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Environmental and Experimental Botany 62 (2008) 45–53

Light environment in pre- and post-dehiscent fruits affectsseed germination in Calotropis procera

Santosh Sharma ∗, Dilip AmritphaleInstitute of Environment Management & Plant Sciences, Vikram University, Ujjain, M.P. 456010, India

Received 15 July 2007; accepted 15 July 2007

bstract

Fruits in Calotropis procera can be distinguished into five discrete but contiguous stages on the basis of diameter and seed color. Seeds fromehisced fruits at stage V germinated >80% on moist substratum in darkness. This was rather unexpected because the seeds developed and maturedn an FR-enriched microenvironment (R:FR ratio ∼0.3) of the chlorophyll-containing maternal tissue and displayed low-fluence response (LFR)

ode of phytochrome action. In contrast to >80% dark-germinating seeds from dehisced fruits at stage V, about 50% seeds from undehisced fruitst that stage were dark germinating, whereas another 30% seeds required light for germination. The light-requiring fraction of the seed populationid not only respond to a very low-fluence R and to a short FR pulse, but also lacked R–FR reversibility thereby indicating to a very low-fluenceesponse (VLFR) mode of phytochrome action. The present study reporting VLFR to non-dormant seed state transition in C. procera suggested that

he state of phytochrome and the subsequent seed germination response in dry-seeded species, besides being determined by the light environmentmmediately before maturation drying, might also be regulated by a post-dehiscence light signal.

2007 Elsevier B.V. All rights reserved.

eywords: Calotropis procera; Chlorophyll; Fruit developmental stage; Low-fluence response; Phytochrome; R:FR ratio; Seed germination; Very low-fluence

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. Introduction

Variation in an individual’s phenotype may be determinedot only by the genotype and environment of that individualut also by maternal effects, which occur via structure or phys-ology (Roach and Wulff, 1987). The light environment of theother plant is known to influence subsequent seed germina-

ion (Gutterman, 2000 and references therein; Galloway, 2001,005). Munir et al. (2001) showed maternal photoperiod at theime of seed maturation to affect seasonal dormancy in Ara-idopsis. Earlier, plants of Arabidopsis grown in light with aow red/far-red ratio were reported to produce seeds requiringight for germination (McCullough and Shropshire, 1970; Hayesnd Klein, 1974). Notably, maternal tissues, the integuments ofhe ovule and the wall of the ovary, which eventually form the

eed coat and fruit wall, respectively, are known to be importanteterminants of seed dormancy, dispersal and germination traits.utterman (2000) identified a number of areas that need further

∗ Corresponding author.E-mail address: santosh ujj@yahoo.com (S. Sharma).

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nvestigation on the effects of maternal environment during seedevelopment on subsequent germination and dormancy includ-ng the influence of chlorophyll content of the maternal tissueurrounding the developing seeds. Berrie et al. (1974) suggestedhat the light reaching to the embryo through chlorophyllousnvesting structures cannot be expected to activate phytochromeue to preferential absorption of red light by chlorophyll. Seedormancy in several species studied by Cresswell and Grime1981) was indeed correlated with the chlorophyll content of theaternal tissue surrounding the embryo. Those species, in which

he chlorophyll level in the investing tissues decreased early dur-ng seed development, tended to produce non-dormant seeds. Inontrast, the seeds produced by species whose extra-embryonicissues remained green until after the seeds had started to dryere light-requiring, presumably because when shed from thelants they contained an inadequate Pfr/Ptotal ratio.

Calotropis procera (Ait.) R. Br. is a perennial shrub repro-ucing primarily by seeds. It is known to act as a key resource

rovider in degraded ecosystems to about 80 animal speciesncluding two predispersal insect seed predators which solelyepend on the plant for completion of their life cycle (Amritphalend Sharma, 2007). The fruit is a follicarium (Spjut, 1994) with

4 al and Experimental Botany 62 (2008) 45–53

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aat6The light simulating the R to FR ratio (R:FR ratio) of typicalmid-day sunlight (1.2:1, henceforth referred to as simulated day-light) was obtained with a bank of compact fluorescent lamps andincandescent bulbs. Photon fluxes for the R and FR and the R:FR

Table 1Fruit diameter, seed color, developmental stages and states of the fruits

Fruit diameter(mm)

Seed color Developmentalstage of the fruit

State of the fruit/seed

≤10 Pale white I Undehisced/immature11–20 Pale white II Undehisced/immature21–30 Pale yellow III Undehisced/immature

6 S. Sharma, D. Amritphale / Environment

relatively thick pericarp, which remains green from the timef fruit-set to fruit dehiscence. In addition, the seed coat alsourns green during the process of maturation before acquir-ng the typical brown color of mature seeds. Amritphale et al.1984) reported more than 70% seed germination in darkness in. procera. Earlier, Van der Veen (1970) showed leaf-filtered

ight to reduce seed germination below dark control valuesn this species. In a preliminary study, we found intermittentar-red light treatment to induce dormancy in mature seedsn C. procera. The dormancy could be reversibly released oreinstated by subsequent brief red or far-red light pulses thusndicating to low-fluence response mode of phytochrome con-rol of seed germination. Indeed, induction of seed germinationy very low-fluence red light (or a pulse of far-red light) andy low-fluence red light is known to occur in several plantpecies including Lactuca sativa, Arabidopsis thaliana, Daturaerox, Caesulia axillaris and Hygrophila auriculata (Blaauw-ansen and Blaauw, 1975; Cone et al., 1985; Scopel et al., 1991;ingh and Amritphale, 1992; Amritphale et al., 1995). Further,s mentioned above, Cresswell and Grime (1981) showed thehlorophyll content of the maternal tissues around the develop-ng embryo and consequent possible lowering of the red–far-redatio to result into reduced capacity of seeds to germinate inarkness in a number of species. The two hypotheses tested inhe present study were thus: seed germination in C. procera isignificantly affected by (i) the spectral composition of lightransmitted through the chlorophyll-rich pericarp during seed

aturation, and (ii) a post-dehiscent light signal.

. Materials and methods

C. procera (Ait.) R. Br. (Asclepiadaceae) is a perennial shrubhat grows as a wasteland weed in and around Ujjain (23◦18′N,5◦77′E) and flowers and fruits profusely every year from Marcho May. The fruit has a three-layered pericarp. The outer layeronsists of the exocarp and the outer mesocarp, the middle layers the fibrous middle mesocarp, whereas the inner layer is com-osed of the inner mesocarp and endocarp (Kuriachen et al.,991). The fruit dehisces along the ventral suture at maturityhile still green dispersing on an average 230 ± 19 silky haired

eeds. Unless otherwise stated, freshly dehisced fruits were col-ected every year in April in the years 2002 to 2005 from theopulation of C. procera in which the coronal scales in the flow-rs had closed stomium (Fig. 1A; Amritphale et al., 1984). Afteremoving the silk, which had little effect on seed germinationn the preliminary studies, the seeds were allowed to desiccaten the paper bags for 3 days at room temperature to a moistureontent of ca. 6% before storing them in air-tight plastic bottlest −10 ◦C until they were used.

Studies with developing seeds/fruits were conducted withreshly harvested fruits in April 2005. Following Ren andewley (1998) in principle, two criteria viz., fruit diameter and

eed color were used for the collection and selection of fruits,

espectively (Table 1). Accordingly, visibly healthy fruits forach diameter class were harvested from 10 randomly selectedruit-bearing plants in the field, labeled and brought to the labo-atory in light-proof, insulated containers lined with moist filter

.5×). Only relevant parts of the coronal scale labeled. (B) The fruit on the righthows the sensor head of the SKR 110/100 inserted to measure R:FR ratio andhe fruit on the left shows an excised strip of the pericarp (not to scale).

aper. In addition to attaining shedding maturity almost syn-hronously, seeds in C. procera do not germinate precociously.his allowed to sample first a small portion of seeds in darkness

rom each fruit of a given diameter, to match the seed color,nd then to pool remaining seeds from the selected fruits ofach stage for the determination of fresh/dry weight, moistureontent and germination.

.1. Light conditions

Light sources, filter combinations and spectral ranges for rednd far-red filters have been described elsewhere (Amritphale etl., 1984; Singh and Amritphale, 1992). Unless otherwise men-ioned, the photon flux at seed level for R was 2 �mol m−2 s−1 at60 ± 5 nm and that for FR was 10 �mol m−2 s−1 at 730 ± 5 nm.

31–40 Light green tolight brown

IV Undehisced/immature

31–40 Brown V Undehisced orDehisced/mature

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atio were measured with a SKR 110/100 (Skye Instrumentsimited, Wales, UK). The photon flux for the simulated day-

ight (10 or 500 �mol m−2 s−1 at 400–700 nm) were measuredith a LI-6200 Primer (Li-Cor, Nebraska, USA).

.2. Germination test

Seeds from fruits at stages I–IV and mature seeds from unde-isced fruits at stage V were tested for germination either freshr after air-drying in darkness for 3 days at room temperature.ecause sowing fixed number of seeds in darkness was not fea-

ible, therefore about 20–30 seeds were used per treatment inriplicate at 25 ◦C. In addition, seeds obtained from dehiscedruits at stage V and stored dry at −10 ◦C were also testedor germination. The seeds were placed in 10-cm glass Petriishes lined with filter paper circles. The light treatment wasiven either before or after moistening each filter paper withml sterilized glass distilled water.

All manipulations of seeds except for explicitly describedxperimental irradiations and germination monitoring were car-ied out in darkness. Using radicle emergence as a criterion,ermination was recorded 5 days (seeds from stage V) or 10ays (seeds from stages other than V) after the last treatmentas initiated. The tests were repeated at least twice and the

verages are presented.

.3. Seed weight and moisture content

Twenty-five seeds were weighed in triplicate (n = 25) forresh weight and dry weight determination as well as for mois-ure content following International Seed Testing Association1999). Results were expressed as percentage on a wet weightasis. To study the effect of reduction in the moisture con-ent of seeds on their germination response to light, the seedsrom undehisced fruits at stage V were allowed to desiccate forarious time intervals at 40 ◦C and at 23% relative humidity.he latter was obtained using a saturated solution of potas-ium acetate (Sun and Gouk, 1999). Seed weight and moistureontent were determined at least thrice and the averages areresented.

.4. R:FR ratio inside the fruits

(i) Selection of fruits: Ten fruit-bearing plants were selectedrandomly in the field. The plant foliage and location offruits could significantly affect the light quality and quan-tity reaching the fruits. For this reason, and because of theconstraint imposed by the size of the sensor head availablehere, fruits located on the outer side of the canopy at moreor less similar height and in the diameter range 31–40 mm(Table 1) were marked for in situ measurement of R:FRratio.

(ii) R and FR reflectance by seeds: It was thought possible

that the selective reflectance of R or FR by seeds mightfurther alter the quality of daylight once it got transmittedthrough the pericarp. To check this possibility, R and FRreflectance by seeds was determined using SKR 110/100

a11i

Experimental Botany 62 (2008) 45–53 47

as follows. Seeds of C. procera obtained from stage IV orundehisced stage V fruits were spread uniformly on a tileso as to completely cover its surface. A tube made up of athick black paper was fixed tightly around the sensor headso that it extended 10 cm beyond its opal white disc. Thesensor was fixed in a stand and was held with its anglerestricting tube 3 cm above the seeds in the sunlight takingcare that it did not shade the area below. The R:FR ratio inthe reflected light was measured. As a reference, a Kodak50% gray photographic card was placed beneath the sensorand the R:FR ratio was noted. The difference in the R:FRratio in the reflected light between the 50% gray card andthe seeds was 5–7% for brown seeds (stage V), whereas itvaried in the range of 15–27% for light green to light brownseeds (stage IV). Hence, in situ R:FR ratio measurementswere made in the fruits in 31–40 mm diameter range afterchecking the seeds for brown color.

iii) Measurement of R:FR ratio: A thick black adhesive tapewas fixed as a precautionary measure on the ventral sutureto prevent it from opening while handling the fruits. Acircular piece of the pericarp was cut out in the medianregion of a fruit on its lateral side so as to allow the 3.5-cmdiameter sensor head of the SKR 110/100. The placentaltissue and seeds were removed after checking the latter forbrown color. The sensor head was then inserted through thehole and its opal white disc was positioned at the level ofthe remaining seeds opposite to the inner epidermis of thepericarp (Fig. 1B). A common glass cover slip that did notalter the quality or quantity of the incident light was fixed ina 2-mm high plastic rim and placed beforehand on the opalwhite disc of the sensor head to protect it from any possibledamage by the plant tissue. The sensor head was securedin place with a thick black adhesive tape that also served tocut-off any stray light. The light incident to the sensor in thisposition was ≤180◦. R:FR ratios were measured inside thefruits (n = 10 per day) and also in the ambient environmentimmediately before and after each in situ measurement onthree different sunny days in April 2005 between 10.00 and14.00 h. Average value of the R:FR ratios based on 30 fruitsin all is presented.

.5. R and FR transmission through pericarp strips

The constraints imposed by the seed reflectance and sensorize, as mentioned above, did not allow measurement of R:FRatio inside the fruits except at stage V. In order to appraisehe light quality inside the fruits at other developmental stagesherefore, excised strips of the pericarp were used. For this, stripsf the pericarp (Fig. 1B) were cut from the median region of aruit with a surgical blade and each was placed on the opal whiteisc of the sensor head of the SKR 110/100 that was protectedith a common glass cover slip as described above. The stripas secured in place with a thick black adhesive tape having

1.5-cm diameter hole in the center. The set-up was placed

0 cm below the source of the simulated daylight (R:FR ratio.2, 500 �mol m−2 s−1) in the laboratory and the R:FR ration the light transmitted through the strip of the pericarp was

4 al and Experimental Botany 62 (2008) 45–53

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Table 2Germination of seeds from dehisced fruits at stage V in response to exposuresof R, FR, or FR followed by R

Light treatment Germination (%)

D 85 ± 5a

1 R 89 ± 3a

1 FR 82 ± 6a

1 FR × 5 days 83 ± 4a

5 FR × 5 days 28 ± 5b

5 FR + 1 R × 5 days 78 ± 2a

Seeds were incubated in water at 25 ◦C in darkness and given one 10 min R orone 10 min FR after 24 h, or one 10 min FR daily for 5 days, or five 10 min FRdaily at hourly intervals for 5 days, or five 10 min FR daily at hourly intervalsand one 10 min R daily as terminal exposure for 5 days. R and FR fluxes were 2and 10 �mol m−2 s−1, respectively. D, dark control. Germination was recordedaad

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8 S. Sharma, D. Amritphale / Environment

easured. Average value of the R:FR ratio for 10 pericarp stripsor each stage is presented.

.6. Chlorophyll content

Discs of 5-mm diameter each were punched from the area ofhe pericarp opposite to the hole made for the sensor head for initu determination of R:FR ratio in undehisced fruits at stage V.iscs were also punched from the pericarp strips used for R:FR

atio measurements in the laboratory. In one set, the discs weresed as such for chlorophyll extraction, whereas in the other set,he outer and inner layers in each pericarp disc were separated byemoving the middle fibrous layer with fine scissors. Chlorophyllontent of the discs was determined following Arnon (1949).

.7. Statistical analysis

Germination percentages were arcsine transformed to nor-alize the variances of binomial data before subjecting them toNOVA (SPSS 10.0 for Windows). When appropriate, Duncan’sultiple range test was used for pair comparisons.

. Results

.1. Effect of light on the germination of seeds fromehisced fruits at stage V

Cumulative percent germination of seeds exposed daily to 5 himulated daylight for 5 days did not differ from those imbibed

n water in darkness (Fig. 2). Exposure to a single 10-min FRulse on day 1 only or daily for 5 days had no suppressive effectn seed germination. However, exposing seeds to five 10-minR pulses daily for 5 days after sowing reduced germination per-

ig. 2. Cumulative germination percentages of seeds in different seed lots inarkness or in light. The seeds obtained from dehisced fruits at stage V werellowed to imbibe in water without or with a 5-h treatment with simulated day-ight (R:FR 1.2, 10 �mol m−2 s−1) given daily for 5 days. Vertical bars represent

eans ± S.E. for nine replicates. F value for seed lots or light treatment notignificantly different at P < 0.05.

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fter further incubation for 4 days in darkness. The mean ± S.E. values thatre followed by the same letter are not significantly different at P < 0.05 asetermined by Duncan’s multiple range test.

entage significantly below the dark control (Table 2) Increasinghe number of intermittent FR pulses to more than five had nodditional effect on the seed germination (data not given). A sin-le 10-min R pulse given daily at the end of the intermittent FRreatment annihilated the inhibitory effect of the latter almostompletely.

.2. Chlorophyll content of the pericarp and the R:FR rationside fruits

The effect of light on seed germination and dormancy isnown to be modulated by the chlorophyll content of the struc-ures surrounding the embryo in a number of species. The

hlorophyll content of the pericarp varied within a narrow rangef 0.3–0.5 mg g−1 dw from the time of fruit-set to dehiscencen C. procera (Fig. 3). The outer layer of the pericarp contained

ig. 3. Chlorophyll contents of the pericarp and its outer or inner layer in fruitst different developmental stages. Fresh tissue was extracted in aqueous acetonend the absorbance was measured at 663 and 645 nm. No meaningful absorbancealues were recorded with the extract of the middle fibrous layer of the pericarpdata not given). Vertical bars represent means ± S.E. for nine replicates. F valuesor the chlorophyll content at different developmental stages not significant at< 0.05.

S. Sharma, D. Amritphale / Environmental and Experimental Botany 62 (2008) 45–53 49

Fig. 4. R:FR ratio inside the fruits of stage V in the field or in the light transmit-ted through the pericarp strips excised from the fruits at the same developmentalswm

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Fig. 5. R:FR ratio in the simulated daylight (500 �mol m−2 s−1) transmittedthrough pericarp strips excised from fruits at different developmental stages.R:FR ratio could not be measured with the pericarp strips from stage I fruits duetFP

otcsfncincreased rapidly first, peaked at stage IV and then declined,whereas the seed dry weight increased gradually to a maxi-mum value at stage IV (mass maturity) and remained unchangedin stage V (shedding maturity). The moisture content of seeds

Fig. 6. Changes in fresh weight, dry weight and percentage moisture contentof seeds from fruits at different developmental stages. The asterisk represents

tage in the laboratory. Photon fluxes for natural daylight and simulated daylightere 1770 ± 50 and 500 �mol m−2 s−1, respectively. Vertical bars representeans ± S.E. for 30 replicates.

hree- to fivefold greater amount of chlorophyll than the innerayer. The chlorophyll content of the middle fibrous layer was not

easurable at any stage of fruit development. Pooled chlorophyllontent of the outer and inner layers ranged between 0.9 and.2 mg g−1 dw during the course of fruit development. The dif-erence between the chlorophyll content of the pericarp and theooled chlorophyll content of the outer and inner layers mighte attributed to the nearly non-chlorophyllous middle fibrousayer. In order to examine whether the quality of the light reach-ng to seeds was altered by the chlorophyll-rich pericarp, the:FR ratio was measured in situ in stage V fruits in the field.he R:FR ratio of the sunlight decreased to about one-fourthfter passing through the pericarp (Fig. 4). Furthermore, simi-ar to the in situ measurements in the field, the R:FR ratio ofhe simulated daylight got reduced from 1.2 to about 0.3 afterts transmission through the excised strips of the pericarp fromtage V fruits in the laboratory. Fig. 5 shows changes in the R:FRatio of the simulated daylight after transmission through theericarp strips excised from the fruits at different developmen-al stages. Although the geometry of the measuring set-up wasonserved during measurements, between-stage comparisons ofhe R:FR ratios cannot be made due to stage-related structuralifferences in the pericarp. Nevertheless, the present data clearlyhow about one-fourth reduction in the R:FR ratio of the inci-ent light after transmission through the pericarp regardless tohe fruit developmental stage.

.3. Effect of light on the germination of seeds from theruits of different developmental stages

It is clear from the data described so far that seeds fromehisced fruits at stage V germinate substantially in darknessespite showing phytochrome control of germination and com-leting their development in an FR-enriched microenvironment

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f the chlorophyll-containing maternal tissue. In order to testhe validity of the much cited negative correlation between thehlorophyll content of the maternal tissue and the capacity ofeeds to germinate in darkness, C. procera seeds obtained fromruits at different developmental stages were tested for germi-ation as well as their fresh weight, dry weight and moistureontent were determined. Fig. 6 shows that fresh weight of seeds

eed moisture content (%) immediately after fruit dehiscence (opening of ven-ral suture initiated). The event of fruit dehiscence is indicated by the arrow.ertical bars represent means ± S.E. for nine replicates when larger than theata symbols.

50 S. Sharma, D. Amritphale / Environmental and Experimental Botany 62 (2008) 45–53

Fig. 7. Cumulative germination percentage of fresh or dried seeds from the fruitsat different development stages in darkness or in light. The seeds were extractedin darkness and allowed to germinate fresh or otherwise in distilled water withoutor with a 4-h treatment with simulated daylight (10 �mol m−2 s−1) given dailyfor 5 days (stage V, undehisced fruits) or 10 days (stages III and IV). Verticalbat

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Fig. 8. Effect of R or FR on the germination of fresh seeds from undehiscedfruits at stage V. Seeds were extracted from the fruits in darkness and exposedimmediately to RVLF (5 nmol m−2 s−1 for 10 s), or RLF (2 �mol m−2 s−1 for10 min) and/or FR (10 �mol m−2 s−1 for 10 min) before placing them in waterfor germination. D, dark control. Germination was recorded after 5 days incu-bation in darkness. The mean ± S.E. values for nine replicates not followed bycb

fswshand, drying the seeds had little effect on their germination indarkness.

Fig. 9. Moisture content (%) or germination (%) of seeds from undehisced fruitsat stage V after desiccation at 23% RH/40 ◦C for 10 or 20 min. After extractionfrom fruits, the seeds were allowed to desiccate in darkness at 23% RH/40 ◦Cfor the indicated time intervals and their moisture content was determined. In theother set, the seeds were desiccated as above and allowed to germinate in water

ars represent means ± S.E. for nine replicates. F values for the light treatmentnd developmental stages significant at P < 0.05, whereas that for the dryingreatment not significant at P < 0.05.

emained high up to stage III, but decreased to 64% at stageV. The seed moisture content, which was about 40% in unde-isced fruits (ventral suture closed) at stage V, did not perceptiblyecrease immediately after the fruit dehiscence (opening of ven-ral suture initiated). Fresh seeds obtained from the developingruits up to stage II were incapable of germination in darknessnd light both (data not given). Although percentage of ger-inable seeds increased a little at stage III, but a marked increaseas observed only at stages IV and V (Fig. 7). Notably, per-

ent germination of fresh and dried seeds both was significantlyreater in light compared with darkness. Since neither drying theeeds caused any perceptible change in the germination responseor there was any significant difference in the cumulative seedermination percentage between stage IV and undehisced stagefruits, hence fresh seeds from the latter stage only were used

n subsequent experiments.In contrast to >80% dark germination of seeds from the

reshly dehisced fruits (Fig. 2, seed lot 2005), >50% seedsrom the undehisced fruits at stage V germinated in darknessFig. 8). A single R pulse (1200 �mol m−2), administered beforeowing, caused a significant increase in percentage germina-ion over the dark control. Germination could also be inducedy exposure to a single 10-min FR pulse alone and was not–FR reversible. Moreover, about 80% seeds germinated in

esponse to a very low-fluence R (50 nmol m−2). In order to ana-yze the observed germination response further, seeds extracted

rom undehisced fruits at stage V were desiccated at 23% rel-tive humidity (RH) and 40 ◦C in darkness for various timentervals before exposing them to a single 10-min pulse ofimulated daylight. Although seed moisture content decreased

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rom 42% to 29% after drying for 10 min (Fig. 9), still 68%eeds germinated in response to the light treatment. However,ith further decrease in the moisture content, the sensitivity of

eeds to a brief pulse of light was completely lost. On the other

ith or without a 10-min exposure to simulated daylight (10 �mol m−2 s−1).he mean ± S.E. values for nine replicates not followed by common lower-ase letters are significantly different at P < 0.05 for moisture content (%) andermination (%) in dark or light as determined by Duncan’s multiple range test.

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. Discussion

.1. Low-fluence response (LFR) in the germination ofeeds from dehisced fruits at stage V

Seeds extracted from the dehisced fruits at stage V in C.rocera showed more or less equal germination percentage inarkness and light (Fig. 2). Similar to several dark-germinatingeeds, for example, Amaranthus caudatus, Cucumis sativus, andeveral cultivars of L. sativa (Mancinelli and Borthwick, 1964;aniv et al., 1967; Kendrick and Frankland, 1969) germinationf mature non-dormant seeds in C. procera was inhibited byntermittent FR, though more frequent daily exposures wereequired (Table 2). Repeated exposure to FR is sometimesequired to prevent germination, since Pfr is formed in the imbib-ng seed from intermediates trapped in the drying seed (Kendricknd Spruit, 1977). The effectiveness of frequent FR exposuresiven daily for 5 days as opposed to the failure of a single FRulse given once or daily for 5 days in suppressing dark ger-ination thus suggested that phytochrome intermediates in C.

rocera seeds did not only generate Pfr over a fairly long periodf time, but also at a relatively rapid rate. Since a brief R pulseould reverse the FR inhibition, it appeared that the effect of FRight be through phytochrome operating in the LFR mode.

.2. Chlorophyll content of the pericarp and R:FR ratioffect seed germination response

Since phytochrome is located in the embryo, hence the opti-al properties of the seed coat and other investing structuresWidell and Vogelmann, 1988) must be taken into account whennterpreting light responses of seeds. Phytochrome is roughlyrrested at the photoequilibrium as existing at the time of seedesiccation (Bewley and Black, 1982; Pons, 2000). This even-ually depends on the light environment of the mother plant andn the chlorophyll content of the seed coat and other investingtructures during seed maturation (McCullough and Shropshire,970; Hayes and Klein, 1974; Cresswell and Grime, 1981;utterman, 2000). Cresswell and Grime (1981) suggested that

etention of chlorophyll till seed maturity in the investing struc-ures and consequent lowering of the R:FR ratio resulted intoark-dormant seeds in a number of species. The chlorophyll con-ent of the pericarp in C. procera (Fig. 3) was more or less equalo that in the investing structures surrounding the embryo in sev-ral species viz., Hypericum perforatum, Myosotis arvensis anduccisa pratensis reported by Cresswell and Grime (1981) toroduce dark-dormant seeds. Since more than 50% seeds wereon-dormant in undehisced fruits at stage V (Fig. 8) despite theigh chlorophyll content of the tissues surrounding the seedsFig. 3), the present data do not seem to be compatible in totoith those of the above workers. While data are not available for

he R:FR ratio inside the chlorophyll-rich investing structures inhe species studied by Cresswell and Grime (1981), or in other

pecies, but the R:FR ratio in the canopy-filtered germination-nhibiting light is known to be in the range of 0.1–0.2 (Frankland,981; Pons, 2000). The R:FR ratio measured inside undehiscedtage V fruits located on the periphery of the canopy in C. pro-

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Experimental Botany 62 (2008) 45–53 51

era was comparatively higher, i.e. about 0.3 (Fig. 4). The ratiof red to far-red light inside the fruits located within the plantanopy was slightly to moderately less than 0.3 depending uponheir location, presumably due to additional FR-enrichment ofhe light on account of transmission through the foliage (data notiven). Further, the R:FR ratio of the light transmitted throughhe pericarpic layer isolated from fruits at other developmen-al stages was also about 0.3 (Fig. 5). Presumably, the R:FRatio inside the C. procera fruits, which is a little higher than theverage range referred above for the canopy-filtered light, mightave resulted into a phytochrome photoequilibrium and/or pho-ostationary photoconversion rate favoring a relatively greaterroportion of non-dormant seeds in the population. It is thusuggested that, in addition to determining the chlorophyll con-ent, R:FR ratio within the investing structures surrounding thembryo may also be monitored while predicting the germinationesponse of phytochrome-containing seeds.

.3. Very low-fluence response (VLFR) induction of seedermination and post-dehiscence light signal

Interestingly, while more than 50% seeds from undehiscedruits at stage V could germinate in darkness, another 30% seedshowed very low-fluence R- and FR-induced germination and–FR irreversibility (Fig. 8). The data are thus consistent with

he VLFR mode of phytochrome action in seeds (Kendrick andone, 1985; Dixit and Amritphale, 1996; Shinomura et al., 1996;ennig et al., 2001). Whether PhyA mediates the VLFR in C.rocera similar to Arabidopsis (Casal and Sanchez, 1998 andeferences therein) is a matter of conjecture. Indeed, the VLFRnduction of seed germination in C. procera can be perhapsxplained based on PhyA mediation and the model proposedy Casal et al. (1998). However, since PhyA mutants are notnown in this or other closely related species, any such attemptay not be worthwhile at present.Quite intriguing is the transition of VLFR germination to

ark germination coincident with fruit dehiscence in C. procera.n other words, it needs to be explained how the seed popula-ion, which comprised of about 30% light-requiring and 50%ark-germinating seeds before fruit dehiscence (Fig. 8), dis-layed more than 80% germination in darkness once the fruitsad dehisced (Table 2). It is known that the minimum hydra-ion level needed in many species to permit complete Pr–Pfrransformations is ca. 15% (Bewley and Black, 1982). The seed

oisture content in undehisced fruits at stage V was about 40%n C. procera (Fig. 6). Irradiation of these seeds with a briefulse of simulated daylight before sowing resulted into about0% increase in germination over the dark control on subse-uent transfer to water (Fig. 9). It is worth mentioning here thathe time interval between the initiation of the splitting of ventraluture in the fruits and the dispersal of first seed varied from 5 to0 min (data not given) under the prevailing conditions of rela-ive humidity (23 ± 3%) and temperature (40 ± 2 ◦C) in the field.

urthermore, (i) the seed moisture content did not perceptiblyecrease (Fig. 6) at least immediately after the fruit dehiscenceopening of the ventral suture initiated), (ii) even after a 10-minesiccation treatment, the seeds were moist enough to respond

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ppreciably to a brief light pulse (Fig. 9), and (iii) phytochromes known to reach equilibrium in 5 s in mid-day sunlight (Smithnd Holmes, 1977). It is thus possible that a post-dehiscenceight signal might have induced in C. procera (i) a VLFR toon-dormant seed state transition (Figs. 2 and 8), and also (ii)he requirement of frequent FR pulses to inhibit germinationue to the generation of Pr–Pfr intermediates (Table 2). Interest-ngly, this may be of some ecological consequence. Cresswellnd Grime (1981) rightly hold that when a seed matures itshytochrome will be arrested in a particular photostationarytate determined by the light environment present immediatelyefore drying out. Thus, seeds which have matured entirelyithin green tissues would have most of their phytochrome in

he inactive Pr form, requiring a light stimulus for germina-ion. The present study reporting VLFR to non-dormant seedtate transition following fruit dehiscence in C. procera indi-ates to yet another possibility. That is, a post-dehiscence lightignal might determine the state of phytochrome and subsequenteed germination response in C. procera and in those dry-seededpecies where the seeds contain phytochrome and remain rela-ively moist following fruit dehiscence and immediately afterispersal.

. Conclusion

Maternal coverings are known to be important determinantsf seed dormancy and germination responses. There is also aide variety of photo-responses in seeds viz., VLFR, LFR andIR, which enable the seed to perceive different aspects of its

ight environment. The results presented here demonstrate theole of the VLFR and LFR in the regulation of seed germina-ion in C. procera before and after fruit dehiscence. Further, theresent data are consistent in general with previous work show-ng the chlorophyll content of the maternal tissue to be negativelyorrelated with the capacity of seeds to germinate in darkness.n addition, however, they also underline the need to monitorhe R:FR ratio within the investing structures surrounding thembryo in predicting the germination response of seeds. Finally,he current findings on the regulation of germination by the lightignal perceived by seeds following fruit dehiscence in C. pro-era may be of general implication in those dry-seeded specieshich disperse light-sensitive seeds in a sufficiently hydrated

tate to allow phytochrome transformations.

cknowledgements

We thank Skye Instruments Limited, Wales, UK for tech-ical assistance in seed reflectance technique and in situ R:FRatio measurements. We also thank Jiwaji Government Observa-ory, Ujjain, India for the field data for temperature and relativeumidity. Financial assistance by UGC, New Delhi, India underhe SAP-DRS Programme is gratefully acknowledged.

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