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Scientia Horticulturae 162 (2013) 242–251

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epa ge: www.elsev ier .com/ locate /sc ihor t i

hort communication

ater-deficit tolerant identification in sweet potato genotypesIpomoea batatas (L.) Lam.) in vegetative developmental stage using

ultivariate physiological indices

uravoot Yooyongwecha, Cattarin Theerawitayab, Thapanee Samphumphuangb,uriyan Cha-umb,∗

Department of Agricultural Science, Mahidol University, Kanchanaburi Campus, Kanchanaburi 71150, ThailandNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailandcience Park, Pahonyothin Road, Khlong Nuang, Khlong Luang, Pathum Thani 12120, Thailand

r t i c l e i n f o

rticle history:eceived 13 May 2013eceived in revised form 25 July 2013ccepted 29 July 2013

eywords:ree prolinerowth performanceet photosynthetic ratesmotic potentialard’s cluster analysis

a b s t r a c t

Sweet potato (Ipomoea batatas [L.] Lam.) is one of three main storage root crops of global importance afterpotato and cassava. It serves as the primary source of carbohydrate for the world population in developingcountries. Sweet potato has been reported as drought sensitive, especially in the rain fed region withextended drought condition. Some cultivars might be more tolerant to drought stress compared to others.We investigated the physiological and morphological responses and storage root yield attributes of 6sweet potato genotypes (cvs. Manphuang and Mankorat, PROC 65-3, Banyang 9, Tainung 57 and Japaneseyellow) to water deficit stress (15% SWC) with an aim to classify the water deficit tolerance using therelationship between free proline and osmotic adjustments. Osmotic potential (� s), chlorophyll b (Chlb),maximum quantum yield of PSII (Fv/Fm), plant height and number of leaves in sweet potato cv. PROC65-3 grown under water deficit condition (15% SWC) were better than those in other cultivars. Theyield reduction, growth inhibition, free proline enrichment, osmotic potential maintaining, chlorophylldegradation, chlorophyll fluorescence diminution, net photosynthetic rate (Pn), stomatal conductance

(gs) and transpiration rate (E) reduction in water deficit stressed plants were subjected to Ward’s clusteranalysis. Mankorat, PROC 65-3 and Japanese Yellow were classified as water deficit tolerance whereasManphuang, Banyang 9 and Tainung 57 genotypes were evaluated as water deficit sensitive. The studyconcludes that free proline accumulation may play a key role as major osmotic adjustment in sweetpotato and negative correlated with osmotic potential of leaf tissues when plants subjected to waterdeficit.

. Introduction

Drought is one of the major abiotic stresses that pose a serioushreat to the world food security, especially in the changing climatecenario (Jovanovic and Stikic, 2012). Most of crop species need toonsume the water throughout their life cycle from germinationo grain harvesting. It has been estimated that agricultural sectorlone accounts for more than two third of total water consumptionFereres and Evans, 2006). Good irrigation practices depending onlant water requirement help in preventing water loss via evapo-

ranspiration (Costa et al., 2007; Huzsvai and Rajkai, 2009). Croproductivity in irrigated agriculture is more than double of that inhe rain-fed area. However, irrigation area still needs to expand by

∗ Corresponding author. Tel.: +66 2 5646700.E-mail address: suriyanc@biotec.or.th (S. Cha-um).

304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2013.07.041

© 2013 Elsevier B.V. All rights reserved.

more than 20% to increase crop yield by 40% and provide the foodfor 8 billion people by 2025 (Lascano and Sojka, 2007). Further,the irrigation management and integrating crop species with lowwater consumption in the rain-fed area of arid and semiarid zonesare one of the major challenges for researchers’ world over.

The photosynthetic ability of the plant is very sensitive todrought stress. Under drought, water in the surface soil is limited,thus deep root system may play a key role as first signal perceptionto find-out available water (Kim et al., 2009; Lewthwaite and Triggs,2012). A limited water supply acts as a signal for ABA (abscisic acid)biosynthesis and regulates stomata closure to prevent the waterloss, leading to limit the CO2 assimilation in the dark reaction(Peuke et al., 2002; van Heerden and Laurie, 2008; Pinheiro and

Chaves, 2010). In the light reaction, low maximum quantum yieldof PSII (Fv/Fm) or water oxidation in the chloroplastic organelle isrestricted (Haimeirong and Kubota, 2003; Yadollahi et al., 2011).Further, the osmoprotectants, i.e., carbohydrates, proline, sugar

S. Yooyongwech et al. / Scientia Hort

Table 1Genotypic background of sweet potato cultivars used in the present study.

Genotypes Origin Leaf form Fresh te xture color

Manphuang Native cultivar Palm Whi te

Mank orat Native cul tivar Heart Cream

PROC 65-3 Breedi ng cul tivar Heart Purple red

Banya ng 9 Breedi ng cul tivar Heart Dark p urple

ascanstCaiest(ttts(Y

stw2wcaistpc2moparw

2

2

i65G(w(g

Tai nung 57 Imported cultivar Palm Yellow

Japan ese Yellow Imported cul tivar Heart Yellow

lcohols, polyamine and glycine betaine, are evidently biosynthe-ized and accumulated to function as osmotic adjustment in theellular level (Sánchez et al., 1998; Babita et al., 2010; Bandurskand Józwiak, 2010). Proline, a small aromatic amino acid, is aeutral molecule without toxicity to the cells. It quickly synthe-izes from glutamate and/or ornithine pathway in the plant cellso play a key role in osmotic adjustment (Hare and Cress, 1997;haitanya et al., 2009; Verslues and Juenger, 2011). When plantsre exposed to drought conditions, non-enzymatic compounds,.e., �-tocopherol, ascorbic acid, glutathione and carotenoids, andnzymatic antioxidants, including catalase, ascorbate peroxidase,uperoxide dismutase and glutathione transferase, are enhancedo reduce the reactive oxygen species (ROS) generated in the cellGuha et al., 2010, 2012; Jan et al., 2012; Kim et al., 2013). Droughtolerance trait is under complex genetic controls that are fruitfulo be overcome by plant breeders. There are many strategieso search drought tolerant plant, i.e., natural selection (droughtcreening), conventional cross, marker aided selection using QTLquantitative trait loci), and transgenic approaches (Ahraf, 2010;ang et al., 2010; Qin et al., 2011; Marguerit et al., 2012).

Sweet potato is storage root crop species with multiple uses,uch as food, green vegetable and animal feed, for biofortifica-ion and bioethanol production, and used in many regions of theorld (Ziska et al., 2009; Mukhopadhyay et al., 2011; Laurie et al.,

012). It has been reported as drought sensitive plant species,hich are reduced water requirement (Gomes and Carr, 2003a),

rop yield (Gomes and Carr, 2003b), dry matter (Gomes et al., 2005)nd storage root (Gomes and Carr, 2001). Over 10,000 accessions,ncluding native cultivars, released cultivars and breeding lines, ofweet potato are available in different germplasm banks aroundhe world. Previously, the drought tolerant identification in sweetotato genotypes was based upon single parameter of physiologi-al, morphological and/or yield character (Haimeirong and Kubota,003; Saraswati et al., 2004). In addition, stress tolerant index andaintenance of yield potential have also been investigated as one

f the criteria for drought tolerant indices in orange-fresh sweetotato (Agili et al., 2012). In the present study, the native, breedingnd imported cultivars of sweet potato were used as initial mate-ial to investigate the physiological and morphological responses toater deficit and then to classify either tolerant or sensitive groups.

. Materials and methods

.1. Plant materials and water deficit treatments

Six sweet potato (Ipomoea batatas [L.] Lam.) genotypes, includ-ng native (cvs. Manphuang and Mankorat), breeding (cvs. PROC5-3 and Banyang 9) and imported cultivars (cvs. Tainung7 and Japanese yellow), procured from Agricultural Extensionroup, Phichit province, Thailand, were used as initial materials

Table 1). Single vine (12 ± 1 cm in length) without leaf bladesas planted into plastic pots (ø = 20 cm) containing 2 kg mixed soil

EC = 2.687 dS m−1; pH = 5.5; organic matter = 10.36%; total nitro-en = 0.17%; total phosphorus = 0.07%; total potassium = 1.19%). The

iculturae 162 (2013) 242–251 243

plants were grown in a greenhouse under 500–1000 �mol m−2 s−1

photosynthetic photon flux density (PPFD) with a 10 h d−1 pho-toperiod, 28 ± 2 ◦C temperature and 80 ± 5% RH, for a period of 4weeks. Thereafter, two groups of plants: well watering (WW; 47%soil water content) and water withholding for 9 days (WD; 15% soilwater content [SWC]) were set as the experimental layout. After9 days, growth characters (length of vine and number of leaves),osmotic potential, free proline content, photosynthetic pigments,chlorophyll fluorescence, net photosynthetic rate, stomatal con-ductance and transpiration rate were measured. In addition, waterdeficit and control sweet potato plants were re-watered and culti-vated prior to storage root harvesting (160 days after cutting). After160 days, the vine weight, root weight and storage root yield weredetermined.

2.2. Soil water content (SWC)

Soil samples were collected at 0, 3, 6, 9 and 12 day after waterwithholding. Soil water content (SWC) was calculated using theweight fraction as: SWC (%) = [(FW − DW)/DW] × 100, where FWwas the fresh weight of a portion of the soil from the internal areaof each pot and DW was the dry weight of the soil portion afterdrying in a hot air oven at 85 ◦C for 4 days (Coombs et al., 1987).

2.3. Free proline determination

Free proline in the leaf tissues was extracted and analyzedaccording to the method of Bates et al. (1973). In brief, fifty mil-ligram of fresh material was ground with liquid nitrogen in amortar. The homogenate powder was mixed with 1 mL aqueoussulfosalicylic acid (3%, w/v) and filtered through Whatman #1 filterpaper. The extracted solution was reacted with an equal volumeof glacial acetic acid and ninhydrin reagent (1.25 mg ninhydrin in30 mL glacial acetic acid and 20 mL 6 M H3PO4) and incubated at95 ◦C for 1 h. The reaction was terminated by placing the containerin an ice bath. The reaction mixture was mixed vigorously with 2 mLof toluene. After cooling to 25 ◦C, the chromophore was measuredat 520 nm on UV-VIS spectrophotometer (HACH DR/4000; Model48000, HACH Company, Loveland, Colorado, USA) using l-prolineas a standard.

2.4. Osmotic potential determination

The osmolarity of leaf tissues was measured according to themethod of Lanfermeijer et al. (1991). In brief, one hundred mil-ligram of fresh leaf tissue was cut into small pieces, transferred to1.5 mL micro tube, and then crushed by stirring with a glass rod. The20 mL of extracted solution was dropped directly onto a filter paperin an osmometer chamber (5520 Vapro®, Wescor, Utah, USA). Then,the osmolarity (mmol kg−1) was converted to osmotic potential(MPa) using conversion factor of osmotic potential measurement.

2.5. Photosynthetic pigment

Chlorophyll a (Chla), chlorophyll b (Chlb) and total chlorophyll(TC) concentrations were analyzed following Shabala et al. (1998)and total carotenoid (Cx+c) concentrations were assayed accordingto Lichtenthaler’s (1987) method. In brief, one hundred milligramof leaf material was collected from the second and third nodesof the shoot tip. The leaf samples were placed in 25 mL glassvial (Opticlear®; KIMBLE, Vineland, NJ, USA) along with 10 mL of95.5% acetone, and blended with a homogenizer (T25 basic Ultra-

Turrax®; IKA, Kuala Lumpur, Malaysia). The glass vials were sealedwith Parafilm® to prevent evaporation and then stored at 4 ◦C for48 h. Chla, Chlb and Cx+c concentrations were measured using a UV-VIS spectrophotometer (DR/4000; Hatch, Loveland, CO, USA) at 662,

244 S. Yooyongwech et al. / Scientia Horticulturae 162 (2013) 242–251

F nder

l how s

6a

2

t(aei(amuscmq(oJ

ig. 1. Vine length (A) and number of leaves (B) of sweet potato genotypes grown uight bar) in the pot culture. Error bars represent ±SE. Different letters in each bar s

44 and 470 nm, respectively. A solution of 95.5% acetone was useds a blank.

.6. Chlorophyll fluorescence

Chlorophyll fluorescence emission from the adaxial surface onhe leaf was measured using a fluorescence monitoring systemFMS 2; Hansatech Instruments Ltd., Norfolk, UK) in the pulsemplitude modulation mode as previously described by Logginit al. (1999). A leaf, adapted to dark conditions for 30 min, wasnitially exposed to the modulated measuring beam of far-red lightLED source with typical peak at wavelength 735 nm). Original (F0)nd maximum (Fm) fluorescence yields were measured under weakodulated red light (<0.5 �mol m−2 s−1) with 1.6 s pulses of sat-

rating light (>6.8 �mol m−2 s−1 PAR) and calculated using FMSoftware for Windows®. The variable fluorescence yield (Fv) wasalculated by the equation: Fv = Fm − F0. The ratio of variable toaximum fluorescence (Fv/Fm) was calculated as the maximum

uantum yield of PSII photochemistry. The photon yield of PSII˚PSII) in the light was calculated as ˚PSII = (F ′

m − F)/F ′m after 45 s

f illumination, when steady state was achieved (Maxwell andohnson, 2000).

47% SWC (WW; well watering a dark bar) and 15% SWC (WD; water deficit stress aignificant difference at p ≤ 0.01 by Tukey’s HSD.

2.7. Net photosynthetic rate (Pn), stomatal conductance (gs) andtranspiration rate (E)

Net photosynthetic rate (Pn; �mol m−2 s−1), stomatal con-ductance (gs; mmol CO2 m−2 s−1) and transpiration rate (E;mmol m−2 s−1) were measured using a Portable PhotosynthesisSystem with an Infra-red Gas Analyzer (Model LI 6400, LI-COR®

Inc., Lincoln, Nebraska, USA). E was measured continuously by mon-itoring the content of the air entering and existing in the IRGAheadspace chamber according to Cha-um et al. (2007).

2.8. Experiment design and statistical analysis

The experiment was arranged as 6 × 2 factorial in a Com-pletely Randomized Block Design (CRBD) with eight replicates(n = 8). Analysis of variance (ANOVA) was calculated and themean values obtained were compared using Tukey’s HSD by SPSSsoftware ver. 11.5. Free proline accumulation, osmotic potential

adjustment, photosynthetic pigment degradation, chlorophyll flu-orescence diminution, Pn, gs and E reduction, growth inhibitionand storage root decrease of sweet potato genotypes grown underwater deficit stress were assessed in order to classify cultivars as

S. Yooyongwech et al. / Scientia Horticulturae 162 (2013) 242–251 245

F t pota( ifferen

ec

3

3

3rtdigl(fiw

3

m

ig. 2. Free proline content (A) and osmotic potential (B) in the leaf tissues of sweeWD; water deficit stress a light bar) in the pot culture. Error bars represent ±SE. D

ither tolerant or sensitive using Ward’s method of Hierarchicalluster analysis in SPSS software ver. 11.5.

. Results

.1. Growth performances

Vine length of all sweet potato genotypes, except cv. PROC 65-, declined significantly under 15% SWC, (Fig. 1A). The percenteduction in vine length ranged from 32.56% (in cv. Japan Yelow)o 46.54% (in cv. Banyang 9) in plant grown under 15% SWC (9ays water withholding) when compared to control or well water-

ng (47% SWC). Likewise, the number of leaves in sweet potatoenotypes, Manphuang, Mankorat, Banyang 9 and Japanese yel-ow, grown under 15% SWC declined significantly (22.65–28.38%)Fig. 1B). Based on growth performances, PROC 65-3 was identi-ed as water deficit tolerant genotype to continuously grow underater deficit.

.2. Free proline and osmotic potential in the leaf tissues

Free proline in the leaf tissues of sweet potato genotypes accu-ulated in relation to reduced SWC. In control, the level of free

to genotypes grown under 47% SWC (WW; well watering a dark bar) and 15% SWCt letters in each bar show significant difference at p ≤ 0.01 by Tukey HSD.

proline was very low (≤0.5 �mol g−1 FW); however, it increasedrapidly when plants were subjected to water deficit (Fig. 2A). Theaccumulation of free proline varied with genotypes, and rangedfrom 4.16 to 39.12 folds greater over that in the control. The greatestaccumulation of free proline was in cv. Japanese Yellow, wherein itwas 39.12 folds (i.e., 6.65 �mol g−1 FW) more of that in the controlwas peaked.

Osmotic potential (� s) in the leaf tissues of cvs., PROC 65-3 (5.62% reduction) and Japanese Yellow (3.43% reduction), wasmaintained when plant were exposed to water deficit (Fig. 2B).In contrast, � s dropped drastically (18.34–25.31% reduction) inother genotypes. A positive relationship was observed betweenfree proline accumulation and osmotic adjustment (Fig. 3). Theenrichment of free proline in Japanese Yellow and PROC 65-3 wasdirectly involved in controlling � s in the water deficit stressedleaves. Further, the reduced � s in the water deficit stressed leavesconsequently damaged the photosynthetic pigment (Fig. 4A).

3.3. Photosynthetic abilities

Chla, Chlb, TC and Cx+c in all six sweet potato genotypes weredegraded (significant at P ≤ 0.05) when plants were subjected towater deficit (Table 2). Chla degradation percentage ranged from

246 S. Yooyongwech et al. / Scientia Hort

y = -0.54 x + 22.8 0

R² = 0.59**

0

5

10

15

20

25

30

0 10 20 30 40 50

Free proline accumul ation (f olds)

Osm

oti

c p

ote

nti

al

ad

just

men

t (%

)

Fas

6C6utcPq˚d

(43.04%) plants grown under water deficit stress. In addition, stor-

Fc(

ig. 3. Relationships between free proline accumulation and osmotic potentialdjustment in the leaf tissues of sweet potato genotypes grown under water deficittress in the pot culture.

0.98% (in cv. PROC 65-3) to 72.10% (in Banyang 9). Interestingly,hlb content in cvs. Mankorat (57.23% reduction) and PROC5-3 (32.86% reduction) grown under 15% SWC was statisticallynchanged (Table 2). Cx+c content in Mankorat (11.99% reduc-ion) and PROC 65-3 (39.30%) were better than those in otherultivars. TC content was very sensitive to water deficit stress.

ositive relationships were observed between Chla and maximumuantum yield of PSII (Fv/Fm), TC and photon yield of PSII (˚PSII),PSII and net photosynthetic rate (Pn) (Fig. 4B–D). Fv/Fm and ˚PSII

eclined significantly under water deficit (Table 3). Diminution of

y = 904 .79x + 1244 .2

R² = 0.61**

0

100

200

300

400

500

600

-1.50 -1.0 0 -0.5 0 0.0 0

y = 0. 0003 x + 0.48 03

R² = 0.53* *

0.00

0.20

0.40

0.60

0.80

1.00

0 10 0 200 30 0 400 50 0 600

Osmotic potential (MPa)

(A)

Total chloroph yll cont ent (μg g-1 FW)

ΦP

SII

(C)

Tota

l chlo

rop

hy

ll con

tent ( μ

g g

-1 FW

)

WW

WD

WWWD

ig. 4. Relationships between (A) osmotic potential and total chlorophyll content, (B) chloontent and photon yield of PSII (˚PSII), and (D) ˚PSII and net photosynthetic rate (Pn) in swater deficit stress; WD) in the pot culture. Error bars represent ±SE.

iculturae 162 (2013) 242–251

Fv/Fm in cvs. Mankorat, PROC 65-3 and Banyang 9, grown underwater deficit stress was lower than 10%, which was better thanother three cultivars (Manphuang [19.97% diminution], Tainung57 [19.42% diminution] and Japanese Yellow [26.19% diminution]).The reduction in ˚PSII activity in Banyang 9 and Japanese Yellowunder water deficit was only 10.40% and 12.27%, respectively.Degradation of photosynthetic pigment and diminution of chloro-phyll fluorescence in water deficit stressed plants directly affectedPn. Pn, stomatal conductance (gs) and transpiration rate (E) inwater deficit stressed plants dropped sharply compared to thosein control plants (well watering). In Japanese Yellow, the E wasunchanged under water deficit condition.

3.4. Yield attributes

Vine yield in all sweet potato genotypes at the harvestingstage (160 days after cutting) declined by 11.34% (Japanese Yel-low) − 41.33% (Tainung 57). The vine yield of Manphuang, Banyang9 and Tainung 57 dropped by 21.35%, 30.43% and 41.33% (signifi-cant at P < 0.05), respectively, in plants grown under water deficitstress and subsequently re-watered prior to storage root harvesting(Table 4). Total root weight in all genotypes also decreased, partic-ularly in Manphuang (42.64%), Banyang 9 (44.08%) and Tainung 57

age root yield trait declined considerably in Manphuang (57.39%),Banyang 9 (46.28%), Tainung 57 (63.58%) and Japanese Yellow(35.83%) (Table 4; Fig. 5).

y = 0.000 9x + 0.61 36

R² = 0.4 2**

0.00

0.20

0.40

0.60

0.80

1.00

0 50 100 150 20 0 25 0 30 0

y = 23.87 x - 10.4 6

R² = 0.42* *

0

2

4

6

8

10

12

0.00 0.2 0 0.4 0 0.6 0 0.8 0 1.00

Fv/F

m

Chloroph yll a content (μg g-1

FW)

(B)

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Net

ph

oto

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WW

WD

WW

WD

rophyll a content and maximum quantum yield of PSII (Fv/Fm), (C) total chlorophyllweet potato genotypes grown under 47% SWC (well watering; WW) and 15% SWC

S. Yooyongwech et al. / Scientia Horticulturae 162 (2013) 242–251 247

Table 2Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC) and total carotenoid (Cx+c) contents in sweet potato genotypes grown under 47% SWC (WW; well watering)and 15% SWC (WD; water deficit stress) in the pot culture. Figures in parentheses represent percent reduction in photosynthetic pigments of water-deficit stressed plants ineach cultivar.

Genotypes Treatment Chla Chlb TC Cx+c

(�g g−1 FW) (�g g−1 FW) (�g g−1 FW) (�g g−1 FW)

Manphuang WW 334.57b 144.31b 478.88b 45.44cWD 97.34d 40.05d 137.39d 25.77e

(70.91%) (72.25%) (71.31%) (43.29%)

Mankorat WW 267.78bc 119.81bc 387.59bc 64.05aWD 101.67d 51.24cd 152.91d 56.37b

(62.03%) (57.23%) (60.55%) (11.99%)

PROC 65-3 WW 238.05c 92.17cd 330.22c 36.97dWD 92.90d 61.88cd 154.78d 22.44e

(60.98%) (32.86%) (53.13%) (39.30%)

Banyang 9 WW 279.05a 240.67a 519.72a 69.77aWD 77.68d 42.95d 120.81d 20.16e

(72.10%) (82.15%) (76.76%) (71.11%)

Tainung 57 WW 252.04c 110.59bc 362.63bc 64.80aWD 98.15d 43.89d 142.04d 34.80d

(61.06%) (60.31%) (60.83%) (46.30%)

Japanese WW 240.68c 142.34b 383.02bc 56.06bYellow WD 90.82d 37.38d 128.20d 23.17e

(62.27%) (73.74%) (66.53%) (56.67%)

Significant levelGenotype (G) ** ** ** **Treatment (T) ** ** ** **G × T ** ** ** **

D ’s HS

3

�d

TM(s

D

ifferent letters in each column show significant difference at p ≤ 0.01 (**) by Tukey

.5. Cluster ranking analysis

Yield reduction, growth inhibition, free proline enrichment,s maintaining, chlorophyll degradation, chlorophyll fluorescence

iminution, Pn, gs and E reduction in water deficit stressed plants

able 3aximum quantum yield of PSII (Fv/Fm), photon yield of PSII (˚PSII), net photosynthetic ra

WW; well watering) and 15% SWC (WD; water deficit stress) in the pot culture. Figures in

tressed plants in each cultivar.

Genotypes Treatment Fv/Fm ˚PSII

Manphuang WW 0.661cd 0.590abc

WD 0.529e 0.482d

(19.97%) (18.31%)

Mankorat WW 0.759ab 0.652a

WD 0.711bcd 0.554bcd

(6.32%) (15.03%)

PROC 65-3 WW 0.728bcd 0.566bc

WD 0.655d 0.480d

(10.03%) (15.19%)

Banyang 9 WW 0.812ab 0.654a

WD 0.733bc 0.586abc

(9.73%) (10.40%)

Tainung 57 WW 0.824a 0.629ab

WD 0.664cd 0.546cd

(19.42%) (13.29%)

Japanese WW 0.779ab 0.546cd

Yellow WD 0.575e 0.479d

(26.19%) (12.27%)

Significant levelGenotype (G) ** **

Treatment (T) ** **

G × T ** **

ifferent letters in each column show significant difference at p ≤ 0.01 (**) by Tukey’s HS

D.

were subjected to Ward’s method in SPSS for cluster ranking group.

Mankorat, PROC 65-3 and Japanese Yellow were classified as waterdeficit tolerant. In contrast, Manphuang, Banyang 9 and Tainung57 genotypes were evaluated as water deficit sensitive cultivars(Fig. 6).

te (Pn) and transpiration rate (E) in sweet potato genotypes grown under 47% SWCparentheses represent percent reduction in photosynthetic abilities of water-deficit

Pn gs E(�mol m−2 s−1) (mmol CO2 m−2 s−1) (mol H2O m−2 s−1)

7.75bc 35.30a 1.13ab0.87g 4.30e 0.23d(88.77%) (87.82%) (79.65%)

8.81ab 27.48bc 1.31a3.35e 13.03d 0.37cd(61.98%) (52.58%) (71.76%)

10.25a 34.00ab 1.02b7.31bc 23.95c 0.38cd(28.68%) (29.56%) (62.75%)

10.08a 39.48a 1.40a3.00ef 4.78e 0.22d(70.24%) (87.89%) (84.29%)

8.81ab 32.38ab 0.88b1.68fg 3.03e 0.28d(80.93%) (90.46%) (68.18%)

8.42bc 32.68ab 0.59c5.35d 20.28cd 0.36cd(36.46%) (37.94%) (38.98%)

** ** **** ** **** ** **

D.

248 S. Yooyongwech et al. / Scientia Horticulturae 162 (2013) 242–251

Table 4Vine fresh weight, root fresh weight and storage root yield of sweet potato genotypes grown under 47% SWC (WW; well watering) and 15% SWC (WD; water deficit stress)in the pot culture subsequently re-watering prior to storage root harvesting (160 days after cutting). Figures in parentheses represent percent reduction in photosyntheticabilities of water-deficit stressed plants in each cultivar.

Genotypes Treatment Vine weight Root weight Storage root yield(g) (g) (g)

Manphuang WW 21.92cd 23.22a 26.40aWD 17.24ef 13.32cd 11.25d

(21.35%) (42.64%) (57.39%)

Mankorat WW 32.57ab 20.96ab 29.88aWD 27.18bc 17.11bc 25.36ab

(16.55%) (18.37%) (15.13%)

PROC 65-3 WW 24.50cd 16.50bc 16.24cdWD 20.34cd 13.12cd 12.53cd

(16.98%) (20.49%) (22.84%)

Banyang 9 WW 20.21cd 22.55a 25.65abWD 14.06ef 12.61cd 13.78cd

(30.43%) (44.08%) (46.28%)

Tainung 57 WW 21.85cd 23.00a 30.29aWD 12.82f 13.10cd 11.03d

(41.33%) (43.04%) (63.58%)

Japanese WW 37.05a 16.93bc 29.42aYellow WD 32.85ab 12.02cd 18.88bc

(11.34%) (29.00%) (35.83%)

Significant levelGenotype (G) ** ** **Treatment (T) ** ** **

D y’s HS

4

oPvRrfi((tiawFapTtd

saoi(dpnstdtsw

G × T **

ifferent letters in each column show significant difference at p ≤ 0.01 (**) by Tuke

. Discussion

Growth characters, including vine length and number of leaves,f sweet potato genotypes were significantly dropped, except inROC 65-3, which was identified as water deficit tolerant. In pre-ious report, vine length of sweet potato cultivars, Isondlo andesisto (drought sensitive check) retarded by 67.53% and 56.41%,espectively, over the control, when plants were subjected to 30%eld capacity (FC) in field trial for 155 days after stress initiationLaurie et al., 2009). In contrast, reduction of vine length in cv. W119drought tolerant check), was only 33.42% when compared to con-rol (100% FC) (Laurie et al., 2009). Likewise, biomass, vine length,nternodal diameter, internodal length, number of leaves, leaf areand root weight of 15 cultivars of sweet potato were decreasedhen plants were exposed to water withholding in the pot culture.

or the reduction in vine length in sweet potato cultivars, Hawaiind Lole, was the lowest, i.e., 13.73% and 14.31%, respectively, whenlants were subjected to water deficit stress (Saraswati et al., 2004).he reduction in overall growth performance subsequently affectedhe total yield and marketable yield of sweet potato, especially inrought sensitive cultivars (Laurie et al., 2009).

We observed an accumulation of free proline in the leaf tis-ues of water deficit stressed plants (15% SWC). These findingsre supported by similar observation in S13 and BC2-59 genotypesf mulberry under water deficit stress and it related to decreasen water potential in the leaf tissues (from −0.75 to −2.25 MPa)Chaitanya et al., 2009). Also, the activity of enzymes, viz., glutamateehydrogenase (GDH), proline-5-carboxylase synthetase (P5CS),roline-5-carboxylate reductase (P5CR) and ornithine transami-ase (OT) related to proline biosynthesis, was regulated when plantubjected to water deficit stress. On the other hand, a down regula-ion in the activity of proline dehydrogenase (PDH) activity in water

eficit stressed plants was observed, resulting in greater accumula-ion of free proline in the leaf tissues (Chaitanya et al., 2009). Earliertudies have demonstrated increase in free proline content underater deficit stress. For example, free proline in Festuca rubra grown

** **

D.

under water withholding for 18 days was accumulated greater thanin Lolium parenne, leading to maintain the water content. A negativerelationship was observed between free proline content and watercontent (Bandurska and Józwiak, 2010). In the present study, freeproline accumulation in the water deficit stressed plants of sweetpotato correlated negatively to osmotic potential in the leaf tis-sues. Previously, free proline in parental lines and their hybrids incastor plant has been reported to to be 4–6 folds higher of the con-trol, when subjected to water deficit stress (−2.25 MPa soil waterpotential) for 33 days (Babita et al., 2010). The free proline accumu-lation in sweet potato grown under water deficit stress may play akey role as major osmotic adjustment (OA). For example, the freeproline has been reported to contribute ∼10–15% to control the OAin water deficit stressed castor plants (Babita et al., 2010). More-over, the free proline enrichment in 49 pea genotypes grown underwater deficit stress has been elucidated while the function of freeproline on OA was only 1%. The major OA solute in pea genotypesgrown under water deficit condition was identified as soluble sugar(Sánchez et al., 1998).

Photosynthetic pigment degradation was one of the most sensi-tive indices in plant responses to water deficit stress. In the presentstudy, Chla, Chlb, TC and Cx+c contents in sweet potato declined sig-nificantly when plants were exposed to 15% SWC. These findings arecorroborated by an earlier observation in tall fescue, wherein Chla,Chlb and TC were degraded by 35.71%, 47.62% and 40.12%, respec-tively, when cultivated under water deficit field trial (Ebrahimiyanet al., 2013). In sweet potato, Cx+c in cultivars Taoyuan (fresh leafvegetable) and Simon 1 (enriched K vitamin) declined by 2.96%and 6.09%, respectively, when plants were subjected to water with-holding for 14 days; whereas Cx+c in Sushu 18 (drought tolerant)was retained (Lin et al., 2006). In the present study, the photosyn-thetic abilities, i.e., Fv/Fm, ˚PSII and Pn, declined significantly when

subjected to 15% SWC, except in some cultivars where these werestabilized. Previously, the Fv/Fm and ˚PSII in cultivar Koganesen-gan of sweet potato, were maintained better than in other cultivars(Beniaka, Okinawa-100 and Tsurunasigenji), leading to high level

S. Yooyongwech et al. / Scientia Horticulturae 162 (2013) 242–251 249

F er 47%p er cut

oIi(Lda(diP

ig. 5. Storage root morphological character of sweet potato genotypes grown undot culture subsequently re-watering prior to storage root harvesting (160 days aft

f Pn under the drought stress (Haimeirong and Kubota, 2003).n addition, the gs and E have been reported as key physiolog-cal indices for drought tolerance classification in sweet potatoHaimeirong and Kubota, 2003; van Heerden and Laurie, 2008;aurie et al., 2009). For example, the gs in cultivar Koganesenganropped by 60% when subjected to drought stress, whereas in Beni-ka, Okinawa-100 and Tsurunasigenji, it declined by 82.44–90.08%

Haimeirong and Kubota, 2003). In addition, the gs in ‘Resisto’, arought sensitive cultivar of sweet potato, decreased sharply to

nhibit the CO2 assimilation by stomatal closure, and leading to lown rate and yield loss (>60%). In contrast, the gs in drought tolerant

SWC (Control; well watering) and 15% SWC (Drought; water deficit stress) in theting).

cultivar ‘A15’ was maintained to assimilate CO2 and prevent H2Oloss through stomata (van Heerden and Laurie, 2008).

Morphological and physiological changes among sweet potatogenotypes grown under 15% SWC were subjected to classify theseinto groups, i.e., water deficit tolerant as Mankorat, PROC 65-3and Japanese Yellow, and water deficit sensitive as Manphuang,Banyang 9 and Tainung 57. In sweet potato, the physiological

and morphological characters have been used as effective indicesfor drought tolerance identification. For example, the Fv/Fm (5%diminution), ˚PSII (33.22% diminution), Pn (49.41% reduction) andgs (60% reduction) in Koganesengan cultivar were retained and

250 S. Yooyongwech et al. / Scientia Horticulturae 162 (2013) 242–251

F ensitiv6

iPnratevd(ptA

irlatdgyJMa

R

A

A

B

ig. 6. Ward’s dendrogram for sweet potato genotypes to classify as water-deficit s5-3 and Japanese Yellow using multivariate cluster analysis.

dentified as drought tolerant (Haimeirong and Kubota, 2003).lant morphological characters, i.e., biomass, vine length, inter-ode diameter, internode length, number of leaves, leaf area andoot weight, have been used as a criteria to select ‘Lole’ cultivars drought tolerant (Saraswati et al., 2004). Likewise, the stressolerant index and high yield potential have been successfullymployed in the classification of orange-fresh sweet potato lines,iz., 1945739, 420014, 440286, 189135.9, 187017.1 and 441725, asrought tolerant in the same group with a positive check ‘Marooko’Agili et al., 2012). Recently, the multivariate cluster analysis andrincipal component analysis (PCA) have been employed to classifyhe drought and heat tolerant cultivars, A3026, A3027, A2316 and46, in sweet potato (Laurie et al., 2013).

In conclusion, our study suggests that free proline (an osmolyte)n water deficit stressed plants of sweet potato may play a keyole as osmotic adjustment to control the osmotic potential in theeaves, leading to prevent the photosynthetic pigment degradationnd loss of chlorophyll fluorescence activities. Net photosyn-hetic rate and prevention of water loss via stomatal closure inrought tolerant genotypes were maintained leading to retainrowth characters such as vine length and number of leaves, andield attributes. Sweet potato cultivars, Mankorat, PROC 65-3 andapanese Yellow, were classified as water deficit tolerant, whereas

anphuang, Banyang 9 and Tainung 57 genotypes were evaluateds water deficit sensitive using multivariate cluster analysis.

eferences

gili, S., Nyende, B., Ngamau, K., Masinde, P., 2012. Selection, yieldevaluation, drought tolerance indices of orange-fresh sweet potato(Ipomoea batatas Lam.) hybrid clone. J. Nutr. Food Sci. 2, 138,http://dx.doi.org/10.4172/2155-9600.1000138.

hraf, M., 2010. Inducing drought tolerance in plants: recent advances. Biotechnol.Adv. 28, 169–183.

abita, M., Maheswari, M., Rao, L.M., Shanker, A.K., Rao, D.G., 2010. Osmotic adjust-ment, drought tolerance and yield in castor (Ricinus communis L.) hybrids.Environ. Exp. Bot. 69, 243–249.

e, Manphuang, Banyang 9, Tainung 57, and water-deficit tolerant, Mankorat, PROC

Bandurska, H., Józwiak, W., 2010. A comparison of the effects of drought on prolineaccumulation and peroxidases activity in leaves of Festuca rubra L. and Loliumperenne L. Acta Soc. Bot. Pol. 79, 111–116.

Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline forwater-stress studies. Plant Soil 39, 205–207.

Chaitanya, K.V., Rasineni, G.K., Reddy, A.R., 2009. Biochemical responses to droughtstress in mulberry (Morus alba L.): evaluation of proline, glycine betaine andabscisic acid accumulation in five cultivars. Acta Physiol. Plant. 31, 437–443.

Cha-um, S., Supaibulwatana, K., Kirdmanee, C., 2007. Glycinebetaine accumulation,physiological characterizations and growth efficiency in salt-tolerant and salt-sensitive lines of indica rice (Oryza sativa L. ssp. indica) in response to salt stress.J. Agron. Crop. Sci. 193, 157–166.

Coombs, J., Hall, D.O., Long, S.P., Scurlock, J.M.O., 1987. Techniques in Bioproductivityand Photosynthesis. Pergamon, Oxford, UK.

Costa, J.M., Otuno, M.F., Chaves, M.M., 2007. Deficit irrigation as a strategy to savewater: physiological and potential application to horticulture. J. Integr. PlantBiol. 49, 1421–1434.

Ebrahimiyan, M., Majidi, M.M., Mirlohi, A., Noroozi, A., 2013. Physiological traitsrelated to drought tolerance in tall fescue. Euphytica 190, 401–414.

Fereres, E., Evans, R.G., 2006. Irrigation of fruit trees and vines. Irrig. Sci. 24, 55–57.Gomes, F., Carr, M.K.V., 2001. Effects of water availability and vine harvesting fre-

quency on the productivity of sweet potato in southern Mozambique. I. Storageroot and vine yields. Exp. Agric. 37, 523–537.

Gomes, F., Carr, M.K.V., 2003a. Effects of water availability and vine harvesting fre-quency on the productivity of sweet potato in southern Mozambique. II. Cropwater use. Exp. Agric. 39, 39–54.

Gomes, F., Carr, M.K.V., 2003b. Effects of water availability and vine harvesting fre-quency on the productivity of sweet potato in southern Mozambique. III. Cropyield and water use response functions. Exp. Agric. 39, 409–421.

Gomes, F., Carr, M.K.V., Squire, G.R., 2005. Effects of water availability and vine har-vesting frequency on the productivity of sweet potato in southern Mozambique.IV. Radiation interception, dry matter production and partitioning. Exp. Agric.41, 93–108.

Guha, A., Sengupta, D., Rasineni, G.K., Reddy, A.R., 2010. An integrated diagnosticapproach to understand drought tolerance in mulberry (Morus indica L.). Flora205, 144–151.

Guha, A., Sengupta, D., Rasineni, G.K., Reddy, A.R., 2012. Non-enzymatic antioxida-tive defense in drought-stressed mulberry (Morus indica L.) genotypes. Trees 26,903–918.

Haimeirong, Kubota, F., 2003. The effects of drought stress and leaf ageing on leafphotosynthesis and electron transport in photosystem 2 in sweet potato (Ipo-

moea batatas Lam.) cultivars. Photosynthetica 41, 253–258.

Hare, P.D., Cress, W.A., 1997. Metabolic implications of stress-induced proline accu-mulation in plants. Plant Growth Regul. 21, 77–102.

Huzsvai, L., Rajkai, K., 2009. Modeling of plant adaptation to climatic drought inducewater deficit. Biologia 64, 536–540.

Hort

J

J

K

K

L

L

L

L

L

L

L

L

L

S. Yooyongwech et al. / Scientia

an, A.T., Singhal, P., Haq, Q.M.R., 2012. Plant abiotic stress: deciphering remedialstrategies for emerging problem. J. Plant Inter. 8, 97–108.

ovanovic, Z., Stikic, R., 2012. Strategies for improving water productivity andquality of agricultural crops in an era of climate change. In: Lee, T.S. (Ed.),Irrigation Systems and Practices in Challenging Environments. InTech,Croatia, ISBN: 978-953-51-0420-9. DOI: 10.5772/29275. Available from:http://www.intechopen.com/books/irrigation-systems-and-practices-in-challenging-environments/strategies-for-improving-water-productivity-and-quality-of-agricultural-crops-in-an-era-of-climate-change

im, S.H., Song, W.K., Kim, Y.H., Kwon, S.Y., Lee, H.S., Lee, I.C., Kwak, S.S.,2009. Characterization of full-length enriched expressed sequence tags ofdehydration-treated white fibrous roots of sweet potato. BMB Rep. 42,271–276.

im, Y.H., Jeong, J.C., Lee, H.S., Kwak, S.S., 2013. Comparative characterization ofsweet potato antioxidant genes from expressed sequence tags of dehydration-treated fibrous roots under different abiotic stress conditions. Mol. Biol. Rep. 40,2887–2896.

anfermeijer, F.C., Koerselman-Kooij, J.W., Borstlap, A.C., 1991. Osmosensitivity ofsucrose uptake by immature pea cotyledons disappears during development.Plant Physiol. 95, 832–838.

ascano, R.J., Sojka, R.E., 2007. Preface. In: Lascano, R.J., Sojka, RE. (Eds.), Irrigationof Agricultural Crops. , 2nd ed. Agronomy Monograph No. 30. ASA-CSSA-SSSPublishing, Madison, Wisconsin USA, p. 664.

aurie, R.N., du Plooy, C.P., Laurie, S.M., 2009. Effect of moisture stress on growthand performance of orange fleshed sweet potato varieties. Afri. Crop Sci. Conf.Proc. 9, 235–239.

aurie, S.M., Calitz, F.J., Adebola, P.O., Lezar, A., 2013. Characterization and evaluationof South African sweet potato (Ipomoea batatas (L.) Lam) land races. South Afri.J. Bot. 85, 10–16.

aurie, S.M., Faber, M., van Jaarsveld, P.J., Laurie, R.N., du Plooy, C.P., Modisane,P.C., 2012. �-Carotene yield and productivity of orange-fleshed sweet potato(Ipomoea batatas (L.) Lam.) as influenced by irrigation and fertilizer applicationtreatments. Sci. Hortic. 142, 180–184.

ewthwaite, S.L., Triggs, C.M., 2012. Sweet potato cultivar response to prolongeddrought. Agron. New Zealand 42, 1–10.

ichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosyntheticbiomembranes. Methods Enzymol. 148, 350–380.

in, K.H., Chao, P.Y., Yang, C.M., Cheng, W.C., Lo, H.F., Chang, T.R., 2006. The effects of

flooding and drought stresses on the antioxidant constituents in sweet potatoleaves. Bot. Stud. 47, 417–426.

oggini, B., Scartazza, A., Brugnoli, E., Navari-Izzo, F., 1999. Antioxidant defense sys-tem, pigment composition, and photosynthetic efficiency in two wheat cultivarssubjected to drought. Plant Physiol. 119, 1091–1099.

iculturae 162 (2013) 242–251 251

Marguerit, E., Brendel, O., Labon, E., van Leeuwen, C., Ollat, N., 2012. Rootstock controlof scion transpiration and its acclinmation to water deficit are controlled bydifferent genes. New Phytol. 194, 416–429.

Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence – a practical guide. J. Exp.Bot. 51, 659–668.

Mukhopadhyay, S.K., Chattopadhyay, A., Chakraborty, I., Bhattacharya, I., 2011. Cropsthat feed the world. 5. Sweet potato. Sweetpotatos for income and food security.Food Sec. 3, 283–305.

Peuke, A.D., Schraml, C., Hartung, W., Rennenberg, H., 2002. Identification ofdrought-sensitive beech ecotypes by physiological parameters. New Phytol. 154,373–387.

Pinheiro, C., Chaves, M.M., 2010. Photosynthesis and drought: can we makemetabolic connection from available data? J. Exp. Bot. 62, 869–882.

Qin, F., Shinozaki, K., Yamaguchi-Shinozaki, K., 2011. Achievements and challengesin understanding plant abiotic stress responses and tolerance. Plant Cell Physiol.52, 1569–1582.

Sánchez, F.J., Manzanares, M., de Andres, E.F., Tenorio, J.L., Ayerbe, L., 1998. Turgormaintenance, osmotic adjustment and soluble sugar and proline accumulationin 49 pea cultivars in response to water stress. Field Crops Res. 59, 225–235.

Saraswati, P., Johnston, M., Coventry, R., Holtum, J., 2004. Identification of droughttolerant sweet potato (Ipomoea batatas (L.) Lam) cultivars. In: New Direction forAdverse Planet: Proceedings of the 4th International Crop Science Congress, 26September–1 October 2004, Brisbane, Australia.

Shabala, S.N., Shabala, S.I., Martynenko, A.I., Babourina, O., Newman, I.A., 1998.Salinity effect on bioelectric activity, growth, Na+ accumulation and chlorophyllfluorescence of maize leaves: a comparative survey and prospects for screening.Aust. J. Plant Physiol. 25, 609–616.

van Heerden, P.D.R., Laurie, R., 2008. Effects of prolonged restriction in water supplyon photosynthesis, shoot development and storage root yield in sweet potato.Physiol. Plant. 134, 99–109.

Verslues, P.E., Juenger, T.E., 2011. Drought, metabolites, and Arabidopsis natural vari-ation: a promising combination for understanding adaptation to water-limitedenvironments. Curr. Opin. Plant Biol. 14, 240–245.

Yadollahi, A., Arzani, K., Ebadi, A., Wirthensohn, M., Karimi, S., 2011. The responseof different almond genotypes to moderate and severe water stress in order toscreen for drought tolerance. Sci. Hortic. 129, 403–413.

Yang, S., Vanderbeld, B., Wan, J., Huang, Y., 2010. Narrowing down the targets:towards successful genetic engineering of drought tolerant crops. Mol. Plant

3, 469–490.

Ziska, L.S., Runion, G.B., Prior, S.A., Torbet, H.A., Sicher, R., 2009. An evaluationof cassava, sweet potato and field corn as potential carbohydrate sourcesfor bioethanol production in Alabama and Maryland. Biomass Bioenergy 33,1503–1508.