Plants of wild type, cv. Craigella, and the near isogenic mutant line, Craigella ls, were grown in a climate controlled green house (21 deg. C/19 deg. C day/night). Pollen was collected by vibration from at least ten individual flowers and suspended in liquid germination medium. The germination medium was modified BK medium (Brewbaker and Kwack, 1963) and contained 127 uM Ca(NO3)2, 99 uM KNO3, 162 uM H3BO3, 81 uM MgSO4 and 35 mM sucrose. Medium with quercetin was produced by diluting a stock of 0.1 M dissolved in dimethylsulphoxide (DMSO) in germination medium to a final concentration of 0.1, 1.0 and 10 uM quercetin with respectively 0.014, 0.14 and 1.4 uM DMSO. Germination medium with 1.4 uM DMSO served as control. Triplicate samples of 250 ul were placed in 24-wells tissue culture plates and incubated at 25 deg.C. Germination was stopped after four hours by adding 250 ul 96% ethanol and germination frequency was determined by sampling 100 pollen grains in each of the triplicate samples. Pollen tube length was determined, with the use of a microscope, drawing tube and an electromagnetic digitizing tablet connected to a computer equipped with the software program Sigma Scan (Jandel Scientific, Corte Madera, CA, USA).
As shown in Table 1, germination of wild type pollen in control medium was rather low in both experiments. As expected, germination of mutant pollen was even lower. The low germination frequency of wild type pollen might be due to the composition of the germination medium. In the original Brewbaker and Kwack medium, which is adequate for tomato pollen germination, salt concentrations are ten times higher. However, the suboptimal germination medium used in our experiments allowed the detection of a stimulation of pollen germination by quercetin. The in vitro germination of both wild type and mutant pollen was improved by addition of quercetin in the germination medium. Optimum stimulation was at a concentration of 1.0 uM and no differences were found between wild type and mutant. Pollen tube growth was not stimulated by quercetin.
Stimulation of pollen germination by flavonoids has been reported for a mutant of maize that was deficient in flavonoid synthesis (Coe et al., 1981) and transgenic petunia plants blocked in the synthesis of flavonoids. Germination and tube growth of in vitro matured tobacco pollen could also be stimulated by quercetin (Ylstra et al. 1992), with an optimum at a concentration of 1 uM.
This is the first report on the sensitivity of 'normal developed' pollen towards stimulation by a flavonoid. However, the results also show that pollen of the ls mutant can not be complemented with flavonoids, indicating that depletion of flavonoids is not the cause of the reduced fertility.
Table 1. Influence of quercetin on germination of wild type and mutant pollen in vitro. Data represent means and standard deviations of two independent experiments. Tube length of germinating pollen was determined in the second experiment for triplicate samples of 20 tubes per treatment.
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genotype quercetin germination (%) pollen tube
__________________ length (mm)
exp.1 exp. 2 exp. 2
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wild type 0.0 uM 40 29 0.16
0.1 uM 53 40 0.15
1.0 uM 62 49 0.16
10.0 uM 53 46 0.15
s.e.d. 2 2 0.13
ls mutant 0.0 uM 24 27 0.17
0.1 uM 38 33 0.16
1.0 uM 39 37 0.16
10.0 uM 36 35 0.15
s.e.d. 2 2 0.13
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Brewbaker J.L. and B.H. Kwack, 1963. American Journal of Botany 50: 589-865.Brown A.G., 1955. Report of the Tomato Genetics Cooperative 5: 6-7.
Coe E.H., S.M. McCormick and S.A. Modena, 1981. The Journal of Heredity 72:318- 320.
Van der Meer I.M., M.E. Stam, A.J. van Tunen, J.N.M. Mol and A.R. Stuitje. 1992. The Plant Cell 4: 253-262.
Ylstra B., A. Touraev, R.M.B. Moreno, E. Stoger, A.J. van Tunen, O. Vicente, J.N.M. Mol and E. Heberle-Bors. 1992. Plant Physiology 100:902-907.