Results and Discussions

In vitro Regeneration of Stevia rebaudiana ‘Bertoni’

Initiation or establishment of culture In vitro propagation studiesshow that plant hormones play an important role in regenerationof culture. For establishment of culture, different cytokinin (BAPand Kn) was used. The axillary bud explants were cultured on MSmedia supplemented with different concentration (0.5, 1.0, 1.5 and2.0 mg/l) of BAP and Kn. BAP at 0.5 mg/l stimulated proliferationof the bud meristems to form bud clusters and maximum frequencyof bud break (FBB) reached 90% and bud elongation reached 95%as compared to higher concentrations of BAP where slow growthwas observed. Similar result was obtained by Abd-Alhady [13]. Thestimulating effect of BAP on bud break has been reported earlier by Debnath [14]. In the initiation stage, Kn also showed maximumfrequencies of bud break (85%) and bud elongation (92%) at 0.5 mg/lKn (Figure 1a; Figure 2a) but lower than BAP while at concentrationgreater than 0.5 mg/l treatment resulted decline in all parameters.

Figure 1: In vitro regeneration of Stevia rebaudiana Bertoni under aseptic medium a) Initiation or establishment of culture at different concentration (0.5, 1.0, 1.5 and 2.0mg/l) of BAP and Kn b) Multiplication of explants at different concentration and combination of BAP and Kn c) Rooting of plantlets at different concentrationof IBA (0.5, 1.0, 1.5 and 2.0mg/l).

Multiplication and elongation of Shoots The proliferation and multiplication of the shoots were also influenced by hormones whenadded singly or in combination. The mean number of shoot (MNS) per explant and mean shoot length (MSL) varied under various cytokinin concentrations and combinations (Figure 1b; Figure 2b, c). The effect of different concentration of BAP used was found to be significantfor increasing the number of shoots (mean number of shoot, MNS) after 3-4 weeks of inoculation. Maximum number of shoots (14±0.94) and shoot length (10.2±0.83) was observed at 0.5mg/l concentration while at concentration higher than 0.5 mg/l treatment, the shoot number declined. It was clear that among different concentration of Kn used, only at 0.5 mg/l Kn concentration, the highest numberof shoots (12±0.65) and shoot length (10.4±0.14) after 3-4 weeks was observed while at higher concentration than 0.5 mg/l Kn, the shoots number declined. Between the two cytokinins tested, BAP was more effective than Kn for shoot multiplication. When cultures were subjected to different combinations and concentrations of BAPand Kn, significant changes were observed. Our data revealed that Stevia rebaudiana, Bertoni cultures treated with 1.0 mg/l BAP in combination with 0.5 mg/l Kn found significant increase in shoot number (30±1.83) and shoot length (12.2±1.83) while concentrationlower or higher declined MNS and MSL (figure 1b). Abd-Alhady [13] reported highest shoot multiplication in medium supplementedwith 1.0 mg/l BAP + 0.5 mg/l Kn (36.9 shoots per explant) and lowest shoot multiplication at 0.5 mg/l + 0.5 mg/l Kn in Stevia rebaudiana, Bertoni. Supplemented Kn to the medium enhanced the elongation of shoots.

Figure 2: In vitro regeneration of S. rebaudiana, ‘Bertoni’ a) Initiation and/or establishment of culture in MS supplemented with 0.5 mg/ l BAP b, c) multiplication and elongation of culture in MS supplemented with 1 mg/l BAP + 0.5 mg/l Kn d) rooting of plantlet on ½ strength MS medium supplemented with 2.0 mg/l IBA and e) Hardening of plantlet in pot containing sand andpeat mixture (1: 1).

Root Induction and acclimatization of plant After two cycles of sub-culturing, elongated shoots of 2-3 cm in length were excised and cultured on MS medium having different concentration of IBA. The experiments were conducted twice, with 3 replications (with 3shoots per tubes). Initiation of rooting took place after 1-2 week of inoculation. Single and multiple roots were formed from the base and the nodal portions within 2-3 weeks. The highest number of root (MNR; 9.7±0.45) and root length (MRL; 9.2±0.17) was observed on ½ strength MS medium supplemented with 2.0 mg/l IBA whilelowest number of root (6.4±0.45) and root length (7.2±0.60) occurred in ½ strength MS medium supplemented with 0.5 mg/l IBA (Figure 1c; Figure 2d ). Similar results were observed by Debnath [14]. Abd-Alhady et al. [15] also stated that IBA gave better response for rootingof Periploca angustifolia than NAA.

Plantlets with well-developed root systems were transferred to pots containing sand and peat mixture (1:1) and covered with translucent plastic bags to ensure high humidity around the plants. The use ofthis procedure during the acclimatization phase ensured that most of the plantlets transplanted to ex vitro conditions continued to grow vigorously. After two months when the plastic bags were completelyremoved, 70% of the plantlets survived in the greenhouse and showed normal growth of the plant.

The result of the experiment and other earlier research reports clearly support the possibility of propagating S. rebaudiana, Bertoni, by adopting in vitro techniques. Thus, In vitro propagation can become an important alternative to conventional propagation andbreeding procedures for wide range of plant species.

In vitro salinity and drought stress studies

Fundamentally, plants require energy (light), water, carbon and mineral nutrients for growth. Abiotic stress is defined as environmental conditions that reduce growth and yield below optimum levels. Plant responses to abiotic stresses are dynamic and complex [16]; they are both elastic (reversible) and plastic (irreversible).

Effect of NaCl and mannitol on plant growth Plant growth inhibition is a phenomenon often occurring in glycophyte or saltsensitive plant species under salinity and drought conditions. In the present study, salt and drought stress induced significant differenceson plant growth during the experimental period. The significant decrease in the shoot number and shoot length was observed with the increasing concentration of NaCl (25, 50, 75 and 100mM) and significant increase in shoot number and shoot length with increasing number of days (10, 20 and 30) (Supplementary file 1A; Figure 3a,b;Figure 4). The highest number of shoot (~25) and shoot length (10.7cm) was observed at 25mM after 30 days and lowest number of shoot (~7) and shoot length (6.5cm) at 100mM after 10 days of treatment as compared with control. The plants grown under control(no salinity application) exhibited the maximum values for shootnumber (~28) and shoot length (12.6cm) as compared to remainingtreatments after 30 days of culture, which indicated that salinity isresponsible for reduction in shoot number and shoot length. Shahidet al. [17] observed the reduction in shoot length with increasingconcentration of NaCl. Ashraf et al. [18] also reported that salt stressaffects the plant growth and development by influencing fresh anddry weight of roots, shoots along with shoot length. This might beexplained as the inadequate photosynthesis caused by stomatalclosure and the reduction of carbon assimilation rate under salt stress[6].

Figure 3: In vitro effect of different concentrations (0, 25, 50, 75 and 100mM) of NaCl and mannitol stress on Stevia rebaudiana ‘Bertoni’ a) Mean number of shoot after 10, 20 and 30 days of culture b) Mean shoot length after 10, 20 and 30 days of culture c) Mean number of root after 10, 20 and 30 days of culture d) Mean root length after 10, 20 and 30 days of culture.

Figure 4: Effect of salinity stress on plant growth and development atdifferent concentrations 25 (a), 50 (b), 75 (c) and 100mM (d) of NaCl.

Drought is a multidimensional stress affecting plants at variouslevels of their organization [19]. The response to drought at the wholeplant and crop levels is complex because it reflects the integration of stress effects and responses at all underlying levels of organizationover time and space. In the present investigation, drought stress exhibited significant differences on shoot number and shoot length during the experimental conditions. After 20 stress days, decreases inshoot number and shoot length was observed in plants grown with 25, 50 75 and 100mM mannitol (Figure 3a, b; Figure 5). In case of shoot number, significant (p < 0.05) differences were not observed with the increasing number of days (Supplementary file 1A). Themeans values of shoot number and shoot length were 16, 14.3, 10.3,7.3, 3.7 and 10.5, 10.3, 9.2, 7.4, 7.1 cm for treatments of 0, 25, 50, 75, and 100mM mannitol, respectively.

Figure 5: Effect of drought stress on plant growth and development atdifferent concentrations 25 (a), 50 (b), 75 (c) and 100mM (d) of mannitol.

Salinity and drought stress also exhibited significant differenceson root number. With the increasing concentration of NaCl andmannitol significant decrease in root number were observed after 10,20 and 30 days. Interestingly, at 100mM Mannitol, decrease in rootnumber 4, 3 and 2 were observed with increasing number of days 10, 20and 30 days, respectively (Supplementary file 1A). Moreover, salinitystress significantly increases the root number after 10, 20 and 30 dayswhile drought stress significantly decreases the root number duringthe experimental condition. Similar result was observed by Abdel-Hussein [20] who evaluated MM106 and Omara apple rootstocks forsalt tolerance in vitro. The reduction in rooting percentage might bedue to the inhibitory effects of salt on the metabolic activities whichassociated with cell division, differentiation and elongation [21].

By following the root length of the plants under salinity anddrought stress during the treatment periods, Fig 3 it appears there isa general trend for the decrease in the root length of the plant usingthe concentration 25, 50, 75 and 100mM while significant increasein the root length after 10, 20 and 30 days. The means values of rootlength were 7.4, 5.9, 5.1, 4.7 and 4.4 cm and 5.5, 4.6, 4.1, 3.9 and 3.2cm for treatments of 0, 25, 50, 75, and 100mM NaCl and mannitol,respectively. Ashraf et al. [18] report that plants grown under control(no salinity application) exhibited the maximum values (10.11cm) for root length as compared to remaining treatments, whichindicated that salinity is responsible for reduction in root length.Thiam et al. [22] reported that root length was adversely affected witha significant reduction of NaCl concentration (50 and 100 mM) and reached 6.95cm and 6.33 cm, respectively. Their findings suggest thathigh NaCl concentration significantly affect the root growth.

Moreover, salinity and drought stress resulting significantdecrease in the leaf FW, leaf DW, stem DW, shoot DW and root DWafter 25, 50, 75 and 100mM treatment. The significant reduction ofleaf FW 36% and 54%, leaf DW 79% and 55%, stem DW 66% and60%, shoot DW 38% and 55%, root DW 72% and 51% were observedat 100mM NaCl and mannitol treatment, respectively. Furthermore,the significant percentage difference in between the salinity anddrought stress conditions were 25%, 21% and 6% of leaf DW, rootDW and stem DW whereas no significant difference were observed inshoot DW and leaf FW (Supplementary file 1B; Table 1, Table 2).

Table 1: Effect of different concentration of NaCl on growth parameters of S. rebaudiana ‘Bertoni’.

Table 2: Effect of different concentration of mannitol on growth parameters of S. rebaudiana ‘Bertoni’.

Zeng et al. [23] reported that plants treated with 90 and 120 mMNaCl showed significantly lower plant height, leaf number, branchlength, stem dry weight, leaf fresh/dry weight, and shoot dry weightthan control. They also found that high salt treatment (120 mM NaCl)drastically decreased the total dry weight of S. rebaudiana by 40% incomparison to control. In addition, shoot/ root ratios of salt-treatedplants were inhibited markedly by 30-50% compared with that of thecontrol. The results obtained in the present experiment verified thisconclusion that as the salinity increases, the biomass content of S.rebaudiana significantly reduces than control. Srivastav and Srivastav[24] also presented the similar findings that severe drought stressconditions reduce the plant biomass of S. rebaudiana. These resultswere similar to the findings of Pirzad et al. [25] and Mohammadianet al. [26].

Leaf is the main plant organ responsible for light absorptionand transformation and plays an important role in providing energyand nutrients for the growth and development of plants. The growthinhibition caused by salt and drought stress mainly originated fromthe damage to leaf function. Results presented in Table 1 and Table 2 showed that higher levels of salinity and drought decrease leafnumber throughout the experiment. In the present study, the numberof fully expanded leaves decreased dramatically with increasingsalt level, which could be seen as the combined effect of increasingthe senescence rate of older leaves and decreasing the productionof new leaves. Zeng et al. [23] reported leaf chlorosis, wilting, andnecrosis were in plants treated with 90 and 120 mM NaCl for 28 days,respectively. It was found that the general trend of the treatmentreflects a gradual decrease in the number of plant leaves with the increase of NaCl and mannitol concentration, compared with theplants of the control experiment.

Leaf number significantly reduced as increasing concentrationof NaCl and mannitol. It was found that the general trend of thetreatment reflects a gradual decrease in the number of plant leaveswith the increase of salt concentration, compared with the plantsof the control experiment. The maximum number of leaves 44.8and 43.5 were found at 25mM and minimum number of leaves 22.5and 26 at 100mM NaCl and manitol stress treatments, respectively.According to Silva et al. [27] leaves were more sensitive than plantheight after 7 days of treatment. After 36 days, significant reductionin leaves number at all NaCl levels was observed. Shao et al. [28]reported that reduction in number of leaves under water stress ismay be due to decline in cell enlargement and more leaf senescenceresulting from reduced turgor pressure. On the other hand, significantreduction in shoot/root ratio was also observed as NaCl and manitolconcentration increase (Table 1). Shoot growth was probably moresusceptible to salt than root, which generally led to a lower shoot/rootratio in salt treatments. Zeng et al. [23] found significant reduction inshoot/root ratios in all NaCl treatments than in control. Moreover,Nejad [29] studied on effect of drought stress on shoot/root ratio withfour levels of water stress as the first factor and plant growth periodsas the second factor with four replications. Their findings suggestthat increasing drought stress with each period of growth, shoot androot weight decreases per plant. Previous studies suggested that thissensitivity could be due to an imbalance among cations as a result ofthe complex interaction in the xylem transport system.

Effect of salinity and drought stress on protein content Figure 6b and Figure 7b results indicate a reverse effect of sodium chlorideand mannitol using various concentrations on total protein of S.rebaudiana plant after 10, 20 and 30 days. It appears from the datathat there was a general decrease in protein content that correspondedwith the increase in NaCl and mannitol concentrations as comparedwith the control plant. Statistical analysis did show a significantdifference during the three measurement periods (Supplementary file 2A, B).

Figure 6: Effect of different concentrations (0, 25, 50, 75 and 100mM) of NaCl on biochemical content of Stevia rebaudiana ‘Bertoni’ a) Effect of salinity stress on proline content of plant b) Effect of salinity stress on protein content and c) Effect of salinity stress on chlorophyll content.

Chen et al. [30] found that exposing Vigna unguiculata (L.) plants,at the age of 14 days, to salt treatment using 75mM of NaCl, reducedsoluble protein content in the plant. The results of the previous studywas confirmed by Cheruth et al. [31], with their study on Cathranthusroseus (L.) and Khosravinejad et al. [32] with their study on barley Hordeum vulgare L. On the contrary, Sibole et al. [33] reported thatthe treatment of clover plant (Medicago citrna L.) for 30 days withconcentrations of 0, 1, 50, 100, 200 mM of NaCl increased solubleprotein content in the seedlings, compared with control plants.Another study by Kapoor and Srivastava [34] on Vigna mungo (L.)supports the previous results.

In contrast to effect of drought stress on protein concentration,there were no significant differences observed with the increasingconcentration of mannitol as compared to control plants. However,the amount of protein (mg/gm) increases after 10, 20 and 30 daysof culture in control plant. Several researches indicated that solubleprotein decreases with increasing drought stress time [35,36]. Thewater stress injury causes damage to protein synthesizing mechanism.The possible reason for decreased protein content under water stressmay be due to increased activity of protease and also it may be dueto proteolysis or decreased synthesis or both. Leaf proteins undergoaccelerated hydrolysis as water stress develops.

Effect of salinity and drought stress on proline content We analyzedthe proline content of plant grown at 0, 25, 50, 75 and 100mM NaCland mannitol. Results showed that amount of proline increased withthe increasing concentration of NaCl and mannitol (Figure 6a; Figure 7a). Statistically significant differences in proline accumulation weredetermined between the control and treatment periods of NaCl whilein drought stress condition no significant differences were observed(Supplementary file 2A, B). Proline accumulation in salt stressedplants is a primary defense response to maintain the osmotic pressurein a cell, which is reported in salt tolerant and salt sensitive cultivarsof many crops [37-39]. Zhang et al. [23] reported that proline contentenhanced significantly with increasing concentration of NaCl andwas 17-, 31-, and 42-fold higher in 60, 90, and 120 mM NaCl than inthe control, respectively. Several studies have indicated that prolinecontent increases during drought stress [40,41].

Figure 7: Effect of different concentrations (0, 25, 50, 75 and 100mM) of mannitol on biochemical content of Stevia rebaudiana ‘Bertoni’ a) Effect of drought stress on proline content of plant b) Effect of drought stress on protein content and c) Effect of drought stress on chlorophyll content.

Effect of salinity and drought stress on chlorophyll content Decrease in chlorophyll content was determined as a result of increasing NaCl and mannitol concentration. The highest content of chlorophyll was found at 25mM after 10 days of culture while lowest content wasfound at 100mM after 30 days in both salinity and drought treatment conditions. The significant decreasing percentage of chlorophyll was 50 % at 100mM NaCl treatment after 10 days of culture that further reduced (16 %) after 30 days of stress treatment. Moreover, significantreduction (54 %) was observed at 25mM after 100 days of mannitol that decrease (10 %) further after 30 days of culture (Figure 6C; Figure 7C). Statistical analysis shows that significant reduction in chlorophyll content was found at each concentration level (25, 50, 75 and 100mM)but data obtained after 10, 20 and 30 days of culture was found no significant in both salinity and drought stress plants (Supplementary file 2A, B). The decrease in chlorophyll content may be due to an increase of chlorophyll degradation or to a decrease of chlorophyllbiosynthesis. Change in chlorophyll contents due to salinity is the most evident biochemical response [42]. Amira et al. [43] also found the similar results on propagation of plant Cathranthus roseus under salinity and drought conditions where they reported that reduction in chlorophyll associated with reduced photosynthetic activity andtranspiration rate in plants at all salinity levels is considered a defense mechanism against damaging reactive oxygen species by diminishing light absorbing capacity that reduces the flow of electrons throughthe photosysteme. Moreover, disruption of the thylakoid and stromal membranes may result in leaf chlorosis and necrosis, Qureshi et al. [44] which indicated that salt concentration above 90 mM NaCl could impair the chloroplast structure of S. rebaudiana. In contrast to drought stress condition, decrease in the chlorophyll was observed which is reported first time in S. rebaudiana. No such study was previously reported in S. rebaudiana.

Summarizing, salinity and drought stress significantly affect the growth and development as well as biochemical content of S. rebaudiana. As the concentration of NaCl and mannitol increased physiomorphologic and biochemical components i.e. proline, protein and chlorophyll contents negatively affects the growth and development of plants but 50 % of plant was survived at the concentration (50mM) of NaCl and mannitol. These results indicatethe mutant development at such concentration. Further research is in progress to decipher the expression profile of four major genes involved in steviol glycoside pathway under salinity and drought conditions and compare with HPLC results for stevioside and rebaudioside i.e. responsible for sweetening property, content withcontrol plant.