increased oxygen bioavailability improved vigor and germination of

HORTSCIENCE 47(12):1714–1721. 2012.

Increased Oxygen BioavailabilityImproved Vigor and Germinationof Aged Vegetable SeedsGuodong Liu1

Horticultural Sciences Department, IFAS, University of Florida, 1117 FifieldHall, Gainesville, FL 32611-0690

D. Marshall PorterfieldDepartment of Agricultural and Biological Engineering, Department ofHorticulture and Landscape Architecture and Weldon School of BiomedicalEngineering, Purdue University, 225 South University Street, W. Lafayette,IN 47907-2093

Yuncong LiTropical Research and Education Center and Water and Soil ScienceDepartment, IFAS, University of Florida, 18905 SW 280th Street,Homestead, FL 33031

Waldemar KlassenTropical Research and Education Center and Entomology and NematologyDepartment, IFAS, University of Florida, 18905 SW 280th Street, Homestead,FL 33031

Additional index words. corn, squash, tomato, oxygen fertilizer, seed imbibition, seed oxygenconsumption, alcohol dehydrogenase activity

Abstract. Large and/or aged seeds are prone to hypoxic conditions during germination.Germination of selected vegetable seeds including corn (Zea mays L.), squash (Cucurbitapepo L.), and tomato (Solanum lycopersicum L.) was studied in water with differentconcentrations of hydrogen peroxide (H2O2) solution ranging from 0, 0.06% to 3.0% (v/v)or in aeroponics, all with 0.5 mM CaSO4. Imbibition, oxygen consumption, proton extrusion,and alcohol dehydrogenase (ADHase) activity of corn seeds were measured gravimetrically,electrochemically, and colorimetrically as appropriate. The results showed that 0.15%H2O2 provided the optimum oxygen concentration for seed germination. The germinationpercentage of aged corn seeds treated with H2O2 was significantly greater than those withoutH2O2 treatment. Corn embryo orientation in relation to a moist substrate also significantlyimpacted oxygen bioavailability to the embryo and hence ADHase activity. Corn seedswithout H2O2 imbibed significantly more slowly than those with oxygen fortification by0.15% H2O2. Increased oxygen bioavailability improved the metabolism of the seeds, whichextruded 5-fold more protons from the embryos. Each treated embryo consumed twice theamount of oxygen as compared with the untreated one and likewise for treated and untreatedendosperms. Increased oxygen bioavailability may be used to improve production of thetested crops. The results from this research imply that consideration should be given toincluding oxygen fortification in seed coatings for aged seeds and for large seeds regardless ofage. The artificial provision of bioavailable oxygen might be effective in rescuing thegermplasm in aged seeds in plant breeding and in crop production.

A high seed germination percentage isa prerequisite for successful commercialvegetable production. Seed vigor, appropriate

temperature, and oxygen and water bioavail-ability are key factors in high percentages ofseed germination (Bewley and Black, 1994).Seed vigor and bioavailable oxygen are closelyassociated. Particularly, aged or large seedsare especially prone to lack of oxygen bio-availability (Geigenberger et al., 2000). How-ever, the sufficiency of oxygen bioavailabilityis not easily secured in soil or water. Forexample, water saturated with air contains�250 mM oxygen (Lide, 1998), which is toolittle to supply sufficient bioavailable oxygento support the germination of relatively largeor aged seeds. In fact, seed hypoxia causedby flooding is a worldwide problem (Jacksonet al., 2009) and a detrimental factor in

plant growth and development of vegetable(Edelstein et al., 1995). The rainy periods inthe spring often introduce much uncertaintyinto growers’ decision-making process per-taining to corn and vegetable planting. Be-cause a corn seed has a volume six to 10 timeslarger than that of wheat or rice, corn seedis more likely to suffer from oxygen de-ficiency than the latter two species as mea-sured (data not shown). Specific surface areais a material property of solids, which mea-sures the total surface area per unit of bulkvolume. It is defined by surface area dividedby the volume (units of m2/m3). Specificsurface area of corn seeds is only 0.1% to0.5% that of the other two types of seedsbased on our calculations. Additionally,waterlogging (i.e., oxygen bioavailabilitydeficiency) sometimes causes up to 50% lossof crop yield (Dennis et al., 2000). Such yieldlosses have usually resulted from failure bothof seed germination and plant growth anddevelopment.

Oxygen bioavailability deficiency can becaused by different factors including water-logging or aging and may be responsible forfailures of seed germination. Oxygen bio-availability is, therefore, a significant factorin determining seed vigor and seed germina-tion (Bewley and Black, 1994). Early agingor degeneration of seeds may also result inlosses of seed vigor and vegetable yield as aresult of unfavorable cultivation or storageconditions (Hong, 2001). Heavy rainfall aftersowing often causes oxygen deficiency in theseedbed sufficiently severe to hamper emer-gence (Hakansson et al., 2012). Aged seedsare characterized by poor germination andslow post-germination growth, and they havelow plasma membrane H+-ATPase activity.This low enzyme activity may partially explainpoor germination and low post-germinationroot elongation and growth of seedlings de-veloped from aged seeds (Sveinsdottir et al.,2009). The essence of slow and/or low germi-nation percentage is the retardation of bio-chemical and physiological reactions in agedseeds. Energetically, such retardation is re-lated to a deficiency of active chemical en-ergy, adenosine triphosphate (ATP), becauseaged seeds may have reduced content of non-symbiotic hemoglobins. These hemoglobinsact in plants to maintain the energy status ofcells in hypoxic conditions such as duringflooding, and they accomplish this by in-creasing substrate-level phosphorylation. Non-symbiotic hemoglobins likely sequester oxygenin hypoxic environments, providing a sourceof oxygen to oxidize NADH to produce ATPfor cell growth and development (Sowa et al.,1998). ATP deficiency may be common bothin aged and flooded seeds. Therefore, newtechnologies are needed to coat large seedslike corn or other vegetable seeds with oxygenfertilizers to improve seed germination andcrop success. Such technologies may be alsoadvantageous in accelerating crop breedingfor rescuing rare plant germplasm from agedor improperly stored seeds. However, thereare no published accounts of an adequateunderstanding of the relationship between

Received for publication 4 Sept. 2012. Acceptedfor publication 15 Oct. 2012.This project was financially supported by the U.S.Department of Agriculture, Grant No. NRICGP2001-35100-10751.We gratefully acknowledge Illinois FoundationSeeds Inc. for providing the corn seeds. We thankProfessor Daniel Cantliff and Professor EdwardHanlon at the University of Florida for their valu-able comments and suggestions.1To whom reprint requests should be addressed;e-mail [email protected].

1714 HORTSCIENCE VOL. 47(12) DECEMBER 2012

oxygen bioavailability and seed germination,and no effective methods are available forimproving germination of large seeds and/oraged seeds.

The objectives of this research were to1) clarify the relationship between the con-centration of bioavailable oxygen in theliquid imbibed by the germinating seedand seed vigor; 2) define the optimum oxy-gen bioavailability for improving germina-tion percentages of aged and waterloggedseeds; and 3) provide a method for rescu-ing plant germplasm from aged or improp-erly stored seeds for use in plant breedingprograms.

Materials and Methods

Seeds. Corn (Zea mays L.) seeds (geno-type, ‘FR27 3 FRM017’) were kindly pro-vided by Illinois Foundation Seeds Inc.,Tolono, IL. Two different age categories ofseeds were used: in one, the seeds were threeyears old and in the other, the seeds were lessthan one year old. Seeds of squash (Cucurbitapepo L.) and tomato (Solanum lycopersicumL.) genotypes were both collected from abreeding research program at the Universityof Florida. The squash seeds (genotype, 125X)were four years old and the tomato seeds(genotype, 6132-HBK) were six years old,respectively. The seeds had been stored atambient room temperature (24 ± 1 �C) andhumidity (78.3% ± 6.9%). The seed lots areall pure lines.

Aeroponics treatment for corn seeds.Twenty-five liters of deionized (DI) wateradjusted to 0.5 mM CaSO4 (Lynch et al.,1987; Nadeem et al., 2012) were poured intoa 38-L square tank (61 3 61 3 30 cm3)(Growgenie, San Jose, CA). Calcium is essen-tial to the intactness and selectivity of bi-ological membranes. Aged seeds have veryfragile biological membranes. To protect themembrane, 0.5 mM CaSO4 was added. Thetank was covered with a 5-mm thick plasticsheet containing 60 evenly spaced holes, each48 mm in diameter. One plastic basket (50 mmhigh and external diameter of 55 mm at thetop and 37 mm at the bottom) was suspendedin the space above the water with a wiresecured through a hole in the cover. Six cornseeds were placed into each basket. A 24-Welectric pump (Danner Mfg. Inc., Islandia,NY) was installed on the tank bottom and anozzle was attached to the pump and posi-tioned in the center of the tank above thesurface of the water. The nozzle created a mistfor treating the seeds in the baskets. The tankwas put in a growth chamber (PercivalScientific, Inc., Perry, IA) at 25 ± 1 �C and16-h photoperiod with fluorescent lamps (in-tensity programmed to 1250 mmol·m–2·s–1 oflight irradiance) and 22 ± 1 �C and 8-hscotoperiod for 3 d. The numbers of germi-nated and non-germinated seeds were alwayscounted at 48 h after treatment.

Hydrogen peroxide treatments of allseeds. Thirty corn seeds were soaked for 24 hin 50 mL of 0.5 mM Ca (as CaSO4) containing

0.06%, 0.08%, 0.10%, 0.15%, 0.3%, or 3.0%(v/v) H2O2 in a 95 3 15-mm petri dish atroom temperature (25 ± 1 �C). The seedswere carefully rinsed four times with DIwater to remove the remaining H2O2. Nextin another 95 3 15-mm petri dish, theserinsed seeds were placed on a napkin satu-rated with 0.5 mM CaSO4 solution. The petridish was sealed with parafilm to retain themoisture. Squash and tomato seeds weretreated in exactly the same manner as thecorn seeds but with or without 0.15% H2O2,because the assay data of corn seeds hadproved that 0.15% H2O2 worked best andsquash and tomato seeds were assumed torespond similarly.

Aeration treatment for corn seeds. Thirtyseeds were put into a plastic basket of thesame type as described previously, whichwas immersed in 300 mL 0.5 mM CaSO4 ina 500-mL beaker. The solution was aeratedfor 24 h. The seeds were placed in a 95 315-mm petri dish on a napkin saturated with0.5 mM CaSO4 solution.

Imbibition measurement. Ten corn seedswere put in a vial with 20 mL of the soaking0.5 mM CaSO4 solution with or without0.15% H2O2 at room temperature (25 ± 1 �C).The weight of the seeds was determinedgravimetrically every 24 h for 96 h afterunbound moisture had been removed fromthe seed surface with an absorbent napkin.

Microelectrode fabrication. Ten-centimeterlengths of untreated 1.5-mm diameter boro-silicate glass capillaries (cat no. TW150-4;World Precision Instruments, Sarasota, FL)were each pulled into two micropipettesthrough a Sutter P-97 micropipette puller(Sutter Instrument Company, Novato, CA) at545 �C. The freshly pulled micropipettes weresilanized at 200 �C with N, N-dimethyltrime-thylslylamine according to Smith’s method(Smith et al., 1999). Micropipettes were back-filled with H+ probe backfilling solution of50 mM KCl and 50 mM HK2PO4. Then hydro-gen ionophore I-Cocktail B (cat no. 95293;Fluke Chemika, Switzerland) was drawn intothe tip with a minimal negative pressure andviewed by a binocular compound microscope(Smith et al., 1999).

Measurement of net ion fluxes. Forty-fivegrams of Sylgard 184 silicone elastomer and5 g Sylgard 184 curing agent (Dow Corpora-tion) were poured onto the bottom of a 10-cmdiameter Pyrex� dish to secure the treatedseed for scanning. One seed was secured onthe Sylgard in the center of the dish with fourto five Minucie stainless steel needles. Mi-croelectrodes were calibrated before and aftereach experiment. Calibrations were done instandard pH 6, 7, and 8 solutions (FisherScientific, Pittsburgh, PA) at 25 �C. TheNernst slopes (in mV/decade) were equal to59. After calibration, the microelectrodewas positioned both on the embryo and onthe endosperm of the targeted seed, respec-tively. Then, a 100 mm 3 100-mm area ofeach embryo and endosperm was scannedby means of the self-referencing ion selec-tive electrode (Smith et al., 1999). Ten em-bryos and endosperms were scanned in each

treatment. Extrusions of protons were al-ways calculated and averaged based on the10 measurements.

Measurements of oxygen consumption.The corn seeds were soaked in 0.5 mM CaSO4

solution with 0% or 0.15% H2O2 for 1 dbefore any measurements were made. Eachseed was secured on Sylgard as describedpreviously. Platinum–iridium oxygen elec-trodes were used. The microelectrode wascalibrated before and after each experiment.Calibrations were performed in DI watersaturated with air as 21% oxygen concentra-tion and in DI water through which nitrogengas had been bubbled for 30 min to reducethe oxygen concentration to 0% (Wu et al.,2002). Scans were done on 10 embryos andon 10 endosperms in each treatment. Oxygenconsumption per embryo or endosperm wasalways calculated and averaged based on the10 measurements.

Alcohol dehydrogenase activity. ADHaseactivity levels were measured in corn em-bryos held at different concentrations of bio-available oxygen for 48 h after germination at25 ± 1 �C. Before germination, the corn seedswere soaked in 0.5 mM CaSO4 solution, inaeroponics with 0.5 mM CaSO4, or in 0.15%H2O2 with 0.5 mM CaSO4 for 24 h. Aftertreatment each corn seed was cut into twohalves so the embryo was also cut into halves.Four embryo halves were homogenized in anextraction buffer composed of 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mg·mL–1

DTT, and 12 mM b-mercaptoethanol. The ex-traction solution of the ADHase enzyme wascentrifuged twice at 4 ± 0.1 �C at 15,000 rpmfor 5 min: first to separate the corn oil onthe top and the pellet on the bottom and thento get the ADHase supernatant used forADHase measurement. The ADHase activityassay was performed according to the pro-cedures described in Xie and Wu (1989). Onehundred microliters of enzyme solution and900 mL of reaction solution composed of50 mM Tris-HCl (pH 9.0), 1 mM EDTA, and1 mM NAD were incubated in 1.5-mL micro-centrifuge tubes in a water bath at 30 �C for3 min. Then, 100 mL 15% ethanol and thepreviously described 1000 mL enzyme andreaction solution were added directly to acuvette. After a reaction time of 1 min in thecuvette, a reading at 340 nm was made witha spectrophotometer (DU 64; Beckman In-struments, Fullerton, CA) to determine theconcentration of NADH. The ADHase activ-ity was calculated and averaged from ninemeasurements. A unit of ADHase activity isdefined as the production of 1 nmol NADH/min·mg–1 protein.

Protein measurement. Protein contentsof the samples were colorimetrically deter-mined according to Lowry’s method (Lowryet al., 1951; Peterson, 1977). Ten microlitersof supernatant were mixed with 990 mL LowryA [equal volumes of copper–tartrate–carbonatesolution consisting of 0.1% CuSO4·5H2O,0.2% KNa-tartrate, and 10% NaCO3; 10%sodium dodecyl sulfate (95%, Sigma # L-5750); 0.80 N NaOH and DI water], and 15 minlater, 500 mL Lowry B [one part of 2.0 N

HORTSCIENCE VOL. 47(12) DECEMBER 2012 1715

Folin & Ciocalteu’s Phenol Reagent Solution(Sigma # F-9252) diluted in five parts of DIwater] was added. Bovine serum albumin(Sigma # A-2153) was used to prepare thestandards (Arcan and Yemsnicioglu, 2007;Harris, 1987). All measurements were re-peated three times.

Statistical analysis. The data were ana-lyzed by one-way analysis of variance (SASInstitute, 2009). Results were considered sig-nificant at P < 0.05. After running the SASprogram, the critical ranges (least significantdifference2, 0.05) of Duncan’s multiple rangetest were used to identify significantly differ-ent means (Hubbard, 2001).

Results

Effects of different oxygen bioavailabilitieson germination percentages of corn seeds.The germination percentage of corn seedstreated with H2O2 was significantly greaterthan that with aerated water but withoutH2O2. Among all of the treatments withH2O2, germination percentages with 0.15%and 0.30% H2O2 were significantly higherthan in the other treatments. There was nosignificant difference between 0.15% H2O2

and 0.30% H2O2 (Fig. 1). This effect ofoxygen fortification from H2O2 on seed ger-mination indicates that, up to a point, i.e.,0.15% in this study, the higher the concen-tration of bioavailable oxygen used, thegreater the seed germination percentage.There is a sigmoid relationship (r2 = 0.81)between H2O2 concentration (%) and cornseed germination (G) in this range of tested

H2O2 concentrations from 0.06% to 3.0% asshown in Eq. (1):

Gð%Þ =93

1 + e�½H2O2 �+ 0:0185

0:0382

(1)

Nevertheless, this effect suggests thatconcentrations of bioavailable oxygen some-what greater than 0.3% may be detrimental tocorn seed germination and subsequent de-velopment. For example, the average lengthof the roots from seeds treated with 3% H2O2

was only 6.8 ± 1.7 mm 3 d after germination.In contrast, the corresponding average rootlength was 34.8 ± 1.7 mm in the 0.15% (v/v)H2O2 treatment.

The treatment with 0.15% H2O2 resultedin the greatest germination percentage ofboth aged and unaged seeds, whereas corre-sponding germination percentages with thewater treatment germinated poorly (Fig. 2).Aged seeds are defined as those seeds thatwere stored at room temperature for morethan two years and otherwise are consideredas unaged seeds. The germination percent-ages of both aged and unaged seeds seemedto be closely correlated with the concentrationof bioavailable oxygen. Moreover, Figure 2shows that when treated with 0.15% H2O2

and CaSO4 or aeroponics with CaSO4, thedifferences in the germination percentagesbetween the aged and unaged corn seedsbecame insignificant.

However, in the 0.5-mM CaSO4 soakingtreatment, which had the lowest concentra-tion of bioavailable oxygen, the germinationpercentage of unaged seeds was more than2.5-fold greater than that of aged seeds

(76.7% ± 15.3% of unaged seeds and 30.0% ±11.5% of aged seeds). The aged seeds weremuch more sensitive to the concentration ofbioavailable oxygen than the unaged seeds.

At 24 h, all of the unaged corn seeds hadbegun to germinate in both the 0.15% H2O2

and the aeroponics treatments but only fewseeds had begun to germinate in the DI watersoaking treatment. Further after 72 h, eachseed in the oxygen-sufficient 0.15% H2O2

treatment had both a shoot and a root 1 to 2 cmin length, but only one-third of the seeds inthe 0.5-mM CaSO4 treatment had begun togerminate (Fig. 3).

Seed position also impacted germinationsignificantly. Keeping corn embryo up to airresulted in a 3.1-fold greater seed germina-tion percentage than positioning the seedwith the embryo beneath and away from air(Fig. 4). This result indicates that the cornseeds in water were lacking oxygen and hencetheir germination was retarded.

Effects of oxygen bioavailability ongermination percentages of selected vegetableseeds. Aged seeds had very poor germinationpercentages regardless of crop type but oxy-gen fortification with 0.15% H2O2 signifi-cantly improved the germination percentagesof the selected vegetable seeds (Fig. 5).

Rate of imbibition by corn seeds. From thefirst day, the imbibition rate of seeds in the0.15% H2O2 treatment was always 11% to13% greater that than in the water treatment.The kinetics of seed imbibition indicate thatcumulative water uptake of corn seeds in the0.15% H2O2 treatment was 14% to 20%faster than that in the water treatment. These

Fig. 1. Germination percentages of corn seeds at 48 h after treatment with different concentrations of bioavailable oxygen. (A) Aeration; (B) aeroponics; (C)0.06%; (D) 0.08%; (E) 0.10%; (F) 0.15%; (G) 0.30%; (H) 3.00% hydrogen peroxide (H2O2). The bars sharing the same letter are not significantly different.Vertical bars indicate least significant difference at a = 0.05.


data demonstrate that the increased bioavail-ability of oxygen accelerates water uptake bycorn seeds (Fig. 6).

The cumulative water uptake (CWU) perseed can be fitted with time (t, day) in thefollowing formula:

CWU =a

1 +t

t0

� �b(2)

where a is the maximum CWU value per cornseed and b is the constant of water uptake rateby the seed with or without H2O2, and t0 isthe initiation time of seed imbibition in this

study. Thus, the CWU value of the seed canbe calculated by using Eq. (3) with H2O2 orEq. (4) without H2O2.

The CWU value per seed treated with0.15% H2O2:

CWUH2O2=

192:1

1 +t

0:5146

� ��1:5118

=192:1

1 +0:5146

t

� �1:5118(3)

r2 = 0:99

The CWU value per seed treated with H2O:

CWUH2O =180:9

1 +t

0:4744

� ��1:1206

=180:9

1 +0:4744

t

� �1:1206(4)

r2 = 0:99

Based on Eq. (2), a is the maximum CWUvalue when t is infinite. Therefore, Eqs. (3)and (4) define the maximum CWU of theseeds with or without H2O2 as 192.1 or 180.9,respectively. These calculations showed thatthe seed with H2O2 imbibed 11.2 mg morewater per seed than that without H2O2.

Based on mathematics, the derivative ofEq. (3) or (4) is the rate of water uptake(WUR) of the corn seed with or without H2O2.

WURH2O2= 1540:7t�2:5118 + 282:2t�1

+ 408:0t�0:5118 (5)

WURH2O = 958:4t�2:1206 + 213:6t�1

+ 221:8t�0:1206 (6)

The H2O2 treatment greatly facilitatedand increased the seed imbibition. For exam-ple, based on Eqs. (5) and (6), the WUR of theseed was 60.1% faster in the H2O2 treatmentthan in the DI water treatment on Day 1.

Consumption of oxygen by corn seeds.The oxygen consumption rate of both theembryo and the endosperm of a corn seed thathad been soaked for 24 h to the 0.15% H2O2

treatment was �2-fold greater than the cor-responding rate in the DI water treatment.The oxygen consumption rate of embryosin the DI water treatment seemed slightlyfaster than that of the endosperm in the0.15% H2O2 treatment (Fig. 7). Thus, thelowest oxygen consumption rate of embryoswas greater than the highest oxygen con-sumption rate of endosperms, regardless ofthe treatments involved.

Influx and efflux of protons with respect tocorn seed embryos and endosperms. Protonflux is characteristic of metabolism in livingorganisms. Figure 8 demonstrates that oxy-gen bioavailability determines the directionof flux of protons. Under hypoxia, bothembryos and endosperms imbibed protons(Yan et al., 1998) and, hence, net decreasesof protons were measured in the seeds.However, in corn seeds in the 0.15% H2O2

treatment, strong proton efflux occurred inboth the embryos and the endosperms. Figure8 also shows that metabolic activity of de-veloping embryos is always much greaterthan that of endosperms irrespective of thetreatment.

Alcohol dehydrogenase activities of cornembryos. It is well known that ADHase en-ables plants to adapt to hypoxic stress. Figure9 shows that H2O2, aeroponics, and orienta-tion of the embryo in the corn seed stronglyaffected the activity of the ADHase. Whencorn seeds had lain with the embryos beneath

Fig. 2. Effects of oxygen fortification with hydrogen peroxide (H2O2) on seed germination of three-year-old and new corn seeds. (A) Seeds were soaked in 0.15% H2O2 with 0.5 mM CaSO4 for 24 h; (B) seedswere treated in aeroponics with 0.5 mM CaSO4 for 24 h; (C) seeds soaked in 0.5 mM CaSO4 solution for24 h. Vertical bar indicates least significant difference at a = 0.05.

Fig. 3. Effect of bioavailable of oxygen fortification on the germination percentage of new corn seeds.Seeds in the two dishes on the left were treated with deionized (DI) water containing 0.5 mM CaSO4 andthose on the right were treated with 0.15% hydrogen peroxide (H2O2) as an oxygen fertilizer with0.5 mM CaSO4 at 24 h (in the upper pairs). At 72 h (in the lower pairs), the root was emerging from onlya few seeds in the DI water treatment, whereas in the H2O2 treatment, a 1- to 2-cm long root and a shootwere growing out of all of the seeds.


and pressed directly on the substrate, theADHase activity of the embryos was almostdouble that of embryos of seeds with theembryo facing up to the air. In the aeroponicstreatment, the seeds had been suspended ina mist and with ample air. However, the H2O2

treatment with concentration of 0.15% re-duced the ADHase activity to �40% of thatof embryos from the aeroponics treatment.At 25 �C, the dissolved oxygen level in wateris only �250 mM. This small supply ofdissolved oxygen might be consumed on theouter cell layers of the seeds.

Discussion

Oxygen bioavailability and improvementof seed vigor and for prolonged storage ofseed. The seeds of many crop species arestored for extended periods of time, and theirgermination percentages and other vital pro-cesses tend to deteriorate progressively whenthe seeds are stored for periods greater thanone year at room temperature and at highrelative humidity (Desai, 2004). As seedsage, much more oxygen is needed (Benamaret al., 2003). Corn seed coatings usually

include nutrients, plant hormones, insecti-cides, and fungicides, but no oxygen fertilizeris included. Oxygen bioavailability is essen-tially ignored because oxygen in its gaseousstate is not easily manipulated. Corn seedshave a relatively large size and a relativelysmall specific surface area. A small specificsurface area of a seed is not conducive toobtaining sufficient oxygen when the seedgerminates. The data from this study provedthat bioavailable oxygen is crucial for seedsto imbibe and germinate regardless of vege-table species (Figs. 1 through 9). Particularly,embryos consumed much more oxygen thanendosperms. This phenomenon indicates thatembryos metabolize faster than endospermsregardless of the bioavailability of oxygenbecause an embryo is a metabolic center.Therefore, the oxygen component should beadded to seed coatings because better oxygenbioavailability improves vigor and germina-tion of seeds. Sufficient bioavailable oxygenis helpful in resisting pathogens and fosteringseedling establishment (Lynch et al., 1981),which may lead to larger harvests. In addi-tion, an oxygen component would makepossible the use of seed that had been held inlong-term storage. Finally, an oxygen com-ponent may facilitate rescuing the germplasmof very old seeds (Desai, 2004), and thiscould be of great importance to plant breedersand curators of seed collections. For conve-nient manipulations, peroxides such as cal-cium peroxide or hydrogen peroxide maybe more readily manipulated and more ap-plicable to improved seed germination thangaseous oxygen. Clearly, seed coatings withoxygen fertilizers should be developed toimprove the establishment of field popula-tions of corn and various other large seededcrop species. This effect of oxygen bioavail-ability on ADHase activity means that theembryos of corn seeds, which are large relativeto seeds of many other species, are vulnerableto mild hypoxia even in aeroponics. How-ever, 0.15% H2O2 can supply �80 timesmore bioavailable oxygen than aeroponics.This concentration of oxygen appears to besufficient to reach all of the cells of the cornembryo. Thus, the seeds did not suffer fromlow-oxygen stress in the 0.15% H2O2 treatment.

Oxygen bioavailability and apertures ofwater gates of corn seeds. Temperature,moisture, and oxygen combine to determinethe basic conditions for germination of mostseeds (Desai, 2004). That temperature im-pacts water uptake is well known. However,the result of this study proves that oxygenbioavailability also influences the imbibitionrate of corn seeds. Tournaire-Roux et al. (2003)recently elucidated the whole root and cellbases for inhibition of water uptake by anoxiaand linked them to cytosol acidosis. Theyproved that one early response of plants tooxygen deficiency is the downregulation ofwater uptake of roots. The present study in-directly showed that corn seeds downregulatedimbibition in hypoxic stress (Fig. 6). Theremight be two possible reasons responsible forthis. On one hand, the water channel, namely,the water gate (Holbrook and Zwieniecki,

Fig. 4. Effect of seed position on germination of new corn seeds. (A) Each seed was soaked in deionized(DI) water with 0.5 mM CaSO4 for 24 h and then positioned with the embryo facing up to the air; (B)each seed was soaked in DI water with 0.5 mM CaSO4 for 24 h and positioned with the embryo beneathand away from the air. Vertical bar indicates least significant difference at a = 0.05.

Fig. 5. Effects of oxygen bioavailability on germination percentages of aged corn, squash, and tomatoseeds. Corn seeds had been aged three years, squash seeds four years, and tomato seeds six years.Vertical bars indicate least significant difference at a = 0.05.


2003), may have been downregulated andtherefore the rate of imbibition was curtailed.Probably this downregulation is an adaptiveresponse of plants to hypoxic stress. On theother hand, sufficient oxygen favored thenormal germination of seeds and hence, thegrowth of seedlings, which consumed moreoxygen (Fig. 7). Consequently, the seeds

needed to use more water (Fig. 6). Maurel(1997) reported that mercury can downregu-late the water gate sites of plant roots. Inanother experiment, we treated seeds with1 mM mercury (Hg) (as HgCl2) but seedimbibition was 8.7% ± 4.4% greater than thatof the untreated control. Moreover, wateruptake of corn seeds treated with both Hg

and H2O2 was 12.4% ± 7.5% greater than thatof the control. Again, H2O2 increased imbi-bition of corn seeds. Thus, the effect of Hgon seeds was different from that reported onroots, and this difference may reflect anorgan-specific response to the metal ions.However, the addition of a small quantity ofH2O2 to the water medium invariably en-hanced the imbibition of the seeds. Clearly,the supply of sufficient bioavailable oxygenis an effective way to improve germination ofcorn seeds. Thus, provision of the appropriateconcentration of bioavailable of oxygen maybe a potent measure to rescue aged and/orimproperly stored seeds for use in crop pro-duction, plant breeding, or maintenance ofgermplasm collections.

Oxygen bioavailability and cellularrespiration. Every step of plant growth anddevelopment depends directly on active chem-ical energy, i.e., ATP. ATP in seeds is producedfrom decomposition of glucose includingglycolysis and oxidative phosphorylation inthe cellular respiration chain. Under hypoxicconditions, one mole of glucose can onlygenerate 2 mol of ATP through glycosis. Asa byproduct from fermentation of glucose,ethanol, which is toxic to embryos and seed-lings, can be produced and accumulated (Drew,1997). Thus, germination percentage is low.However, the same mole of glucose can pro-duce up to 38 mol of ATP when there issufficient oxygen (Rich, 2003). No ethanolcan be produced under oxygen enrichmentwith 0.15% H2O2. Hence, seed germinationpercentage is high and seedlings grow faster(Figs. 1 and 5). Oxygen consumption isclosely related to metabolic rate. This phe-nomenon can be found from Figure 7. Em-bryos metabolize and consume oxygen fasterthan endosperms regardless of the bioavail-ability of oxygen (Fig. 8). When no H2O2 wasincluded in the liquid medium, the corn seedssuffered from lack of oxygen bioavailability(Figs. 6 to 8).

Oxygen bioavailability has a crucial rolein regulating germination of vegetable seeds.This research was conducted with seeds ofcorn, squash, and tomato treated with orwithout H2O2. The age of the seeds rangedfrom less than one year to six years. Seedgermination percentages, imbibition, oxygenconsumption, and ADHase activity weregravimetrically, electrochemically, and col-orimetrically determined as appropriate. Thebest concentration of H2O2 to produce anexcellent germination percentage is 0.15%for both aged and unaged seeds of the threetested species. The amounts of oxygen con-sumed by corn embryos and endosperms weresignificantly greater in 0.15% H2O2 than inwater. The ADHase activity of corn seedswith the embryo lying against the moist sub-strate was significantly greater than that of theseeds with the embryo facing upward in theair. Also, the ADHase activity of the corn seedsin an aeroponics treatment was significantlygreater than that of the seeds treated with0.15% H2O2. Physical orientation of cornseeds had, accordingly, an important effecton the germination percentage of the corn

Fig. 6. Time course of imbibition of corn seed at 30 �C treated either with 0.15% hydrogen peroxide (H2O2)(curve A) or with water (curve B). Weight of seeds was determined gravimetrically after unboundmoisture had been removed from the seed surface. Asterisk indicates significantly (P < 0.05) different.Inset: Changes in relative seed weight (%) calculated by using the seed weight in the deionized water.X axis donates time (day).

Fig. 7. Oxygen consumption rates of corn seeds that had been soaked for 24 h either in 0.15% hydrogenperoxide (H2O2) (A) or in deionized (DI) water (B). Vertical bars indicate least significant difference ata = 0.05.


seeds. The germination percentage of seedswith the embryos facing up to the air was�50% greater than that of seeds that had theembryo pressed against the underlying moistsubstrate. Dilute H2O2 greatly improved thegermination percentages of the tested vege-table seeds as old as six years and which had

not been stored under optimum conditions.This concentration significantly acceleratedthe imbibition of corn seeds and boosted seedgermination. As already noted, this sameconcentration of H2O2 was highly effectivein rescuing aged vegetable seeds of squashand tomato as well. Bioavailable oxygen

controlled water uptake, namely, imbibitionof the tested corn seeds. Oxygen fortificationwith dilute H2O2 greatly improved seedvigor and accelerated establishment of seed-lings of the three tested species. Oxygenbioavailability enrichment with dilute H2O2

significantly increased seed germination per-centage and seedling growth of the testedspecies and has great potential to enhance theproductivity of commercial crop production.

Literature Cited

Arcan, I. and A. Yemsnicioglu. 2007. Antioxidantactivity of protein extracts from heat-treatedor thermally processed chickpeas and whitebeans. Food Chem. 103:301–312.

Benamar, A., J. Grelet, C. Tallon, E. Teyssier,M. Renard, P. Satour, F. Duval, F. Montrichard,P. Richomme, and D. Macgerel. 2003. Someobservations on seed quality and mitochondrialperformance, p. 243–250. In: Nicolas, G., K.J.Bradford, D. Come, and H.W. Pritchard (eds.).The biology of seed. CABI Publishing, Cam-bridge, MA.

Bewley, J.D. and M. Black. 1994. Seeds: Physiol-ogy of development and germination. 2nd Ed.Plenum Press, New York, NY.

Dennis, E.S., R. Dolferus, M. Ellis, M. Rahman,Y. Wu, F.U. Hoeren, A. Grover, K.P. Ismond,

A.G. Good, and W.J. Peacock. 2000. Molecularstrategies for improving waterlogging toler-ance in plants. J. Expt. Bot. 51:89–97.

Desai, B.B. 2004. Seeds handbook: Processing andstorage. 2nd Ed. Marcel Dekker, Inc., NewYork, NY.

Drew, M.C. 1997. Oxygen deficiency and rootmetabolism: Injury and acclimation under hyp-oxia and anoxia. Annu. Rev. Plant Physiol.Plant Mol. Biol. 48:223–250.

Edelstein, M., F. Corbineau, J. Kigel, and H. Nerson.1995. Seed coat structure and oxygen avail-ability control low temperature germination ofmelon (Cucumis melo) seeds. Physiol. Plant.93:451–456.

Geigenberger, P., A.R. Fernie, Y. Gibon, and M.Christ. 2000. Metabolic activity decreases as anadaptive response to low internal oxygen ingrowing potato tubers. Biol. Chem. 381:723–740.

Hakansson, I., T. Keller, J. Arvidsson, and T.Rydberg. 2012. Effects of seedbed propertieson crop emergence. 4. Inhibitory effects ofoxygen deficiency. Acta Agr. Scandinavica,Section B-Soil and Plant Sci. 62:166–171.

Harris, D.A. 1987. Spectrophotometric assays,p. 59–60. In: Harris, D.A. and C.L. Bashford(eds.). Spectrophotometry and spectrofluorom-etry. I.R.L. Press, Oxford, UK.

Holbrook, N.M. and M.A. Zwieniecki. 2003. Watergate. Nature 425:361.

Hong, F. 2001. Study on the mechanism of ceriumnitrate effects on germination of aged rice seed.Biological Trace Element Research 87:191–200.

Hubbard, M.R. 2001. Statistical quality control forthe food industry. 2nd Ed. Aspen Publishers,Inc., Gaithersburg, MD.

Jackson, M.B., K. Ishizawa, and O. Ito. 2009. Evol-ution and mechanisms of plant tolerance toflooding stress. Ann. Bot. (Lond.) 103:137–142.

Lide, D.R. 1998. Handbook of chemistry andphysics. CRC Press, Boca Raton, FL.

Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J.Randall. 1951. Protein measurement with theFolin phenol reagent. J. Biol. Chem. 193:265–275.

Fig. 8. Proton efflux and influx with respect to corn seed embryos and endosperms treated with either0.15% hydrogen peroxide (H2O2) or deionized (DI) water for 1 d. The embryos had much strongermetabolism than the endosperms regardless of oxygen bioavailability and hence had much apparentproton efflux. Vertical bars indicate least significant difference at a = 0.05.

Fig. 9. Alcohol dehydrogenase (ADHase) activities in corn embryos at 48 h after germination andmaintained in media at 25 �C with different concentrations of bioavailable oxygen. (A) 0.15%hydrogen peroxide (H2O2) for 24 h; (B) aeroponics; (C) every seed was positioned on a on a napkinsaturated with 0.5 mM CaSO4 solution with the embryo oriented to the top of the seed and adjacent tothe air; (D) each seed was positioned with the embryo against the napkin saturated with 0.5 mM CaSO4

solution. Vertical bar indicates least significant difference at a = 0.05.


Lynch, J., G.R. Cramer, and A. Lauchli. 1987. Salinityreduces membrane-associated calcium in cornroot protoplasts. Plant Physiol. 83:390–394.

Lynch, J.M., S.H.T. Harper, and M. Sladdin. 1981.Alleviation, by a formulation containing calciumperoxide and lime, of microbial inhibition ofcereal seedling establishment. Curr. Microbiol.15:27–30.

Maurel, C. 1997. Aquaporins and water perme-ability of plant membranes. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 48:399–429.

Nadeem, M., A. Mollier, C. Morel, A. Vives, L.Prud’homme, and S. Pellerin. 2012. Maize (Zeamays L.) endogenous seed phosphorus remobi-lization is not influenced by exogenous phos-phorus availability during germination andearly growth stages. Plant soil. <http://www.springerlink.com/content/b747127159k36g82/>.

Peterson, G. 1977. A simplification of the proteinassay method of Lowry et al. which is more

generally applicable. Anal. Biochem. 83:346–356.

Rich, P.R. 2003. The molecular machinery ofKeilin’s respiratory chain. Biochem. Soc. Trans.31:1095–1105.

SAS Institute. 2009. SAS/STAT user’s guide.Version 9.1.3. SAS Inst., Cary, NC.

Smith, P.J.S., K. Hammar, D.M. Porterfield, R.H.Sanger, and J.R. Trimarchi. 1999. Self-referencing,non-invasive, ion selective electrode for single celldetection of trans-plasma membrane. Microsc.Res. Tech. 46:398–417.

Sowa, A.W., S.M.G. Duff, P.A. Guy, and R.D. Hill.1998. Altering hemoglobin levels changesenergy status in maize cells under hypoxia.Proc. Natl. Acad. Sci. USA 95:10317–10321.

Sveinsdottir, H., F. Yan, Y. Zhu, T. Peiter-Volk,and S. Schubert. 2009. Seed ageing-inducedinhibition of germination and post-germinationroot growth is related to lower activity of

plasma membrane H+-ATPase in maize roots.J. Plant Physiol. 166:128–135.

Tournaire-Roux, C., M. Sutka, H. Javot, E. Guout, P.Gerbeau, D.T. Luu, R. Bligny, and C. Maurel.2003. Cytosolic pH regulates root water trans-port during anoxic stress through gating ofaquaporins. Nature 425:393–397.

Wu, F.H., G.C. Zhao, and X.W. Wei. 2002.Electrocatalytic oxidation of nitric oxide atmulti-walled carbon nanotubes modified elec-trode. Electrochem. Commun. 4:690–694.

Xie, Y. and R. Wu. 1989. Rice alcohol dehydro-genase genes: Anaerobic induction, organ spe-cific expression and characterization of cDNAclones. Plant Mol. Biol. 13:53–68.

Yan, F., R. Feuerle, S. Schaffer, H. Fortmeier, andS. Schubert. 1998. Adaptation of active protonpumping and plasmalemma ATPase activity ofcorn roots to low root medium pH. PlantPhysiol. 117:311–319.


increased oxygen bioavailability improved vigor and germination of

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