Since Johnston 1971 first reported the poten- tial stratospheric ozone reduction over two
decades ago, UV-B effects on higher plants have been the subject of considerable research Cald-
well et al., 1995. A wide range of biochemical, physiological,
morphological, anatomical
and growth responses have been extensively investi-
gated, but little is known about UV-B effect on reproductive biology Demchik and Day, 1996;
Van de Staaij et al., 1997; Torabinejad et al., 1998. In some species an effect of elevated UV-B
radiation on total plant seed production has been found Van de Staaij et al., 1997. Although pol-
len of open flowers appears to be well shielded from solar UV-B when still within the anther sacs
Flint and Caldwell, 1983, it may be exposed to natural UV-B radiation following dehiscence until
successful germination and stigma penetration oc- cur. The exposure time of individual pollen tubes
to natural UV-B flux rates depends on the time required for individual pollen tube to grow from
the germinated pollen grains up to penetration of stigma. This time in vivo varies considerably
among species and pollen grain types, averaging 13 – 57 min summarized by Torabinejad et al.,
1998. Moreover, pollen grain walls can transmit as much as 20 of the UV-B Stadler and Uber,
1942, therefore, most of the studies concerning UV-B effects on reproductive characteristics of
higher plants have been focused on pollen.
Pollen germination and tube elongation in most species or cultivars are inhibited by enhanced
UV-B radiation in vitro. For instance, Flint and Caldwell 1984 reported partial inhibition of in
vitro pollen germination in Scrophularia pere- grina, Geranium 6iscosissimum, Papa6er rhoeas,
and Cleome lutea under elevated UV-B radiation. Musil 1995 reported reduced pollen germination
in two out of eight species tested. Decrease of in vitro pollen tube growth under UV-B has been
observed for Nicotiana tabacum and Petunia hy- brida Feder and Shrier, 1990. For four dicotyle-
donous Asteraceae and four monocotyledonous Iridaceae tested, pollen tube growth of all di-
cotyledonous species and one Iridaceae species was reduced Musil, 1995. In a recent report,
Torabinejad et al. 1998 detected differences among 34 species under two levels of UV-B expo-
sure to in vitro pollen grains, and found that the length of pollen tube of more than 50 of the
taxa tested was significantly reduced and the pol- len germination of five taxa was significantly in-
hibited. In comparison with other physiological andor ecological effects of enhanced UV-B irra-
diance, less than 70 species or cultivars of higher plants have been examined regarding the effect of
elevated UV-B on pollen. More studies in this field are obviously necessary to gain a better
understanding of the effect of increased UV-B radiation.
Previous research has often been conducted un- der low levels or in the absence of visible light.
This prohibits the process of photorepair of the UV-B induced damage to the DNA Pang and
Hays, 1991. Therefore a radiation regime in which UV-B radiation was combined with suffi-
ciently visible light was used in this experiment.
At present it is not clear whether UV-B damage accumulates over time. Zea mays Pfahler, 1973,
Brassica rapa Demchik and Day, 1996 and Di- morphotheca sinuata Musil, 1996 only have been
studied. In the present study we subjected mature pollen from 19 plant taxa to different UV-B
regimes to determine: i the interspecific response of pollen germination and tube elongation in vitro
to enhanced UV-B radiation, and ii whether there was a cumulative effect of UV-B exposure
duration on pollen germination and tube growth. Here we hypothesize that the cumulative effect of
UV-B over time exists and that as a consequence, pollen germination and tube elongation under
longer exposure to UV-B will be inhibited more seriously than under shorter exposure time.
2. Material and methods
2
.
1
. Plant materials Plants were grown in the botanical garden of
Lanzhou University Lanzhou, China 36.04°N, 1550 m except for Fritillaria cirrhosa which was
collected from alpine shrubs or meadows at an elevation 3200 m above sea level. The species used
are listed in Table 1. Many species are woody except C. bursa-pastoris, Carex heterostachya and
Table 1 Effect of enhanced UV-B radiation on pollen germination and percent change of 19 taxa from Lanzhou 36.04°N, 1550 m, China
means 9 S.D.
d
UV-B radiation taxa Pollen germination
Exposure time Percentage change
c h
Low High
Low c High c
Control 63.4 9 1.6
b
5.6 9 1.2
a
75.1 9 5.6
a
19.2 18.3
Salix matsudana f. tortosa Vilm. Rehd. 1
Salicaceae 75.8 9 1.5
a
75.1 9 2.1
a
2 19.4
63.4 9 1.6
b
18.3 63.4 9 1.6
b
76.8 9 2.1
b
77.4 9 3.4
a
21.1 22.1
3 43.8 9 2.0
c
46.6 9 1.1
b
Juglans regia L. Juglandaceae −
18.4 1
− 13.2
a
53.7 9 2.0
a
43.9 9 3.3
b
43.2 9 1.5
b
− 18.2
53.7 9 2.0
a
− 19.6
b
2 53.7 9 2.0
a
3 43.5 9 1.0
b
30.9 9 1.5
c
− 19.1
− 42.6
c
25.1 9 1.1
a
20.1 9 1.4
b
Capsella bursa-pastoris L. Medic. 15.4 9 1.2
c
1 −
19.9 −
38.6 Brassicaceae
21.4 9 0.7
b
17.4 9 0.5
c
2 −
14.6 −
30.6 25.1 9 1.1
a
22.4 9 0.8
b
20.8 9 1.3
c
− 10.6
− 17.1
3 25.1 9 1.1
a
89.8 9 1.0
a
79.3 9 1.0
b
77.2 9 0.9
c
1 −
11.7 Philadelphus incanus Koehne Sax-
− 14.1
ifragaceae 89.8 9 1.0
a
79.6 9 0.5
b
78.4 9 1.9
b
− 11.3
− 12.7
2 76.6 9 0.9
b
78.2 9 1.5
b
− 14.4
89.8 9 1.0
a
− 12.9
3 1
Chaenomeles speciosa Sweet. Nakai 41.5 9 0.9
a
36.6 9 2.1
b
33.5 9 1.1
c
− 11.9
− 19.3
Rosaceae 35.1 9 1.1
b
31.9 9 1.6
c
− 15.5
41.5 9 0.9
a
− 23.2
2 41.5 9 0.9
a
3 34.7 9 1.2
b
33.1 9 1.4
b
− 16.4
− 20
71.5 9 1.5 1
71.6 9 1.1 71.9 9 1.6
2.0 0.6
Kerria japonica L. DC. Rosaceae 71.5 9 1.6
72.5 9 1.0 0.1
71.5 9 1.5 4.2
2 71.6 9 1.6
73.6 9 1.1 0.1
3.0 3
71.5 9 1.5 8.0 9 0.6
6.8 9 1.0 −
3.3 8.3 9 1.9
− 18.3
Pyrus bretschneideri Rehd. Rosaceae 1
8.3 9 1.9 2
6.7 9 1.0 4.6 9 1.5
− 19.2
44.5 6.6 9 1.4
5.2 9 1.1 −
20.7 −
37.0 3
8.3 9 1.9 61.8 9 1.4
a
54.1 9 2.1
b
50.4 9 1.6
b
1 −
12.5 Wisteria sinensis Sims. Sweet. Fa-
− 18.6
baceae 61.8 9 1.4
a
51.2 9 1.3
b
52.9 9 1.6
b
− 17.1
− 14.4
2 50.3 9 0.9
b
43.8 9 1.2
c
− 18.6
− 29.1
3 61.8 9 1.4
a
27.2 9 0.7
b
25.2 9 0.5
c
− 21.5
a
34.7 9 1.4
a
− 28.8
a
Forsythia giraldiana Lingelsh. Oleaceae 1
34.7 9 1.5
a
2 23.3 9 2.8
b
22.6 9 2.4
b
− 32.8
b
− 44.4
b
19.8 9 1.1
b
18.2 9 0.9
b
− 42.9
c
− 49.1
b
3 34.7 9 1.5
a
46.6 9 1.1
a
35.5 9 1.0
b
24.7 9 1.2
c
1 −
23.7
a
Forsythia suspensa Thunb. Vahl. −
46.3
a
Oleaceae 46.6 9 1.1
a
25.3 9 1.1
b
24.5 9 1.2
b
− 45.7
b
− 47.5
a
2 3
46.6 9 1.1
a
19.1 9 1.5
b
18.6 9 0.8
b
− 59
c
− 60.0
b
52.5 9 1 52.5 9 1.1
− 2.7
54 9 1.3 −
2.8 1
Fraxinus chinensis Roxb. Oleaceae 54 9 1.3
2 49.7 9 1.3
52.4 9 2.1 −
7.9 −
2.6 52.3 9 1
51.6 9 0.7 3.1
− 4.3
3 54 9 1.3
30.6 9 2.5
a
27.0 9 1.6
b
− 6.3
a
32.6 9 1.1
a
− 9.1
1 Syringa oblata Lindl. Oleaceae
32.6 9 1.1
a
2 26.0 9 1.4
b
25.8 9 1.5
b
− 20.4
b
− 13.1
32.6 9 1.1
a
27.1 9 1
b
26.9 9 1.5
b
− 17.1
b
− 17.5
3 11.6 9 1.1
9.3 9 0.6 10.6 9 1
1 −
29.8 Syringa oblata var. affinis Henry Lin-
− 9.1
gelsh. Oleaceae 11.6 9 1.1
8.9 9 1.4 10.1 9 0.6
− 23.5
− 13.1
2 3
11.6 9 1.1 9.0 9 0.3
9.0 9 1 −
22.3 −
22.4
Table 1 Continued Exposure time
UV-B radiation taxa Pollen germination
Percentage change c
h Control
Low High
Low c High c
45.7 9 1.0
a
37.5 9 2.1
b
Syringa pinnatifolia Hemsl. Oleaceae 35.9 9 0.8
b
1 −
18.0 −
21.4
a
45.7 9 1.0
a
38.1 9 0.9
b
36.2 9 0.5
b
2 −
16.7 −
20.7
a
45.7 9 1.0
a
36.7 9 0.6
b
33.8 9 1.1
c
3 −
19.7 −
26.2
b
Paulownia tomentosa Thunb. Steud. 1
71.8 9 1.1
a
70.7 9 1.2
a
66.4 9 1
b
− 1.5
a
− 7.5
a
Scrophulariaceae 71.8 9 1.1
a
67.2 9 1.9
b
61. 9 1.5
c
2 −
6.4
b
− 15.0
b
71.8 9 1.1
a
57.3 9 0.7
b
44.9 9 1.1
c
− 20.1
c
3 −
37.5
c
1 71.1 9 1.7
a
68.8 9 1.1
a
48.2 9 1.1
b
Lonicera maackii Rupr. Maxim. Capri- −
3.3 −
32.2
a
foliaceae 2
71.1 9 1.7
a
67.7 9 1.0
a
47.6 9 1.6
b
− 4.7
− 33.8
a
71.1 9 1.7
a
67.6 9 1.2
a
39.5 9 1.4
b
− 3.6
− 44.4
b
3 1
47.3 9 2.1
a
42.4 9 1.6
b
Weigela florida Bge. A.DC. Caprifoli- 31.6 9 1.5
c
− 10.4
b
− 33.2
b
aceae 47.3 9 2.1
a
38.2 9 0.9
b
31.8 9 1.1
c
2 −
19.5
b
− 32.8
b
47.3 9 2.1
a
44.6 9 3.0
b
40.1 9 0.9
b
− 5.7
a
3 −
15.2
a
21.7 9 1.4 22 9 1.1
26.7 9 5.4 1
0.13
b
Carex heterostachya Bge. Cyperaceae 23.3
2. 21.7 9 1.4
b
28.8 9 1.1
a
31.7 9 1
a
33.9
a
46.5 21.7 9 1.4
b
22.4 9 1.5
b
26.9 9 1.1
a
3 3.3
b
24.2 2
F. cirrhosa D. Don Liliaceae 16.7 9 1.8
a
10.5 9 1.1
b
7.3 9 1
b
− 37.1
− 56.3
d
Significantly difference at PB0.05 with LSD test. Values with different letter in the same row the row with symbol within a species show the dose effect and in the same column the column with c symbol show the accumulative effects at PB0.05 levels
F. cirrhosa. Plants chosen in this study flowered between the middle of April and the end of May.
At that time ambient UV-B approximates 70 – 85 of that occurring with a clear sky at the
summer solstice in Lanzhou Jiang et al., 1998; personal communication from G.L. Ji.
2
.
2
. UV-B radiation treatments and growth chamber conditions
Enhanced UV-B radiation was provided by filtered Qin brand Baoji Lamp Factory, China
30-W fluorescence sun lamps in a controlled envi- ronment chamber Yue et al., 1998. The lamps
were suspended above and perpendicular to the Petri dishes and filtered with either 0.13-mm thick
cellulose diacetate transmission down to 290 nm for UV-B irradiance or 0.13-mm polyester plastic
films absorbs all radiation below 320 nm as a control. Cellulose diacetate filters were presolar-
ized. The desired irradiation was obtained by changing the distance between the lamps and the
Petri plates. The spectral irradiance from the lamps was determined with an Optronics Model
742 Optronics Laboratories, Orlando, FL, USA spectroradiometer and weighted with the general-
ized plant action spectrum Caldwell, 1971 nor- malized to 300 nm to obtain the biologically
effective UV-B radiation UV-BBE. The two lev- els of UV-B irradiation were 350 and 500 mWm
2
, which simulated 8 and 20 stratospheric ozone
depletion, respectively, with a clear sky at the summer solstice Lanzhou, using the model of
Green et al. 1980, and no UV-B radiation was regarded as the control. In addition to UV-B
radiation, visible radiation photosynthetically ac- tive radiation, PAR 400 – 700 nm 220 mmolm
2
per s was also supplied. The air temperature and relative humidity in the growth chamber were
maintained at 23 9 2°C and 75 9 5, respectively.
2
.
3
. Pollen culture Pollen was collected from individual flowers of
the same plant while the anthers were beginning
to dehisce. Most species used in this survey typi- cally release pollen in the morning or midday
hours. The pollen grains were brushed with a clear camel’s hair brush and cultured at the den-
sity of 50 grains mm
2
on a solid medium according to Brewbaker and Kwack 1963 in
9-cm diameter Petri plates, containing 15 su- crose, 1.5 bacto-agar, 0.01 H
3
BO
3
, 0.03 CaNO
3 2
.4H
2
O, 0.02
MgSO
4
.7H
2
O, 0.01
KNO
3
and 0.01 KH
2
PO
4
. The seeded plates were exposed to two levels of UV-B radiation.
After 1, 2 or 3 h of exposure, the plates were incubated under white light for 7, 6 or 5 h,
respectively. Control group plates were cultured for 8 h only under visible light. Growth of pollen
was stopped by adding some drops of killing and preserving solution comprising water, glycerine,
formaldehyde and glycerol acetic acid 72:20:5:3, v:v to each plate. Plates were stored at 4°C until
pollen germination and tube growth could be scored and measured. Three to five plates were
used for each treatment per experiment, and each experiment was repeated two or three times. At
least 1000 pollen grains per treatment were scored for germination and 50 were measured under a
light microscope for tube length when their elon- gation was greater than pollen diameter.
2
.
4
. Statistical analysis Data were statistically analyzed using a one-
way analysis of variance ANOVA to test the significance of treatment and duration exposure
effect of UV-B except for F. cirrhosa because of insufficient samples. Data of germination was
transformed using the arcsin transformation be- fore the analysis.
3. Results
