Biologia Plantarum 18(1): 26-30 (Jan 1976)
Effect of calcium and sucrose concentration on pollen germination in vitro of six Rosa species
Marie N. Končalová, Dagmar Jičínská, Olga Sýkorov
First Online: 26 June 2008

Abstract The germination of the pollen from 11 individuals of six wild Rosa species was studied. The presence of calcium resulted in increased pollen germination, longer pollen tubes, and in a decrease of the requirement of sucrose concentration in cultivation media. Pollen germination in the medium with Ca reached the values of pollen viability estimated by tetrazolium test in all cases except with roses with balanced heterogamy, of the section Caninae. The stimulating effect of calcium was generally most pronounced in the pollen from roses of hybrid nature, such as R. jundzillii, R. canina, and especially in the case of the calciphilous species R. eglanteria.

*It might be worth considering that quality of pollen varies from flower to flower on a single plant (Yeamans et al., 2014), and sometimes even in different anthers of one flower (Beaton 1861).

A great variability* of rose pollen germination in vitro has been observed (WOHLERS and MOREY 1963, SÝKOROVÁ et al. 1976). The common condition for successful germination of rose pollen in vitro seems to be a relatively high requirement of sucrose concentration in the cultivation media (TÄCKHOLM 1922, MAMELI CALVINO 1951, KONČALOVÁ, 1975, JIČINSKÁ et al. 1976). Temperature is also of importance for pollen germination in roses (KONČALOVÁ, 1975): for some species reproducible results were obtained only at 28 to 35 °C The dependence of pollen germination on sucrose concentration in germination media was proved in several wild species of Czechoslovak roses (JIČINSKÁ, et al. 1976). However, for some species, especially from the section Caninae, Gallicanae, and Synstylae, the optimum germination conditions were not achieved even in this way.

WOHLERS and MOREY (1963) tried 52 different media for pollen germination of rose cultivars and did not obtain reproducible results. Only the complex nutrient media for tissue cultures, especially the HRD medium (Krusberg), were more promising. The authors suggested that kinetin was the main stimulating factor in these media. The essential role of calcium in pollen germination has been stated (BREWBACKER and KWACK 1963) and studied in various plants (KWACK 1965) but not in roses. The HRD nutrient medium used by WOHLERS and MOREY (1963) also contains a relatively high amount of calcium. We decided therefore to test the medium with calcium, recommended by KWACK (1965), for pollen germination of several wild Rosa species.

It is known that for the promotion of pollen growth by Ca, the presence of other cations, especially Mg and K, is essential. These ions alone are, however, inactive unless Ca is present (KWACK and KIM 1967, KWACK 1967). In this paper therefore, whenever the effect of Ca is mentioned, the combined influence of Ca, Mg, and K ions contained in the specified cultivation medium is understood.

Materials and Methods

The following 11 individuals of 6 species of wild Rosa from the rhodological collection of the Botanical Institute, Czechoslovak Academy of Sciences, Pruhonice, were examined: Rosa arvensis HUDS. (2), R. canina L. (2), R. eglanteria L. (2), R. gallica L. (3), R. jundzillii BESS. (1), R. pendulina L. (1). Ten to thirty flower buds about to open were collected from each shrub for pollen analysis. During 24 h at room temperature the anthers dehisced and the mixture of pollen from each individual was scattered in Petri dishes of 4 cm in diameter. The concentration of pollen grains varied from 2 x 103 to 2 x 105 per cm3 of cultivation medium.

Sucrose solution in 0.01% boric acid was used as the control cultivation medium (–Ca). Different sucrose concentrations were used in the treatments, increasing from 5 to 50% by 5% intervals. The series with calcium (+Ca) contained in addition the mixture of salts according to KWACK (1965): 0.03% Ca(NO3)2 . 4H2O, 0.02% MgSO4 . 7H2O, 0.01% KNO3. The acidity was measured before cultivation and was found to be the same, pH = 5.3, in all media. As in our previous work (KONČALOVÁ 1975, JIČINSKÁ et al. 1976), the pollen was incubated at 28°C during 24 h, fixed in ethanol-acetic acid (3:1, v/v), and stained with acetocarmine.

Five hundred pollen grains per dish were taken into account, the results in the media +Ca and –Ca with the same sucrose concentrations were compared with random expectations in χ-square tests. The mean length of pollen tubes was established from 30 tubes in each variant.

For the examination of pollen viability, the TTC-test was used: 1% 2,3,5-triphenyltetrazoliumchloride in Sorensen's phosphate buffer pH = 6.8, incubation at 22 to 25°C for one to five hours according to the requirements of the species (JIČINSKÁ et al. 1976, SÝKOROVÁ et al. 1976).

Results and Discussion

The effect of calcium on the germination of rose pollen was in most cases evident without counting. Not only that more pollen grains were germinating, but the pollen tubes were also much longer in the media +Ca (Fig. 1).

In a comparison of pollen germination in –Ca media with the germination estimates obtained by the TTC-test, the results corresponded only for one shrub of Rosa arvensis (Fig. 2) in 35% sucrose. The pollen germination in media +Ca was in good conformity with the TTC-test for the other shrub of R. arvensis, for all three shrubs of R. gallica, for R. pendulina, and R. jundzillii. The species of the section Caninae did not attain the values shown by the TTC-test even in media +Ca. The hybrid nature of these species may have been responsible for this failure. Meiotic irregularities known as "caninae meiosis", described by TÄCKHOLM (1922) as balanced heterogamy, result in the formation of a large number of abortive pollen grains. During the heterotypic meiotic division in PMC, only 14 chromosomes pair to 7 bivalents and then form nuclei of morphologically normal pollen grains whereas the remaining 14, 21 or 28 univalents for 2n = 28, 35 or 42 usually constitute 4 to 12 nuclei of abortive pollen. But sometimes the nucleus of a normal-looking tetrad contains fewer or more than 7 chromosomes (KONČALOVÁ, unpublished observations). It is probable that not all morphologically normal pollen grains in media +Ca. KWACK (1967) and and KIM (1967) explain the effect of Ca ions on pollen tube growth by the ability of calcium to control the permeability of the cell walls. Negatively charged groups in the pectin of the pollen cell walls link with Ca ions to compose a more strongly and effectively united colloidal system. The Ca effect on pollen growth appears to be the result of an increased rigidity of the pollen cell walls, which protects them from bursting in a hypotonic environment. The above authors compared the pollen germination in 10% sucrose solution. In the wide range of concentrations used in our experiments, the effect of Ca was particularly distinct, as in the presence of Ca the rose pollen germinated well even in media poor in sucrose. The most marked increase in pollen germination by Ca was attained in media with the lowest sucrose concentrations. The values were six times higher in R. arvensis and R. gallica; in R. canina, they were on the average 9 x and in R. pendulina and R. eglanteria 18 x higher than in media –Ca. In R. jundzillii, the germination in 5% sucrose +Ca surpassed the germination in –Ca medium as much as 31 times.

Distinct differences appear between maxima of germination in different media as they do not occur at the same sucrose concentration. The ratio +Ca max/–Ca max. is equal to 1 only in R. arvensis. In the other species, this ratio is higher: R. pendulina 1.5, R. gallica 1.6, R. jundzillii 2.6, R. canina 3.1, and R. eglanteria 3.6. From this list, it is clear that the stimulation effect of calcium is the highest in those species which have a balanced heterogamy, R. eglanteria being the most striking example. It is not without interest that, in comparison with the other species examined, Rosa eglanteria distinctly prefers calcareous soils in its native habitats, occurring almost exclusively on limestone, dolomite, or basalt.

The presence of Ca in the germination medium results also in more rapid growth of pollen tubes in all six species investigated. This effect is most evident in R. arvensis where the pollen tubes are on the average 18 x longer in 5% sucrose +Ca than in the medium –Ca. In a –Ca medium, the pollen tubes were the longest in 15% sucrose, but even here, they were 5.5 x shorter. In the other species, the following ratios of the pollen tube length (+Ca/–Ca) were recorded: R. jundzillii 11, R. pendulina 10, R. canina 7, R. gallica 7, R. eglanteria 4. A study of pollen-tube length after 24 h of incubation as related to the length of styles in the respective species seems to prove the mutual dependence between these two characters. The growth of pollen tubes is most rapid in R. arvensis, with the longest styles, and it is the slowest in R. eglanteria with styles relatively short.

Study on the influence of different Ca concentrations in the cultivation medium with respect to their promoting effect on pollen germination appears promising in species with different demands on Ca content in the soil. Such study could help to clear the calciphilous nature of some species. The question of whether the stimulating effect of calcium on pollen germination in roses is generally valid or is caused by calcium deficiency in the mineral nutrition of the plants studied still remains to be verified.