Soybean (Glycine max) Pollen Germination Characteristics, Flower and Pollen Morphology in Response to Enhanced Ultraviolet-B Radiation

Soybean (Glycine max) Pollen Germination Characteristics Flower and PollenMorphology in Response to Enhanced Ultraviolet-B Radiation

S KOTI1 K R REDDY1 V G KAKANI1 D ZHAO1 and V R REDDY2

1Department of Plant and Soil Sciences Box 9555 117 Dorman Hall Mississippi State University

Mississippi State MS 39762 USA and 2USDA-ARS Alternate Crops and Systems Laboratory Bldg 001

Rm 342 BARC-W 10300 Baltimore Avenue Beltsville MD 20705 USA

Received 21 April 2004 Returned for revision 29 July 2004 Accepted 19 August 2004 Published electronically 5 October 2004

Background and Aims Ultraviolet-B (UV-B) radiation effect on reproductive parts of the plants has received littleattention We studied the influence of UV-B radiation on flower and pollen morphology pollen production andin vitro pollen germination and tube growth of six genotypes of soybean (Glycine max) Methods Soybean genotypes were investigated by growing them under four levels of biologically effective UV-Bradiation of 0 (control) 5 10 and 15 kJ m2 d1 in sunlit controlled-environment chambers Key Results Reductions in lengths of flower standard petal and staminal column along with reduced pollenproduction germination and tube growth were observed in all genotypes with increasing UV-B radiation Combinedresponse index (CRI) the sum of percentage relative responses in flower size pollen production pollen germinationand tube growth due to UV-B radiation varied with UV-B dosage 67 to 152 with 5 kJ m2 d1 90 to 212with 10 kJ m2 d1 and 118 to 248 with 15 kJ m2 d1 of UV-B compared to controls Genotypes wereclassified based on the UV-B sensitivity index (USI) calculated as CRI per unit UV-B where D 90-9216 DG5630RR and D 88-5320 were classified as tolerant (USI gt743) and DP 4933RR Stalwart III and PI 471938 weresensitive (USI lt743) in their response to UV-B radiation Pollen grains produced in plants grown at 15 kJ m2 d1

UV-B radiation were shrivelled and lacked apertures compared to control and other UV-B treatments in bothsensitive and tolerant genotypes and the differences were more conspicuous in the sensitive genotype (PI 471938)than in the tolerant genotype (D 90-9216) The number of columellae heads of the exine was reduced with increasingUV-B radiation Conclusions Soybean genotypes varied in their reproductive response to UV-B radiation The identified UV-Btolerant genotypes could be used in future breeding programmes ordf 2004 Annals of Botany Company

Key words Floral morphology Glycine max pollen germination pollen morphology ultraviolet-B radiation

INTRODUCTION

Significant trends of reduction in the total column of ozoneare apparent in both northern and southern hemispheres atboth mid- and high latitudes (Herman et al 1999) Recentprojections suggest that ozone depletion will reach its max-imum in the coming years and forecasted to recover slowlyover the next several decades Relative to the 1970spresent-day losses in stratospheric ozone are estimated tobe 50 in the spring over Antarctica and 5 in the mid-latitudes during the year (Madronich et al 1999) Howevera number of uncertainties exist including interactions withother projected changes in global climate such as globalwarming (Schindell et al 1998 Madronich et al 1999)The measurable attenuation of the stratospheric ozone layerand consequent increase in the terrestrial UV-B radiationshowed a 6ndash14 increase since 1970s (Kerr and McElroy1993 UNEP 2002) which has raised interest in understand-ing the deleterious effects of UV-B radiation on higherplants (for reviews see Searles et al 2001 Kakani et al2003a Krupa 2003) Global terrestrial UV-B radiationlevels range between 2 and 12 kJ m2 d1 on a givenday with near equator and mid-latitudes receiving higherdoses (total ozone mapping spectrometer 2002 httptomsgsfcnasagovery-uveuvhtml) The UV-B levels

under soybean growing conditions in the USA rangedfrom 42 to 87 kJ m2 d1 during JunendashAugust 2002(httpuvbnrelcolostateeduUVB)

Many studies evaluating the impact of enhanced UV-B oncrop yields have been carried out in both field and green-house conditions (Kakani et al 2003a) Almost half ofthem showed that enhanced UV-B radiation decreasedyield and the other half showed no effect on the yieldThe UV-B supplied in these studies varied considerably(25ndash63 kJ m2 d1) and some studies conducted in thefield on soybean (Glycine max) showed that UV-A waseffective in mitigating the response to UV-B damage(Caldwell et al 1994) Recent studies showed genotypicvariation in the physiological responses of 20 soybean cultivarsto UV-B radiation of about 5 kJ m2 d1 (Zu et al 2003)which decreased grain yield by 15ndash92 (Yuan et al 2002Ambasht et al 2003) Understanding mechanisms andcauses for the yield losses in crops when they are exposedto enhanced UV-B radiation is necessary

Pollen release and viability were shown to be themajor limiting factors for fruit set under several otherenvironmental stresses such as high-temperature stress intomato (Lycopersicon esculentum Mill) (Peet et al 1998)and groundnut (Arachis hypogaea L) (Prasad et al 1999)High temperatures have been reported to affect micro-sporogenesis more than megasporogenesis and post-anthesis

Annals of Botany 946 ordf Annals of Botany Company 2004 all rights reserved

For correspondence E-mail krreddyramsstateedu

Annals of Botany 94 855ndash864 2004

doi101093aobmch212 available online at wwwaoboupjournalsorg

reproductive developmental processes in tomato(Monterroso and Wien 1990 Peet et al 1998) Alongwith high temperatures other stresses such as water-deficit(Shen and Webster 1986) and low night temperatures(Mercado et al 1997) reduced pollen germination andtube lengths Therefore it is necessary to understand UV-Beffects on pollen morphology germination and tube growth

Some studies where pollen collected from healthy plantswas directly exposed to UV-B by exposing the germinationmedia to UV-B showed that UV-B has reduced pollengermination (Torabinejad et al 1998 Musil et al 1999Feng et al 2000) In natural growth conditions howeverthe plant itself will be exposed to UV-B radiation and thismay result in both direct morphological disturbances onpollen that may later reduce pollen germination and pollentube growth Therefore studies on pollen collected fromUV-B-irradiated plants are necessary to gain knowledgeon the effect of increased UV-B radiation on pollenmorphology along with germination and other relatedcharacteristics The objectives were to examine the effectsof UV-B radiation on flower and pollen morphology pollenproduction and in vitro pollen germination and tube growthof soybean genotypes and also to understand the intraspe-cific variation of soybean in response to UV-B radiation

MATERIALS AND METHODS

Soilndashplantndashatmosphere research chambers

The experiment was conducted at the RR Foil PlantScience Research Facility Mississippi State University(33280N 88470W) Mississippi State Mississippi USAin 2003 using four soilndashplantndashatmosphere research (SPAR)chambers The SPAR facility has the capability to preciselycontrol temperature and CO2 concentration ([CO2]) at pre-determined set points for plant growth studies under nearambient levels of photosynthetically active radiation (PAR)Details of the operation and control of SPAR chambers havebeen described by Reddy et al (2001) and Zhao et al(2003) Each SPAR unit consists of a steel soil bin (1 mdeep middot 2 m long middot 05 m wide) and a Plexiglas chamber(25 m tall middot 2 m long middot 15 m wide) to accommodate aerialplant parts a heating and cooling system and an environ-ment monitoring and control system The Plexiglas chamberis opaque to solar UV radiation of below 385 nm buttransmits 966 6 05 of incoming PAR (wavelength400ndash700 nm) (Zhao et al 2003) During the experimentthe ambient total solar radiation (285ndash2800 nm) measuredwith a pyranometer (Model 4-48 Eppley Laboratory IncNewport RI USA) was 212 6 05 MJ m2 d1 The airtemperature and [CO2] in each SPAR unit were monitoredand adjusted every 10 s throughout the day and night

Plant culture

Six soybean genotypes were selected from 45 genotypesvarying in maturity groups that included glyphosate-tolerantand conventional varieties The selection of genotypes wasbased on the amount of phenolics (ranging from 517 to1312 mg cm2) accumulated under normal growing

conditions as they are screening compounds for UV-Bradiation tolerance (Cockell and Knowland 1999) Geno-types D 88-5320 (matu D 88-5320 (maturity group VInon-glyphosate-tolerant phenolic content 967 mg cm2)D 90-9216 (maturity group VII non-glyphosate-tolerantphenolic content 1208 mg cm2) Stalwart III (maturitygroup III non-glyphosate-tolerant phenolic content 1312mg cm2) Plant Introduction (PI) 471938 (maturity group Vnon-glyphosate-tolerant phenolic content 911 mg cm2)Delta Grow (DG) 5630RR (maturity group V glyphosate-tolerant phenolic content 725 mg cm2) and Delta Pine(DP) 4933RR (maturity group IV glyphosate-tolerantphenolic content 578 mg cm2) were sown on 5 August2003 in 75-cm-diameter pots filled with fine sandThirtypots (five pots of each genotype) were arranged randomly ineach SPAR chamber Emergence was 5 d after sowing(DAS) The temperatures were maintained at 3022 C(daynight) in all the units and the measured temperatureswere 297 6 024207 6 025 C (control) 294 6 0282136 023 C (5 kJ UV-B treatment) 2946 0212126022 C (10 kJ UV-B treatment) and 287 6 028213 6023 C (15 kJ UV-B treatment) The differences were notsignificant between the units Plants were watered threetimes a day with half-strength Hoaglandrsquos nutrient solutiondelivered at 0800 1200 and 1700 h to ensure favourablenutrient and water conditions for plant growth through anautomated and computer-controlled drip system Variable-density black shade cloths around the edges of plants wereadjusted regularly to match plant height in order to simulatenatural shading in the presence of other plants First flower(FF) (R1 stage) was recorded on all the genotypes in all thetreatments

Treatments

Four treatments of biologically effective UV-B (280ndash320 nm) radiation intensities of 0 (control) 5 10 and15 kJ m2 d1 were imposed from emergence Square-wave UV-B supplementation systems were used to providerespective UV-B radiation under near-ambient PAR ThePlexiglas of the SPAR chamber was opaque to solarUV-B radiation The UV-B radiation was delivered to plantsfor 8 h each day from 0800 to 1600 h by eight fluorescentUV-313 lamps (Q-Panel Company Cleveland OH USA)driven by 40 W dimming ballasts The lamps were wrappedwith presolarized 007 mm cellulose diacetate film to filterUV-C (lt280 nm) radiation The cellulose diacetate film waschanged at 3 to 4 d intervals The biologically effectiveUV-B energy delivered at the top of the plant canopy waschecked daily at 0900 h with a UVX digital radiometer(UVP Inc San Gabriel CA USA) and calibrated againstan Optronic Laboratory (Orlando FL USA) Model 754Spectroradiometer which was used initially to quantifylamp output The lamp output was adjusted as neededto maintain the respective UV-B radiation levels A distanceof 05 m from lamps to the top of plants was maintainedthroughout the experiment The actual biologically effectiveUV-B radiation was measured during the crop growth per-iod at six different locations in each SPAR unit correspond-ing to the pots arranged in rows The weighted total UV-B

856 Koti et al mdash Effects of UV-B Radiation on Soybean Pollen

radiation levels received at the top of the plants beneaththe lamps were 0 48 6 005 98 6 016 and 143 6011 kJ m2 d1 for 0 5 10 and 15 kJ m2 d1 treatmentset points respectively using the generalized plant responseaction spectrum (Caldwell 1971) normalized at 300 nmAlthough square wave supplementation system in controlledenvironments provide disproportionate spectral conditionson cloudy days they are useful for quantifying the growthand developmental responses of plants to UV-B

Measurements

Floral morphology Length of the flower standard petaland staminal column were measured on 20 fresh flowersrandomly picked from five plants of each genotype Soy-bean has a typical papilionaceous flower with a tubularcalyx of five unequal sepal lobes and a five-parted corollaconsisting of posterior standard petal two lateral wingpetals and two anterior keel petals in contact with eachother but not fused (Carlson and Larsten 1987) Flowerlength was measured from the tip of the standard petal tothe base of the calyx The standard petal was stretchedbefore measuring the length and the length was measuredfrom the point of insertion to the distal end Staminalcolumn was separated from flower and its length wasmeasured

Pollen number Mature anthers were collected from fivedifferent inflorescences from five plants a day beforeanthesis to determine the number of pollen grains producedper anther Pollen was counted by placing a single anther ina water drop on a glass slide and squashed with a needle andthe pollen grains dispersed in the drop of water werecounted (Bennett 1999)

Pollen germination and pollen tube lengths Flowerswere randomly selected from all the five plants in eachgenotype in the morning between 0900 and 1000 h fromfive plants for pollen germination Flowers were air-driedfor 2 h and fresh pollen was then dusted onto modifiedin vitro germination medium (Gwata et al 2003) Thegrowth medium was prepared with 15 g sucrose(C12H22O11) 003 g calcium nitrate [Ca(NO3)24H2O]001 g boric acid (H3BO3) and 06 g agar in 100 mL ofdistilled water and boiled for 10 min and then 10 mL of themedium was poured into five 5-cm-diameter Petri dishes offor each genotype at each UV-B level (Salem et al 2004)The pollen was dusted onto the solidified medium to allow auniform distribution of grains on the surface of the mediumThe plates were then covered and incubated at 30 C(Precision Instruments New York USA) for 8ndash12 h Pollengrain was considered germinated when its tube length atleast equalled the grain diameter (Luza et al 1987) using aNikon SMZ 800 microscope (Nikon InstrumentsKanagawa Japan) with a magnification of middot63 (five fieldsin each Petri dish) Pollen germination was determined as apercentage of total pollen Pollen tube length was obtainedby measuring 20 randomly selected pollen from each Petridish using a microscope The lengths were measured with anocular micrometer fitted to the eye-piece of the microscope

A total of 100 pollen tubes were measured for each genotypeat each UV-B level

Combined response index (CRI) and UV-B sensitivityindex (USI) A combined response index (CRI) based onthe concept of Dai et al (1994) was calculated to evaluatethe overall reproductive response of soybean to enhancedUV-B radiation using the following equation

CRI =PGtPGceth THORN

PGc

+PTLtPTLceth THORN

PTLc

+PNtPNceth THORN

PNc

+FLtFLceth THORN

FLc

middot 100

where CRI = combined response index PG = pollengermination percentage PTL = pollen tube length PN =pollen number anther1 and FL = flower length under t(treatment) and c (control) levels of UV-B radiation AUV-B sensitivity index (USI) was calculated as the slopeof the curve when CRI was regressed against UV-B radia-tion treatment Based on the USI values the genotypes wereclassified as tolerant and sensitive

Pollen morphology After the classification of genotypesbased on USI two genotypes one tolerant (D 90-9216) andone sensitive (PI 471938) were selected for pollen morpho-logical studies Fresh flowers were collected between 1900and 2100 h a day before anthesis and stored in FAA(formaldehydendashglacial acetic acidndashethyl alcohol) solutionfor scanning electron microscopy (SEM) Flower buds wereremoved from FAA and were fixed overnight in 3 glu-taraldehyde in 01 M phosphate buffer at pH 72 and 4 Cfor SEM After fixation specimens were rinsed in buffer post-fixed in 2 osmium tetroxide (OsO4) in 01 M phosphatebuffer for 2 h rinsed in distilled water dehydrated in anethanolic series and critical-point dried in a Polaron E 3000Critical Point Dryer (Quorum Technologies NewhavenUK) Specimens were mounted on aluminium stubssputter-coated with gold in a Polaron E 5100 sputter coater(Quorum Technologies) and viewed in a LEO Stereoscan360 SEM (LEO Electron Microscopy Thornwood NYUSA) at an accelerating voltage of 15 kV Images wererecorded on Polaroid Type 55 film (Polaroid CambridgeMA USA)

Statistical analysis

Four treatments were randomly arranged in four identicalSPAR units Except for the treatment factors of UV-B radia-tion the other growth conditions were the same in all unitsData were statistically analysed using a two-way analysis ofvariance (ANOVA) to test the significance of UV-B andgenotype effect on flower morphological features and pollengermination percentage and pollen tube lengths by Genstat 6for Windows (Genstat 6 Committee 1997) The least sig-nificant difference (LSD) tests at P = 005 were employed todistinguish treatment differences Data of pollen germina-tion percentages were transformed using the arcsin transfor-mation before statistical analysis Differences betweengenotypes were determined by testing the heterogenity of

Koti et almdashEffects of UV-B Radiation on Soybean Pollen 857

slopes and comparison of intercepts of the linear models ofCRI against UV-B dosage using Genstat 6 for Windows(Genstat 6 Committee 1997) Genotypes were classifiedas tolerant [(gtminimum USI + 1 sd] and sensitive(ltminimum USI + 1 sd)

RESULTS

Floral morphology

Genotypic variation was observed for soybean in reachingthe R1 stage whereas the UV-B treatments did not modifythis phenostage Averaged over the UV-B treatments firstflower (FF) was produced on 33 43 44 46 49 and 52 d afteremergence (DAE) in Stalwart III DP 4933 RR PI 471938DG 5630 RR D90-9216 and D88-5320 respectively Theeffects of UV-B radiation on flower morphological charac-teristics are shown in Fig 1With increasingUV-B radiationthere were significant (P lt 0001) reductions in flowerlengths (Fig 1A) in all genotypes and genotypes variedsignificantly for flower lengths A reduction of about78 was observed in Stalwart III at 15 kJ m2 d1

UV-B treatment but the reduction was only 20 in DG5630RR (Table 1) The longest flowers with longer standardpetals and staminal column lengths were observed in DG5630RR while the shortest flowers with shorter standardpetal and staminal columns were observed in Stalwart IIIand PI 471938 genotypes under control conditions (Figs 1Band 2A)

Pollen number per anther

Anthers borne on plants exposed to high levels of UV-Bradiation had significantly (P lt 0001) less pollen comparedwith those on control plants (Fig 2B) Of all the genotypesStalwart III produced 543 461 358 and 338 pollenanther1at 0 5 10 and 15 kJ m2 d1 UV-B respectivelysignificantly more than any other genotype at thoserespective treatments There were no significant differencesin the number of anthers produced per flower between UV-Btreatments (data not shown) in all the genotypes The reduc-tion in pollen production due to UV-B radiation was greaterin DP 4933RR where the relative response ranged from60 to 75 across the UV-B radiation treatments whereasthe response was 8 to 21 in DG 5630RR (Table 1)

Pollen germination and tube lengths

All the genotypes exhibited considerable sensitivity toUV-B in terms of pollen germination and pollen tube length(Fig 3) There were significant reductions (P lt 0001) inpollen germination and tube lengths of the genotypes underhigh levels of UV-B radiation but genotypes differedsignificantly (P lt 0001) in the degree of their response toUV-B radiation (Fig 3) The percentage of pollen germinationranged from 72 (D 88-5320) to 92 (PI 471938 and DP4933RR) and tube lengths ranged from 187 (DP 4933RR)to 329mm(PI 471938) among treatments Relative responsesof pollen tube lengths were more in PI 471938 (46 to63)and less in D 90-9216 (7 to 21) with high UV-Bradiation levels when compared with the control (Table 1)

Combined response index (CRI)

The CRI an integration of UV-B effects on pollen pro-duction pollen germination pollen tube lengths and flowerlengths showed that soybean is sensitive to enhanced UV-Bradiation In this study all the genotypes had a negative CRI(Table 1) indicating deleterious effects of UV-B The CRIranged from 67 (D 90-9216) to 152 (DP 4933RR) at5 kJ90 (DG 5630RR) to212 (DP 4933RR) at 10 kJ and118 (D 90-9216) to 248 (DP 4933RR) at 15 kJ m2 d1

of UV-B (Table 1) The genotypes were classified based onUSI the slope when CRI is plotted against UV-B (Fig 4)The genotypes with USI gt 743 were classified as tolerant(D 90-9216 DG 5630RR and D 88-5320) and those withUSI lt 743 as sensitive (DP 4933R Stalwart III and PI471938) The genotypes differed significantly (P lt 0001)between slopes and intercepts of the linear models

Pollen morphology

Although no gross morphological differences in thepollen grown under control conditions were visible betweenthe sensitive (PI 471938) and tolerant (D 90-9216) genotypes

D 88-5320 UV-B G UV-B times G

UV-B G UV-B times G

D 90-9216 DG 5630RRPI 471938

DP 4933RRStalwart III

14

A

B

12

10

8

6

4

2

0

8

6

4

2

00 5 10

UV-B dosage (kJ mndash2 dndash1)

Stan

dard

pet

al le

ngth

(m

m)

Flow

er le

ngth

(m

m)

15

F I G 1 Influence of UV-B radiation (UV-B) on (A) flower length and (B)standard petal length of six soybean genotypes (G) (D 90-9216 Stalwart IIIPI 471938 DP 4933RR D 88-5320 and DG 5630RR) Bars representstandard errors (n = 25 for flower and standard petal lengths)

Significance levels P lt 0001


TABLE 1 Genotypic sensitivity to UV-B radiation based on percentage relative responses in flower length pollen productiongermination and pollen tube length of six genotypes of soybean in comparison to control

UV-B (kJ m2 d1) Flower length Pollen production Pollen germination Pollen tube length Combined response index Rank

5 DG 5630RR (10) DG 5630RR (8) D 90-9216 (30) DP 4933RR (0) D 90-9216 (68) 1D 90-9216 (13) PI 471938 (11) D 88-5320 (32) D 88-5320 (5) DG 5630RR (72) 2PI 471938 (20) Stalwart III (15) DG 5630RR (35) D 90-9216 (7) D 88-5320 (80) 3D 88-5320 (22) D 90-9216 (18) PI 471938 (40) DG 5630RR (19) PI 471938 (117) 4DP 4933RR (40) D 88-5320 (21) Stalwart III (45) Stalwart III (24) Stalwart III (128) 5Stalwart III (44) DP 4933RR (60) DP 4933RR (52) PI 471938 (46) DP 4933RR (152) 6

10 DG 5630RR (0) DG 5630RR (13) D 90-9216 (42) D 90-9216 (14) DG 5630RR (90) 1D 90-9216 (13) PI 471938 (23) PI 471938 (45) D 88-5320 (15) D 90-9216 (93) 2D 88-5320 (22) D 90-9216 (24) D 88-5320 (51) DG 5630RR (19) D 88-5320 (117) 3PI 471938 (30) D 88-5320 (29) Stalwart III (54) DP 4933RR (26) PI 471938 (156) 4DP 4933RR (50) Stalwart III (34) DG 5630RR (58) Stalwart III (45) Stalwart III (189) 5Stalwart III (56) DP 4933RR (71) DP 4933RR (65) PI 471938 (58) DP 4933RR (212) 6

15 DG 5630RR (20) DG 5630RR (21) D 90-9216 (48) D 90-9216 (21) D 90-9216 (121) 1D 90-9216 (25) D 90-9216 (27) D 88-5320 (53) DG 5630RR (24) DG 5630RR (132) 2D 88-5320 (33) D 88-5320 (32) DG 5630RR (67) D 88-5320 (35) D 88-5320 (153) 3PI 471938 (50) PI 471938 (36) PI 471938 (72) DP 4933RR (37) PI 471938 (221) 4DP 4933RR (60) Stalwart III (38) Stalwart III (73) Stalwart III (48) Stalwart III (237) 5Stalwart III (78) DP 4933RR (75) DP 4933RR (76) PI 471938 (63) DP 4933 RR (248) 6

Ranking 1ndash6 is in the order of increasing sensitivity to UV-B radiation based on combined response index (the sum of relative responses)Values in parentheses represent percentage relative responses

10A

B

8

6

4

2

0

500

400

300

200

100

00 5 10

UV-B dosage (kJ mminus2 dminus1)

Polle

n pr

oduc

tion

(no

ant

herminus1

)St

amin

al c

olum

n le

ngth

(m

m)

15

D 88-5320UV-B G UV-B times G

UV-B G UV-B times G

D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III

F I G 2 Influence of UV-B radiation (UV-B) on (A) staminal column length(mm) and (B) pollen production (pollen anther1) of six soybean genotypes(G) (D 90-9216 Stalwart III PI 471938 DP 4933RR D 88-5320 and DG5630RR) Bars represent standard errors (n = 9 for pollen production andn = 25 for staminal column lengths) Significance levels P lt 0001

UV-B G UV-B times G

D 88-5320D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III

UV-B G UV-B times G


0 5 10 15

300

0

20

40

60

80

100

200

100

0

Polle

n ge

rmin

atio

n (

)Po

llen

tube

leng

th (

microm)

Α

Β

F I G 3 Influence of UV-B radiation (UV-B) on (A) pollen germinationpercentage and (B) pollen tube lengths (mm) of six soybean genotypes(G) (D 90-9216 Stalwart III PI 471938 DP 4933RR D 88-5320and DG 5630RR) Bars represent standard errors (n = 25 for pollengermination percentage and n = 100 for pollen tube lengths)



pollen abnormalities were observed in both genotypes withincreasing UV-B radiation levels The abnormalities weremore evident in PI 471938 compared with D 90-9216At the highest UV-B treatment of 15 kJ m2 d1 largenumbers of the pollen grains were shrivelled comparedwith the pollen from plants grown in control conditions(Fig 5D H) There were no apertures on the pollen grainsfrom 15 kJ m2 d1 UV-B irradiated plants while the pollenproduced in plants grown under control conditions weretriporate ie they had three protruding apertures (Fig 5Aand E) Differences in pollen exine structure were alsoobserved (Fig 6AndashH) The columellae heads of the exinewere less at high UV-B levels in both sensitive and tolerantgenotypes and the differences in the columellae heads weremore pronounced in the sensitive genotype resulting in analtered appearance of the exine

DISCUSSION

This is the first report to show that soybean plants exposed tohigher levels of UV-B radiation produce smaller flowerswith fewer pollen grains with altered pollen morphologyand germination percentage suggesting that UV-B damagesreproductive efficiency The smaller flowers in all the gen-otypes at higher levels of UV-B radiation were the result offlower-component part reductions such as standard petaland staminal column lengths Similar reductions in flowerlengths at elevated UV-B radiation levels were observed incotton (Gossypium hirsutum) (Kakani et al 2003b)

Flowers produced fewer pollen grains under enhancedUV-B radiation The considerable genotypic differencesin pollen produced under different levels of UV-B radiation

in our study might be due to different adaptive mechanismsof the genotypes to UV-B radiation In natural environ-ments such large reductions in pollen production couldhave far-reaching consequences on the reproductive successof plants Reductions observed in germination and inhibi-tion of pollen tube growth caused by high levels of UV-Bradiation might decrease the effectiveness of pollination andfertilization and consequently change the quantity andquality of seed (Demchik and Day 1996 Van de Staaijet al 1997) Similar reductions in the amount of pollenproduced were observed in Brassica rapa with enhancedUV-B radiation simulating 16 and 32 ozone depletions(Demchik and Day 1996) An alteration in plant carbonallocation increased allocation of resources towards repairmechanisms such as photoreactivation excision repair(Taylor et al 1996) and biosynthesis of UV-B flavonoidand related phenolic compounds (Caldwell et al 1983) atthe expense of reproductive structures may cause a reduc-tion in flower production (Sampson and Cane 1999) andflower size (Kakani et al 2003b)

Generally it is thought that the reproductive systemwith developing pollen grains is well protected fromUV-B (Martin 1970 Flint and Caldwell 1983) becauseperianths of most plants provide complete screening ofreproductive organs from UV-B radiation since the corollaexhibits low UV-B transmittance (Flint and Caldwell1983) This may not be true in the natural conditionsbecause UV-B can cause an indirect effect on pollenwhen the plant itself is exposed to high UV-B radiationsIn our study UV-B irradiation reduced pollen qualityReductions in in vitro pollen germination were shownwhen pollen from normal plants was directly irradiatedwith UV-B radiation (Torabinejad et al 1998 Musil

ndash50

ndash100

ndash150

ndash200

ndash250

ndash300


5 10 15

Com

bine

d re

spon

se in

dex

D 90-9216 y = ndash5middot1x ndash 41middot5 r2 = 0middot99DG 5630RR y = ndash5middot98x ndash38middot3 r2 = 0middot95

D88-5320 y = ndash7middot42x ndash 42middot5 r2 = 1

PI 471938 y = ndash10middot62x ndash58middot8 r2 = 0middot99

Stalwart III y = ndash10middot8x ndash76middot7 r2 = 0middot99DP 4930RR y = ndash9middot6x ndash108middot6 r2 = 0middot98

F I G 4 Combined response index a sum of the relative responses in pollen germination tube growth pollen production and flower lengths due to UV-Bradiation regressed over respective levels of UV-B radiation in six soybean genotypes (D 90-9216 Stalwart III PI 471938 DP 4933RR D 88-5320 and

DG 5630RR)


et al 1999 Feng et al 2000) Pollen grain walls appear totransmit as much as 20 of UV-B (Stadler and Uber1942) This relatively high transmittance may explain whyseveral researchers have found reductions in in vitro ger-mination of pollen when exposed to UV-B

The CRI calculated is considered as a good indicator toassess plant sensitivity to UV-B radiation (Dai et al 1994Li et al 2000 Yuan et al 2002) Although parametersused in calculating CRI in this study were different from

those used in previous studies we found that the ranking ofthe genotypes for UV-B sensitivity was generally similarregardless of whether ranking was assessed using individualparameters or an index We classified all the six genotypesinto tolerant and sensitive types based on the value of USIOut of all the genotypes tested D 90-9216 DG 5630RR andD88-5320 were found to be tolerant and DP 4933R StalwartIII and PI 471938 were found to be sensitive Similar geno-type classifications based on CRI were performed for

A E

B F

C G

D H

PI 471938(Sensitive genotype)

D 90-9216(Tolerant genotype)

F I G 5 Scanning electronmicroscopy images of pollen grains of two soybean genotypes PI 471938 (UV-B sensitive) (AndashD) andD90-9216 (UV-B tolerant)(EndashH) grown under four levels of UV-B radiation of 0 (control) (A and E) 5 (B and F) 10 (C andG) and 15 kJm2 d1 (D andH) Arrows indicate shrivelled

pollen grains without apertures at 15 kJ m2 d1 UV-B treatment Scale bars = 20 mm


20 wheat (Triticum aestivum) and soybean cultivars by Liet al (2000) and Yuan et al (2002) respectively whilestudying enhanced UV-B radiation effects on vegetativeand yield characteristics

The present study is the first to report pollenabnormalities at high levels of UV-B radiation The SEMresults show that both sensitive (PI 471938) and tolerant

(D 90-9216) genotypes grown under 15 kJ m2 d1 UV-Bproduced flattened and shrivelled pollen with abnormalexine surface ornamentation but the differences weremore pronounced in the sensitive genotype In the controltreatment pollen was equipped with three apertures whichact as exits for the germinating pollen tube and also allowrecognition of protein signals that initiate development

A E

B F

C G

D H



F I G 6 Scanning electronmicroscopy images of pollen grain surface of soybean genotypes PI 471938 (UV-B stress sensitive) (AndashD) andD 90-9216 (UV-Bstress tolerant) (EndashH) grown at four levels of UV-B radiation 0 (control) (A and E) 5 (B and F) 10 (C and G) and 15 kJ m2 d1 (D and H) Arrows indicate

disturbed exine ornamentation of pollen from 15 kJ m2 d1 UV-B treatment Scale bars = 2 mm


of a pollen tube (Heslop-Harrison 1971) The missingapertures could be the main reason for the reduction inpollen germination and tube growths observed under highUV-B radiation treatments The inhibition of exine forma-tion could be due to limitations in sporopollenin productiontransport or deposition The exine sporopollenin originatesfrom the tapetum (Dickinson and Potter 1976) and normalpollen (gametophyte) development depends on a close inter-action with the tapetal (sporophytic) tissue that composes ofthe innermost layer of the anther which plays an importantlayer in microsporogenesis The tapetum supplies nutrientsnecessary for pollen development (Dickinson 1992) andprovides the precursors of exine formation (Shivanna andJohri 1985) Abnormalities observed in this study such asaltered exine ornamentation with reduced columellae headsare indicative of the abnormal tapetum function Althoughpremature degeneration of the tapetal layer (Ahmed et al1992) could explain the abnormalities observed the role ofthe tapetum was not described definitively All these andother unknown factors may have contributed to the poorpollen germination observed in plants grown under UV-Bradiation The UV-B stress-induced morphological changesare intriguing and necessitate further investigation

The experiment was conducted in sunlit-SPAR chambersunder controlled environments using the square-wave UV-Bdelivery system On cloudy days the square-wave UV-Bsystem usually overestimates UV-B dosages (Musil et al2002) Although evidence from published reports indicatesno systematic differences among the chambers (Kakaniet al 2003b Zhao et al 2003) one chamber for eachUV-B treatment and square-wave UV-B system mightlimit our strong conclusions in this study

In summary enhanced UV-B radiation had a significanteffect on the reproductive biology of soybean Genotypicvariability was evident in response to UV-B radiationalthough all the genotypes were significantly affectedThe altered floral morphology and decreased pollen produc-tion along with altered pollen morphological characters andpollen germination capacity will have a direct impact on thefertilization process and fruit set in sensitive genotypes Thedifferences in UV-B sensitivity identified among genotypesimply the possibility of selecting soybean genotypes withtolerance to elevated UV-B radiation The UV-B tolerantgenotypes identified in our study could be used as possibledonors in breeding programmes However their geneticbases for these differencesmust be further examined Furtherstudies to relate pollen germination to fruit set are needed

ACKNOWLEDGEMENTS

The project was funded in part by the USDA UV-B Mon-itoring Program and the RSTC-NASA We thank D BrandK Gourley and W Ladner for their technical support andBill Monroe Electron Microscopy Center for his help dur-ing SEM studies We thank Drs D C Gitz L G HeatherlyM M Peet and C S Tara for reviewing the manuscript andfor their helpful comments We acknowledge Drs R Smithand T Carter for providing the seed of genotypes used inthis study Contribution from the Department of Plant and

Soil Sciences Mississippi State University MississippiAgricultural and Forestry Experiment Station paper no J10477

LITERATURE CITED

Ahmed FE Hall AE DeMason 1992 Heat injury during floral develop-ment in cowpea (Vigna unguiculata Fabaceae) American Journal ofBotany 79 784ndash791

Ambasht M Agarwal M Agarwal M 2003 Interactive effects of ozoneand ultraviolet-B radiation on physiological and biochemical charac-teristics of soybean plants Journal of Plant Biology 30 37ndash45

Bennett SJ 1999 Pollen-ovule ratios as a method of estimating breedingsystems in Trifolium pasture species Australian Journal ofAgricultural Research 50 1443ndash1450

CaldwellMM 1971 Solar UV irradiation and the growth and developmentof higher plants In Giese AC ed Photophysiology New YorkAcademic Press 131ndash137

CaldwellMMRobberechtRStephanDF1983 Internalfilters Prospectsfor UV-acclimation in higher plants Physiologia Plantarum 58445ndash450

Caldwell MM Flint SD Searles PS 1994 Spectral balance and UV-Bsensitivity of soybean a field experimentPlant Cell and Environment17 267ndash276

Carlson JB Larsten NR 1987 Reproductive morphology In Wilcox JRed Soybeans Improvement Production and Uses Wisconsin ASA-CSSA-SSSA Inc Publishers 97ndash102

CockellCSKnowlandJ 1999Ultraviolet radiation screeningcompoundsBiological Reviews 74 311ndash345

DaiQJPengSBChavezAQVergaraBS1994 Intraspecific responsesof188 rice cultivars to enhanced UV-B radiation Environmental andExperimental Botany 34 422ndash433

Demchik SM Day TA 1996 Effect of enhanced UV-B radiation on pollenquantity quality and seed yield in Brassica rapa (Brassicaceae)American Journal of Botany 83 573ndash579

Dickinson HG Potter U 1976 The development of patterning in thealveolar sexine of Cosmos bipinnatus New Phytologist 76 543ndash550

Dickinson HG 1992 Microspore derived emobryogenesis InCresti M Tiezzi A eds Sexual Plant Reproduction Berlin SpringlerBerlag 1ndash5

Feng H An L Tan L Hou Z Wang X 2000 Effect of enhancedultraviolet-B radiation on pollen germination and tube growth of19 taxa in vitro Environmental and Experimental Botany 4345ndash53

Flint SD Caldwell MM 1983 Influence of floral optical properties in theultraviolet radiation environment of pollen American Journal ofBotany 70 1416ndash1419

Genstat 6 Committee 1997 Genstat 6 Release 3 Reference ManualOxford Clarendon Press

Gwata ETWofford DS Pfahler PL Boote KJ 2003 Pollen morphologyand in vitro germination characteristics of nodulating and nonnodulat-ing soybean (Glycine max L) genotypes Theoretical and AppliedGenetics 106 837ndash839

Herman JRMcKenzie RL Diaz S Kerr JMadronich S SeckmeyerG1999 Ultraviolet radiation at the earthrsquos surface In Albritton DPAucamp P Megies G Watson R eds Scientific assessment of ozonedepletion Geneva World Meteorological Organization chapter 91ndash46

Heslop-Harrison JS 1971The pollenwall structure and development InHeslop-Harrison J S ed Pollen Development and PhysiologyLondon Butterworths 75ndash98

Kakani VG Reddy KR Zhao D Koti S 2003a Field crop responsesto ultraviolet-B radiation A review Agricultural and ForestMeteorology 120 191ndash218

Kakani VG Reddy KR Zhao D Mohammed AR 2003b Effects ofultraviolet-B radiation on cotton (Gossypium hirsutumL)morphologyand anatomy Annals of Botany 91 817ndash826

Kerr JB McElroy CT 1993 Evidence for large upward trends ofultraviolet-B radiation linked to ozone depletion Science 2621032ndash1034

Krupa SV 2003 Joint effects of elevated levels of ultraviolet-B radiationcarbon dioxide and ozone on plantsPhotochemistry and Photobiology78 535ndash542


Li Y Zu YQ Chen HY Chen JJ Yang JL Hu ZD 2000 Intraspecificresponses in crop growth and yield of 20 wheat cultivars to enhancedUV-B radiation under field conditions Field Crops Research 6725ndash33

Luza JG Polito VS Weinbaum SA 1987 Staminate bloom andtemperature responses of pollen germination and the growth oftwo walnut (Juglans nigra) species American Journal of Botany74 1308ndash1403

Madronich S VeldersG Daniel J LalMMcCullochA SlaperH 1999Halocarbon scenarios for the future ozone layer and related conse-quences InAlbrittonDAucampPMegieGWatsonR edsScientificassessment of ozone depletion Geneva World MeteorologicalOrganization chapter 11 1ndash38

Martin FW 1970 The ultraviolet absorption profile of the stigmaticextracts New Phytologist 69 425ndash430

Mercado JAMar TregoM ReidMS Valpuesta V QuesadaMA 1997Effects of low temperature on pepper pollen morphology and fertilityevidence of cold induced exine alterations Journal of HorticulturalScience 72 217ndash226

Monterroso VA Wien HC 1990 Flower and pod abscission due to heatstress in beans Journal of the American Society of HorticulturalScience 115 631ndash634

Musil CF Midgley GF Wand SJE 1999 Carry-over of enhancedultraviolet-B exposure effects to successive generations of a desertannual Interaction with atmospheric CO2 and nutrient supply GlobalChange Biology 5 311ndash329

Musil CF Bjorn LO Scourfield MWJ Bodeker GE 2002 How sub-stantial areultraviolet-Bsupplementation inaccuracies inexperimentalsquare-wave delivery systems Environmental and ExperimentalBotany 47 25ndash38

Peet MM Sato S Gardner RG 1998 Comparing heat stress effects onmale-fertile and male-sterile tomatoes Plant Cell and Environment21 225ndash231

Prasad PVV Craufurd PQ Summerfield RJ 1999 Fruit number in rela-tion to pollen production and viability in groundnut exposed to shortepisodes of heat stress Annals of Botany 84 381ndash386

Reddy KR Hodges HF Read JJ McKinion JM Baker JT Tarpley LReddy VR 2001 Soil-plant-atmosphere-research (SPAR) facility Atool for plant research and modeling Biotronics 30 27ndash50

Salem MA Kakani VG Reddy KR 2004 Temperature effects onin vitro pollen germination and pollen tube growth of soybeangenotypes Annual meetings of the Southern Branch of theAmericanSocietyofAgronomy 27ndash29 June2004BiloxiMississippiMS USA

SampsonBJCane JH 1999 Impact of enhancedultraviolet-B radiation onflower pollen and nectar production American Journal of Botany 86108ndash114

Schindell RT Rind D Lonergan P 1998 Increased polar stratosphericozone losses and delayed eventual recovery owing to increasinggreenhouse-gas concentrations Nature 392 589ndash592

Searles PS Flint SD Caldwell MM 2001 A meta-analysis of plantfield studies simulating stratospheric ozone depletion Oecologia127 1ndash10

ShenXYWebster BD 1986Effects of water stress on pollen ofPhaseolusvulgarisL Journal of American Society for Horticultural Science 111807ndash810

Shivanna KR Johri BM 1985 The Angiosperm Pollen Structure andFunction New Delhi Wiley Eastern Limited 5ndash83

Stadler LJ Uber FM 1942 Genetic effects of ultraviolet radiation inmaize IV Comparison of monochromatic radiation Genetics 2784ndash118

Taylor RM Nikaido O Jordan BR Rosamond J Bray CM Tobin AK1996 Ultraviolet B-induced DNA lesions and their removal inwheat (Triticum aestivum L) leaves Plant Cell and Environment19 171ndash181

Torabinejad J Caldwell MM Flint SD Durham S 1998 Susceptibilityof pollen to UV-B radiation an assay of 34 taxa American Journalof Botany 85 360ndash369

UNEP 2002Executive Summary Final of UNEPWMOScientific Assess-ment Panel of the Montreal Protocol on Substances that Deplete theOzone Layer UNEP Nairobi

VanDe Staaij JWMBolinkE Rozema J ErnstWHO 1997The impactof elevated UV-B (180ndash320 nm) radiation levels on the reproductivebiologyofahighlandanda lowlandpopulationofSilene vulgarisPlantEcology 128 173ndash179

Yuan L Yanqun Z Jianjun C Haiyan C 2002 Intraspecific responsesin crop growth and yield of 20 soybean cultivars to enhancedultraviolet-B radiation under field conditions Field Crops Research78 1ndash8

Zhao D Reddy KR Kakani VG Read JJ Sullivan JH 2003 Growthand physiological responses of cotton (Gossypium hirsutum L)to elevated carbon dioxide and ultraviolet-B radiation undercontrolled environmental conditions Plant Cell and Environment26 771ndash782

Zu Y Yuan L Haiyan C Jianjun C 2003 Intraspecific differencesin physiological response of 20 soybean cultivars to enhancedultraviolet-B radiation under field conditions Environmental andExperimental Botany 50 87ndash97


reproductive developmental processes in tomato(Monterroso and Wien 1990 Peet et al 1998) Alongwith high temperatures other stresses such as water-deficit(Shen and Webster 1986) and low night temperatures(Mercado et al 1997) reduced pollen germination andtube lengths Therefore it is necessary to understand UV-Beffects on pollen morphology germination and tube growth

Some studies where pollen collected from healthy plantswas directly exposed to UV-B by exposing the germinationmedia to UV-B showed that UV-B has reduced pollengermination (Torabinejad et al 1998 Musil et al 1999Feng et al 2000) In natural growth conditions howeverthe plant itself will be exposed to UV-B radiation and thismay result in both direct morphological disturbances onpollen that may later reduce pollen germination and pollentube growth Therefore studies on pollen collected fromUV-B-irradiated plants are necessary to gain knowledgeon the effect of increased UV-B radiation on pollenmorphology along with germination and other relatedcharacteristics The objectives were to examine the effectsof UV-B radiation on flower and pollen morphology pollenproduction and in vitro pollen germination and tube growthof soybean genotypes and also to understand the intraspe-cific variation of soybean in response to UV-B radiation

MATERIALS AND METHODS

Soilndashplantndashatmosphere research chambers

The experiment was conducted at the RR Foil PlantScience Research Facility Mississippi State University(33280N 88470W) Mississippi State Mississippi USAin 2003 using four soilndashplantndashatmosphere research (SPAR)chambers The SPAR facility has the capability to preciselycontrol temperature and CO2 concentration ([CO2]) at pre-determined set points for plant growth studies under nearambient levels of photosynthetically active radiation (PAR)Details of the operation and control of SPAR chambers havebeen described by Reddy et al (2001) and Zhao et al(2003) Each SPAR unit consists of a steel soil bin (1 mdeep middot 2 m long middot 05 m wide) and a Plexiglas chamber(25 m tall middot 2 m long middot 15 m wide) to accommodate aerialplant parts a heating and cooling system and an environ-ment monitoring and control system The Plexiglas chamberis opaque to solar UV radiation of below 385 nm buttransmits 966 6 05 of incoming PAR (wavelength400ndash700 nm) (Zhao et al 2003) During the experimentthe ambient total solar radiation (285ndash2800 nm) measuredwith a pyranometer (Model 4-48 Eppley Laboratory IncNewport RI USA) was 212 6 05 MJ m2 d1 The airtemperature and [CO2] in each SPAR unit were monitoredand adjusted every 10 s throughout the day and night

Plant culture

Six soybean genotypes were selected from 45 genotypesvarying in maturity groups that included glyphosate-tolerantand conventional varieties The selection of genotypes wasbased on the amount of phenolics (ranging from 517 to1312 mg cm2) accumulated under normal growing

conditions as they are screening compounds for UV-Bradiation tolerance (Cockell and Knowland 1999) Geno-types D 88-5320 (matu D 88-5320 (maturity group VInon-glyphosate-tolerant phenolic content 967 mg cm2)D 90-9216 (maturity group VII non-glyphosate-tolerantphenolic content 1208 mg cm2) Stalwart III (maturitygroup III non-glyphosate-tolerant phenolic content 1312mg cm2) Plant Introduction (PI) 471938 (maturity group Vnon-glyphosate-tolerant phenolic content 911 mg cm2)Delta Grow (DG) 5630RR (maturity group V glyphosate-tolerant phenolic content 725 mg cm2) and Delta Pine(DP) 4933RR (maturity group IV glyphosate-tolerantphenolic content 578 mg cm2) were sown on 5 August2003 in 75-cm-diameter pots filled with fine sandThirtypots (five pots of each genotype) were arranged randomly ineach SPAR chamber Emergence was 5 d after sowing(DAS) The temperatures were maintained at 3022 C(daynight) in all the units and the measured temperatureswere 297 6 024207 6 025 C (control) 294 6 0282136 023 C (5 kJ UV-B treatment) 2946 0212126022 C (10 kJ UV-B treatment) and 287 6 028213 6023 C (15 kJ UV-B treatment) The differences were notsignificant between the units Plants were watered threetimes a day with half-strength Hoaglandrsquos nutrient solutiondelivered at 0800 1200 and 1700 h to ensure favourablenutrient and water conditions for plant growth through anautomated and computer-controlled drip system Variable-density black shade cloths around the edges of plants wereadjusted regularly to match plant height in order to simulatenatural shading in the presence of other plants First flower(FF) (R1 stage) was recorded on all the genotypes in all thetreatments

Treatments

Four treatments of biologically effective UV-B (280ndash320 nm) radiation intensities of 0 (control) 5 10 and15 kJ m2 d1 were imposed from emergence Square-wave UV-B supplementation systems were used to providerespective UV-B radiation under near-ambient PAR ThePlexiglas of the SPAR chamber was opaque to solarUV-B radiation The UV-B radiation was delivered to plantsfor 8 h each day from 0800 to 1600 h by eight fluorescentUV-313 lamps (Q-Panel Company Cleveland OH USA)driven by 40 W dimming ballasts The lamps were wrappedwith presolarized 007 mm cellulose diacetate film to filterUV-C (lt280 nm) radiation The cellulose diacetate film waschanged at 3 to 4 d intervals The biologically effectiveUV-B energy delivered at the top of the plant canopy waschecked daily at 0900 h with a UVX digital radiometer(UVP Inc San Gabriel CA USA) and calibrated againstan Optronic Laboratory (Orlando FL USA) Model 754Spectroradiometer which was used initially to quantifylamp output The lamp output was adjusted as neededto maintain the respective UV-B radiation levels A distanceof 05 m from lamps to the top of plants was maintainedthroughout the experiment The actual biologically effectiveUV-B radiation was measured during the crop growth per-iod at six different locations in each SPAR unit correspond-ing to the pots arranged in rows The weighted total UV-B



Measurements







PGc

+PTLtPTLceth THORN

PTLc

+PNtPNceth THORN

PNc

+FLtFLceth THORN

FLc

middot 100







RESULTS

Floral morphology










Pollen morphology



UV-B G UV-B times G

D 90-9216 DG 5630RRPI 471938


14

A

B

12

10

8

6

4

2

0

8

6

4

2

00 5 10


Stan

dard

pet

al le

ngth

(m

m)

Flow

er le

ngth

(m

m)

15










10A

B

8

6

4

2

0

500

400

300

200

100

00 5 10


Polle

n pr

oduc

tion

(no

ant

herminus1

)St

amin

al c

olum

n le

ngth

(m

m)

15


UV-B G UV-B times G

D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III


UV-B G UV-B times G

D 88-5320D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III

UV-B G UV-B times G


0 5 10 15

300

0

20

40

60

80

100

200

100

0

Polle

n ge

rmin

atio

n (

)Po

llen

tube

leng

th (

microm)

Α

Β





DISCUSSION





ndash50

ndash100

ndash150

ndash200

ndash250

ndash300


5 10 15

Com

bine

d re

spon

se in

dex






DG 5630RR)





A E

B F

C G

D H









A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED



















































Measurements







PGc

+PTLtPTLceth THORN

PTLc

+PNtPNceth THORN

PNc

+FLtFLceth THORN

FLc

middot 100







RESULTS

Floral morphology










Pollen morphology



UV-B G UV-B times G

D 90-9216 DG 5630RRPI 471938


14

A

B

12

10

8

6

4

2

0

8

6

4

2

00 5 10


Stan

dard

pet

al le

ngth

(m

m)

Flow

er le

ngth

(m

m)

15










10A

B

8

6

4

2

0

500

400

300

200

100

00 5 10


Polle

n pr

oduc

tion

(no

ant

herminus1

)St

amin

al c

olum

n le

ngth

(m

m)

15


UV-B G UV-B times G

D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III


UV-B G UV-B times G

D 88-5320D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III

UV-B G UV-B times G


0 5 10 15

300

0

20

40

60

80

100

200

100

0

Polle

n ge

rmin

atio

n (

)Po

llen

tube

leng

th (

microm)

Α

Β





DISCUSSION





ndash50

ndash100

ndash150

ndash200

ndash250

ndash300


5 10 15

Com

bine

d re

spon

se in

dex






DG 5630RR)





A E

B F

C G

D H









A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED



















































RESULTS

Floral morphology










Pollen morphology



UV-B G UV-B times G

D 90-9216 DG 5630RRPI 471938


14

A

B

12

10

8

6

4

2

0

8

6

4

2

00 5 10


Stan

dard

pet

al le

ngth

(m

m)

Flow

er le

ngth

(m

m)

15










10A

B

8

6

4

2

0

500

400

300

200

100

00 5 10


Polle

n pr

oduc

tion

(no

ant

herminus1

)St

amin

al c

olum

n le

ngth

(m

m)

15


UV-B G UV-B times G

D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III


UV-B G UV-B times G

D 88-5320D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III

UV-B G UV-B times G


0 5 10 15

300

0

20

40

60

80

100

200

100

0

Polle

n ge

rmin

atio

n (

)Po

llen

tube

leng

th (

microm)

Α

Β





DISCUSSION





ndash50

ndash100

ndash150

ndash200

ndash250

ndash300


5 10 15

Com

bine

d re

spon

se in

dex






DG 5630RR)





A E

B F

C G

D H









A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED
























































10A

B

8

6

4

2

0

500

400

300

200

100

00 5 10


Polle

n pr

oduc

tion

(no

ant

herminus1

)St

amin

al c

olum

n le

ngth

(m

m)

15


UV-B G UV-B times G

D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III


UV-B G UV-B times G

D 88-5320D 90-9216

DG 5630RRPI 471938

DP 4933RR

Stalwart III

UV-B G UV-B times G


0 5 10 15

300

0

20

40

60

80

100

200

100

0

Polle

n ge

rmin

atio

n (

)Po

llen

tube

leng

th (

microm)

Α

Β





DISCUSSION





ndash50

ndash100

ndash150

ndash200

ndash250

ndash300


5 10 15

Com

bine

d re

spon

se in

dex






DG 5630RR)





A E

B F

C G

D H









A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED



















































DISCUSSION





ndash50

ndash100

ndash150

ndash200

ndash250

ndash300


5 10 15

Com

bine

d re

spon

se in

dex






DG 5630RR)





A E

B F

C G

D H









A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED





















































A E

B F

C G

D H









A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED





















































A E

B F

C G

D H









ACKNOWLEDGEMENTS



LITERATURE CITED





















































ACKNOWLEDGEMENTS



LITERATURE CITED












































































Soybean (Glycine max) Pollen Germination Characteristics, Flower and Pollen Morphology in Response to Enhanced Ultraviolet-B Radiation

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