Steviol Glycoside Content Dynamics during the Growth Cycle of Stevia rebaudiana Bert

The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia is a polyannual crop (5 7 years) with a vegetative period lasting from April-May until October, during which time the crop can be harvested two or three times. This research focuses on the need for knowledge about Stevia’s response to temperate climates in Southern Europe. Two field assays were established from June to October 2013 at two sites in Navarra (Spain). The biomass and content of the two major SG, stevioside (ST) and rebaudioside A (RA), were measured using High Performance Liquid Chromatography (HPLC) in 66 cloned plants, at different developmental stages. Although the results from the two sites showed different SG leaf content dynamics during the plant growth, the optimum harvest date at both sites coincided with the bud-flowering stage at the beginning of September (around 96 days after planting), when a ST yield of 27 g·m was reached. These results show Stevia’s potential as a commercial crop for stevioside production in northern Spain.


Introduction
The number of people suffering cardiovascular diseases, diabetes, hypercholesterolemia and obesity has increased globally [1]. For this reason, the demand for non-caloric sweeteners has increased while sugar consumption has decreased [2]. In this context Stevia plays an important role. Besides being a natural sweetener, stevia leaf extracts have other interesting properties, including decreasing much work has been done on variety selection, particularly in China, Korea and India.
The European Commission approved the use of SG as a food additive (E-960) in 2011 (Commission Regulation EU No 1131/2011). As this new market opened up, the demand for Stevia increased and its introduction into Europe as a crop was encouraged, especially in the Mediterranean region. In Spain, the area dedicated to Stevia cultivation is estimated to be around 70 -80 ha.
Among the steviol glycosides (SG), stevioside (ST) and rebaudioside A (RA) are present in the highest concentrations [8]. In the biosynthetic pathways for the main SGs, ST is a direct precursor to RA [9]. ST represents between 60% -70% of the total SGs and is 300 times sweeter than sucrose, with a slight licorice flavor and a bitter aftertaste [10]. In contrast, RA lacks the bitter aftertaste and is the sweetest SGs, being from 250 up to 400 times sweeter than sucrose [10]. SGs are mainly found in the leaves, with higher levels in younger than older leaves [8] [11] [12] [13], with less in shoots, roots, flowers and seeds [14]. For this reason, the leaves are used for obtaining SG extracts, while shoots are discarded because of the difficulty of extracting SGs from lignified shoots. SG yield per dried leaf varies from 5% to 22% [15] and SG content depends on plant variety or population [9] [16], propagation methods [17] Stevia is a short-day species with a critical photoperiod of 13 to 14 hours needed to induce flowering [3]. In general, SG content is highest just before flowering, during the flower bud formation phase [24], making this the optimum time for harvesting [25]. After this phase, photoassimilates are allocated to reproductive organs and SG content tends to either stagnate [26] [27] or decrease [28]. Temperate areas, with long summer days (more than 13 -14 hours of sunlight), provide the ideal conditions for obtaining high yields of SG [8]. A longer vegetative period before flowering and harvest promotes higher leaf biomass production and a higher SG yield.
In various temperate areas, the cultivation of Stevia is long-term (5 -7 years) and the vegetative period lasts from April-May to October. During winter, above-ground parts of the plant remain inactive, and new stems sprout from the buried rhizome the next spring. In these areas with a temperate climate, Stevia can be harvested two or three times per year [29]. At higher latitudes, with cooler weather, the crop is harvested once a year.
This research focuses on the need for knowledge about Stevia's response to

Study Sites
Field assays were carried out from June to October 2016 at two sites in Navarra,  Table 1 presents climate data (mean temperatures and precipitation) for the two sites during the field assay period. Soils from both sites show similar characteristics: both are classified as sandy-loam with a basic pH and similar N content, although the soil in San Adrián has more P and K content than that of Puente la Reina (Table 2).

Plant Material and Experimental Design
For the assays were used in vitro plant clones, obtained from a single previously-selected plant. On June 5th, at each site, 10 cm-tall specimens of Stevia were manually planted in one row using a planting pattern of 40 × 35 cm. Black padding was installed to minimize weed growth. Nitrogen, phosphorus and potassium were supplied three times through fertigation (COMPO, 7% N, 5% P 2 O 5 , 6% K 2 O, and microelements).
Every two weeks, four plants were cut five cm above soil level and the shoots and leaves were separated. A subsample of fresh leaves from each plant was used to measure SG content and the rest of the sample was air dried, being turned over every two days. Both the fresh and dried biomass of the shoots and leaves were recorded.
In total, 66 plants (32 in San Adrián and 34 in Puente la Reina) were analyzed over 9 sampling dates corresponding to different developmental stages, from early vegetative growth (V3) to the crop flowering stage (R2) ( Table 3). The description of plant developmental stages followed Carneiro [30].  Table 3. The transition from one developmental stage to the next lasts a few days.

Data Analysis
Scatterplots of dry leaf biomass, leaf-to-stem ratio, ST and RA versus time after planting were performed and a polynomial surface for each site was fitted using local polynomial regression fitting with the loess function from the statistical software R [31].

Results
Leaf biomass increased with time at both sites but peaked at different dates ( Figure 1(a)). The plants in San Adrián presented greater leaf biomass and earlier than those at Puente la Reina. In San Adrián, leaf biomass reached a peak (63.3 g/plant) 96 days after planting, between the end of the late-V4 and early R1 stages (grey square point in Figure 1(a)). In Puente la Reina, the highest yield (53.6 g/plant) was reached 102 days after planting, at the R1 stage (black square point in Figure 1(a)). The leaf to stem ratio decreased throughout the plant growth cycle, from 4.5 down to 0.5 at both sites (Figure 1(b)).
The dynamic of ST percentage in leaves throughout plant development differed between the two sites (Figure 1(c)). In San Adrián, the ST percentage reached a peak (6.22%) in the late-V4 stage, 87 days after planting (grey square point in Figure 1(c)). In Puente la Reina, the ST percentage increased with time and was its highest (8.67%) in the R2 stage, 120 days after planting (black square point in Figure 1(c)). Taking into account biomass production, total ST leaf content per plant peaked in San Adrián (3.72 g/plant) at the end of the late-V4 stage, 92 days after planting (grey square point in Figure 1(d)), whereas the peak in Puente la Reina (4.03 g/plant) was reached in the R1 stage, 108 days after planting (black square point in Figure 1(d)).
A. B. Guerrero et al. The dynamic of RA percentage in leaves throughout plant development also differed between the sites (Figure 1(e)). In San Adrián, the RA percentage was highest (5.03%) between the end of the late-V4 stage and the beginning of R1, 97 days after planting (grey square point in Figure 1(e)). In Puente la Reina, the RA percentage peaked (6.55%) in the V4 stage, 70 days after planting (black square point in Figure 1(e)). Taking into account biomass production, total RA leaf content per plant was at its highest level between the end of the late-V4 and beginning of the R1 stages at both sites. In San Adrián, RA content reached 3.12 g/plant 95 days after planting (grey square point in Figure 1(f)) and in Puente la Reina, RA content reached 2.43 g/plant 97 days after planting (black square point in Figure 1(f)).

Discussion
The observed decrease in leaf biomass during the reproductive stages (R1 and A leaf to stem ratio decrease was observed throughout the plant growth cycle. In the early stages, the stems were green and soft with a high water content and leaf weight was greater than stem weight (L:S > 1). In later stages, the stems enlarged and lignified, and consequently their dry weight increased considerably.
This meant that stem weight was greater than leaf weight during the reproductive stages. Decreased leaf biomass in the later stages due to reproductive development has been observed previously [33]. Although a similar L:S pattern was seen at both sites throughout the growth stages, L:S was greater in Puente la Reina plants than in San Adrián plants during the vegetative stages. Similar studies in Israel also showed a decreased L:S ratio with time and a leaf biomass peak in September [22].

Conclusion
In conclusion, this paper addresses the viability of growing Stevia under the cli-