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Scientia Horticulturae 119 (2009) 98–102

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romotion by 5-aminolevulenic acid of pepper seed germination and seedlingmergence under low-temperature stress

hmet Korkmaz *, Yakup Korkmaz

ahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Horticulture, Kahramanmaras 46060, Turkey

R T I C L E I N F O

rticle history:

eceived 30 January 2008

eceived in revised form 9 July 2008

ccepted 11 July 2008

eywords:

apsicum annuum

ow-temperature stress

eed treatment

eed storage

A B S T R A C T

The effects of incorporating 5-aminolevulenic acid (ALA) into the priming solution on low-temperature

germination and emergence percentage performance of red pepper (Capsicum annuum cv. Sena) seeds

before and after seed storage were investigated. Seeds were primed in 3% KNO3 solution for 6 days at

25 8C in darkness containing 0 ppm, 1 ppm, 10 ppm, 25 ppm, 50 ppm or 100 ppm ALA. Following priming,

seeds were either immediately subjected to germination and emergence tests at 15 8C or stored at 4 8C or

25 8C for 1 month after which they were subjected to germination and emergence tests at 15 8C. Priming

pepper seeds in the presence of ALA improved final germination percentage (FGP) and germination rate

(MGT) at 15 8C compared to non-primed seeds. The highest FGP was obtained from seeds primed in the

presence of 25 ppm and higher ALA concentrations while the highest MGT was obtained from seeds

primed in KNO3 supplemented with 10 ppm ALA. Emergence percentages were the highest for the seeds

primed in the presence of 25 ppm ALA and 50 ppm ALA while non-primed seeds had the lowest

emergence percentage. Highest emergence rates (MET) and heaviest seedlings were also obtained from

seeds primed in KNO3 supplemented with 50 ppm ALA. Although all priming treatments improved

germination and emergence performance of pepper seeds at 15 8C following 1 month of storage under

two different temperatures, inclusion of 25 ppm and 50 ppm ALA into the priming solution resulted in

higher germination and emergence percentages and faster germination and emergence compared to

seeds primed in KNO3 only and non-primed seeds. These results indicate that priming seeds in 25 ppm

and 50 ppm ALA incorporated into the KNO3 solution can be used as an effective method to improve low-

temperature performance of red pepper seeds and that these seeds can be stored for 1 month at 4 8C or

25 8C and still exhibit improved germination and emergence performance at 15 8C.

� 2008 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Scientia Horticulturae

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1. Introduction

Pepper has a prolonged germination period and an optimumgermination temperature requirement of 30 8C (O’Sullivan andBouw, 1984). When pepper is sown directly in the field, soiltemperatures can often be suboptimal, causing delayed and non-uniform seedling emergence. For example, in the Kahramanmarasprovince, one of the commercial red pepper production areasin Turkey, the mean soil temperature ranges from 10 8C to 20 8C atthe planting depth on typical seeding dates (Korkmaz, 2005).Lorenz and Maynard (1988) reported that emergence from 1.2 cmsoil depth took 8–9 days at soil temperatures of 25 8C and wasprevented totally at temperatures lower than 15 8C. As theemergence is delayed, the problem is exacerbated becausethe probability of soil crust formation becomes greater and the

* Corresponding author. Tel.: +90 344 223 7666; fax: +90 344 223 0048.

E-mail address: akorkmaz@ksu.edu.tr (A. Korkmaz).

304-4238/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

oi:10.1016/j.scienta.2008.07.016

chances of germinating seeds and seedlings to be infected bydamping-off causing pathogens such as Pythium increase (Hendrixand Campbell, 1973). Thus, obtaining ideal plant stands requiresfast and uniform emergence to avoid these problems.

The 5-aminolevulenic acid (ALA) is a key precursor in thebiosynthesis of all porphyrins compounds such as chlorophyll,heme and phytochrome (Wang et al., 2005). Exogenous applica-tions of ALA have been found to regulate plant growth anddevelopment and increase crop yield (Hotta et al., 1997). It is alsoknown to enhance plant’s tolerance to cold (Hotta et al., 1998;Wang et al., 2004) and salinity stresses (Watanabe et al., 2000;Nishihara et al., 2003) in low concentrations and exhibit herbicidaleffects if used at concentrations over 5 mM (Kumar et al., 1999) allof which suggest that ALA has a great application potential inagricultural production as a new non-toxic endogenous substance(Wang et al., 2003).

Seed priming or osmoconditioning is a pre-sowing treatmentthat consists of the incubation of seeds in an osmoticum, usually asalt or polyethylene glycol (PEG) solution, in order to control their

Table 1Final germination percentage (FGP) and its angular transformation (8) and mean

germination time (MGT) of ‘Sena’ red pepper seeds germinated in darkness at 15 8Cfollowing priming for 6 days at 25 8C in 3% KNO3 supplemented with 5-

aminolevulenic acid (ALA)

Treatments FGP MGT (days)

% (8)

Non-primed seeds 51 45d 8.33a

KNO3 64 53c 5.93b

KNO3 + 1 ppm ALA 82 64b 4.93bc

KNO3 + 10 ppm ALA 86 68ab 4.70c

KNO3 + 25 ppm ALA 91 73a 4.90bc

KNO3 + 50 ppm ALA 91 73a 4.85bc

KNO3 + 100 ppm ALA 91 73a 5.83b

LSD0.05 6 1.08

Significances *** ***

***Significant at P � 0.001.

A. Korkmaz, Y. Korkmaz / Scientia Horticulturae 119 (2009) 98–102 99

water uptake and prevent radicle protrusion (Pill, 1995). Thistechnique has been used in some vegetable seeds to increase thegermination rate, total germination and seedling uniformity, mainlyunder unfavorable environmental conditions (Bradford et al., 1990;Ozbingol et al., 1998; Nascimento and Arago, 2004; Korkmaz, 2006).The objectives of this study were: (1) to investigate incorporation ofALA into priming solution would improve pepper seed germinationand emergence at low temperature and (2) to determine whethergermination and emergence enhancements are retained in seedsprimed in the presence of ALA and stored under two differenttemperature regimes.

2. Materials and methods

2.1. Plant material

Seeds of ‘Sena’ red pepper (Capsicum annuum), all from the sameseed lot, were obtained from Agricultural Research Institute,Kahramanmaras, Turkey. The standard germination test (ISTA,1996) was conducted and their initial germination percentage wasdetermined as 95%.

Seeds were disinfested in 1% (active ingredient) sodiumhypochlorite solution for 10 min to eliminate possible seed-bornemicroorganisms, rinsed for 1 min under running water prior todrying for 30 min at room temperature (Korkmaz, 2005).

2.2. Treatments

Priming was accomplished by imbibing 5 g of seed for 6 days at25 8C in darkness in 3% KNO3 (Korkmaz, 2005) containing 1 ppm,10 ppm, 25 ppm, 50 ppm or 100 ppm ALA (Sigma Aldrich, St. Louis,MO, USA). Seeds were placed in covered transparent polystyrenegermination boxes (10 cm � 10 cm � 4 cm) on double layers offilter paper (Whatman #1) saturated with 15 ml priming solutionsupplemented with above-mentioned concentrations of ALA. Thepriming solutions were changed 3 days after the onset of theexperiment to maintain a constant osmotic potential. Followingpriming, the seeds from each box were washed in a sieve andrinsed under running tap water for 1 min and left to surface dry onpaper towels under room conditions (22 8C and 60% relativehumidity) for 2 h to make the singulation of the seeds easier. Seedsprimed in 3% KNO3 solution without ALA and non-primed seedswere also included in the germination and emergence tests.

2.3. Seed germination at low temperature

Germination test was carried out in darkness in a temperature-controlled incubator held at 15 � 1 8C. Fifty seeds were placed ontwo layers of filter paper moistened with 5 ml of distilled water incovered 9 cm petri dishes. To prevent fungal contamination, 1 ml of%0.5 Captan was added to the water. Treatments were arranged incompletely randomized design with four replications. Radicleprotrusion to 1 mm was scored as germination. Germination wasrecorded daily until the numbers stabilized (for 15 days) andgerminated seeds were removed from the petri dishes. From the totalnumber of seeds germinated, final germination percentage (FGP), itsangular transformation (arcsineHFGP) and mean germination time(MGT) were calculated. MGT, an inverse measure of germination rate,was calculated according to the following formula (Cantliffe, 1991):

MGT ¼ A1D1 þ A2D2 þ � � �AnDn

A1 þ A2 þ � � �An

where A is the number of seeds germinating per day, D is the timecorresponding to A in days and n is the number of days to finalcount.

2.4. Seedling emergence at low temperature

Seeds were primed as described above and 20 seeds from eachtreatment were planted into 1.5 cm depth in 18 cm � 9 cm � 4 cm(length �width � height) plastic cups filled with growth mediumconsisting of peat and perlite in the ratio of 4:1. The cups werewatered and placed in a growth chamber (Buzkar, Model No:BT610, Kahramanmaras, Turkey) at 15 � 1 8C and under coolfluorescent lamps providing a photosynthetic photon flux densityof 100 mmol m�2 s�1 for 14 h day�1 at the seedling level. Thetreatments were replicated four times and all the cups were arrangedin a randomized complete block design in the growth chamber.Emergence counts (hypocotyl arc visible) (Korkmaz, 2005) weremade daily until the percentage of emerging seedlings had stabilizedin all treatments and final emergence percentage (FEP) and meanemergence time (MET) were calculated. Twenty-eight days afterplanting, shoot fresh (cut at the medium surface) and dry weights(dried at 70 8C for 48 h) were determined.

2.5. Seed germination and seedling emergence at low temperature

following storage

Seeds were primed as described above; however, only ALAconcentrations (25 ppm and 50 ppm) that resulted in highestgermination and emergence (see Tables 1 and 2) were included inseed storage experiment.

Following the priming, seeds were rinsed as described aboveand left to dry on paper towels for 2 days under room conditions(18–23 8C and 50–60% relative humidity). Non-primed seeds werealso subjected to the same drying conditions. Primed and non-primed seeds then were placed in screw-top plastic bottles andstored in darkness at 4 8C or 25 8C for 1 month. At the end ofstorage, seeds were subjected to germination and emergence testsat 15 � 1 8C. Germination and emergence test conditions were thesame as described above.

To determine seed moisture content following storage, fourreplications of 50 seeds from all treatments were weighed, thenplaced in glass petri dishes and oven-dried at 130 8C for 1 h. At theend of 1 h, seeds were weighed again, and their moisture contentsexpressed as percentages of oven-dry weight were calculatedaccording to wet weight basis.

2.6. Statistical analysis

Data from all experiments were subjected to analysis ofvariance and mean separation was performed by Fisher’s leastsignificant difference (LSD) test if F-test was significant at P = 0.05.

Table 2Final emergence percentage (FEP) and its angular transformation (8), mean

emergence time (MET) and shoot fresh and dry weights of ‘Sena’ red pepper seeds

emerged at 15 8C following priming for 6 days at 25 8C in 3% KNO3 supplemented

with 5-aminolevulenic acid (ALA)

Treatments FEP MET (days) Shoot weight

(mg/plant)

% (8) Fresh Dry

Non-primed seeds 35 36d 18.15a 15.8e 3.8

KNO3 60 51c 14.95b 23.2d 3.8

KNO3 + 1 ppm ALA 85 68b 13.53bc 29.2ab 4.1

KNO3 + 10 ppm ALA 89 71ab 13.60bc 24.8cd 3.9

KNO3 + 25 ppm ALA 95 81a 13.65bc 26.8bc 4.0

KNO3 + 50 ppm ALA 94 78a 12.85c 32.0a 4.4

KNO3 + 100 ppm ALA 85 69b 13.63bc 27.8bc 3.9

LSD0.05 11 1.79 3.3 –

Significances *** *** *** NS

***,NSSignificant at P � 0.001 or not significant, respectively.

A. Korkmaz, Y. Korkmaz / Scientia Horticulturae 119 (2009) 98–102100

Experiments were repeated twice and since there was nosignificant difference between the results of two experiments,data from both experiments were combined and the mean valuesare presented.

3. Results

3.1. Seed germination at low temperature

All priming treatments significantly improved pepper seedgermination at 15 8C compared to non-primed seeds which had anFGP of 51% (Table 1). Priming pepper seeds regardless of ALAconcentrations added to the priming solution also enhancedgermination compared to seeds primed in KNO3 only which had anFGP of 64%. The highest FGP (91%) was obtained from seeds primedin the presence of 25 ppm and higher ALA concentrations. Eventhough all priming treatments improved the MGT of pepper seedscompared to the non-primed seeds (MGT = 8.33 days), the highestgermination rate was obtained from seeds primed in KNO3

supplemented with 10 ppm ALA (MGT = 4.70 days).

3.2. Seedling emergence at low temperature

All priming treatments improved FEP of pepper seedlings at lowtemperature compared to non-primed seeds which had an FEP of35% (Table 2). Additionally, inclusion of ALA in increasingconcentrations into the priming solution enhanced further low-temperature emergence of pepper seedlings compared to seedsprimed in KNO3 only (60%). The highest FEP was obtained when25 ppm ALA (95%) and 50 ppm ALA (94%) were added to the

Table 3Final germination percentage (FGP) and its angular transformation (8), mean germinatio

darkness at 15 8C following priming for 6 days at 25 8C in 3% KNO3 supplemented wit

Treatments FGP

4 8C 25 8C

% (8) %

Non-primed seeds 44 41c 43

KNO3 59 50b 55

KNO3 + 25 ppm ALA 88 70a 87

KNO3 + 50 ppm ALA 91 73a 89

LSD0.05 6

Significances ***

***,NSSignificant at P � 0.001 or not significant, respectively.a Wet weight basis, oven-dried (130 8C, 1 h).

priming solution. Even though all priming treatments regardless ofALA concentrations added improved the MET of pepper seedscompared to non-primed seeds (MET = 18.15 days), the fastestMET was obtained from seeds primed in KNO3 supplemented with50 ppm ALA (MET = 12.85 days). Moreover, priming in thepresence of ALA significantly affected shoot fresh weight andthe heaviest seedlings were obtained from seeds that were primedin KNO3 supplemented with 50 ppm ALA. On contrary, primingregardless of ALA concentrations added to the priming solution didnot affect shoot dry weight.

3.3. Seed germination and seedling emergence at low temperature

following storage

All priming treatments significantly improved FGP and MGT ofpepper seeds at low temperature following storage under twotemperature regimes compared to non-primed seeds, indicatingthat beneficial effects of priming in the presence of ALA wereretained after 1 month of storage (Table 3). Moreover, priming inthe presence of 25 ppm ALA and 50 ppm ALA led to higher FGP thanpriming in KNO3 only. Seeds primed in the presence of 25 ppm ALAor 50 ppm ALA also exhibited higher germination rates when theywere stored in 25 8C compared to seeds primed in KNO3 only;however, there were no significant differences in MGT when theywere stored in 4 8C. Additionally, seed moisture content was notaffected by seed priming followed by storage for 1 month at 4 8C or25 8C.

One month after the storage in two different temperatures,seeds primed in the presence of both concentrations of ALA hadsignificantly higher FEP and MET than those primed in KNO3 onlyor non-primed seeds even though storing the seeds in 25 8Cresulted in slightly lower FEP and lower emergence rates than 4 8C(Table 4). Regardless of storage temperature, the heaviest shootswere obtained from the seeds that were primed in the presence of25 ppm ALA but shoot dry weight was unaffected by the primingtreatments.

4. Discussion

In this study, evidence is provided that ALA improves low-temperature germination and emergence performance of pepperseeds and to the best of our knowledge, this is the first report tosuggest that ALA imbibed into seeds during priming imparttolerance to low temperatures that may occur during seedgermination and emergence.

Recent studies have documented that pre-treatment of plantswith ALA enhanced plant’s tolerance to low-temperature stress.ALA is known to induce the activity of antioxidant enzymes such assuperoxide dismutase and ascorbate peroxidase all of which are

n time (MGT) and seed moisture content of ‘Sena’ red pepper seeds germinated in

h 5-aminolevulenic acid (ALA) after 1 month storage at 4 8C or 25 8C

MGT (days) Seed moisturea (%)

4 8C 25 8C 4 8C 25 8C

(8)

41b 8.38a 8.28a 5.9 5.8

48b 5.68b 6.13b 6.1 6.1

69a 5.62b 5.48c 6.2 5.9

71a 5.43b 5.40c 6.0 6.0

8 0.69 0.58 – –

*** *** *** NS NS

Table 4Final emergence percentage (FEP) and its angular transformation (8), mean emergence time (MET) and shoot fresh and dry weights of ‘Sena’ red pepper seeds emerged at 15 8Cfollowing priming for 6 days at 25 8C in 3% KNO3 supplemented with 5-aminolevulenic acid (ALA) after 1 month storage at 4 8C or 25 8C

Treatments FEP MET (days) Shoot fresh weight

(mg/plant)

Shoot dry weight

(mg/plant)

4 8C 25 8C 4 8C 25 8C 4 8C 25 8C 4 8C 25 8C

% (8) % (8)

Non-primed seeds 40 39b 35 36c 19.68a 21.08a 14.2c 15.5b 3.2 2.8

KNO3 55 48b 54 47b 15.95b 18.33b 20.6b 18.1b 3.4 2.8

KNO3 + 25 ppm ALA 81 65a 73 59a 13.53c 15.98c 29.0a 23.4a 3.5 3.7

KNO3 + 50 ppm ALA 78 62a 71 58a 14.38c 16.48c 21.2b 18.2b 4.1 2.8

LSD0.05 12 10 1.2 1.79 5.9 3.7 – –

Significances ** ** *** *** ** ** NS NS

***, **,NSSignificant at P � 0.001, 0.01 or not significant, respectively.

A. Korkmaz, Y. Korkmaz / Scientia Horticulturae 119 (2009) 98–102 101

associated with plant’s tolerance to stressful environments(Nishihara et al., 2003). Hotta et al. (1998) reported that rootsoaking of rice seedlings in 1 ppm ALA solution increased toleranceto subsequent low-temperature stress at 5 8C for 5 days. Moreover,Wang et al. (2004) found that melon seedlings treated with10 mg l�1 ALA solution exhibited increased growth and photo-synthetic capacity as well as improved chilling tolerance withoutany adverse effect.

On the contrary, very little information is available about theinvolvement of ALA during seed germination under stressfulconditions. It has been proposed that exposure of plants to low andhigh temperatures is associated with blockage of ALA biosynthesissuggesting that ALA biosynthesis is a temperature-sensitiveprocess (Hodgins and van Huystee, 1986; Tewari and Tripathy,1998). ALA is the first precursor in the biosynthesis of porphyrinscompounds such as chlorophyll and heme which is a key elementfor the activity of cytochrome c in the respiration chain of themitochondrion (Gonzalez et al., 2000). During seed germination,respiration which is a temperature-dependent and heme-requiringprocess increases in order to supply the germinating seed withnecessary energy. Wang et al. (2005) reported that exogenousapplication of ALA resulted in increased respiration and promotedgermination of pakchoi seeds that were under salinity stresswhereas non-ALA-treated seeds had significantly lower respirationrates and germination. They concluded that enhanced seedgermination under salinity stress was associated with increasedheme biosynthesis and respiration. Moreover, ALA is also known toserve as a protectant against oxidative damages of membranes byreducing the malondialdehyde content of tissues, a decompositionproduct from the peroxidation of polyunsaturated fatty acids ofbiomembranes in plants under such stresses as chilling, droughtand salinity (Zhang et al., 2006). In our study, the reason forsuperior performance of seeds that were treated with ALA beforesowing may be that similar mechanisms may have operated insuch a way and protected the germinating pepper seeds fromdamaging actions of low temperatures.

In seed priming studies, seeds usually are transferred directlyfrom the priming solution to the germination media, generallyresulting in very rapid germination compared to non-primed seeds(Pill, 1995). Since primed seeds have completed phase I (hydration)and phase II (lag phase) of germination, they only require a favorablewater potential gradient for water uptake in order to begin radiclegrowth. Primed seeds have the advantage of entering immediatelyinto phase III of germination, while seeds which are dried afterpriming must repeat phase I and often at least a short phase II beforegrowth occurs (Pill, 1995). In order to maintain high quality inprimed seeds for extended storage periods, seeds should be stored atlow temperature and low moisture content. In general, storingprimed seeds reduces germination or seedling emergence and there

are conflicting results on the effect of storage life of primed seeds. Forexample, slower and less synchronous emergence from primed anddried-back seeds has been reported for such species as asparagusand tomato (Pill et al., 1991) and muskmelon (Oluoch and Welbaum,1996). On the contrary, Thanos et al. (1989) reported that primedsweet pepper seeds retained their improved vigor after a storageperiod of 6 months at 5 8C. Korkmaz (2005) also reported that 1month storage at 4 8C did not reduce the performance of pepperseeds that were primed in the presence of salicylic acid.

In this study, although there was a slight reduction ingermination and emergence percentages and rates of the primedpepper seeds stored for 1 month compared to their germinationperformance before the storage, seeds from all priming treatmentsperformed better than non-primed seeds. Higher germinationpercentages and germination rates indicated that beneficial effectsof priming especially in the presence of ALA were retained in theseeds without any significant loses after a month of storage undertwo different temperature regimes.

5. Conclusion

The result of the present study revealed that inclusion of 25 ppmand 50 ppm ALA significantly improved pepper germination andemergence compared to seeds primed in KNO3 only and non-primedseeds. Priming with the addition of ALA may be an effective way toshorten the time of emergence and increase stand establishment inpepper at low temperatures. The fact that ALA, an endogenous plantgrowth regulator, could be used to prevent crop losses due to low-temperature stress may have a significant practical application.Moreover, this study also revealed that beneficial effects of seedpriming in the presence of ALA were retained after 1 month ofstorage under two different temperature regimes and that primedseeds exhibited improved germination and emergence performanceat low temperature compared to non-primed seeds. The mechanismby which ALA increases tolerance to low temperatures during seedgermination and seedling emergence has not yet been fullyelucidated. However, our efforts are currently underway to betterunderstand the role of ALA in enhancing tolerance of germinatingpepper seeds and seedlings to low temperatures.

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