Factors influencing pollen germination in three Explorer roses

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1 Factors influencing pollen germination in three Explorer roses C. Richer 1, M. Poulin 1, and J.-A. Rioux 2 1 Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Blvd., Saint-Jean-sur-Richelieu, Quebec, Canada J3B 3E6 ( richerc@agr.gc.ca; melissa.poulin@mapaq.gouv.qc.ca); 2 Département de Phytologie, Université Laval, Quebec City, Quebec, Canada G1K 7P4 ( Jacques-Andre.Rioux@fsaa.ulaval.ca). Received 22 December 2005, accepted 14 August Richer, C., Poulin, M. and Rioux, J.-A Factors influencing pollen germination in three Explorer roses. Can. J. Plant Sci. 87: Pollen germination is useful for the estimation of pollen viability. For three cultivars from the Explorer TM series, Champlain, Nicolas and Frontenac, a liquid medium with a ph of 5.6 and a saccharose concentration of 15% provides optimal germination conditions. Four flower stages were determined and evaluated according to the following scale: flower bud at stage 0 (closed bud), stage 1 (closed petals but open sepals), stage 2 (bud with petals three-quarters open) and stage 3 (fully open flower). Pollen germination was higher when the pollen was collected at stages 0 and 1 for Champlain, at stages 1 and 2 for Frontenac and at stage 2 for Nicolas. Based on the results of the evaluation of pollen drying techniques, it is recommended that pollen be dried for 24 h in darkness before use in crossing. Key words: Rosa, rose, rose breeding, pollen harvesting, pollen drying, pollen germination test Richer, C., Poulin, M. et Rioux, J.-A Facteurs influençant la germination du pollen de trois rosiers Explorateur. Can. J. Plant Sci. 87: La germination est un outil qui permet de déterminer la viabilité du pollen. Pour trois cultivars de rosiers de la série Explorateur MC Champlain, Nicolas et Frontenac, la germination du pollen est optimale dans un milieu liquide dont la concentration en saccharose est de 15% et le ph à 5,6. Quatre stages du développement floral ont été établis selon l échelle suivante : bouton floral au stade 0 (bouton fermé), stade 1, (pétales fermés avec sépales ouvertes), stade 2 (boutons avec pétales au trois-quarts ouverts) et stade 3 (fleurs complètement ouvertes). Le taux de germination du pollen était plus grand lorsqu il était récolté aux stades 0 et 1 pour le cultivar Champlain, aux stades 1 et 2 pour le cultivar Frontenac et au stade 2 pour le cultivar Nicolas. Basés sur les résultats obtenus lors d essais effectués sur différentes techniques de séchage du pollen, il est recommandé de sécher le pollen pour une période de 24 heures à la noirceur avant de l utiliser pour les croisements. Mots clés: Rosiers, amélioration génétique, récolte du pollen, séchage du pollen, test de germination du pollen 115 In most modern breeding programs, the creation of new rose hybrids involves a series of steps that include controlled pollination followed by the fertilization of the ovary, fruit set and hip formation, seed maturation, the extraction of achenes and the germination process resulting in new hybrid plants. Additionally, the compatibility of the two parents must be considered a factor. The literature on rose breeding does not provide specific information on the optimal period for collecting Explorer TM rose pollen nor on the ideal storage conditions, and there are conflicting recommendations among authors. Gudin et al. (1991) conducted a study in a greenhouse on Rosa hybrida L. and found that pollen germination is highest in April and October. However, their results cannot be directly transferred to the shrub rose group (Explorer roses) because the latter are not produced in greenhouses and also present a different genetic pool than the hybrid tea rose group. Pollen germination capacity varies from cultivar to cultivar (Erlanson 1934). The success or failure of germination depends on such factors as the period of the year during which the pollen is collected, the stage of development of the flower from which the pollen is collected, and the drying and storage techniques (Marchant et al. 1993). Depending on the study, it is recommended that the pollen be collected when the sepals are open (Pearson and Harney 1984), when the outside petals begin to open (Marchant et al. 1993) or when the flowers are fully open (Rajasekharan and Ganeshan 1994). The literature reports wide variations in the techniques used for drying the anthers and storing the pollen (Calvino 1951; Khan 1987; Marchant et al. 1993; Visser et al. 1977). Good pollen germination has been reported with drying at ambient temperature for 24 h (Jicinska et al. 1976; Koncalova 1975) and 48 h (Gudin et al. 1991), whereas Voyiatzi (1995) prefers a period of 1 h at 60% relative humidity. The germination of rose pollen can be estimated by germinating the pollen grains on liquid media. Several studies report that the saccharose concentration in the media could stimulate or inhibit the germination percentage. Visser et al. (1977) and Voyiatzi (1995) obtained the best pollen germination percentage with a saccharose concentration of 15% for hybrid tea roses. Khan (1987) suggested a concentration of 20% for R. rugosa Thunb. Jicinska et al. (1976) studied

2 116 CANADIAN JOURNAL OF PLANT SCIENCE Czechoslovak Rosa species and established that the more favorable concentrations were between 10 and 35%. However, Koncalova (1975) reported a higher germination percentage for R. hugonis Hemsl. with 30, 35 and 40% saccharose. The effects of medium ph and stigmatic ph were clearly demonstrated by Gudin and Arene (1991) on six varieties of R. hybrida: a medium ph of 5.0 resulted in a higher germination percentage and greater lengthening of the pollen tubes than a ph of 3.0 or 9.0, and a stigmatic ph of 5.0 resulted in higher achene numbers per hip than a stigmatic ph of 9.0. Voyiatzi (1995) observed no significant differences for ph values between 5.0 and 7.5 in hybrid tea roses. The main objective of this study was to determine the optimal stage of flower development for collecting viable, high-germination pollen for three cultivars of the Explorer TM series of roses. The secondary objectives were to determine the optimal germination medium in terms of saccharose concentration and ph in order to assess the germination percentage of the pollen, and to develop pollen drying conditions with a view to maintaining pollen effectiveness. MATERIALS AND METHODS Three separate studies were conducted to characterize the optimal pollen germination conditions for three Rosa cultivars from the Explorer TM series of roses. These tests were performed to determine the effects of four saccharose concentrations, four ph of the liquid medium on pollen germination, as well as the effect of two pollen drying period. Furthermore, pollen germination was characterized at four different stages of flower development. Plant Material and General Procedure for Test All tests were carried out with Rosa Champlain (Svejda 1982), Frontenac (Ogilvie and Arnold 1995) and Nicolas (Richer and Davidson 2004). These cultivars are often used in the Agriculture and Agri-Food Canada (AAFC) rose breeding program at Saint-Jean-sur-Richelieu, QC, and were developed with the objective of producing long-flowering roses that can withstand winter temperatures of 35 C without winter protection and are resistant or tolerant to foliar diseases. For each of the three cultivars, a group of ten 4-yr-old plants was identified for flower selection from the plant beds at the Horticulture Research and Development Centre (HRDC) in Saint-Jean-sur-Richelieu (45 22 N, W). Between 5 and 10 buds per cultivar were collected when the petals were three-quarters open (except for the experiment that tested the germination of pollen collected at different stages of flower development). In the laboratory, the petals of all the flowers were removed, and the anthers were removed and divided into six replicates (six Petri dishes). After the anthers were dried 24 h (except for the experiment that tested the pollen drying time) at ambient temperature, the pollen released was collected in each Petri dish under a laminar flow hood with a small metal spatula that could be used to pick up the pollen grains that stuck to the sides of the dish. The pollen was then placed on the liquid culture medium [basic medium suggested by Jackson (1968)] in six 3-cm Petri dishes (six replicates), which were closed, sealed with Parafilm and wrapped in aluminum foil as recommended by several authors (Gudin and Arene 1991; Gudin et al. 1991; Rajasekharan and Ganeshan 1994; Voyiatzi 1995). After a 24-h germination period at 28 C, both germinated and ungerminated pollen grains in each dish were counted with a hemacytometer (SPotlite TM Hemacytometer Cat. B3175, Hausser Scientific, USA) under an optical microscope (Leica Microsystems, 10 eyepiece and 10 objective). The medium was stirred with a pipette to prevent segregation of the germinated and ungerminated pollen grains. A drop was placed on the grid of the hemacytometer, and the grains were counted. Each grain that had developed a pollen tube whose length was equal to or greater than twice the diameter of the grain was considered germinated (Gudin et al. 1991). Saccharose Concentration in the Medium Using the basic medium suggested by Jackson (1968), four concentrations of saccharose (ultra pure, BioShop Canada Inc., Burlington, ON) were compared: 5, 10, 15 and 20% (by weight). The media were prepared with g boric acid (Jicinska et al. 1976; Pearson and Harney 1984; Gudin et al. 1991a, b; Marchant et al. 1993) dissolved in 200 ml water. Saccharose was added and the volume was adjusted to 300 ml. The ph was adjusted to 5.6 with the addition of small droplets of potassium hydroxide (concentrations of 0.10 mol L 1 and 0.01 mol L 1 ). This solution was autoclaved for 20 min and maintained at 15 C for less than a week. The test was carried out with 10 flower buds from each of the three cultivars, all harvested at stage 2 (to reflect the conventional rose breeding practices at Saint-Jean-sur- Richelieu) between Jun. 25 and 29 and dried for 24 h in Petri dishes in darkness. Medium ph A second experiment was conducted at the same time using a similar protocol to compare the four ph values of the germination media: 4.5, 5.0, 5.6 and 6.0. The ph was adjusted with potassium hydroxide as previously described. The test was conducted with 10 flower buds from each of the three cultivars, all harvested at stage 2 between Jul. 02 and 04 and dried for 24 h in Petri dishes in darkness. For this test, the saccharose concentration in the germination medium was 15% (by weight). Pollen Drying Time In an effort to optimize the pollen germination percentage, two drying periods (24 h and 48 h) with and without light were tested. A first test was carried out in darkness, and a second test was conducted with pollen placed in light provided by two 52-W spot lights located 30 cm under the Petri dishes (about 1000 lm each). The tests were carried out with five flower buds from each of the three cultivars, all harvested at stage 2 between Jul. 14 and 19. In the laboratory, the petals were removed and the anthers were removed and dried. For these tests, the saccharose concentration in the liquid media was 15% and the ph was adjusted to 5.6. The germination tests were performed as previously described.

3 RICHER ET AL. POLLEN GERMINATION IN THREE EXPLORER ROSES 117 Characterization of the of Pollen Collected at Different Stages of Flower Development For the last experiment, conducted between Jul. 29 and Aug. 02, four flower stages were determined and evaluated according to the following scale: flower bud at stage 0 (closed bud), stage 1 (closed petals but open sepals), stage 2 (bud with petals three-quarters open) and stage 3 (fully open flower). So that contamination of the pollen from stage 3 buds (fully open) could be avoided, small paper bags were placed on the stage 2 buds 1 or 2 d before harvesting. The anthers were dried for 24 h in darkness, and the pollen was extracted and placed on a germination medium with a saccharose concentration of 15% and a ph of 5.6. The air relative humidity and air temperature were measured 12 and 24 h before the pollen was collected. Five flower buds were harvested at each of the four stages from each cultivar. Statistical Analysis In each experiment, the treatments were assigned in a completely randomized design with six replicates (Petri dishes). For each replicate of each treatment, three samples (pollen drops) were successively counted and averaged to determine the germination rate. For each cultivar and each experiment, analysis of variance (ANOVA) was performed using the linear model of SAS software (SAS Institute, Inc. 2001) with the GLM procedure. Differences among treatments were further analyzed using LSMEANS when the F values were significant at P < Normality of residuals and homogeneity of variance were tested before the data were pooled. RESULTS Effect of Saccharose Concentration on Pollen The germination percentage of pollen from Champlain was higher in the medium that had a 15% saccharose concentration than in the media with concentrations of 5 and 20% (Fig. 1, P = ). There were no statistical differences among the germination percentages measured on the saccharose concentrations for Frontenac (Fig. 1, P = ). The germination percentage of pollen from Nicolas was higher in the medium that had a 15% saccharose concentration than in the media with concentrations of 5 and 10% but did not differ from the germination percentage in the medium with a concentration of 20% (Fig. 1, P < 0.001). Effect of Liquid Medium and ph on Pollen Champlain was not affected by the medium ph (Fig. 2, P = ). A ph of 5.6 improved pollen germination for Frontenac (Fig. 2, P < 0.001), whereas a ph of 4.5 reduced pollen germination for Nicolas (Fig. 2, P = ). Effect of Pollen Drying Time on Pollen For each cultivar, there were no significant differences in germination percentage between the pollen dried for 24 h and the pollen dried for 48 h, regardless of whether the pollen was dried in light or in darkness (Table 1). Influence of Stage of Flower Development on Pollen The pollen germination percentage was statistically highest when the pollen was collected at stages 0 and 1 for Champlain (Fig. 3, P = ), at stages 1 and 2 for Frontenac (Fig. 3, P = ), and at stage 2 for Nicolas (Fig. 3, P = ). There was no pollen on Nicolas at stage 0. Influence of Climatic Conditions on Pollen Climatic data observed 12 and 24 h before pollen collection showed that the pollen germination percentage was not related to the air temperature or the air humidity (data not presented). However, there was a trend toward a higher germination percentage when the temperature was more than 25 C at the time of collection. Interestingly, the pollen of all cultivars was found to change color as it deteriorated, a change that facilitates the elimination of non-viable pollen. DISCUSSION Based on the fact that not all pollen will potentially have the same germination requirements, these results showed that a saccharose concentration of 15% and a ph of 5.6 provide optimal conditions for pollen germination in the three cultivars studied. Our results are similar to those of Visser et al. (1977) and Voyiatzi (1995). They also correspond closely to those of Calvino (1951), who obtained better results with a saccharose concentration of 20% for six species of roses. However, they are very different from those obtained by Koncalova (1975), who recommended a saccharose concentration of 30 to 45% for R. hugonis. Of the three cultivars tested, pollen from Frontenac was more responsive to a specific ph than pollen from the other cultivars. With a ph of 5.6, an acceptable pollen germination percentage was obtained for all cultivars. Some studies have found that overly long drying or exposure to lamps can dry out pollen grains, reducing their germination capacity (Linskens 1964). In addition, protecting pollen from light has been found to favor germination, because a small quantity of the ultraviolet rays emitted by fluorescent lights could be harmful (Stanley and Linskens 1974). We found no effects of drying time or light on pollen germination, and for the purposes of time and energy savings, a drying time of 24 h in darkness is recommended. The recommended stage of flower development for collecting pollen in a breeding program differs from cultivar to cultivar. Pollen quality is highest when pollen is collected from flowers at stages 0 and 1 for Champlain, at stages 1 and 2 for Frontenac and at stage 2 for Nicolas. These results differ from those obtained for hybrid tea roses by Visser et al. (1997), who suggested collecting pollen at the freshly open flower stage (stage 3). Pearson and Harney (1984) reported better results for R. hybrida when buds are fully colored and one or two petals are open (stage 1). Of the

4 118 CANADIAN JOURNAL OF PLANT SCIENCE Fig. 1. Determination of the germination percentage of pollen from the rose cultivars Champlain, Frontenac and Nicolas harvested at the three-quarters open stage (stage 2) and germinated on media with various saccharose concentrations. For each cultivar, columns with the same letters are not significantly different at P = Pollen germination (%) Champlain Frontenac Nicolas A Frontenac ph 4.5 ph 5.0 ph 5.6 ph 6.0 Fig. 2. Determination of the germination percentage of pollen from the rose cultivars Champlain, Frontenac and Nicolas harvested at the three-quarters open stage (stage 2) and germinated on media with different ph values. For each cultivar, columns with the same letters are not significantly different at P = Table 1. Evaluation of the pollen germination percentage in the roses Champlain, Frontenac and Nicolas under different drying conditions Drying time Cultivar Light treatment 24 h 48 h Champlain Dark 18.0a 20.7a Light 11.0a 12.8a Frontenac Dark 28.0a 30.9a Light 12.8a 18.5a Nicolas Dark 17.7a 12.1a Light 12.6a 8.9a a Values with the same letter within each row are not significantly different at P = three cultivars, Champlain has the greatest number of petals on its flowers, at close to 30 petals (Svejda 1982). It is also the only cultivar in our study to benefit from being harvested early in its flower development stage, i.e., at stage 0 or 1. The flower takes longer to open (more petals) (Richer, personal observation), and the pollen reaches maturity at an early stage of flower development. In conclusion, it is important to collect pollen when it is at its maximum germination capacity. High-quality pollen will allow a larger number of seeds to mature and give rise to genetically unique plants, thus providing a broader range

5 RICHER ET AL. POLLEN GERMINATION IN THREE EXPLORER ROSES 119 Fig. 3. percentage of pollen from the rose cultivars Champlain, Frontenac and Nicolas harvested at different stages of flower development. Stage 0: unopened flower bud; stage 1: bud with open sepals; stage 2: bud with petals three-quarters open; and stage 3: fully open flowers. For each cultivar, bars with the same letters are not significantly different at P = of possibilities for selection. The findings presented here should improve efficiencies in breeding programs. ACKNOWLEDGMENTS The authors would like to thank Valentin Taquet, a summer student, and Caroline Lafond, a research assistant with AAFC, for their contribution to this research project. Calvino, E Ricerche sul polline del genere Rosa. Ann. Sper. Agrar. 5: Erlanson, E. W Pollen analysis for rose breeders. American rose annual. The 1934 year-book progress Gudin, S. and Arene, L Influence of the ph of the stigmatic exudates on male female interaction in Rosa hybrida L. Sex. Plant Reprod. 4: Gudin, S., Arene, L. and Bulard, C Influence of season on rose pollen quality. Sex. Plant Reprod. 4: Jackson, G. A. D Hormonal control of fruit development, seed dormancy and germination with particular reference to Rosa. Soc. Chem. Ind. Monogr. 31: Jicinska, D., Koncalova, M. N. and Skyorova, O Studies in rose pollen. III. Pollen viability and germinability in eight Czechoslovak Rosa species. Preslia 48: Khan, M. A and storage of Rosa pollen. Pak. J. Agric. Res. 8: Koncalova, M. N Studies in rose pollen. I. In vitro germination of pollen grains of Rosa hugonis. Preslia 47: Linskens, H. F Pollen physiology. Annu. Rev. Plant Physiol. 15: Marchant, R., Power, J. B., Davey, M. R., Chartier-Hollis, J. M. and Lynch, P. T Cryopreservation of pollen from two rose cultivars. Euphytica 66: Ogilvie, I. and Arnold, N The Explorer series of roses from Ottawa and L Assomption. HortScience 30: 2, 175. Pearson, H. and Harney, P Pollen viability in Rosa. HortScience 19: Rajasekharan, P. E. and Ganeshan, S Freeze preservation of rose pollen in liquid nitrogen: Feasibility, viability and fertility status after long-term storage. J. Hortic. Sci. 69: Richer, C. and Davidson, C Winter-hardy roses: Explorer, Parkland and Prairie series. Agriculture and Agri-Food Canada, AAFC Publication 1922 E. 56 pp. SAS Institute, Inc SAS/STAT user s guide. Version 8.2. SAS Institute Inc., Cary, NC pp. Stanley, R. G. and Linskens, H. F Pollen: biology, biochemistry, and management. Springer-Verlag, New York, NY. 307 pp. Svejda, F Charles Albanel and Champlain roses. HortScience 17: Visser, T., De Vries, D. P., Welles, G. W. H. and Scheurink, J. A. M Hybrid tea-rose pollen. I. and storage. Euphytica 26: Voyiatzi, C. I An assessment of the in vitro germination capacity of pollen of five tea hybrid rose cultivars. Euphytica 83:

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