Exploring the Potential of PEG and Sucrose as Growth Retardant of Geranium wallichianum

doi:10.21203/rs.3.rs-3762498/v1

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Exploring the Potential of PEG and Sucrose as Growth Retardant of Geranium wallichianum

https://doi.org/10.21203/rs.3.rs-3762498/v1

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Geranium wallichianum is a medicinal plant belonging to the Geraniaceae family. It is mainly found in the East Asian Himalayas. Because of its numerous ethnomedicinal uses in the community, this plant is threatened by over-exploitation and over-harvesting. Pakistan has already lost its population by more than 75%. Considering its medicinal importance, this plant must be conserved before it becomes extinct. This study aimed to provide the protocol for in vitro conservation of G. wallichianum. Owing to the plant conservation applications of osmotic stress inducers such as polyethylene glycol (PEG) and sucrose, their ability to regulate the in vitro growth of this medicinally significant plant was evaluated. Using nodal segments as explants, the effect of Murashige and Skoog (MS) medium supplemented with different concentrations of PEG (0, 0.5, and 1.5%) and sucrose (0, 4, and 8%) on various growth parameters was investigated. At 45-day intervals, PEG (0.5 and 1.5%) treatment was found to be more effective than sucrose treatment in reducing in vitro growth (0.92 and 0.57 cm), with minimum fresh (0.06 and 0.04 g) and dry weight (0.04 and 0.02 g), respectively. Total soluble sugars (4.07 and 4.12 mg/g) and total phenolic content (0.96 and 0.19 mg/g) were significantly increased by PEG treatment (0.5 and 1.5%, respectively), whereas total chlorophyll content (0.17 and 0.14 mg/g) and total protein content (2.38 and 2.32 mg/g) were decreased. In addition, Pearson correlation analysis confirmed that total phenolic content was negatively correlated with chlorophyll a, chlorophyll b, and total chlorophyll content, whereas shoot length was negatively correlated with total phenolic content but positively correlated with fresh weight and dry weight. According to the principal component analysis, total phenolics and total sugars, which clustered distinctly from the other parameters, indicated relatively higher osmotic stress responses to PEG and sucrose. Based on these findings, it appears that PEG was more effective than sucrose at slowing in vitro growth during short-term conservation.

G. wallichianum

In vitro conservation

Indigenously threatened plant

Osmotic stress inducer

This study provides an effective in vitro conservation protocol for threatened G. wallichianum. Osmotic stress inducers, such as polyethylene glycol (PEG) and sucrose, were used at different concentrations to establish a short-term in vitro conservation protocol in nodal explant cultures, and PEG was found to be more effective than sucrose in regulating G. wallichianum growth in vitro. This protocol will help establish the future medium- and long-term in vitro conservation of G. wallichianum.

In developing countries such as Pakistan, more than 80% of people rely on herbal medicines for essential healthcare, and up to 25% of the suggested drugs are derived from wild plants (Hamilton et al. 2009). Globally, the use of medicinal plants is increasing with the demand for natural health products and secondary metabolites derived from these medicinal plants (Islam et al. 2001; Jamshidi et al. 2018). Pakistan is diverse in climate, geography, altitude, and phytogeography and hosts approximately 1572 genera and 6000 species of wild plants, most commonly in the Indus, Himalayas, and Karakoram regions, with 428 endemic species (Bano et al. 2014). North Pakistan is rich in medicinal plants and traditionally consumed by local communities. Plants are the only primary source of revenue for local people. Significant risks to Pakistan's biodiversity include habitat damage, desertification, grazing, illegitimate trade, industrial pollution, high demand for natural resources, and deficiency of required knowledge (Asad and Abdur 2014).

Geranium wallichianum D. Don ex Sweet belongs to the Geraniaceae family and is locally called 'Ratanjot' in Pakistan and generally 'Shepherd's needles. It is also called the Wallach cranesbill, known as Sra zeal (Razzaq et al. 2015), ratinloog, and rattenjot (Ishtiaq et al. 2021). G. wallichianum is tall and erect, with several branches found mainly in the Himalayas, at 7000–10000 feet from Nepal to Kashmir. It is distributed across Hazara, Gilgit, Dir, Chitral, Lowari Top, Murree Hills, Swat, Shogran Valley, and Baluchistan in Pakistan. Worldwide, it is distributed in the East Asia-Himalayas, including Afghanistan, India, Nepal, Bangladesh, and Bhutan (Malik et al. 2015). G. wallichianum possesses great ethnobotanical value. Its floral extract is used for vision problems and blood purification, whereas its leaves are effective against liver problems. Plant rhizomes are used for various health issues such as constringent, toothache, ear infections, and eye infections (Rahul Vikram 2014). G. wallichianum is endemic to Pakistan (Asad and Abdur 2014). The major threats to this plant are over-exploitation and over-harvesting because of its high ethnomedicinal importance (Shaheen et al. 2017).

Several conservation efforts have been undertaken to preserve important medicinal plants; however, many long-term and multiple conservation approaches are required to protect such threatened and endangered species. Conventional procedures (in situ and ex situ) and modern biotechnological methods, particularly micropropagation, cryopreservation, and in vitro conservation, are available to safeguard plant biodiversity (Agrawal et al. 2019). Ex situ plant conservation depends on the species, methods used, and desired storage time, which have costs, difficulties, and scientific obstacles. In contrast, in vitro conservation allows for the simultaneous preservation of plant genetic resources, availability of disease-free planting material to farmers all year, and control of gene integrity, as determined by molecular analysis. Slow growth methods, particularly with fewer subcultures, reduce the danger of losing germplasm owing to handling errors, such as contamination issues, and reduce the hazards of genetic instability, human labor, and space required for germplasm conservation.

This approach is sometimes called "slow growth storage" or "minimal growth storage" because of the application of various physical, chemical, or nutritional conditions that limit plantlet growth. In commercial micropropagation laboratories, the slow growth approach is used as an appropriate strategy for the short- to medium-term storage of plant materials in a limited space, providing a market with high-quality commercial cultivar output at a lower cost (Gupta et al. 2020). Microplants are grown on Murashige and Skoog (1962) (MS) media supplemented with growth retardants or osmotic stress inducers and incubated under low temperature, low light intensity, and altered photoperiod to reduce the frequency of sub-culturing (Gopal and Sukh 2010). Slow growth techniques have been used to protect various plant species such as Glycyrrhiza glabra (Uzundzhalieva et al. 2014), Coleus forskohlli Briq (Dube et al. 2011), Ullucus tuberosus (Hammond et al. 2019), Rhazya stricta and Lavandula dentata (Attia et al. 2017).

The present study used osmotic regulators to slow growth and extend the subculturing time. The primary functions of osmotic stress inducers in culture media are to decrease water potential, reduce photosynthesis through stomatal closure, and decrease turgor pressure when used at appropriate concentrations. Several osmotic inducers, such as sucrose, sorbitol, mannitol, ribose, polyethene glycol (PEG), and 2.2–dimethyl hydrazide, have been reported to be effective (Dubois and Inze 2020). Sucrose is the most appropriate energy source for in vitro culturing (Gago et al. 2014). It has also been used as an osmotic agent to reduce the growth rate of mature explants in vitro. High sucrose levels have been reported to reduce the growth rate of several in vitro-cultured plant species (Sanghamitra et al. 2019). Moreover, most in vitro and in vivo studies on osmotic stress have used PEG as an ideal, inert and nonionic material that can exert a more negative water potential in tissues by trapping water molecules and cannot be transmitted through cellular walls (Akte et al. 2016).

Thus, the purpose of this study was to evaluate the potential of using PEG and sucrose to slow the growth of G. wallichianum with the goal of establishing an in vitro conservation system that promotes a slower growth rate for this species. This is necessary because the increasing demand for G. wallichianum due to its high medicinal value has led to a significant decline in its population, posing a serious threat to its survival.

2.1. Plant material

The nodal segments of the G. wallichianum plants (Fig. S1) were used as explants, and the research was carried out in the Department of Biotechnology at the CUI's Abbottabad Campus in Pakistan. In July 2017, a medicinal plant (G. wallichianum) was collected during the flowering stage in Galyat, Pakistan. After verification by a plant taxonomist, it was acclimatized to the nursery area of the Department of Biotechnology, CUI, Pakistan.

2.2. Surface sterilization of explants

The nodal explants were surface sterilized following the procedure of (Munir et al. 2015) with some modifications, as mentioned in Supplementary Table S1. The nodal explants were cut into small segments, treated with 70% ethanol (Sigma-Aldrich) for 1 min and 2% sodium hypochlorite (Sigma-Aldrich) for 5 min, and washed three times with sterilized distilled water. The explants were then placed on a Petri plate containing autoclaved filter paper for 1 min to air dry before complete inoculation in the culture medium. After surface sterilization, the explants were transferred to a simple MS medium (Murashige and Skoog 1962) under aseptic conditions. The culture medium was kept in an air-conditioned culture chamber at 25 ± 2°C with a 16 h photoperiod to establish the culture. The light intensity was 3000 lx. Different parameters, such as contamination, mortality (browning or blackening of explants), and survival percentage, were recorded after one week of culture (Table S1).

2.3. Application of PEG and sucrose treatments

After two weeks, a well-established culture was sub-cultured on Murashige and Skoog (1962) (MS) media supplemented with different concentrations of PEG (CAS 25322-68-3) and sucrose. Sucrose was the only source of carbon added to the medium. MS media were prepared with varying concentrations of PEG (0, 0.5, and 1.5%) and sucrose (0, 4, and 8%), and the pH was adjusted to 5.6. Agar (10 mg/ml) (Sigma-Aldrich, Bacteriological grade) was used as a solidifying agent, and the solution was autoclaved for 20 min at 121°C and 15 psi pressure. The explants were inoculated on a sterile MS medium before being transferred to a controlled growth chamber at 23 ± 2°C under a light/dark cycle of 16/8 h. The culture of PEG and sucrose treatments was observed for morphological and biochemical parameters after 15 days for 45 days.

2.4. Morphological analysis

At regular intervals of 15, 30, and 45 days of culture, the length of the shoot (cm) per nodal explants, fresh weight (g), dry weight (g), and survival percentage (%) of nodal explants were measured. The heights of the nodal explants were evaluated using simple visual measurements with a ruler. To ascertain the fresh and dry weights of the nodal explants, they were carefully extracted from the culture medium and wiped with tissue paper. The fresh weight of each explant was recorded using an electronic balance. After determining the fresh weight, the samples were placed on clean aluminum foil and desiccated at 100°C in an oven. The desiccated explants were weighed for dry weights and recorded using the same electronic scale. The survival of the explant cultures was evaluated as dead, or brown nodal explants were considered non-surviving, whereas those with growth and good health were assessed as surviving. The number of surviving explants was divided by the total number of initially cultured explants and multiplied by 100 to calculate the explant survival rate.

2.5. Biochemical analysis

Using the following experimental procedures, various factors, including total phenolics, soluble sugars, chlorophyll, and protein content, were analyzed after 15, 30, and 45 days of cultivation.

2.5.1. Determination of total soluble sugars

Ethanol (80%) was used to prepare the nodal explant extracts, which were added to 25 ml anthrone reagent (0.15 g anthrone (Merck) in 100 ml of 72% H₂SO₄ (Sigma-Aldrich). The mixture was heated at 97^°C for 15 min, and the absorbance was measured at 625 nm using a spectrophotometer (IRMECO Model U2020). The total soluble sugar content was determined using a glucose standard (Riazi et al. 1985).

2.5.2. Determination of total phenolics

Total phenolic content was determined using the method described by Ainsworth and Gillespie (2007). Explant extracts were prepared in 80% methanol (Sigma-Aldrich) and were centrifuged at 10,000 rpm for 3 min. Then, 700 µL of Folin and Ciocalteu reagent (Merck) (10%) was added to 300 µL of extract, and the final mixture was prepared by adding 700 mM sodium carbonate (Na₂CO₃) (BioWorld), followed by incubation at room temperature (25^°C) for 60 min. The absorbance of the sample was measured at 765 nm using a spectrophotometer (IRMECO Model U2020) and the phenolic content was determined using gallic acid (Sigma-Aldrich) as a standard (Gill and Tuteja 2010; Vicas et al. 2019).

2.5.3. Quantification of chlorophyll contents

The extract was prepared by grinding fresh explants in 10 ml of acetone (Sigma-Aldrich) (80%, Sigma-Aldrich) for 12 h. The supernatant was collected by centrifuging the extract at 10,000 rpm for 3–5 mins and chlorophyll a and chlorophyll b contents were calculated spectrophotometrically (IRMECO Model U2020) at absorbances of 645 and 663 nm, respectively (Lichtenthaler 1987). The average chlorophyll and chlorophyll a and b contents were calculated from the readings of three replicates using the following formula (Arnon, 1949):

Chlorophyll a content (mg/g) = 12.7 (A663) – 2.69 (A645) ˟ W/1000 ˟ V

Chlorophyll b content (mg/g) = 22.9 (A645) – 4.68 (A663) ˟ W/1000 ˟ V

Total chlorophyll content (mg/g) = Chlorophyll (a + b)

Where V is the total volume of the extract (mL), W is the fresh sample weight (g), and A is the absorbance of wavelength.

2.5.4. Quantification of total proteins

The total protein content was estimated using the Bradford method (1976). Extracts (0.5 g) were ground in phosphate buffer (pH 7.4) and centrifuged at 8000 rpm for 20 min. Then, 2.9 ml of Coomassie blue dye (Sigma-Aldrich) was mixed with the collected supernatant and incubated for one hour at room temperature. Absorbance was measured at 600 nm using a spectrophotometer (IRMECO Model U2020), and the standard curve was calculated using BSA (bovine serum albumin) (BSA; Sigma-Aldrich) as a standard.

2.6. Experimental design and statistical analysis

The experiment was carried out in a completely randomized design (CRD) with three biological replicates, each with eight explants (n = 8). The collected data were subjected to two-way analysis of variance (ANOVA), with treatments and days as the main factors. The treatment means were compared using the least significant difference (LSD) test at p < 0.05, and the linear relationship between parameters was investigated using Pearson correlation analysis. Principal component analysis (PCA) was performed using the IBM-SPSS software (version 26).

3.1. Morphological analysis

Morphological parameters, including shoot length, survival percentage, fresh weight, and dry weight of shoots, in response to osmotic stress inducers are shown in Fig. 1.

3.1.1. Effects of PEG and sucrose concentrations on shoot length of nodal explant

The results in Fig. 1a show the effects of different concentrations of PEG and sucrose on the explant shoot length. Shoot length data were subjected to analysis of variance, and the results revealed that both treatments had a significant effect over time, but their interaction was found to be non-significant. In terms of sucrose (8%), the maximum reduction in the shoot length was (0.93, 1.03 and 1.12 cm) at the interval of 15, 30 and 45 days, respectively (Fig. 2). With an interval of 15, 30 and 45 days, at PEG (0.5%) the shoot length of explants usually decreased (0.84, 0.88 and 0.92 cm) respectively compared with control (1.52, 1.55 and 1.57 cm) but at higher concentration of PEG (1.5%), the shoot length reduced significantly (0.61, 0.62 and 0.57 cm, respectively) (Fig. 2). The medium with sucrose (4%) provided the highest shoot length (1.45 cm) at the 45 days interval (1.14 cm), which was significantly different from the other comparison treatments and day intervals, whereas PEG (1.5%) produced the lowest shoot length (0.60 cm).

3.1.2. Survival percentage of nodal explant under PEG and sucrose treatments

The survival percentage data were subjected to analysis of variance, and the results showed that only the treatments had a significant effect. However, the interaction between treatments over time was not significant. The survival rate decreased with increasing concentrations of PEG and sucrose. The survival rate of nodal explants under PEG treatment was greater than that under sucrose treatment. The media containing PEG (0.5%) and sucrose (4%) resulted in the maximum survival rates (87.5 and 83.33%, respectively) after 15 days, (83.33 and 79.16%, respectively) after 30 days, and (79.16%) after 45 days. However, the medium with the highest concentration of PEG (1.5%) and sucrose (8%) showed the lowest survival rates (66.66 and 62.5%, respectively) after 15 days, (66.66 and 54.16%, respectively) after 30 days, and (62.5 and 54.16%, respectively) after 45 days compared with the control (Fig. 1b). As the data from Fig. 1b illustrate, the medium containing PEG (0.5%) and 15 days interval gave the highest survival percentage (84.72 and 79.16%, respectively), compared to other treatments and day intervals; however, the medium with sucrose (8%) and 45 days interval provided the lowest survival percentage (56.94 and 71.66%, respectively).

3.1.3. Effect of PEG and sucrose concentrations on the fresh and dry weight of nodal explant

The fresh weight and dry weight data were analyzed by analysis of variance and the results indicated that the treatments over a time interval and their interaction had a significant effect (Table S2).

As shown in Fig. 1c and 1d, there was often a reduction in the fresh and dry weight of the explant with increasing concentrations of PEG and sucrose. For 15 days interval, the maximum fresh and dry weight (0.03 and 0.01 g) of the explant, respectively, was observed with PEG (0.5%) in the medium, whereas the lowest fresh and dry weight of the explant was 0.02 and 0.01 g, respectively, observed at PEG (1.5%). A similar trend for the results from the fresh and dry weight of the explant (0.03 and 0.02 g) was recorded after 15 days on the medium containing sucrose (4%), while the lowest value of fresh and dry weights (0.02 and 0.01 g, respectively) was observed with sucrose (8%) in the medium.

At 45 days, the results obtained with the control and sucrose (4%) media were acceptable. The highest fresh and dry weights of explants were 0.08 and 0.02 g, grown in the control medium, respectively, while the fresh and dry weights of explants were (0.09 and 0.04 g, respectively) grown in the sucrose (4%) medium. However, the lowest fresh and dry weight (0.04 and 0.02 g, respectively) was obtained from the medium supplemented with PEG (1.5%) while the fresh and dry weight of explants were (0.05 and 0.03 g, respectively) in the medium mended with sucrose (8%).

Figure 1c and 1d indicate that the medium supplemented with sucrose (4%) produced the highest fresh and dry weights of the nodal cultures (0.06 and 0.03 g), respectively, at 45 days interval (0.06 and 0.03 g), while the medium with PEG (1.5%) and a 15-day interval gave the lowest values of fresh and dry weights (0.03 and 0.02 g) and (0.02 and 0.01 g), respectively.

3.2. Biochemical analysis

3.2.1. Total soluble sugars in nodal explant under PEG and sucrose concentrations

The results of total soluble sugar analysis are shown in Fig. 3a. The data for total soluble sugars were subjected to analysis of variance, and the results revealed that the treatments, days, and their interaction all had a significant effect on total soluble sugars. The explants cultured in the medium containing (1.5%) PEG showed an increase (3.96, 4.06, and 4.12 mg/g) in total soluble sugars at 15-, 30- and 45-days interval. Compared with the control, explants were grown on a medium with sucrose (4%) and (8%) also showed increases (2.61, 2.77 and 2.95 mg/g) and (2.64, 2.95, and 3.20 mg/g), respectively in total soluble sugars with gradually increased concentrations and days interval (15, 30 and 45). At the same time, total soluble sugars were greatly increased (3.63, 3.93 and 4.07 mg/g) in a medium containing 0.5% PEG at 15, 30 and 45-days interval.

3.2.2. Effects of PEG and sucrose concentrations on total phenolics contents in nodal explant

The results of the total phenolic content assay are revealed in Fig. 3b. The results showed that treatment, days, and their interaction had a significant effect. Compared to the control, medium supplemented with 4% and 8% sucrose resulted in a slight increase (0.15, 0.21 and 0.40 mg/g) and (0.18, 0.33 and 0.65 mg/g) respectively, of the total phenolics content at 15-, 30- and 45-days interval, while in the medium containing (0.5%) PEG, total phenolics content was highly increased (0.33, 0.78 and 0.96 mg/g). However, the medium with a PEG concentration (1.5%) led to a two-fold increase (0.47, 0.95, and 1.19 mg/g) in the total phenolic content compared to the control.

3.2.3. Effects of Different PEG and sucrose concentrations on the total protein contents in nodal explant

The treatments, days, and their interaction had a significant effect on the total protein content of the nodal explants (Fig. 3c). Compared to the control, the medium supplemented with sucrose (4%) and (8%) showed a slight decrease (3.12, 3.39 and 3.60 mg/g) and (2.47, 2.69 and 2.89 mg/g) respectively, of the total phenolics content at 15-, 30- and 45-days interval, while in medium containing (0.5%) PEG, the total phenolics content were highly reduced (2.08, 2.28 and 2.38 mg/g). However, the medium with PEG concentration (1.5%) resulted in a twofold decrease (1.96, 2.17 and 2.32 mg/g) in the total phenolics content compared to the control.

3.2.4. Effect of PEG and sucrose concentrations on chlorophyll contents in nodal explant

The results revealed that treatments, days, and their interaction all had a significant effect on chlorophyll a content, whereas in the case of chlorophyll b, the number of days was not significant, but the treatments and their interaction with days had a significant impact (Fig. 3e and 3f). Compared to the control, at 15-, 30- and 45-day intervals, chlorophyll b was found to be in the highest quantity in medium supplemented with (4%) sucrose (0.19, 0.23 and 0.27 mg/g), followed by (8%) sucrose (0.12, 0.12 and 0.11 mg/g), (0.5%) PEG (0.14, 0.12 and 0.07 mg/g) and (1.5%) PEG (0.12, 0.11 and 0.06 mg/g) respectively. Similarly, chlorophyll a was found to be the second highest amount in medium supplemented with (4%) sucrose (0.16, 0.18 and 0.21 mg/g), followed by (8%) sucrose (0.17, 0.15 and 0.15 mg/g), (0.5%) PEG (0.16, 0.13 and 0.10 mg/g) and (1.5%) PEG (0.14, 0.12 and 0.08 mg/g) respectively.

When the total chlorophyll content data were subjected to analysis of variance, the results showed that the treatments, days, and their interaction also had a significant effect. The medium with (4%) and (8%) sucrose resulted in the lowest decrease (0.34, 0.40 and 0.47 mg/g) and (0.29, 0.28 and 0.26 mg/g) respectively, of total chlorophyll content compared to the control, while the medium with (0.5%) and (1.5%) PEG led to the highest decrease (0.29, 0.25 and 0.17 mg/g) and (0.27, 0.23 and 0.14 mg/g) of total chlorophyll content at 15-, 30- and 45-days intervals (Fig. 3d).

3.3. Pearson correlation analysis

The results in Table 1 show that shoot length was significantly negatively correlated (p < 0.01) with total phenolic content. Total phenolic content was significantly negatively correlated (p < 0.01) with chlorophyll a, b, and total chlorophyll contents. Shoot length was positively and significantly correlated with shoot fresh and dry weights (P < 0.01).

Table 1

Pearson correlation analysis of physiological parameters (shoot length, survival rate, fresh weight and dry weight) and biochemical parameters (soluble sugars, phenolic content, protein content and chlorophyll content) of *G. wallichianum*
	Shoot length (cm)	Fresh weight (g)	Dry weight (g)	Survival rate (%)	Soluble sugar (mg/g)	Phenolic content (mg/g)	Protein content (mg/g)	Total chlorophyll (mg/g)	Chlorophyll a (mg/g)	Chlorophyll b (mg/g)
Shoot length (cm)	1
Fresh weight (g)	0.640^**	1
Dry weight (g)	0.572^**	0.950^**	1
Survival rate (%)	0.419^**	0.190	0.210	1
Soluble sugar (mg/g)	-0.858^**	-0.317^*	-0.243	-0.227	1
Phenolic content (mg/g)	-0.626^**	0.025	0.126	-0.245	0.812^**	1
Protein content (mg/g)	0.934^**	0.769^**	0.689^**	0.288	-0.798^**	-0.469^**	1
Total chlorophyll (mg/g)	0.871^**	0.556^**	0.446^**	0.428^**	-0.793^**	-0.676^**	0.859^**	1
Chlorophyll a (mg/g)	0.584^**	0.288	0.147	0.153	-0.607^**	-0.701^**	0.532^**	0.770^**	1
Chlorophyll b (mg/g)	0.877^**	0.593^**	0.508^**	0.483^**	-0.769^**	-0.585^**	0.882^**	0.969^**	0.587^**	1
*. Correlation is significant at the 0.01 level (2-tailed); . Correlation is significant at the 0.05 level (2-tailed)

3.4. Principal component analysis

As indicated in Fig. 4, the first principal component (PC1) accounted for 47.97% of the variance, while the second component (PC2) accounted for 35.08%. Thus, the first two components account for 83.77% of the variance.

G. wallichianum faces a threat to extinction due to overexploitation and harvesting because of its medicinal value, particularly in northern Pakistan. Ex situ conservation has been used to study several endangered plant species (Rodrigues et al. 2020). However, little is known regarding the conservation and protection of this species. Based on the findings of this study, osmotic stress inducers were used to manage nodal explant growth for short-term in vitro conservation, which could be further extended for long-term in vitro conservation.

PEG and sucrose were used as osmotic stress inducers in this study, and both reduced the growth rate and fresh and dry weights of the nodal explants. Importantly, the reductions in growth rate, fresh weight, and dry weight under specific concentrations of PEG and sucrose were independent of day intervals (15, 30, and 45 days, respectively), similar to the results reported by Sakthivelu et al. (2008). At both concentrations of PEG (0.5 and 1.5%), the growth rate, fresh weight, and dry weight were significantly reduced compared with sucrose (4 and 8%) concentrations. This is because of a high increase in osmotic pressure in the media, which lowers water and nutrient uptake owing to low turgor pressure and strongly suppresses cell elongation. By comparing both PEG and sucrose, it could be suggested that PEG has a greater influence on the growth of G. wallichianum than does sucrose. Similar results have been observed for Lathyrus sativus (Piwowarczyk et al. 2014), Milk thistle (Bekheet et al. 2016), and four varieties of durum wheat (Bouiamrine and Diouri 2012). According to Razavizadeh et al. (2019), adding PEG to the medium induced osmotic stress in cultured Thymus vulgaris explants, and similar conditions were found in all plants subjected to drought stress via a reduction in the fresh weight and length of thyme explants, which was consistent with other studies on several aromatic plants (Bettaieb et al. 2009; Lutts et al. 2004). Reduced growth parameters, such as weight and height, are typical of stressed plants and may be caused by alterations in plant metabolism (Hund et al. 2009). Studies have shown that plants modify their morphological, physiological, and anatomical features to enhance tolerance to PEG-induced stress. Similar to other plants under these conditions, G. wallichianum reduced its total biomass to reduce consumption and increase water uptake, as found in this study. The decrease in fresh weight of plants under drought stress could be attributed to the significantly reduced canopy structure, photosynthetic capacity, and plant growth under water deprivation conditions (Shao et al. 2008).

According to Hessini et al. (2009), increased total soluble sugar content in plants can aid stress tolerance. The ability of plants to regulate osmotic conditions and maintain water balance under stressful conditions results in the accumulation of nutrients, such as soluble sugars, proline alcohols, organic acids, and glycine betaine, among others. Water pressure can increase the total amount of soluble sugars in several plant species (Hernandez et al. 2021). The primary function of soluble sugars is to maintain turgor pressure and increase the soluble sugar concentration for increased starch hydrolysis via the action of hydrolytic enzymes under stress (Bartels and Sunkar 2005). Other stressors include non-photosynthetic sugar synthesis, reduced leaf transport, and increased leaf sugar levels under water stress (Hu et al. 2009). Sugar concentrations in the shoots of stressed plants increased drought severity by preventing dissociation and regulating osmotic balance, according to Ramak et al. (2004) when studying Onobrychis radiate and Onobrychis goneifoli. Similarly, Kumari et al. (2022) obtained similar results for medicinal herbs such as Sinopodophyllum hexandrum and beans by Subbarao et al. (2000), soybeans by Niakan and Ghorbanli (2007), and Heidary et al. (2007). The current study supports these findings, with a significant increase in soluble sugars in G. wallichianum treated with PEG compared to the controls. Under osmotic stress, the high concentration of total soluble sugars might be due to starch breakdown and amylase activity (Villadsen et al. 2005). Increased sugar and other osmolyte accumulation contribute to growth regulation under stress by expelling reactive oxygen species (ROS) via an increased antioxidant system (Ahanger et al. 2017; Keunen et al. 2013). In this study, soluble sugars increased under osmotic stress, possibly due to increased glucose and fructose from the increased hydrolysis of sucrose and starch. Other cellular and nutritional functions of soluble sugars include as an energy source and osmotic protector, which protect plants under stressful conditions.

Phenolic compounds are important for osmotic homeostasis and accumulate significantly under osmotic stress conditions (Minocha et al. 2014), as observed in the current study. Plant phenolic content plays a role in stress tolerance. Citrus sinensis showed a significant increase in phenolic content and antioxidant activity (Huang et al. 2008). According to Razavizadeh et al. (2019), T. vulgaris culture with PEG treatment had a significant effect on phenolic compound induction because phenolic compounds play an important ecological role in plant defense and protection, and it has been proposed that applied stress may increase the biosynthesis of phenolics in response to oxidative stress (Karuppusamy 2009). In addition, Zhuang et al. (2011) reported a significant correlation between phenolics and 1,1-diphenyl-2-picrylhydrazine, as well as total phenolic content and reducing iron or antioxidants. In the present study, explants accumulated the maximum concentration of phenolic content in their tissues when treated with both PEG concentrations (0.5 and 1.5%) compared to sucrose concentrations. The significant accumulation of phenolic compounds could be a protective mechanism that maintains stable steady-state protein and reactive oxygen species (ROS) in levels.

The accumulation of chlorophyll in plant tissues is an excellent indicator of the negative effects of various stressors. High stress causes hydrogen peroxide accumulation, which can lead to protein and chlorophyll deficiencies. Stress factors can also induce chloroplast structural degradation by activating chlorophyllase (Noreen et al. 2009). The reduction in chlorophyll concentration may be related to increased activity of this enzyme or degradation of the plastid envelope and thylakoids by the formation of extreme reactive oxygen species (ROS). Plants under stress are less likely to synthesize chlorophyll, an indicator of oxidative stress. Consequently, the instability of ion homeostasis reduces chlorophyll synthesis, and a decrease in chlorophyll concentration may correspond to a decrease in chlorophyll metabolism (Siddiqi and Husen 2016). In our study, PEG (0.5 and 1.5%) treatments resulted in a significant decrease in chlorophyll content in explant cultures when compared to sucrose (4 and 8%) treatments. In addition, Tátrai et al. (2016), PEG enrichment in the culture medium resulted in the production of reactive oxygen species (ROS) involved in the degradation of chlorophyll through lipid peroxidation. PEG enhancement has also been shown to have a negative effect on the phytochemical, physiological, and morphological characteristics of in vitro cultures of Tagetes erecta seedlings (Liao et al. 2012), Hordeum vulgare (Hellal et al. 2018) and Allium hirtifolium (Ghassemi et al. 2018). According to Sobhkhizi et al. (2014), the effect of stress on chlorophyll a and b in node cultures induces chloroplast proteolysis, which reduces and destroys chlorophyll as a fundamental factor. Water stress has been linked to decreased chlorophyll levels in plants such as Carthamus tinctorius (Siddiqi et al. 2019), Paulownia imperialis (Ayala and Alcaraz 2010) and beans (Beinsan et al. 2009). Martínez-Santos et al. (2021) found that increased PEG concentrations decreased the total chlorophyll content of V. planifolia shoots. Gao et al. (2020) observed a reduction in the chlorophyll levels of D. officinale shoots subjected to 0, 1, 3, and 5% PEG in an in vitro Orchidaceae culture. Suis et al. (2015) reported a decrease in the chlorophyll content in Aranda Broga Blue Bell protocorm-like bodies treated with 15% PEG. Ex vivo studies have also revealed a decrease in the chlorophyll content of V. planifolia under osmotic stress (Chandran and Puthur 2009).

Compared with sucrose, cultures exposed to 0.5 and 1.5% PEG had lower total protein contents in our study. PEG treatment has been reported to reduce the total soluble protein content in Vigna unguiculata and a few species of Nicotiana tabacum (Priyanka et al. 2011). PEG treatment induces oxidative stress in plants, leading to degradation of active proteins and membranes. Therefore, protein modification reduced the total protein content of the PEG-treated G. wallichianum. According to Martínez-Santos et al. (2021), soluble protein content in V. planifolia shoots decreased as PEG concentration increased, but the protein content was significantly higher in all PEG treatments than in the controls. The increase in soluble proteins at 1% PEG compared to the control (no PEG stress) is most likely due to the synthesis of hydrophilins (Battaglia et al. 2008), aquaporins (Hussain et al. 2011), dehydrins (Chiappetta et al. 2015), antioxidant enzymes (Sewelam et al. 2016), and chaperonin (Zia et al. 2021). Plants produce this protein to combat oxidative and osmotic stress (Li et al. 2020). In several crops, including rice (Sikuku et al. 2010), legumes (Ashraf and Foolad 2005), soybean leaves (Niakan and Ghorbanli 2007), and onions (Arvin and kazemipour 2001), a decrease in protein content in the shoot organs has a synergistic effect with an increase in stress levels. Razavizadeh et al. (2019) observed a reduction in the protein content of T. vulgaris seedlings grown with 2, 4, 6, and 8% PEG. Similarly, Gao et al. (2020) observed a decrease in the protein content of D. officinale shoots cultured under 1, 3%, and 5% PEG-induced osmotic pressure.

The present study investigated the morphological and biochemical responses of an established nodal culture of G. wallichianum to different concentrations of PEG and sucrose over a short period ( 45 days). Explant growth factors revealed that low concentrations of PEG (0.5%) and sucrose (4%) had no negative effect, whereas higher concentrations, specifically PEG, significantly reduced growth compared to the control. As a result, this study can be extended to examine the long-term effects of osmotic inducers on medicinal plant conservation in vitro.

Considering the threat of extinction from over-harvesting and consumption of the medicinally important plant G. wallichianum, the present study was conducted to establish a protocol for in vitro conservation using osmotic stress inducers such as PEG and sucrose to regulate the growth of G. wallichianum in a controlled environment. Growth attributes were significantly reduced at both concentrations of PEG (0.1 and 0.5%) compared to sucrose concentrations (4 and 8%), but a high survival rate was observed at low concentrations. Additionally, PEG treatment resulted in higher concentrations of total phenolics and total soluble sugars than sucrose treatment, followed by a decrease in the total chlorophyll and total protein content, suggesting that the in vitro cultured nodal segments were under osmotic stress. The results showed that PEG was more effective than sucrose at regulating the in vitro growth of G. wallichianum. For short- to medium-term in vitro conservation of this plant, we recommend using an in vitro conservation method based on slow growth, with a limited number of in vitro subcultures.

Author Contributions: ZA performed experiment and involved in the writeup of manuscript, YS and SA planned the study and did the analysis, while AB, GA and IH edited and revised the original draft.

Funding: No external fundings

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Available data are presented in the manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

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Exploring the Potential of PEG and Sucrose as Growth Retardant of Geranium wallichianum

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Abstract

Figures

Key Message

1. Introduction

2. Materials and methods

2.1. Plant material

2.2. Surface sterilization of explants

2.3. Application of PEG and sucrose treatments

2.4. Morphological analysis

2.5. Biochemical analysis

2.5.1. Determination of total soluble sugars

2.5.2. Determination of total phenolics

2.5.3. Quantification of chlorophyll contents

2.5.4. Quantification of total proteins

2.6. Experimental design and statistical analysis

3. Results

3.1. Morphological analysis

3.1.1. Effects of PEG and sucrose concentrations on shoot length of nodal explant

3.1.2. Survival percentage of nodal explant under PEG and sucrose treatments

3.1.3. Effect of PEG and sucrose concentrations on the fresh and dry weight of nodal explant

3.2. Biochemical analysis

3.2.1. Total soluble sugars in nodal explant under PEG and sucrose concentrations

3.2.2. Effects of PEG and sucrose concentrations on total phenolics contents in nodal explant

3.2.3. Effects of Different PEG and sucrose concentrations on the total protein contents in nodal explant

3.2.4. Effect of PEG and sucrose concentrations on chlorophyll contents in nodal explant

3.3. Pearson correlation analysis

3.4. Principal component analysis

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

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