Osmopriming improves tomato seed vigor under aging and salinity stress

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1 Africn Journl of Biotechnology Vol. 11(23), pp , 20 Mrch, 2012 Avilble online t DOI: /AJB ISSN Acdemic Journls Full Length Reserch Pper Osmopriming improves tomto seed vigor under ging nd slinity stress Mengyu Zhng #, Zhoufei Wng #, Lingling Yun, Congfei Yin, Jinping Cheng, Ling Wng, Ji Hung nd Hongsheng Zhng* The Lbortory of Seed Science nd Technology, Stte Key Lbortory of Crop Genetics nd Germplsm Enhncement, Nnjing Agriculturl University, Nnjing , PR Chin. Accepted 1 Mrch, 2012 This study investigtes the effects of osmopriming on tomto (Lycopersicon esculentum M.) hybrid seed vigor under ging nd slinity stress. Tomto seeds of the ZZ1 hybrid vriety, stored for four yers under nturl (ged) or -20 C (unged) s were primed in 10% (w/v) polyethylene glycol (PEG) solution for 2 d t 20±1 C in the drk. The seed vigor ws evluted t 25±1 C for 7 d under norml (wter) nd 100 mm NCl s, respectively. Results show tht the germintion percentge (GP), germintion index (GI) nd men germintion rte (MGR) of primed, ged seeds were significntly enhnced with substntil increse in the rdicle length (RL), shoot length (SL) nd totl fresh weight (FW) compred with unprimed ged seeds. Similrly, the GI, MGR nd FW significntly incresed in primed seeds compred with unprimed seeds under slinity stress, while GP, RL nd SL did not show significnt differences. Furthermore, decline in the reltive electrolyte lekge (REL) nd in mlondildehyde (MDA) ws detected in primed seeds during the imbibition stge compred with unprimed seeds under ging nd slinity stress. The negtive correltions between seed vigor nd REL nd MDA were observed which suggests tht seed priming improves seed vigor under stress s ssocited with decrese in seed lipid peroxidtion. Key words: Tomto, seed priming, osmopriming, seed vigor, seed ging, slt tolernce. INTRODUCTION Tomto (Lycopersicon esculentum M.) is one of the most importnt vegetble crops throughout the world. Rpid nd uniform seed germintion is essentil for incresing tomto crop yield nd qulity. However, seed germintion might be problemtic due to the decrese in seed vigor cused by deteriortion during storge (Coolber et l., 1984) nd differences in seed vigor (non-uniformity; Ismil et l., 2005) nd effects of biotic stress (Wng et l., 2011). In tomto, slinity hs become gret thret to *Corresponding uthor. E-mil: hszhng@nju.edu.cn. Tel: Abbvrevittions: PEG, Polyethylene glycol; GP, germintion percentge; GI, germintion index; MGR, men germintion rte; RL, rdicle length; SL, shoot length; FW, totl fresh weight; REL, reltive electrolyte lekge; MDA, mlondildehyde. tomto growth since tomto cultivtion ws switched to greenhouses (Chen et l., 2009). Therefore, low nd non-uniform seed vigor s well s slinity stress, if not properly mnged might become limiting fctors for synchronized stnd seedling estblishment in tomto. Developing n elite vriety with high level seed vigor is the best wy to solve the foregoing mentioned problems; while this strtegy is difficult nd time-consuming. However, seed priming tretment is n effective nd low-cost method to improve seed vigor. Seed priming is technique tht controls seeds hydrtion nd drying to their originl moisture content. After seed priming, the first step of seed germintion is completed but rdicle emergence does not occur. Mostly, seeds re prtilly hydrted to hve wter content tht cn be controlled by priming osmotic solutions (osmopriming) or by limiting the imbibition time (hydropriming; Schwember nd Brdford, 2010). Previous

2 6306 Afr. J. Biotechnol. Tble 1. Seeds pre-germintion tretments nd germintion s. Tretment Aging tretment Priming tretment Germintion T1 Unged Unprimed Wter Norml T2 Unged Primed Wter Aging Slinity T3 Aged Unprimed Wter T4 Aged Primed Wter T5 Unged Unprimed Slinity T6 Unged Primed Slinity studies hve indicted tht seed priming constitutes successful strtegy for improving seed longevity (Butler et l., 2009) nd seed germintion under stress s (Gun et l., 2009). In tomto, previous studies hve reported tht priming benefits seed storbility (Liu et l., 1996), seed germintion under drought (Muromicle nd Cvllro, 1995) nd low temperture stress (Muromicle nd Cvllro, 1997; Ozbingol et l., 1998). Hybrid tomto vrieties re populr for commercil cultivtion becuse they hve mny dvntges compred with open pollinted vrieties. However, little is known bout the priming effects on hybrid tomto seed vigor under ging nd slinity s. During the seed hydrtion period, vriety of physiologicl nd biochemicl chnges tke plce in seeds. In summry, the benefits of seed priming include the dvncement of germintion metbolism (Frooq et l., 2010), enhnced ntioxidnt ctivities (Billy, 2004; Chen nd Aror, 2011) nd improved repir processes (Sivritepe nd Dourdo, 1995), nd so on. Under stress s, the rective oxygen species (ROS) cn cuse lipid peroxidtion, which results in cell membrne dmge nd mlondildehyde (MDA) production (Fu nd Hung, 2001; Mei nd Song, 2010). Thus, the reltive electrolyte lekge (REL) nd concentrtion of MDA re good indictors for investigting the effects of priming under stress s (Gun et l., 2009). However, few studies hve considered the chnges of lipid peroxidtion in primed tomto seeds t imbibition stge, little re known bout the effects of lipid peroxidtion t seed imbibition stge on tomto successful seed germintion nd seedling growth. The objectives of this study were to explore the beneficil effects of osmopriming using PEG s priming regent on the vigor of tomto hybrid seeds. Therefore, severl seed vigor trits such s germintion percentge (GP), germintion index (GI), men germintion rte (MGR), shoot length (SL), rdicle length (RL) nd totl fresh weight (FW) were nlyzed under norml, nturl ging nd slinity s, respectively. Furthermore, using the REL nd MDA indictors, the chnges of lipid peroxidtion in primed hybrid tomto seeds during the seed germintion process were further nlyzed. These results my provide vluble informtion for improving seed vigor of tomto under stress s by PEG osmopriming tretment. MATERIALS AND METHODS Plnt mterils The ZZ1 hybrid vriety of tomto (L. esculentum M.) ws used in this study. Seeds were produced nd hrvested t mture stge by the Chin Ntionl Seed Group Co. LTD (Beijing, Chin) in After ripening t 30 C, 85% RH for 7 d, the originl moisture content nd germintion of seeds were pproximtely 9.0 nd 100% by seed testing, respectively. Then, seeds were stored in krft pper bgs under nturl (ged) nd -20 C (unged) s in Nnjing (Jingsu Province, Chin; E118 50, N32 02 ) for four yers. Six tretments were conducted in this study (Tble 1). Seed priming Seeds were surfce sterilized with 0.1% HgCl 2 for 5 min, then plced in Petri dish (9-cm dimeter) on two sheets of filter pper with 10 ml of 10% (w/v) polyethylene glycol (PEG) 6,000 (moleculr weight) solution, seled with prfilm nd stored t 25 C in the drk for 48 h. The primed seeds were then wshed twice with distilled wter nd dried to their originl moisture content (9.0%) t 25 C to perform germintion tests. Seed germintion Seeds were germinted under norml wter or 100 mm NCl s t 25±1 C for 7 d with 12-h light/drk photoperiod. Briefly, 100 seeds per repliction were surfce sterilized with 0.1% HgCl 2 for 5 min. Then, they were plced in Petri dish (9-cm dimeter) on two sheets of filter pper with 10 ml of distilled wter or 100 mm NCl. Seeds were considered to hve germinted when their rdicle length reched more thn 2 mm. The germinted seeds were observed ech dy. The percentge of germinted seeds t 7 d ws referred to s the GP tht represents seed germinbility. The GI nd MGR were clculted s GI=Σ (Gt/t) nd MGR=1/MGT (men germintion time), MGT=Σ (Gt t)/σgt, respectively, where Gt is the number of germinted seeds on dy t (Gun et l., 2010 ). Here, the GI represents germintion speed nd uniform nd the

3 Zhng et l Tble 2. The effects of priming on seed germintion under norml, ging nd slinity s. Tretment GP (%) b GI b MGR b Norml T1 100±0.0 A 24.9±0.2 A 0.500±0.000 A T2 100±0.0 A 13.0±0.5 B 0.414±0.020 B Aging T3 61.1±4.0 C 4.3±1.1 C 0.233±0.006 C T4 87.8±6.9 B 11.7±0.9 B 0.417±0.002 B Slinity T5 99.3±1.2 A 12.5±0.4 B 0.244±0.004 C T6 98.9±1.9 A 21.0±0.5 A 0.500±0.000 A T1: Unged seeds germinted under wter ; T2: Primed unged seeds germinted under wter ; T3: Aged seeds germinted under wter ; T4: Primed ged seeds germinted under wter ; T5: Unged seeds germinted under slinity stress; T6: Primed unged seeds germinted under slinity stress. b GP: Germintion percentge; GI: Germintion index; MGR: Men germintion rte; Mens ± SD (stndrd devition); Smple size n = 100, replictions r = 4; Mens within the sme column followed by the sme letters re not significntly different between tretments t the 1% level of probbility ccording to Fisher s lest significnt difference (LSD) test (P 0.01, LSD). MGR represents germintion speed. The RL, SL nd SW were mesured fter 3 nd 7 d. The seed germintion process ws replicted four times. Mesurement of membrne permebility nd MDA After seeds germinted t 25 C for 1 d nd 7 d in 10 ml of wter or 100 mm NCl, respectively they were wshed with deionized wter for the mesurement of membrne permebility nd MDA. Membrne permebility ws estimted by mesuring cell electrolyte lekge. Seeds or seedlings were plced in test tube with 80 ml of deionized wter nd incubted t 25 C for 24 h. Subsequently, the electrolyte lekge ws determined s E1 ccording to the method of Gun et l. (2009) using conductometer (DDS-11A, Shnghi, Chin). Then, test tubes were kept in wter t 100 C for 10 min nd were cooled to 25 C when the second electrolyte lekge ws mesured s E2. The electrolyte lekge of deionized wter ws nmed E0. Finlly, the REL of seeds ws clculted ccording to the following formul: REL (%) = (E1 E0)/ (E2 E0) 100 (Gun et l., 2009). The MDA content of seeds ws mesured by the thiobrbituric cid (TBA) rection method (Gun et l., 2009). The seeds or seedlings were mixed with 3 ml of 0.1% (w/v) trichlorocetic cid (TCA) nd centrifuged t 10,000 g for 10 min; the superntnt (1 ml) ws then mixed with 4 ml of 20% (w/v) TCA. The resulting mixture ws heted t 95 C for 30 min nd then quickly cooled on ice for 5 min. After centrifugtion t 10,000 g for 10 min t 4 C, the bsorbnce of the rection mixture ws mesured t 450, 532 nd 600 nm. The MDA concentrtion (nmol/g DW) ws clculted using the following eqution: [MDA] = 6.45 (A 532 A 600) 0.56 A 450, where A 532, A 600, nd A 450 represent the bsorbnce of the mixture t 450, 532, nd 600 nm, respectively. The experiment ws replicted four times nd 100 seeds per replicte were used. Sttisticl nlysis Experimentl dt were nlyzed using the Sttisticl Anlysis System (SAS) softwre, nd tretment mens were compred using Fisher s lest significnt difference (LSD) test t 1% level of probbility. The correltion of different trits ws computed using PROC CORR in the SAS softwre. RESULTS Effects of priming on seed germintion Compred to unged seeds under norml (T1), there were significnt effects of long-term nturl storge (ged) on tomto seed germintion (Tble 2); the GP, GI nd MGR of unprimed ged seeds (T3) decresed to 61.1, 4.3 nd fter four yers of storge, respectively. In contrst, under slinity, the GI nd MGR of unprimed seeds (T5) significntly decresed to 12.5 nd compred to T1, respectively but non-significnt effects on GP. Bsed on the curve of seed germintion, the pek germintion of T3 nd T5 occurred on the 6 th dy; however, tht of T1 occurred on the 2 nd dy (Figure 1). The seed germintion could be improved by PEG priming under ging nd slinity s, respectively. Under ging, significnt differences were observed in GP, GI nd MGR between unprimed (T3) nd primed seeds (T4) (Tble 2); the GP, GI nd MGR of T4 incresed by 26.7, 7.4 nd 0.184, respectively compred with T3. Under slinity, significnt differences were lso observed in GI nd MGR between unprimed (T5) nd primed seeds (T6) but non-significnt difference in GP; the GI nd MGR of T6 incresed by 8.5 nd 0.256, respectively compred with T5. Bsed on the seed germintion curve, the priming tretment reduced the number of dys to the pek germintion of seeds under ging nd slinity s; the pek germintion of T4 nd T6 occurred on the 5 th dy nd 2 nd dy (Figure 1). However, non-significnt benefits of priming were observed under norml ; the GI nd MGR of T2 significntly decresed by 11.9 nd 0.086, respectively compred with T1.

4 6308 Afr. J. Biotechnol (% ) te r n 60 tio in 40 e r m G Germintion dys (d) T1 T2 T3 T4 T5 T6 Figure 1. The effects of priming on seeds germintion process under norml, ging nd slinity s. Effects of priming on seedling growth Likewise, four yers of nturl storge (T3) significntly reduced seedling growth compred with the unged seeds (T1); the SL, RL nd FW of T3 on the 3 rd nd 7 th d were reduced, respectively (Tble 3). However, under slinity, only the FW of T5 significntly decresed on the 3 rd nd 7 th d compred with T1; non-significnt differences in SL nd RL. Similrly, the seedling trits lso could be improved by PEG priming under ging nd slinity s, respectively. Under ging, the priming tretment significntly incresed the SL, RL nd FW of T4 on the 3 rd nd 7 th d compred to T3 (Tble 3). Under slinity, priming tretment significntly incresed the FW of T6 compred with T5, but non-significnt difference in SL nd RL. However, non-significnt effects of priming were observed under norml ; the SL, RL nd FW of T2 were non-significnt difference compred with T1. In generl, fter priming, the seedling performnces under ging nd slinity s were improved, which lso reflected seed germintion speed ws enhnced by priming. the REL of unprimed seeds (T3 nd T5) significntly incresed compred with tht of the control (T1) t imbibition stge (Tble 4). However, t seedling stge, the REL of T3 nd T5 ws significntly decresed nd incresed, respectively compred to T1. Furthermore, the MDA content significntly incresed only in the ging tretment (T3) t both imbibition nd seedling stges, the MDA content ws pproximtely double tht of T1; wheres in the slinity tretment (T5), the MDA content ws comprble to tht of T1. Under norml, ging nd slinity s, primed seeds (T2, T4 nd T6), in generl, exhibited lower REL nd MDA thn unprimed seeds (T1, T3 nd T5) (Tble 4). Under norml, only the REL of T2 t imbibition stge ws significntly decresed s compred to T1. Under ging, the REL nd MDA of T4 significntly decresed t imbibition stge compred with T3, however, only the REL of T4 ws significntly decresed t seedling stge. Upon exposure to slinity stress, only the REL of T6 ws significntly decresed nd incresed s compred to T5 t imbibition nd seedling stges, respectively wheres the MDA content of T6 ws comprble to tht of T5. Effects of priming on the level of REL nd MDA during seed germintion process To investigte the effects of priming on lipid peroxidtion subject to ging nd slinity stress, chnges of REL nd MDA t seed imbibition nd seedling stges were evluted. Upon exposure to either ging or slinity stress, Correltions between the REL nd MDA in imbibition seeds with seed vigor In order to cler the effects of lipid peroxidtion t seed imbibition stge on tomto successful seed germintion nd seedling growth by seed priming tretment, the correltions between REL nd MDA in imbibition seeds

5 Zhng et l Tble 3. The effects of priming on seedling growth under norml, ging nd slinity s. Tretment 3 d b 7 d b SL (cm) (RL) (cm) FW (mg/per line) SL (cm) RL (cm) FW (mg/per line) Norml T1 3.7±0.3 A 4.3±0.5 A 19.5±1.7 A 10.2±1.4 A 8.8±0.7 A 40.4±1.2 A T2 3.4±0.2 A 5.1±0.3 A 20.1±1.0 A 10.8±2.8 A 6.4±0.5 A 38.3±4.6 A Aging T3 0.0±0.0 C 0.0±0.0 C 0.0±0.0 C 3.5±0.9 B 2.6±0.4 B 13.3±2.8 B T4 4.8±0.4 A 6.1±0.2 A 21.3±1.7 A 9.4±0.7 A 7.2±0.7 A 41.6±2.9 A Slinity T5 3.5±0.9 A 3.9±0.9 B 17.2±1.2 B 8.9±1.6 A 6.8±1.4 A 33.3±1.3 B T6 3.9±0.9 A 5.2±1.0 A 23.8±2.9 A 9.0±1.4 A 7.1±1.0 A 37.0±1.4 A T1: Unged seeds germinted under wter ; T2: Primed unged seeds germinted under wter ; T3: Aged seeds germinted under wter ; T4: Primed ged seeds germinted under wter ; T5: Unged seeds germinted under slinity stress; T6: Primed unged seeds germinted under slinity stress. b SL: Shoot length; RL: Rdicle length; FW: Totl fresh weight; Mens ± SD (stndrd devition); Smple size n = 100, replictions r = 4; Mens within the sme column followed by the sme letters re not significntly different between tretments t the 1% level of probbility ccording to Fisher s lest significnt difference (LSD) test (P 0.01, LSD). Tble 4. The effects of priming on the level of REL nd MDA t different germintion stges under norml, ging nd slinity s. Tretment REL (%) b MDA (nmol/g DW) b 1 d 7 d 1 d 7 d T1 53.8±2.9 C 69.8±4.0 C 15.5±0.7 B 12.5± 0.6 B Norml T2 44.0±0.7 D 68.4±3.4 C 13.9±1.2 B 9.8±1.5 B Aging T3 95.8±7.2 A 48.4±2.0 D 31.0±1.6 A 34.2±1.7 A T4 62.5±6.5 B 35.7±1.7 E 15.5±0.8 B 33.5±1.5 A Slinity T5 69.3±2.7 B 79.9±2.0 B 14.0±0.7 B 7.8±0.3 B T6 13.8±1.5 E 92.9±0.5 A 13.0±0.7 B 7.9±0.4 B T1: Unged seeds germinted under wter ; T2: Primed unged seeds germinted under wter ; T3: Aged seeds germinted under wter ; T4: Primed ged seeds germinted under wter ; T5: Unged seeds germinted under slinity stress; T6: Primed unged seeds germinted under slinity stress. b REL: Reltive electrolyte lekge; MDA: Mlondildehyde; Men ± SD (stndrd devition); Smple size n = 100, replictions r = 4; Mens within the sme column followed by the sme letters re not significntly different between tretments t the 1% level of probbility ccording to Fisher s lest significnt difference (LSD) test (P 0.01, LSD). with seed vigor trits under ging nd slinity s were nlyzed, respectively. Under ging, correltion nlyses showed tht the REL nd MDA were significntly nd negtively correlted with GP, GI, MGR, SL, RL nd FW, respectively (Tble 5). Under slinity, significnt nd negtive correltions were observed in the reltionships between REL with MGR nd between MDA with GP, GI nd MGR. The significnt nd negtive correltions between REL nd MDA with seedling trits were lso observed, except between REL nd SL nd between MDA nd FW t 7 d. DISCUSSION Seed deteriortion is mjor problem in griculturl production. It hs been estimted tht 25% of the nnul vlue of seeds in inventories might be lost becuse of poor seed qulity (Schwember nd Brdford, 2010). The rte of ging is strongly influenced by severl environmentl nd genetic fctors such s storge temperture, storge time, storge fungi, seed moisture content nd seed qulity (Hrmn nd Mttick, 1976; Wlters, 1998; Rjjou nd Debeujon, 2008). In Nnjing (Jingsu Province, Chin; E118 50, N32 02 ), the verge nnul temperture is pproximtely 15 C, with the extreme lowest nd highest vlues reching -10 nd 40 C, respectively nd the precipittion period rnges from mid-june to July. In this study, fter four yers of storge under nturl s in Nnjing, hybrid tomto seeds showed decresed seed germinbility, germintion speed nd germintion uniformity.

6 6310 Afr. J. Biotechnol. Tble 5. Correltion between the level of REL nd MDA t imbibition stge with seed vigor under ging nd slinity s. 3 d 7 d SL RL FW SL RL FW REL ** ** ** ** ** ** ** ** ** MDA ** ** ** ** ** ** ** ** ** REL ** ** ** ** ** ** MDA ** ** ** ** ** ** ** ** Tretment Trit GP GI MGR Aging Slinity REL: Reltive electrolyte lekge; MDA: Mlondildehyde; GP: Germintion percentge; GI: Germintion index; MGR: Men germintion rte; SL: Shoot length; RL: Rdicle length; FW: Totl fresh weight ** Significnt t 0.01 level. These results revel tht either protection of seed longevity or usge of ged seeds hve significnt impcts on crop production nd conservtion of plnt biodiversity in hot nd moist climte regions such s Nnjing. However, if deteriortion dmges do not rech criticl level, the seed vigor cn be improved by priming tretment (Goel et l., 2003). For exmple, Dell'Aquil et l. (1984) indicted tht the germintion performnce of ged whet seeds could be improved by priming tretment. Similr results were found in this study tht priming cn effectively improve the vigor of ged tomto seeds. Slinity dversely ffects seeds germintion nd seedling growth, s reported in most crops (Sivritepe et l., 2003; Wng et l., 2011). In this study, similr results were observed tht slinity significntly decresed germintion speed, germintion uniformity nd totl fresh weight of seedlings in tomto. However, priming could improve seed vigor by enbling rpid nd uniform germintion nd improving seedling fresh weight. Similr results of the improvement of seed germintion nd seedling growth due to priming were observed in mny crops under other stress s, such s chilling (Gun et l., 2009), het (Schwember nd Brdford, 2010) nd drought stress (Kur et l., 2002). Seed germintion includes three phses, nmely, imbibition, lg phse nd protrusion of rdicle through test. Seed priming is the controlled hydrtion of seeds to llow the completion of imbibition nd lg phse of germintion. The benefits of seed priming re relted to morphologicl, physiologicl nd biochemicl chnges tht occur in seeds during priming process, such s reducing the physicl resistnce of the endosperm during imbibition (Toorop et l., 1998), promoting cell membrne stbility nd integrity (Gun et l., 2009), fcilitting repir of chromosoml dmge (Sivritepe nd Dourdo, 1995) nd incresing the ctivity of enzymes relted to seed vigor (Billy et l., 2000). Therefore, the significntly improved vigor of tomto seeds under ging nd slinity stress by priming might be due to the reduction of germintion lg phse, the erly reserve brekdown nd the increse in metbolic nd recovery ctivities. For exmple, the beneficil effects of priming on improving germintion speed nd uniform under ging nd slinity stress could prtilly be explined by the increse in imbibition speed, reduction in lg phse nd ccelertion of metbolic ctivities in primed seeds. However, detiled investigtion is required to revel the priming mechnisms in seed germintion processes such s the seed imbibition step. Indeed, erly imbibition is simple nd rpid physicl process, nd lrge mount of ROS is produced during this process (Liu et l. 2007). In this study, we investigted the effects of priming on the reduction of lipid peroxidtion during seed imbibition stge under ging nd slinity stress in tomto. In generl, the extent of lipid peroxidtion ws lower in primed seeds thn tht of unprimed seeds. Further correltion nlyses showed tht n increse in seed vigor under ging nd slinity stress by priming ws significntly correlted with decrese in the levels of REL nd MDA. These results suggest tht priming effects on tomto seed vigor under stress s might be closely relted to the reduction of lipid peroxidtion during seed imbibition stge. However, the bility to understnd, mnipulte nd control the seed priming process t the moleculr level is one of the gretest chllenges of modern seed science (Gllrdo et l., 2001; Schwember nd Brdford, 2010). Further studies re required to clrify the moleculr mechnisms of seed priming effects on seed vigor under stress s in tomto. ACKNOWLEDGEMENTS Project supported by the Ntionl Nturl Science Foundtion of Chin (No ), the Nturl Science Foundtion of Jingsu Province (No. BK ) nd the Ntionl Key Technology R nd D Progrm in the 11th Five Yer Pln (No. 2008BADB1B06), Chin. REFERENCES Billy C (2004). Active oxygen species nd ntioxidnts in seed biology. Seed Sci. Res. 14: Billy C, Benm, A, Corbineu F, Côme D (2000). Antioxidnt systems

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