May 26, 1992 - Treatment of pollen with the Alexander (1969) stain did not give meaningful ... baker and Kwack medium (Leduc et al. 1990). This medium.
270
Short Communication / Kart Mededeling
Stimulation of in vitro pollen germination in Audouinia capitata by the simultaneous use of several carbohydrates J.H. de Lange National Botanical Institute, Private Bag X7, Claremont, 7735 Republic of South Africa and
C. Boucher Botany Department, University of Stellenbosch, Stellenbosch, 7600 Republic of South Africa Received 26 May 1992; revised 16 November 1992
Sucrose, fructose, galactose, raffinose, mannitol and sorbitol, when used as single exogenous carbohydrate sources, resulted in hardly any in vitro germination of Audouinia capit~ta pollen. Germination was increased significantly using either glucose or glycerol, the latter being the most effective carbohydrate. Sucrose: fructose: glycerol used in a 65 : 10 : 25 molar ratio with a total molarity of 0.3, resulted in good germination of pollen. Compared to the levels of germination when using fructose, sucrose and glycerol alone, the composite medium produced approximately 30-, 20- and 2.5fold increases, respectively. The composite pollen germination medium provides a useful means to sUNey pollen fertility of individual plants. Met die gebruik van sukrose, fruktose, galaktose, raffinose, mannitol en sorbitol, elk as die enigste eksogene koolhidraatbron, was daar feitlik geen in vitro-ontkieming van Audouinia capitata stuifmeel. Ontkieming is betekenisvol verhoog deur sowel glukose as gliserol, laasgenoemde synde die mees effektiewe koolhidraat. Die gebruik van sukrose : fruktose : gliserol in 'n 65: 10 : 25 molare verhouding met 'n totale molariteit van 0.3, het goeie ontkieming van stuifmeel tot gevolg gehad. In vergelyking met die ontkiemingsvlakke wanneer fruktose, sukrose en gliserol elk afsonderlik gebruik is, hetdie saamgestelde medium respektiewelik tot ongeveer 30-, 20- en 2.5-voudige verhogings gelei. Die saamgestelde medium kan nuttig in opnames van stuifmeelvrugbaarheid van individuele plante aangewend word. Keywords: Audouinia, carbohydrates, glycerol, pollen germination.
In a current autecological study of Audouinia capitata (LJ.) Brongn. (Bruniaceae), a threatened plant in the Cape Floral Kingdom (Hall & Veldhuis 1985), a survey of pollen fertility in the different populations was deemed necessary. Determination of seed set following in vivo pollination, using pollen from a large number of plants, would have been an extremely laborious procedure and would have necessitated the cross-pollination of flowers on many different plants and involved various degrees of sexual incompatibility and
S.AfrJ.Bot., 1993, 59(2): 270 - 272
female infertility (de Lange et al. 1993). Initially, attention was given to chemical stammg of pollen. Treatment of pollen with the Alexander (1969) stain did not give meaningful results. More than 90% of the pollen in different batches stained red, supposedly indicating good viability, although the samples were known to represent extremes in in vitro germination from less than 1% to more than 40% when using 0.3M glucose as the carbohydrate source in the incubation medium. Similar results were obtained following treatment of the pollen with fluorescein diacetate and observing the pollen using a fluorescence microscope (Widholm 1972). The Brewbaker and Kwack (1963) medium, with sucrose as the carbohydrate source, produced marginal germination of most of the pollen investigated. Various substances, such as indole-acetic acid, gibberellic acid, isopentenyladenine, malt extract and yeast extract, added to the medium, were found to be ineffective. It is not known whether A. capitata pollen is bi- or trinucleate. The latter pollen type is considered to be recalcitrant in in vitro germination on the Brewbaker and Kwack medium (Leduc et al. 1990). This medium was not considered suitable for the in vitro germination of A. capitata pollen. Earlier experiments with Agapanthus praecox pollen (de Lange 1989) indicated that a combination of sucrose and glucose in the pollen incubation medium resulted in better germination than either of the carbohydrates on their own. This prompted an evaluation of the sucrose-glucose combination in Audouinia. The effect of various combinations and ratios on a molar basis of several carbohydrates on the germination rates of A. capitata pollen grains was also evaluated. The total carbohydrate molarity in each case was maintained at 0.3. All media were adjusted to pH 6 using HCl or KOH solutions, and contained 0.6% (m/v) Difco Bacto agar and the Brewbaker and Kwack (1963) inorganic salts. The procedure used by de Lange (1989) to prevent the pre-incubation hydrolysis of carbohydrates and induction of toxicity when preparing the media, was followed. Flowers were collected shortly before dehiscence of anthers. The anthers were isolated and left to dehisce in the laboratory after which the pollen was dusted evenly on agarsolidified media in lO-ml transparent plastic containers at a density of approximately 20 grains mm-2 • The pollen was incubated for 18 h at 25°C in the dark under humid conditions. Pollen from different sources was used in a series of experiments, although the pollen tested in each experiment originated from a single plant. A total of 100 randomly selected pollen grains were scored in each replicate. Pollen was recarded as having germinated when the tube length exceeded the grain's diameter. The number of fully randomized replicates in the various experiments illustrated in Figure 1 were: A, 5; B, 5; C, 5; D, 3; E, 3; F, 5; G, 5; and H, 3. The experiments followed a randomized design and were analysed statistically using a t-test to compare treatment means. Logit transformations were applied. Both the transformed and the back-transformed data are given in the relevant graphs (Figures lA, C, D, F & G) with the least significant difference (p = 0.05) indicated in respect of the former. Glucose was found to be superior to sucrose in inducing germination, the rates being 3l.0 and 4.4%, respectively
271
S.Afr.J.Bot., 1993, 59(2)
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Figure 1 A - G: The results of experiments aimed at the determination of an effective carbohydrate medium composition for in vitro germination .of Audouinia capitata pollen. H: Confirmation that sucrose: fructose: glycerol (65 : 10 : 25) is the best mixture in comparison with the other two selected media (arrowed) as well as the component carbohydrates singly, when using pollen of seven different plants, All media were used at a total carbohydrate molarity of 0,3. Ratios are indicated on a molar basis, Differences in the viability between the different batches of pollen used in the various experiments, preclude direct comparisons of results between the individual experiments. A, C, D, F & G: Vertical bars indicate LSD logit transformation (p = 0,05). B, E & H: Treatment means not followed by common letters are statistically different (p < 0,05),
272
(Figure lA). The use of a glucose : sucrose ratio of 9 : 1 resulted in statistically significantly higher germination rates than glucose on its own. Eight different carbohydrates were evaluated. Glycerol and glucose were superior to the other carbohydrates used (Figure IB). Glycerol proved better than glucose. A 25 : 75 glycerol: sucrose ratio resulted in 68 .8% germination while sucrose and glycerol used singly, induced only 6.4 and 31.0% germination, respectively (Figure lC). A 25% glycerol content, in combination with different sucrose and glucose ratios, produced no statistically significant differences (Figure 10). Six additional carbohydrates were evaluated in combination with 25% glycerol and 50% sucrose to determine whether any third carbohydrate component might enhance the beneficial glycerol : sucrose interaction (Figure IE). Fructose, as a third component, was found to increase germination, despite its poor performance when used on its own (Figure IB). Fructose is generally known as a poor carbohydrate source for in vitro germination of pollen (portnoi & Horovitz 1977). The evaluation of all possible combinations of sucrose, glycerol and fructose would have necessitated an impractical number of treatments. After considering results in Figure lC, only the glycerol ratios as shown in Figure IF, were evaluated. A combination of sucrose: fructose: glycerol in a ratio of 65 : 10 : 25, proved to be the best treatment. A higher ratio of fructose might have increased the germination response even more but increases in fructose concentrations are accompanied by increasing suppression of pollen tube growth and growth abnormalities. The importance of fructose in combination with glycerol in the absence of sucrose was subsequently investigated (Picure 1G). Although the inclusion of 10% fructose in the medium tended to improve the germination, the result was not statistically significant. The main findings were consolidated in a single experiment using pollen from seven different plants in the Karbonkelberg population. Pollen from each plant was incubated separately on sucrose, fructose and glycerol, and on the three best formulations identified previously (Figure IH). The results confirmed that a combination of sucrose : fructose : glycerol in a ratio of 65 : 10 : 25 was the most successful carbohydrate formulation. The complete medium at pH 6 included the latter mixture at a total carbohydrate molarity of 0.3M (0.195M sucrose, 0.03M fructose and 0.075M glycerol) plus 0.6% Difco Bacto agar and the inorganic salts used by Brewbaker and Kwack (1963). The 0.3M total carbohydrate molarity used, corresponds closely to the 0.29M used by Brewbaker and Kwack (1963) and the total 0.34M of endogenous fructose, glucose and sucrose found in watermelon stigmatic exudate (Hawker et al. 1983). Of all the carbohydrates evaluated in the present study, glycerol was the most effective in the stimulation of pollen germination. It is not known whether glycerol is a natural constituent of A. capitata stigmas. Three references to the use of glycerol in the in vitro germination of pollen were traced in the literature. O'Kelly (1955) found that this non-nutrient sugar alcohol at 0.32M supported pollen tube growth equally well to sucrose in two
S.-Afr.Tydskr.Plantk., 1993, 59(2)
species but it performed poorly in a third species. No reference was made to its effect on the pollen's germination percentage. Brewbaker and Kwack (1963) mentioned that glycerol could be substituted for sucrose. De Bruyn (1966) found that glycerol alone or in combination with sucrose (total 0.5M) did not promote or inhibit germination of Setaria sphacelata pollen. Another approach to determine suitable chemicals for the in vitro germination of A. capitata pollen would be the biochemical analysis of substances obtained from ripe stigmas. This was not investigated in the present study. According to O'Kelly (1955), however, the suitability of a particular sugar for pollen germination and pollen tube growth appears to have no relation to the natural occurrence of the sugar in plants. Portnoi and Horovitz (1977) suggested that the complete stigmatic exudate, including factors such as vitamins, hormones and minerals influencing membrane permeability and metabolism during germination, should be considered.
Acknowledgements Critical comments on the manuscript by Professor I.A. de Bruyn of the Botany Department, University of Stellenbosch, and statistical analysis of data by Mr. F. Calitz and Mrs. J. Bezuidenhoudt of the Agrimetric Institute in Stellenbosch, are gratefully acknowledced. The results presented, form part of a P~ . D. study under the second author and Prof. IJ.A. van der Walt at the University of Stellenbosch.
References ALEXANDER, M.P. 1969. Different staining of aborted and nonaborted pollen. Stain Technol. 44: 117 - 122. BREWBAKER, J.L. & KWACK, B.H. 1963 . The essential role of calcium ion in pollen germination and pollen tube growth. Am. 1. Bot. 50: 859 - 865. DE BRUYN, J.A. 1966. The in vitro germination of pollen of Setaria sphacelata. 1. Effects of carbohydrates, hormones, vitamins and micronutrients. Physiologia Pl. 19: 365 - 376. DE LANGE, J.H. 1989. Significance of autoclaving-induced toxicity from, and hydrolysis of carbohydrates in in vitro studies of pollen germination and tube growth . S. Afr. 1. Bot. 55: 1 - 5. DE LANGE, J.H., BOUCHER, C. & VAN DER WALT, J.J.A. 1993. Autecological studies on Audouinia capilala (Bruniaceae). 3. Pollination biology. S. Afr. 1. Bot. 59: 135 - 144. HALL, A.V. & VELDHUIS, H.A. 1985 . South African Red Data Book - Fynbos and Karoo Biomes. S. Afr. Nat . Sci. Prog . Rep. No. 117. HAWKER, J.S. , SEDGELY, M. & LOVEYS, B.R. 1983. Composition of stigmatic exudate, nectar and pistil of watermelon, Cilrullus lanatus (Thunb.) Matsum. & Nakai, before and after pollination. Aust. 1. Pl. Physiol. 10: 257 - 264. LEDUC, N., MONNIER, M. & DOUGLAS, G.C. 1990. Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris. Sex. Pl. Reprod. 3: 228 - 235. O'KELLY, J.e. 1955. External carbohydrates in growth and respiration of pollen tubes in vilro. Am. 1. BOI. 42: 322 - 327. PORTNOI, L. & HOROVm, A. 1977. Sugars in natural and artificial pollen germination substrates. Ann. Bot. 4: 21 - 27 . WIDHOLM, J.M . 1972. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47 : 189 - 194.
