Harvesting regimes to optimize yield and quality in annual and perennial Stevia rebaudiana under sub-temperate conditions

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ARTICLE IN PRESSG ModelNDCRO-7556; No. of Pages 9

Industrial Crops and Products xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal home p age: www.elsev ier .com/ locate / indcrop

arvesting regimes to optimize yield and quality in annual anderennial Stevia rebaudiana under sub-temperate conditions

robir Kumar Pala,∗, Mitali Mahajana, Ramdeen Prasadb, Vijaylata Pathaniaa,ikram Singha, Paramvir Singh Ahujac

Division of Natural Plant Products, Council of Scientific and Industrial Research-Institute of Himalayan Bioresource Technology (CSIR-IHBT),ost Box No. 6, Palampur 176 061, HP, IndiaDivision of Hill Area Tea Science, CSIR-IHBT, Post Box No. 6, Palampur 176 061, IndiaDivision of Biotechnology, CSIR-IHBT, Post Box No. 6, Palampur 176 061, India

r t i c l e i n f o

rticle history:eceived 2 June 2014eceived in revised form8 September 2014ccepted 30 September 2014vailable online xxx

eywords:arvesting regimeife cycle

a b s t r a c t

Uses of the perennial crops have been proven to increase the sustainability in agriculture. However,information about the relative efficiency of annual versus perennial stevia (Stevia rebaudiana) underdifferent harvest regimes is unknown in the sub-temperate region of western Himalaya in India. Theharvesting time of stevia is generally controlled by growth behavior and accumulation pattern of thesteviol glycosides (SGs). Our objectives were to standardize the harvesting regimes in both the croppingmodes for higher yield and quality of stevia leaf. Thus a field experiment with six treatment combinationscomprising two life cycles (annual and perennial) and three harvest regimes (single-cut, double-cut andtriple-cut) was conducted. The total dry leaf yield was in the range of 1.40–4.11 t ha−1, and significantly(P ≤ 0.05) highest dry leaf yield was recorded with the perennial stevia under three-cut management

egrowth yieldteviol glycosidestevia rebaudiana

system (PH3) but remained statistically at par with PH2 (perennial with two-cut). However, PH2 producedmaximum SGs (10.29 g plant−1). The results of Principle component analysis reveal that PH2 and PH3 areequally effective and suitable management practices for dry leaf and SGs yield. Thus, our results suggestthat the perennial stevia with two-cut management system is suitable for sustaining the stevia productionin this condition.

. Introduction

Stevia (Stevia rebaudiana Bertoni), a perennial herb of the Aster-ceae family and native to South America, is widely known forweet-tasting and low-calorie diterpenoid steviol glycosides (SGs)ontent in its leaves. Dry leaves of stevia have been used for cen-uries by the indigenous Guarani Indians of South America as

natural sweetener. Now stevia cultivation has been spread tother regions of the world including China, Brazil, Japan, Paraguay,exico, Russia, Indonesia, Korea, USA, Tanzania, Canada, Thailand

nd Argentina (Brandle et al., 1998; Kim et al., 2002; Lemus-ondaca et al., 2012). Indian farmers are also growing stevia since

ast decade, and using it as raw dry leaf or selling it for processed

Please cite this article in press as: Pal, P.K., et al., Harvesting regimrebaudiana under sub-temperate conditions. Ind. Crops Prod. (2014), h

weetener (Pal et al., 2013).More than 30 SGs have been identified in stevia leaves with

arying concentrations (Wolwer-Rieck, 2012). However, the most

∗ Corresponding author. Tel.: +91 1894 233 341; fax: +91 1894 230 428;obile: +91 9805551007.

E-mail addresses: pkpal [email protected], [email protected] (P.K. Pal).

ttp://dx.doi.org/10.1016/j.indcrop.2014.09.060926-6690/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

dominant compound is stevioside, which is about 300 timessweeter than sucrose (Crammer and Ikan, 1986). Unfortunately,this compound is responsible for the bitter aftertaste (Bakal andO’Brien Nabors, 1986). The second most abundant compound isrebaudioside -A (Reb-A), which is better suited than steviosidefor use in foods and beverages due to its pleasant taste (Kinghornand Soejarto, 1991; Tanaka, 1997). Thus, the stevioside/Reb-A ratiois also considered as parameter to measure the quality of stevia.In addition to the sweet tasting compounds, high levels of phe-nol content and high antioxidant activity were observed in stevialeaves and leaf extracts, respectively (Ghanta et al., 2007; Shuklaet al., 2009; Kaushik et al., 2010; Tavarini and Angelini, 2013). It hasalso been reported from toxicological studies that stevioside doesnot have mutagenic, teratogenic or carcinogenic effects (Lemus-Mondaca et al., 2012). Hence SGs are excellent alternative to canesugar and artificial sweeteners, particularly for diabetes patients.

The market of stevia and stevia products has significantly

es to optimize yield and quality in annual and perennial Steviattp://dx.doi.org/10.1016/j.indcrop.2014.09.060

increased, particularly after getting approval of SGs as a foodadditive from worldwide regulatory authorities (European FoodSafety Authority, the US Food and Drug Administration, TheJoint FAO/WHO Expert Committee on Food Additives, and Food

dx.doi.org/10.1016/j.indcrop.2014.09.060


http://www.sciencedirect.com/science/journal/09266690

http://www.elsevier.com/locate/indcrop

mailto:[email protected]

mailto:[email protected]


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tandards Australia New Zealand) (Geuns, 2003; FSANZ, 2008;ECFA, 2009; EFSA, 2010; Tavarini and Angelini, 2013). The keynterest of stevia growers is the concentration of SGs in leaves,

hich generally varies between 4% and 20% on a dry weight basis.owever, the concentration of SGs in stevia leaf depends uponrowing conditions (Pol et al., 2007), cultivar (Bondarev et al.,003), photoperiod (Ceunen and Geuns, 2013), and plant nutri-nts (Pal et al., 2013). Leaf yield and concentration of bioactiveompounds in leaves also depend upon agro-technique (Gardanat al., 2010; Geuns, 2003). The agronomical evaluation of steviaas not been properly evaluated, since it is a new crop. Among thegronomic practices, harvest regime is one of the important factorshich markedly influences on leaf biomass and quality of stevia.

Generally, stevia is commercially cultivated as an annual orerennial crop, depending upon growing conditions. However, therowth pattern of stevia is different under the annual and peren-ial conditions. The agricultural sustainability can be increasedhrough selection of perennial crops (Piper and Kulakow, 1994;lover, 2003) instead of annual crops. In marginal environment,erennial crop improves yield stability (Ploschuk et al., 2001). Itas also been reported that yield can be potentially high in theerennial crops (Moffat, 1996; Pimm, 1997). Moreover, perennialropping system can utilize many resources more efficiently thannnual system (Jordan et al., 2007). Robertson et al. (2000) havelso reported that the perennial crops have capacity to store morearbon in the soil (320–440 kg ha−1 y−1) compared with the annualrops (0–300 kg ha−1 y−1).

The accumulation of SGs reaches its peak during the onset ofowering (Bondarev et al., 2003), which is the ideal time to harvesthe leaves in terms of quality. Stevia has enormous potential to storehe energy in the root for regeneration of the shoot after cutting.he single harvesting at flowering stage may lead to lower leaf yieldompared with multi-cut management system due to defoliationf lower and old leaves. Moreover, regeneration quality of stevias remained unexploited under single-cut (at onset of flowering)

anagement system. Therefore, harvest management of the annualnd the perennial stevia requires a negotiation between quality anduantity of biomass. The regrowth of perennial stevia after everyarvest depends upon energy reserves stored in the roots. In casef the perennial stevia, substantial energy reserve is also neededor the development of cold-hardiness, which allows the plant tourvive during winter and regrowth during spring. The harvestingime of stevia should be decided based on a sound understanding ofrowth behaviors and survival mechanism. However, informationbout the interaction effects of life-cycle and harvesting regime isot available for higher productivity and quality of stevia underub-temperate region in India. The main objective of this researchs to standardize the harvesting regime for increasing the yield anduality of stevia under the annual and the perennial cropping mode

n the western Himalayan region.

. Materials and methods

.1. Experimental location, climate and soil characteristics

This investigation was conducted at the experimental farm ofSIR-Institute of Himalayan Bioresource Technology (32◦06′05′′ N;6◦34′10′′ E), Palampur, India, during the growing seasons of 2010nd 2011, to standardize the harvest management under the annualnd the perennial cropping modes for attaining higher leaf yield anduality in the sub-temperate region in India. The altitude of the site

Please cite this article in press as: Pal, P.K., et al., Harvesting regimrebaudiana under sub-temperate conditions. Ind. Crops Prod. (2014),

s 1393 m from mean sea-level. The experimental farm is charac-erized by high rainfall (250 cm mean annual rainfall), and averagennual temperature is 18 ◦C. The detailed environmental condi-ions viz., maximum and minimum temperature, relative humidity,

PRESSProducts xxx (2014) xxx–xxx

sunshine hours, and rainfall during the investigation years are pre-sented in Fig. 1. The soil of experimental site was silty clay in textureand acidic in reaction with pH 5.86 (1:2). The electrical conduc-tivity (EC) of the soil was very low (0. 088 m mhos cm−2), whereasorganic carbon was quite high (1.75%). Available nitrogen (N), phos-phorus (P), and potassium (K) in the soil were 379.46, 44.93, and508.26 kg h−1, respectively. The AP in the experimental unit is veryhigh since the soil pH is about 6.0. It is a fact that the maximum Pavailability occurs at soil pH between 6.0 and 7.0.

2.2. Layout and application of treatments and crop management

The experimental design was a randomized complete block witha factorial arrangement of treatments and three replications. Sixtreatment combinations comprising two crop cycles (annual andperennial) and three harvest systems (single, two and three-cutsper year) were tested. Harvest systems included cut once per yearat the onset of flowering for single cut management system in bothannual and perennial crops. For two cut management, the first cutwas done at the active vegetative stage, and 2nd cut was done at theonset of flowering. The details of cutting schedule are presented inFig. 2.

Eighteen permanent plots were established during 1st week ofMarch 2010, then after proper field preparation, 90 days old steviaseedlings were transplanted at the end of March with the spacingof 45 cm × 45 cm. The plot size was 3 m × 5 m with 0.6 m borderbetween the plots. During final land preparation, well-rotted farm-yard manure was applied uniformly at the rate of 5 t ha−1. The cropwas also fertilized with N, P, and K at the rate of 100, 21.85, and41.5 kg ha−1 y−1, respectively. Nitrogen, phosphorus, and potas-sium fertilizers were applied in the form of urea (46% N), singlesuper phosphate (16% P2O5), and muriate of potash (60% K2O),respectively. The entire doses of P and K and a half dose of Nwere applied at the time of transplanting for all treatments. Theremaining half quantity of N was applied on the day 60 aftertransplanting (AT) for single-cut management system, whereas theremaining half N was applied after 1st harvesting for two-cut man-agement system. In case of three-cut management system, theremaining half quantity of N was applied into two equal doses atafter 1st and 2nd harvest. The underground parts were left withoutany disturbance for the perennial crop. For the perennial crop 1stdose of fertilizer was applied on 1st April, 2011, while subsequentfertilizer doses were applied as per schedule followed during 1styear. The annual crop was repeated during 2011 with the perennialcrop to eliminate the year effect due to environmental conditions.Thus the age of perennial crop was two years. The irrigation andweeding operations were done as per requirement of the crop. Aproper drainage facility was also provided to avoid water loggingdue to heavy rain during monsoon.

2.3. Growth and yield

For growth observation, two randomly selected plants were cutat ground level for the single-cut management system. However,in case of two and three-cut management system, two randomlyselected plants were cut at 15 cm height from ground level during1st harvest and marked with an aluminum tag for the subse-quent observations. At final harvest, the tagged plants were cutat ground level for both annual and perennial crops. After sepa-ration of leaves from stems, total number of branches (shoots) perplant was recorded. The leaf area of respective treatments was mea-

es to optimize yield and quality in annual and perennial Steviahttp://dx.doi.org/10.1016/j.indcrop.2014.09.060

sured by a leaf-area meter (AM 300, ADC Bio-scientific Ltd., UK)for expression of leaf area index (LAI). After recording the freshleaves and stem weight, these samples were dried as representa-tive samples at 70 ± 2 ◦C in a hot air oven until a constant weight


ARTICLE IN PRESSG ModelINDCRO-7556; No. of Pages 9

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as attained, and the dried samples were weighed to determinehe dry matter (DM) content.

For estimation of leaf and stem yield, five another plants werearvested from each treatment at 15 cm height from ground levelnder two and three-cut management system, whereas, plantsere removed from ground level at final harvest. The fresh leaves

nd stems samples were weighed separately, and the dry leaf andtem yield were calculated by multiplying factors, which were cal-ulated from growth observation samples.

.4. Extraction and analysis of steviol glycosides (SGs)

For estimation of SGs content in stevia leaves, the collected leafamples were washed under running tap water to remove dust andicrobes sticking to the surface. The moisture, which retained on

he surface, was removed by blotting paper. After that, the samplesere dried in a hot air oven at 40 ± 2 ◦C until constant weight was

chieved, and the samples were ground by a laboratory grindingachine. Steviol glycosides were extracted from 50 mg of prepared


eaf sample in 5 ml methanol for overnight and then filtered. Re-xtraction was done twice with the same solvent for 3 h each, andhe pooled extracts were concentrated up to dryness under reducedressure. The extract, which was obtained after de-fractioning with

Fig. 2. Harvesting schedule for stev

fall (cm) and relative humidity (RH %) during the growing season of 2010 and 2011SW) and closing date of 40th MSW are 26th March and 7th October, respectively.

2 ml n-hexane and vacuum drying, was dissolved in 5 ml high-performance liquid chromatography (HPLC)-grade Acetonitrile andwater (8:2) mobile phase. Then the filtrate was used for estima-tion of stevioside and rebaudioside-A by HPLC. The Waters HPLC(Waters, Milford, MA, USA) system, equipped with Waters 717plus auto-sampler, 996 Photodiode Array Detector and Empower2 software (4.01 version), was used for the analysis of steviosideand Reb-A. The extracts of plant samples were separated on a LiChrosphere NH2 100 column (250 mm × 4 mm × 5 �m particles) (E.Merck, Germany). The detection wavelength was 205 nm. Standardstevioside and Reb-A samples (purchased from Chromadex LifeTechnologies, India) were used for the preparation of the standardcurve, which was used for the quantification of stevioside and Reb-A.

2.5. Determination of pH and nutritional status of soil

After completion of two years life cycle (after 3rd cut of peren-nial crop) of stevia, soil samples were collected from the surface


layer (0–15 cm) for determination of pH, organic carbon (OC) andnutritional status of the soil. The pH of soil water suspension(1:2 w/v) was measured by a pH meter (model Eutech Instru-ments pH 510), while the soil OC was determined by using the

ia under different life cycles.


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tandard dichromate oxidation method (Nelson and Sommers,982). Available N status of the soil was estimated as per standardethod suggested by Subbiah and Asija (1956). Available phos-

horus (AP) and available potassium (AK) were estimated using apectrophotometer (model T 90 + UV/vis, PG Instrument Ltd.) and aame photometer (model BWB XP, BWB Technologies UK Ltd., UK)espectively, as per the procedures described by Bray and Kurtz1945) and Mehlich (1984).

.6. Statistical analysis

The data were subjected to analysis of variance (ANOVA) toest the sole effect of life cycle and harvest regime, and cropycle × harvest regime interaction using Statistica 7 software (Statoft Inc., Tulsa, OK, USA). Least significant difference (LSD) test athe P = 0.05 level was used to separate the treatment means. Theata on dry leaf and stem yield for different harvesting stagesre presented as mean ± standard error (SE). Student paired t-est (P = 0.05) was conducted to test the significance of differenceetween the annual and the perennial crop for respective harvest-

ng stages. We performed Principal component analysis (PCA) tovaluate the nature of influences of treatment combinations onield and yield attributes as a bi-plot, and nature of variation amonghe treatment combinations was also projected. The factor loadingalues are the correlations of each variable with the principal com-onent (PC). We also established the correlation matrix among theotal LAI, yield and secondary metabolite accumulation (g plant−1).

. Results

.1. Yield attributes and root biomass

Dynamics of the branches (no. plant−1) and LAI are presented inig. 3. The perennial crop produced significantly (P ≤ 0.05) higherumber of branches (no. plant−1) compared with the annual cropnder all cutting management systems. The maximum number ofranches (72 plant−1) was recorded with the perennial crop at 2ndarvest of two-cut management system; however, least numbersf branches (7.5 and 12.67) were observed at final harvest of three-ut management system under both annual and perennial croppingode (Fig. 3a). Under single-cut management system, the peren-

ial stevia produced 133.35% higher number of branches comparedith the annual stevia. Similar to the branch, the LAI was also signif-

cantly (P ≤ 0.05) affected by crop life-cycle and harvesting regime.n terms of LAI, the overall efficiency of perennial stevia was signif-cantly (P ≤ 0.05) higher than annual stevia. The maximum total LAI5.63) was registered with three-cut management system in casef the perennial crop, whereas almost equal amount of LAI wasbserved with two-cut and three-cut management systems undernnual cropping mode (Fig. 3b). The perennial stevia registered4.34, 91.38, and 96.17% higher total LAI over the annual steviander single-cut, double-cut, and triple-cut management systems,espectively. Root biomass (g plant−1) of stevia was significantlyffected by the crop life cycle (Fig. 3c). Irrespective of harvestingegime, the perennial stevia produced about 88% higher dry rootiomass compared with the annual stevia.

.2. Yield data

Dry leaf yield (t ha−1) and stem yield (t ha−1) at different cuttingtages were presented in Table 1. The data revealed that the overallffect of life cycle on dry leaf and stem yield at all cutting stages was


ignificant (P ≤ 0.05), and the highest values were registered withhe perennial cropping system (Table 1). Under single-cut manage-

ent system, the perennial stevia significantly (P ≤ 0.05) increasedry leaf and stem yield by about 92.85 and 75.93%, respectively,

biomass (g plant−1) of stevia under different cutting managements in both annualand perennial cropping modes. Vertical bars indicate a mean standard error (±).

compared with annual stevia. Under double and triple-cut man-agement systems, the re-growth yields had smaller contributionto total yield than first-cut yields in both cropping modes. The dryleaf yield at third harvesting was nominal; however, significantly(P ≤ 0.05) higher yield (0.23 ± 0.033 t ha−1) was registered with theperennial crop. The variations in leaf: stem ratio were also observedamong the treatments, and the lowest value (1.18) was recordedwith triple-cut management system of the perennial crop.

The analyzed data (Table 2) also revealed that the sole effectof life cycles and harvesting regimes and their interaction effects


on total dry leaf yield (t ha−1) of stevia were significant (P ≤ 0.05).The perennial crop produced about 93.6, 155.9, and 158.5% higherdry leaf compared with the annual crop under single, double and



P.K. Pal et al. / Industrial Crops and Products xxx (2014) xxx–xxx 5

Table 1Dry leaf yield (t ha−1) and dry stem yield (t ha−1) of stevia at different cutting stages in annual and perennial cropping mode.

Treatment Dry leaf yield (t ha−1) Dry stem yield (t ha−1) Leaf: Stem (based ontotal dry weight)

Life cycle Harvestregime

1st cut 2nd cut 3rd cut Total 1st cut 2nd cut 3rd cut Total

Annual Single cut 1.40 ± 0.05a 1.40 2.41 ± 0.07a 2.41 1.72Double cut 1.38 ± 0.08c 0.21 ± 0.03g 1.59 1.55 ± 0.24c 0.76 ± 0.11g 2.31 1.45Triple cut 1.38 ± 0.03e 0.13 ± 0.01i 0.07 ± 0.003k 1.59 1.38 ± 0.04e 0.06 ± 0.01i 0.72 ± 0.22k 2.16 1.36

Perennial Single cut 2.71 ± 0.20b 2.71 4.24 ± 0.15b 4.25 1.56Double cut 3.32 ± 0.17d 0.75 ± 0.04h 4.07 2.99 ± 0.19d 1.87 ± 0.40h 4.86 1.19Triple cut 3.25 ± 0.18f 0.63 ± 0.03j 0.23 ± 0.033l 4.11 3.01 ± 0.12f 0.37 ± 0.04j 1.47 ± 0.50l 4.85 1.18

The values are the mean ± standard error (n = 3 for all cutting stages). The values within each column followed by the different letter indicate significant different (P ≤ 0.05)for the respective cutting stages. For first column, a and b are used for single cut management, c and d for double cut, and e and f are used for triple cut management system.For second column, g and h are used for double cut, i and j are used for triple cut management; whereas, k and l are used for 3rd column. Same notations are used for stemyield.

Table 2Interaction effect of life cycle and harvesting regime on total dry leaf yield (t ha−1) and dry stem yield (t ha−1) of stevia.

Treatment Dry leaf yield (t ha−1) Dry stem yield (t ha−1)

Single cut Double cut Triple cut Mean (life cycle) Single cut Double cut Triple cut Mean (life cycle)

Annual 1.40 1.59 1.59 1.53 2.41 2.31 2.16 2.29Perennial 2.71 4.07 4.11 3.63 4.25 4.86 4.85 4.65Mean (harvesting regimes) 2.06 2.83 2.85 3.33 3.59 3.51SEm(±) For life cycle = 0.07

For harvesting regime = 0.08For interaction (life cycle × harvesting regime) = 0.12

For life cycle = 0.17For harvesting regime = 0.20For interaction (life cycle × harvesting regime) = 0.29

CD (P = 0.05) For life cycle = 0.22For harvesting regime = 0.23

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riple-cut management systems, respectively. Regardless of lifeycle, double and triple-cut management systems produced almostqual amount of dry leaf yield (2.83 and 2.85 t ha−1); however, thesewo treatments produced significantly (P ≤ 0.05) higher leaf yieldompared with single harvesting. The significant (P ≤ 0.05) inter-ction effects of the life cycles and harvesting regimes were alsobserved, and the maximum leaf yield (4.11 t ha−1) was recordedith three-cut management in the perennial cropping mode.owever, this treatment was statistically at par with two-cut man-gement in the perennial cropping mode (Table 2). Irrespective ofarvesting regimes, the perennial stevia produced about 103.06%igher dry stem yield compared with annual crop. On the otherand, regardless of the life cycle, highest dry stem yield (3.59 t ha−1)as recorded with two-cut management system but remained sta-

istically at par with the rest of cutting management systems.

.3. Secondary metabolites

The accumulation pattern of stevioside and Reb-A under differ-nt harvesting regimes in case of both the annual and the perennialropping modes is presented in Fig. 4. There was no significantP ≥ 0.05) effect of life cycle on stevioside content (%) under single-ut management system. Moreover, under multi-cut managementystem, stevioside content was not significantly affected by theife-cycle during 1st harvesting; however, in subsequent harvestingtages, higher percentage of stevioside in leaves was observed in theerennial stevia (Fig. 4a). The perennial crop produced about 71.4%igher stevioside compared with the annual crop at 2nd harvestingnder double-cut management system. In three-cut managementystem, the concentration of stevioside was distinctly declined at


rd harvesting compared with the rest of the harvesting time underoth the annual and the perennial crops. Similar to the steviosideontent, Reb-A content was not significantly (P ≤ 0.05) affected byrop life cycles. However, the Reb-A concentration in leaves was

For interaction (life cycle × harvesting regime) = NS

marginally increased with the perennial crop at both the harvest-ing stages under two-cut management system (Fig. 4b). Total SGsyield (g plant−1) was estimated based on stevioside and Reb-A con-tent in leaves and dry leaf yield of the respective cutting stages.The data indicated that maximum SGs yield (10.29 g plant−1) wasrecorded with the perennial crop under two-cut management sys-tem (Fig. 4d).

3.4. Correlation matrix and Principle component analysis (PCA)

The correlation analysis revealed that dry leaf yield (g plant−1)and stem yield (g plant−1) were significantly (P ≤ 0.05) and posi-tively correlated with total LAI with correlation coefficients of 1.00and 0.97, respectively (Table 3). In this study, the yield of stevioside(g plant−1) and Reb-A (g plant−1) was also significantly (P ≤ 0.05)and positively correlated with dry leaf yield (g plant−1). On theother hand, the negative correlation was found between leaf: stemratio and other growth parameters. Nevertheless, correlation wasnot significant (P ≥ 0.05) in all the cases.

Principal component analysis (PCA) was carried out using theset of 4 growth parameters and SGs yield (g plant−1), and the out-puts from PCA were presented in Fig. 5a and b. In the present study,the first two factors (component), PC1 and PC2, allow us to explain99.5% of the initial variability of the data. The correlation cycle(Fig. 5a) shows a projection of the original variables in the factorsspace and also indicates how each variable contributes to the prin-cipal components (PC1 and PC2). Except leaf: stem ratio (V4), othersfour variables [LAI (V1), leaf yield (V2), stem yield (V3), and stevio-side + Reb-A yield (V5)] are located in the positive coordinate of PC1with markedly high loading values (0.99, 0.99, 0.96 and 0.98). The


PCA bi-plot (Fig. 5b) separated the treatment PH2 (perennial cropwith two harvesting) and PH3 (perennial crop with three harvest-ing) from rest of the treatments along with PC1 placed in positivecoordinate of both PCs, whereas the treatment AH1 (annual crop


Please cite this article in press as: Pal, P.K., et al., Harvesting regimes to optimize yield and quality in annual and perennial Steviarebaudiana under sub-temperate conditions. Ind. Crops Prod. (2014), http://dx.doi.org/10.1016/j.indcrop.2014.09.060


6 P.K. Pal et al. / Industrial Crops and Products xxx (2014) xxx–xxx

Fig. 4. The accumulation pattern of stevioside (a), Rebaudioside-A (b) and total steviol glycoside (c) in stevia leaves under different harvesting regimes in both annual andperennial cropping modes. Stevioside (a), Rebaudioside-A (b) and total steviol glycoside are presented in %, whereas total steviol glycosides yield is presented in g plant−1.(d) Vertical bars indicate a mean standard error (±).

Table 3Correlation matrix among the important variables of stevia. The mean values of the three replications of the corresponding treatments are used (where N = 6). N is the numberof treatments.

Variable Total LAI Total dry leaf yield(g plant−1)

Total dry stemyield (g plant−1)

Leaf:Stem Stevioside yield(g plant−1)

Reb-A yield(g plant−1)

Stevioside + Reb-A(g plant−1)

Total LAI 1.00Total dry leaf yield (g plant−1) 1.00** 1.00Total dry stem yield (g plant−1) 0.97** 0.97** 1.00Leaf: Stem −0.76 −0.76 −0.59 1.00Stevioside yield (g plant−1) 0.97** 0.98** 0.96** −0.71 1.00Reb-A yield (g plant−1) 0.95** 0.94** 0.98** −0.60 0.93** 1.00Stevioside + Reb-A (g plant−1) 0.98** 0.98** 0.98** −0.69 0.99** 0.96** 1.00

LAI is the leaf area index, whereas, Reb-A indicates rebaudioside-A.** Significant at P = 0.01.

Fig. 5. Bi-plot of principal components based on mean value of LAI, dry leaf yield (t ha−1), stem yield (t ha−1), leaf:stem ratio and total steviol glycosides (stevioside andrebaudioside-A) yield (g plant−1). Principal component 1 and 2 (PC1 and PC2) explain 99.5% of the initial variability of the data. (a) and (b) Variable vector distributions andcase distributions (treatment combinations), respectively. The loading values of variables are presented (a) as vectors in the space of the PCA bi-plots. A and P are the lifecycle of annual and perennial, respectively, while H1, H2 and H4 are representing the harvesting regime of single-cut, double-cut and triple-cut, respectively.


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ith single-cut management) was separated by the both PCs, andituated in the negative coordinates.

Thus, the overall PCA results indicate that the treatment PH2perennial crop with two harvesting) and PH3 (perennial cropith three harvesting) are almost equally effective and rep-

esent the appropriate management practices in terms of LAI,ry leaf yield (g plant−1), dry stem yield (g plant−1), and stevio-ide + rebaudioside yield (g plant−1). On the other hand, the annualtevia with single-cut management system is the least effective inerms of dry leaf yield and SGs yield.

.5. Chemical properties of soil after harvest

After completion of two years cropping cycle, the changes inhemical properties (pH, EC, OC, AN, AP, and AK) of the soil were notignificant (P ≥ 0.05) due to cropping mode and harvesting regimesTable 4). Though the effect of life cycle was not significant, the

arginally lower values of AN (296.87 kg ha−1), AP (31.73 kg ha−1),nd AK (492.99 kg ha−1) were registered with the perennial cropompared with the annual crop. On the other hand, the reverseesults were found in the case of pH and OC. Irrespective of crop-ing modes, the three-cut management system maintained highestP (33.53 kg ha−1) and AK (532.54 kg ha−1), whereas the maximumN (315.95 kg ha−1) and OC (2.08) were recorded with the single-ut management system. The interaction effects of life cycles andarvesting regimes were also insignificant (P ≥ 0.05) except AP.

. Discussion

Number of branches plant−1, one of the yield-attributes of ste-ia, was significantly (P ≤ 0.05) affected by the life cycle, and theaximum number was recorded with the perennial crop under all

utting management systems. This result might be attributed to thextensive root systems, which enhanced cytokinin synthesis andheir eventual export to the shoot in the perennial crop. Cytokininsre mostly synthesized in the root tips (Miyawaki et al., 2004).ytokinin plays a vital role for hastening the growth of the dormantxillary buds. In our study, higher root biomass was also observedith the perennial crop, regardless of harvesting regimes. Similar to

he branches, the maximum LAI was also recorded with the peren-ial stevia under all cutting management systems. The increase inAI of the perennial stevia was probably due to higher number ofranches, and it increased the activity of cytokinin that delayed leafenescence. Dong et al. (2012) also reported that local increases inytokinin production by the roots delayed the leaf senescence. Theffect of harvesting regime on total LAI was significant (P ≤ 0.05).owever, the effect was more pronounced under the perennialropping mode, and the highest magnitude was registered withhree-cut management system. On the other hand, three-cut man-gement system did not produce higher LAI compared with two-cutanagement under the annual cropping mode. This result might be

ttributed to reduction of the carbohydrate reserve, which influ-nced the leaf area development and affected the growth rate ofhe plant.

The traditional recommendation for stevia harvest is at the onsetf flowering stage in relation to higher SGs content in leaves; never-heless, the harvesting time of stevia should be decided based on theypes of cropping mode and on a sound understanding of growthehaviors and survival mechanism to growing conditions. In ourtudy, the perennial stevia produced significantly (P ≤ 0.05) higherry leaf and stem yield compared with the annual stevia at all har-


esting stages. These results may be due to the fact that the shootsmerge from rhizomes of the perennial stevia in the early springt least month before seedling transfer of annual stevia. Moraest al. (2013) reported that stevia shoots during re-growth formed a

PRESSProducts xxx (2014) xxx–xxx 7

cluster of shoots at the adjunction of stem and adventitious roots. Ithas also been reported that the dry leaf yield at 1st and 2nd growingseason is less than the yield recorded at 3rd growing season (Moraeset al., 2013). Furthermore, due to extensive living root systems ofthe perennial stevia, rhizome-derived shoots of stevia grow morerapidly compared with newly transplanted small seedlings. Conse-quently, the maximum leaf yield was obtained with the perennialstevia as a result of higher LAI, which enhanced light interceptioncapacity.

It has also been reported that the perennial plants generallyencompass longer growing seasons, and its deeper rooting systemutilizes more of the natural precipitation compared with annuals(Tilman et al., 2006; Dohleman and Long, 2009; Glover et al., 2009).Glover et al. (2010) reported that long-lived plants built up largerand deeper root system compared with short-lived plants, and thedeeper roots facilitated the perennials to mine a larger volume ofsoil. On the other hand, due to earlier canopy development andlonger life of green leaves longer photosynthetic season increaseslight interception efficiency, which is an important factor for plantproductivity (Dohleman and Long, 2009). Moreover, the perennialcrops can absorb nutrients during parts of the year when croplandremains completely bare or partially covered by small seedlingswith shallow roots of the annual crops (Cox et al., 2006).

The responses of stevia biomass yields (dry leaf and stem)to the different harvesting regimes were significantly (P ≤ 0.05)changed under both the cropping modes. However, the effect ofharvesting regime on dry leaf yield was more pronounced underthe perennial cropping system. These results might be due to thefact that the perennial stevia stored high energy reserves in rootand crowns, which hastened the rate of regrowth. In our study,the regrowth yields of two and three-cut management systemcontributed a smaller amount to total yield compared with first-cut yield under both the cropping modes. This result might beattributed to low sunshine hours (Fig. 2) prevailed during rainyseason, which seemed to suppress the regrowth capacity of steviaafter the first-cut. Serfaty et al. (2013) also reported that biomassproduction of stevia at second harvest was greatly reduced whenplants were harvested from June onwards. The contribution ofregrowth yield in three-cut was very nominal, particularly in theannual stevia. This result might be due to insufficient quantity ofenergy reserves stored in the roots and crowns by the perennialstevia for regrowth. On the other hand, the weather conditions,particularly day-length and temperature were not conducive forvegetative growth after the second-cut. It had also been reportedthat regrowth yields of switchgrass had a smaller contribution tototal yield compared with a first-cut yield (Vogel et al., 2002).

In this study, the accumulation pattern of stevioside in leaveswas not greatly affected at first-cutting stage in both the croppingmodes. However, in subsequent stages, the variation was distinct,and higher values were attained with the perennial stevia. Thisresult might be due to the fact that before first-cutting, plantsgot sufficient time for phenotypic expression and utilized longerphotoperiod for accumulating the stevioside. Tavarini and Angelini(2013) also reported that SGs profile in stevia leaves was influ-enced by the age of plant and developmental phase. Since steviais a photoperiod sensitive (Kang and Lee, 1981), its morphologicaldevelopment is largely determined by the length of photoperiod. Ithas been reported that long-day condition increases leaf biomassand stevioside content in stevia leaves (Ceunen and Geuns, 2013).In our study, fluctuation of stevioside content was also observed atdifferent harvesting stages under both the annual and the perennialcropping systems. In case of the annual stevia, total SGs content in


leaves was remarkably reduced during second-cut and third-cut fortwo-cut and three-cut management system, respectively. In caseof the perennial, total SGs content was reduced during third-cutof three-cut management system. This result may be due to the



8 P.K. Pal et al. / Industrial Crops and Products xxx (2014) xxx–xxx

Table 4Effect of life cycle and harvest regime on chemical properties of soil.

Treatment pH (1:2.0) EC25

(m mhos cm−2)Organiccarbon (%)

Available nitrogen(kg ha−1)

Available phosphorus(kg ha−1)

Available potassium(kg ha−1)

Life cycle (C)Annual 6.01 0.064 1.91 323.75 35.70 511.91Perennial 6.16 0.060 2.15 296.87 31.73 492.99SEm(±) 0.13 0.005 0.16 17.89 4.58 25.88CD (P = 0.05) NS NS NS NS NS NSHarvest regime (H)Single cut 5.96 0.065 2.08 315.95 33.45 456.29Double cut 6.22 0.060 2.08 307.32 31.06 518.53Triple cut 6.09 0.060 1.92 307.66 33.53 532.54SEm(±) 0.16 0.01 0.20 21.91 5.61 31.69CD (P = 0.05) NS NS NS NS NS NSInteractionSEm(±) for C × H 0.22 0.01 0.28 3.99 7.94 44.82

N

famcbsGa

(blsnui(Ictac

5

ycmhtrfmoitahpt

A

Dp

CD (P = 0.05) for C × H NS NS NS

S indicates non-significant at P = 0.05.

act that the biosynthesis process of SGs for re-growing leaves isdversely affected by short sunshine hours (Fig. 1). Overall maxi-um total SGs yield (g plant−1) was recorded with the perennial

rop under two-cut management system. This result was proba-ly due to higher concentration of SGs in leaves at both harvestingtages coupled with moderate biomass production. Ceunen andeuns (2013) also observed linear relationship between dry matternd total SGs yield.

In this study, the soil reaction (pH), EC, OC, and nutrient statusAN, AP, and AK) of the soil were not changed significantly (P ≥ 0.05)y the crop cycles and harvesting regimes; however, the slightly

ower values of AN, AP, and AK were observed with the perennialtevia. These decreased nutrient status of soil under the peren-ial stevia may be attributed to the extensive deep roots, whichptakes more nutrients from larger volume of the soil. This result

s confirmed by the significantly higher toot biomass productionFig. 3c) by the perennial stevia compared with the annual stevia.n contrast, the annual stevia reduced the soil OC. This result is inonformity with the finding of Glover (2005) who reported thathe annual crop with less extensive root systems utilized waternd nutrients less effectively and stored less carbon below groundompared with perennial plant communities.

. Conclusion

From our studies, it is clear that the dry leaf yield (t ha−1) and SGsield (g plant−1) of stevia are considerably affected by the crop lifeycles and harvesting regimes. The perennial stevia with three-cutanagement system registered maximum dry leaf yield; however,

ighest total SGs yield was recorded with the perennial stevia underwo-cut management system. The effect of life cycle and harvestingegimes on soil nutrient status was not significant (P ≥ 0.5). There-ore, it can be concluded that the perennial stevia with two-cut

anagement system is most effective under sub-temperate regionf western Himalaya in India to increase the total SGs yield, whichs the key interest of the stevia growers. Further research is neededo understand the physiological basics associated with yield vari-tion between the annual and the perennial stevia under differentarvesting regimes. The standardization of nutritional dose for theerennial stevia is also needed under different harvesting regimeso increase the biomass yield and quality.

cknowledgements


The authors wish to sincerely thank to Dr. R. D. Singh, Head,ivision of Biodiversity, for his constructive suggestions in thereparation of the manuscript. The authors are also grateful to

NS 25.01 NS

Mr. Kuldeep Singh Gill for field management. The authors acknowl-edge the Council of Scientific and Industrial Research (CSIR),Government of India, for financial support. This research work hasbeen undertaken under the CSIR network project- BSC-0110 andMLP 0010.

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Documents

Harvesting regimes to optimize yield and quality in annual and perennial Stevia rebaudiana under sub-temperate conditions