sis of pollen tube pectins (Stanley and Loewus. 1964) ... baker and Kwack 1963). Such a .... Peter JK, Stanley RG (1974) Boron in pollen and pollen cell fractions ...
Sex Plant Reprod (1990) 3:228 235
SexualPlant
Reproduction
9 Springer-Verlag 1990
Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris N. Leduc 1'2, M. Monnier 1, and G.C. Douglas 2 1 Laboratoire d'Histophysiologie Vegetale, Universit6 Pierre et Marie Curie, 12, rue Cuvier, F-75005 Paris, France 2 The Agriculture and Food Development Authority, Kinsealy Research Centre, Malahide Road, Dublin 17, Ireland
Summary. In Brewbaker and Kwack's medium (BK) only 16% of the pollen grains germinated, and these produced pollen tubes having a maxim u m length of 25 gm. With a solution based on Monnier's medium 47% germination and 160-gmlong pollen tubes were observed. Calcium was shown to be essential for germination; the optimal concentration was 880 mg/1 calcium chloride. The optimal concentrations of magnesium sulphate and boric acid were 360 and 50 mg/1, respectively. Germination at pH 4.0 but also pH 8.0 and the presence of vitamins B1 and B6 (1 mg/1 each) were stimulatory. Polyethylene glycol (PEG) was superior to sucrose as an osmoticum and germination and tube length were significantly improved using PEG 4000 at a concentration of 120 g/1 (0.03 M). Equimolar concentrations of PEG 400 and PEG 600 gave inferior results. Combining PEG with sucrose in the medium did not improve germination or increase tube length. Key words: Trinucleate pollen - In vitro germination - Polyethylene glycol.
Introduction The reproductive capacity of a species depends on many factors, but especially on its reproductive system and the viability of its gametes. Because it is difficult to assay the viability of ovules, evaluation of the fertility rate in plant breeding, hybridization and self-compatibility systems is often based on assays of pollen germination. Other viability tests include classical nuclear and cytoplasmic Offprint requests to. N. Leduc, Laboratoire d'Histophysiologie Vegetale, Universit6 Pierre et Marie Curie, 12, rue Cuvier, F75005 Paris, France
staining, enzyme assays and the exclusion of fluorochromes, but in comparative studies the most reliable assay has been obtained by germinating pollen (Heslop-Harrison et al. 1984). In view of recent objectives, genetic transformation via pollen, it is essential to have a good system of supporting pollen germination and the subsequent development of pollen tubes (Ohta 1986, Sanford et al. 1985). In addition pollen viability in vitro should be high in order to facilitate the fertilisation of ovules by direct application of pollen (Zenkteler 1980), electroporation (Mishra et al. 1987) fusion of pollen tubes with liposomes (Gad et al. 1988) and studies on the fine ultrastructural details of tubes (Lancelle etal. 1987; Kandasamy etal. 1988). With respect to pollen germination the essential nature of calcium, boric acid and sucrose has been shown for 79 genera (Brewbaker and Kwack 1963, Vasil 1964). The composition of the medium in these studies was relatively simple and has been used extensively thereafter (Heslop-Harrison et al. 1984, Heslop-Harrison and Heslop-Harrison 1988, Mulcahy and Mulcahy 1988). Despite the general suitability of Brewbaker and Kwack's (1963) medium (BK) for a large range of species with binucleated pollen their study showed that plants with trinucleated pollen in the Crueiferae and Compositae showed relatively poor pollen germination. With Capsella bursa-pastoris (Cruciferae) percentage germination was low (16%) with BK medium, and pollen tubes were abnormally short and reduced to protuberances. Supplementing media with amino acids such as glutamine improved the germination of tobacco pollen (Tupy et al. 1983) while the addition of PEG improved the growth of Petunia (Zhang and Croes 1982). In previous studies with Capsella bursa-pastoris we showed that it was possible to obtain plants
229
from fertilised ovules which were cultured in vitro with attached placentas using a newly developed medium (Lagriffol and Monnier 1983). Our objectives in the study reported here were to modify this new medium further and to formulate an improved medium which would support both the germination and the growth of normal pollen tubes of Capsella. Like Arabidopsis thaliana, Capsella produces many seeds, can be conveniently grown and there is extensive knowledge of its reproductive physiology (Monnier 1974, 1976). Capsella is therefore a useful model system with which to study genetic transformation via gametes and in vitro fertilisation by the direct application of pollen to ovules. The existence of a suitable medium to support pollen development is essential for these studies.
Materials and methods Plants of Capsella bursa-pastoris (Moench) were grown in a growth chamber at a constant temperature of 18 ~ C and under constant fluorescent illumination - conditions which induce flowering all year round. In all of the experiments reported pollen was collected from flowers having a mean ovary length of 1000 gin. Pollen grains at anthesis were stained with a 2.5 gg/ml solution of 4,6-diamino-2-phenyl-indole (DAPI) to reveal vegetative and generative cells under fluorescent light. To reduce genotypic effects in culture, pollen was collected from several different plants (approximately six) and mixed together. A constant number of pollen grains (approximately 100) was placed in a single spot on a dry glass slide (six spots per slide) and 10 btl of pollen germination medium (PGM) was added to cover the grains. The slide was then placed on a U-shaped glass rod in a 9-cm petri dish. A piece of filter paper (Watman no. 1) with a rectangular observation hole cut in it was placed beneath the glass rod and soaked with the same PGM, Brewbaker and Kwack's medium (1963) was used, as was the basal medium of Monnier (1973), modified to give the ME1 and ME2 media described in Table 1. N a O H (1 N) and HC1 (1 N) solutions were used to adjust the pH. In all experiments except that on pH effect, the pH was adjusted to 4.0. A pollen grain was regarded as having germinated if it showed a protuberance the length of which was at least equal to the radius of the grain (Zhang and Croes 1982). Since germination occurred within a short period of time, the percentage germination was measured after a 1-h culture period. The length of the pollen tube was measured with a graduated ocular; per treatment the mean length was calculated over 20 pollen tubes. Percentage germination was computed as : number of germinated pollen grains x 100 total number of pollen grains Each percentage germination is the mean of the percentage measured in 12 different drops. The comparison between two percentages is based on the reduced deviation e choosing a risk of 5%. If e is above 1.96, the difference between two percentages is significant. A significant difference between two points is designated by an arrow on the graphs.
Table 1. Mineral composition of Brewbaker and Kwack's medium (BK) and of three media (ME1, ME2 and ME3) derived from Monnier's medium. ME3 is the new improved medium, pH 4.0. MEI medium is modified from Monnier's (1973) by increasing the concentration of H3BO3 to 100 rag/1 BK (mg/1)
ME1 (rag/l)
ME2 (mg/1)
ME3 (mg/l)
Macronutrients Ca(NO3)2, 4H20
300.00
MgSO4, 7H20
200.00
3700.00
185.00
370.00
KNO3
100.00
950.00
1900.00
950.00
H2PO4K
170.00
85.00
85.00
CaClz, 2HzO
880.00
440.00
880.00
NHgNO3
825.00
412.50
412.50
KC1
350.00
175.00
175.00
NazEDTA
14.90
7.45
7.45
FeSO4, 7H20
11.10
5.55
5.55
Micronutrients 100.00
100.00
50.00
MnSO4, HzO
HaBO3
100.00
33.60
16.80
16.80
ZnSO4, 7H20
21.00
10.50
10.50
KI
1.66
0.83
0.83
Na2MoO4, 2H20
0.50
0.25
0.25
CuSO4, 5H20
0.05
0.025
0.025
COC12, 6H20
0.05
0.025
0.025
Vitamins BI B6 Sucrose 100000.00 PEG 4000 --
--
50.000 -
--
40.000 -
1 . 0 0
1.00 120000.00
Results
At anthesis, pollen grains of Capsella bursa-pastoris are 50 gm long with a sculpted exine. Our cytological observations using fluorescence microscopy revealed that the pollen was trinucleated (Fig. 1). Flowers contain six anthers that dehisce as soon as the flower bud starts to open, and fertilisation is autogamous. The developmental stage of the flowers was categorised on the basis of the length of the ovary (Fig. 2 a, b), and the germination percentage of pollen from these different stages was
230
~ o 12 ~ 10 c
9
8
E (1)
(,9
6
i--
0..
30
i
do 8'0 1;o lbo 1so Numberof PollenGrainsper 10 p.l Drop
i
18o
Fig. 3. Effect of pollen density on pollen germination in BK medium Fig, 1. A trinucleate pollen grain at anthesis of Capsella bursapastoris. Fluorescent microscopy reveals the vegetative nuclei (Vg) and the two sperm cells (Sp). x 340
30 v 25 2o
determined with ME2 medium. Pollen collected from flowers with ovaries I000 pm long gave optimal percentage germination (23%). The germination rate decreased by more than fourfold with pollen collected from older flowers: 5.5% and 4.8% germination for flowers with ovary lengths of 1500 pm and 2000 pm, respectively.
-~=
Effects of pollen density, pH
Fig. 4. Effect of pH on pollen germination in ME2 medium
0 13.-
5'
pH
The effect of pollen density on percentage germination in the range 40 to 180 grains per 10-gl drop is shown in Fig. 3. Using BK medium percentage germination ranged from 6% to 12% with 12% obtainable when the pollen grain density was 150-180 grains per drop. However, the increase in germination at the highest density was not significant (P>0.05). In all subsequent experiments a lower density of 100 grains per drop was used so as to facilitate observations of promotive treatments. Percentage germination of pollen was measured under different pH conditions (3.0-9.0) in ME2
medium (Fig. 4). In the pH range of 5.0 to 7.0 percentage germination varied between 17% and 21%. An abundant precipitate formed in the medium at pH 8.0, and pollen germination in this supernatant was significantly higher (P < 0.05) than that at pH 6.0. Although the supernatant was analysed by flame photometry it was not possible to determine its exact chemical composition in these preliminary tests. At pH 4.0 a non-significant (P_>0.05) increase in germination was recorded. Extreme pH conditions of 3.0 and 9.0 were inhibitory (Fig. 4).
b
9 800prn
@
@
I000pm
1500l~m
2000l~m
Fig. 2. a Four developmental stages of Capsetla flower bud and the respective ovary length. Anthesis occurs at stage 2, the stage used in our experiments. The time interval between each successive floral stage 1 to 4 was approximately 16 h. b Measurement of ovary length
231
optimal germination with 40 g/1 sucrose: percentage germination reached 23% (Fig. 5). This concentration was therefore used in subsequent experiments. When concentrations higher than 50 g/1 were used, the germination percentage decreased dramatically, dropping to as low as 1% at 100 g/1. To determine the effects of specific salts on pollen germination, media were formulated as shown in Table 2. The omission of potassium chloride, ammonium nitrate or potassium nitrate had no significant effect on germination. However, significant (P
