS culture medium supplemented with 0.5 g NaCl plus 0.2 or 0.4 g/l proline and 1 g NaCl plus 0.2 g/l proline which were similar to control treatment. Micropropagation ability of Paulownia tomentosa under salinity stress was optimized when the explants were cultured on MS medium supplemented with BA at 0.2 mg/l, and Kin and IBA at 0.1 mg/l using proline at 0.2 g/l. This study will help the producers to cultivate Paulownia trees in saline soils up to 1000 ppm such as some new lands which are not suitable for growing other crops.


Background
Paulownia trees belong to Scrophulariaceae family involve nine species of fast-growing trees that are native to China and East Asia (Ayan et al. 2006). Paulownia tomentosa has been entered into the USA and Europe as an ornamental plant and is still vastly used for this objective. The average height of trees reaches 12 m and 13.4 cm of average diameter through 7 years in Bulgaria (Kalmukov 1995). The trees can reach up to 30 m at maturity (Bonner 1995). This high yielding tree can be utilized for the production of energy, wooden building materials, and paper pulp (Bergmann and Moon 1997).
The environmental deterioration causes biotic and abiotic stresses in plants and restricted their growth and development (Shao and Chu 2005). Among the abiotic stresses, salinity and drought are the main stresses that damage crop worldwide (Vinocur and Altman 2005). Rising salinity causes severe harm to plants, including growth inhibition, necrosis, impaired metabolism, and reduction of production and quality (Sivritepe and Eris 1999). Salinity stress cause accumulation of reactive oxygen species (ROS) (O 2 , superoxide radicals; OH, hydroxyl radical; H 2 O 2 , hydrogen peroxide; and 1O 2 , singlet oxygen), producing an oxidative stress. Oxidative stress can damage DNA, lipid peroxidation, and inactivate enzymes (Gill and Tuteja 2010).
However, sugars, sugar alcohols, and several other molecules as amino acids, organic acids, or inorganic ions have the ability to improve salt tolerance mechanisms (Munns 2005). Proline acid is a compatible osmolyte that accumulates in many crops under salt stress (Munns and Tester 2008). Proline helps to protect cells of the plant from the negative effects of salt by preserving the osmotic balance, scavenging reactive oxygen species (ROS), and stabilizing subcellular structures, such as proteins and membranes (Ashraf and Foolad 2007). Many reports show the effective role of exogenous proline in osmoregulation of several plant species under salt stress (Lone et al. 1987;Tal and Katz 1980;Wyn et al. 1977).
Plant tissue culture provides beneficial information to illustrate plant response to salt stress. Micropropagation provides greater control than in vivo growth conditions and has the advantage of small scale with monitoring responses of shoot and root in the presence of imposed stress (Shibli et al. 1992). The micropropagation technique of Paulownia species offers a rapid means of producing woody biomass and planting stock for afforestation, and it is an effective method to maintain the genetic gain (Park and Bonga 1992). The success of consecutive micropropagation for many woody plants could be affected by different factors such as plant growth regulators which are the most important ones (Taha et al. 2008).
Paulownia tree is considered as very sensitive to salinity. The aim of the research was to optimize a micropropagation protocol using different plant growth regulators (BA, Kin, and IBA) and improving plant ability to salinity tolerance so that it can be cultivated in the new lands which are not suitable for growing other crops.

Materials and methods
The experimental work was conducted on Paulownia tomentosa during the years 2018 and 2019 at the Tissue Culture Technique Lab., Central Laboratories, Department of Ornamental Plants and Woody Tress, National Research Centre (NRC), Egypt, to evaluate and improve the in vitro propagation ability of plant under the effect of salinity stress.

Explant source and disinfection
Nodal explants of Paulownia tomentosa at the Faculty of Agriculture, Ain Shams University, Shobra El-Khyema, Egypt, were taken and washed in septal soap, then rinsed with running tap water for 1 h. The surface disinfection of explant was done under aseptic condition using 70% ethanol for 1 min, followed by 15% (v/v) of Clorox (NaOCl 5.25%) for 10 min, followed by mercuric chloride at 0.2% (w/v). The explants were rinsed three times with sterile distilled water after each disinfection treatment.

Culture medium
Explants were cultured on MS medium (Murashing and Skoog, 1962) supplemented with 2.5% sucrose and 0.7% agar (w/v) prior to autoclaving at 1.2 kg/cm 2 for 15 min. The pH of the culture medium was adjusted to 5.8. Culturing was done in 200 ml glass jars containing 25 ml of the medium.
For evaluation of in vitro propagation behavior, plant growth regulators were added to MS culture medium at different concentrations: Benzlaminopurine (BAP) at 0.2 and 0.4 mg/l with or without Kin at 0.1 mg/l and indole-3-butyric (IBA) at 0.1 or 0.2 mg/l.
For evaluation of in vitro propagation ability under salinity stress, the in vitro growth (shooting and rooting) behavior was examined through culturing the nodal explants which were obtained from the most suitable multiplication medium (MS + 0.2 mg/l BA + 0.1 mg/l Kin + 0.1 mg/l IBA) under different concentrations of NaCl (0.0, 500, 1000, 2000, 4000 ppm). Two concentrations of proline (0.2 and 0.4 g/l) were examined to improve the in vitro propagation ability under salinity stress.

Incubation conditions
Cultures were incubated under controlled conditions in the growth chamber. The incubation temperature was 24 ± 2°C controlled by a "power" air conditioner. The photoperiod was 16-h light/8-h darkness, controlled automatically. Illumination intensity was 3000 lux from cool fluorescent lamps.

Photosynthetic pigments
Plant material after multiplication stage was collected, and photosynthetic pigments (chlorophyll a and b) as well as carotenoids were determined in shootlets tissues as mg/ 100 g fresh weight by using spectrophotometer, according to the procedure achieved by Saric et al. (1967).

Experimental design and data analysis
The data were analyzed through analysis of variance ANOVA, and the treatments' means were compared for significance by Duncan's new multiple range test at 0.05% level of probability (Duncan 1955) using COSTATV-63.

In vitro propagation behavior
The results presented in Table 1 and Fig. 1 show the in vitro shooting and rooting ability of Paulownia tomentosa under the effect of different plant growth regulators (BA, Kin, and IBA) concentrations. The best results for both shooting and rooting behaviors were obtained when MS culture medium was supplemented with BA at 0.2 mg/l, at Kin and IBA at 0.1 mg/l which lead to the highest survived explants (100%), number of shootlets (5.67), longest shootlets (6.96 cm), and highest rooting percent (98.33%) as compared to control which was MS medium without growth regulators and caused lowest values. The data also revealed that using of low concentration of BA (0.2 mg/l) combined with Kin (0.1 mg/l) and IBA at 0.1 or 0.2 mg/l were favored for improving the in vitro propagation ability of Paulownia comparing with other treatments. This indicates the role of all used plant growth regulators for attaining the internal hormone balance that led to the best results.
Means within a column having the same letters are not significantly different according to Duncan's multiple range test (DMRT) at 5% level

Effect of salinity stress on micropropagated plants
The growth characters as survival, shoot number, shoot length, and leaves' number, also the rooting parameters as rooting percentage, roots number, and root length of Paulownia tomentosa were significantly affected by the NaCl treatments. As shown in Table 2, all in vitro growth characters were decreased with increasing NaCl concentrations from 500 to 4000 ppm. The 500 ppm NaCl did not reduce the survival (100%) and produced an increase of the shoot number (5.67) as compared to control. At 4000 ppm NaCl, the number of shootlets, shoot length, and leaves' number was adversely affected and gave only half values approximately of that of control, whereas the survival percentage was decreased to 61%. The rooting parameters were decreased significantly from 500 and 1000 ppm NaCl and were absent at 2000 and 4000 ppm NaCl. Concentrations of NaCl at 500 and 1000 ppm resulted in a significant reduction in rooting percentage (22.22 and 11.11 %), roots number (0.67 and 0.33), and roots' length (11.78 and 5 mm), respectively, compared with control treatment; however, no rooting occurred when explants were grown on 2000 and 4000 ppm of NaCl.
Means within a column having the same letters are not significantly different according to Duncan's multiple range test (DMRT) at 5% level Table 3 demonstrates the effect of proline on alleviation of salinity stress on the micropropagated plants, with increasing salinity concentrations using different proline level, and all parameters were reduced. The survival was 100 % when the shootlets of Paulownia grown on MS culture medium supplemented with 0.5 g NaCl plus 0.2 or 0.4 g proline and 1 g NaCl plus 0.2 g proline which were similar to control treatment. Applying 0.5 g of NaCl plus 0.2 proline gave the best values after the control for each of the number of shootlets/explant, length of shootlets, and number of nodes/shootlet (1.33, 57.92, and 41.33 mm, respectively). All previous parameters were declined sharply with increasing salinity levels.  The shootlets were produced roots even applying 1 g of sodium chloride and using the higher concentration of it prevents root formation. These results indicate that Paulownia plants can grow at 1000 ppm of NaCl, but the higher concentration of NaCl causes poor growth or death. The use of proline with salt stress improved in vitro growth slightly. Means within a column having the same letters are not significantly different according to Duncan's multiple range test (DMRT) at 5% level Photosynthetic pigments under salinity stress Figure 2 indicates the effect of salt stress by applying different levels of sodium chloride on the chlorophyll content of the Paulownia leaves under study, including chlorophyll a and b. The results show a reverse relationship between salt level and chlorophyll a and b contents. Whenever the level of NaCl was increased, chlorophyll content was decreased. Chlorophyll a and b reach to the lowest contents (71.42 and 25.51 mg/100 g F.W. at 4000 ppm of NaCl) compared to control (271.90 and 175.90 mg/100 g F.W., respectively). A statistical analysis demonstrates that the observed differences were significant. By following carotenoids content through the exposure of the Paulownia explants to salt stress, it appears from Fig. 2 that salt stress (NaCl) was an inhibiting agent for the formation of carotenoids inside the stressed leaves, where the carotenoids' content was decreased. The least carotenoids content was appeared with the level 4000 ppm of NaCl (54.80 mg/100 g F.W.), whereas the highest content (606.7 mg/100 g F.W.) appeared in control leaves. There were gradual decreases with increasing salinity levels.

Photosynthetic pigments under different salinity levels and proline concentrations
Data illustrated in Fig. 3 mention the role of proline concentration in accumulated pigments content in shootlets of Paulownia that grown under salinity stress. The highest chlorophyll a and b contents (528.4 and 273.1 mg/100 g F.W.) were obtained in shootlets grown on MS culture medium supplemented with 0.5 g NaCl plus 0.2 g proline followed by the treatment of NaCl 1.0 g plus 0.2 proline which caused also the highest carotenoids content (397.1 mg/100 g F.W.) comparing with other treatments. This indicates the role of proline when it was used at low concentration in increasing chlorophyll content under salinity stress.

Proline content under salinity stress
The data presented in Fig. 4 shows the response of proline content in the shootlets of Paulownia to salt stress. The content of proline ranged between 0.277 and 0.443 nmol/ mg. The salinity treatments increased the content of proline as compared with the control. A slight increase was recorded with the low concentrations of NaCl (500 and 1000 ppm), then marked increases were recorded using NaCl at 2000 and 4000 ppm. Where the proline content reached its maximum value (0.443 nmol/mg) when the explants were grown on the highest concentration of NaCl, compared to that for the untreated explants.  Proline content under salinity stress and proline concentration Figure 5 shows that the highest proline content (0.377 nmol/mg) resulted in shootlets grown on MS culture medium supplemented with 4 g NaCl plus 0.2 g proline followed by those obtained from NaCl 2.0 g plus 0.2 proline which gave 0.335 nmol/mg as compared to control which gave the lowest value (0.121 nmol/mg).

Discussion
In this study, the response of in vitro propagation could be improved by adding plant growth regulators (PGR) to the culture medium (Table 1). Confirmed results pointed out that different concentrations of BAP, Kin, or IBA showed a significant effect on bud sprouting of Paulownia kawakemii during three repeated subcultures (Abd El-Kader and Abou El-Ghit 2015). They mentioned also that adding IBA to the culture medium had a promotion effect on bud break, especially with a low concentration of BAP. The inhibition of adventitious meristem elongation could reduce the number of shoots as a result of using a high concentration of BAP (Borchetia et al. 2009). Concerning the effect of salinity stress on in vitro propagation ability (Table 2), explants' growth of apple rootstock was severely affected by salinity treatments. High salinity gave the reduction in shoot number, shoot length, rooting percentage, root number, and root length (Bahmani et al. 2012). Shahid et al. (2011) noticed the reduction in the length of the shoot with an increasing level of NaCl. This might be explained as the inadequate photosynthesis caused by stomatal closure and the reduction of carbon assimilation rate under salt stress  Concerning the results in Table 3, using proline with salt stress improved in vitro growth slightly. Proline is a predominant organic molecule that acts as an intercessor of osmotic adjustment under salinity stress, a sink for energy, a stabilizer of sub-cellular structures, and even a stress linked signal. It is also contributed to cell osmoregulation and protection of proteins during dehydration, and under stress conditions, it can act as an enzymatic regulator (Rontain et al. 2002).
As results in Figs. 2 and 3, the pigments (chlorophyll a, b, and β-carotene) in Paulownia imperialis leaves were significantly reduced when exposed to a high level of NaCl (Astorga and Meléndez 2010).
The response of proline to salt stress (Figs. 4 and 5) may due to these osmoprotectants that acts an important role in decrease stress-induced cellular acidification and osmotic adjustments and stabilizes sub-cellular structures for recovery (Tan et al. 2008). Recent studies also suggested that the protective role of proline depends on protecting the protein turnover machinery against stress damage and upregulating stress-protective proteins (Khedr et al. 2003).

Conclusion
MS culture medium that was supplemented with BA at 0.2 mg/l, and Kin and IBA at 0.1 mg/l gave the best results for both shooting and rooting behaviors of Paulownia tomentosa. The plantlets were able to grow in a saline condition (1000 ppm), and the growth could be improved slightly using proline.