CYTOLOGY OF VIOLA ODORATA L. POLLEN GERMINATION SILVICA PĂDUREANU 1. Introduction

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1 Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LIII, s. II a. Biologie vegetală, 27 CYTOLOGY OF VIOLA ODORATA L. POLLEN GERMINATION SILVICA PĂDUREANU 1 Abstract: The paper presents the characteristics of Viola odorata L. pollen germination process. We also mention here other points concerning the length of pollen tubes in the dynamics of germination process and according to the glucide concentration of nutritive mediums used in the experiment. A certain length of pollen tubes was very important, as they ensured ovule fertilization and implicitly, the fructification of this taxon. We have also mentioned other points on the characteristics of pollen tubes and abnormalities that might appear during the germination process of sweet violet pollen. Key words: pollen tube, generative cell, vegetative cell, abnormalities of pollen tubes Introduction Pollen as an organization organ takes part to a very important stage of the vegetal world, the stage of reproduction. Once it reached the surface of stylus, the pollen grain begins to germinate and to form a pollen tube, which crosses the tissues of stigmata and stylus, being directed through chemotropism to an ovule it fertilizes. The pollen tube of angiosperms contains two haploid, immobile spermatic cells, which have the value of male gametes. In fact, the pollen tube is a vector of male gametes. Once reached the ovule micropyle, the pollen tube is resorbed, and its valuable content (the two male gametes) is flowed into ovule for its fertilization: a male gamete is joined to oosphere, resulting the zygote. The other male gamete is joined to central cell of embryo sac from ovule, resulting the endosperm of the future seed. The double fecundation specific to angiosperms is done this way [2, 3, 6]. Pollen is finally found in direct correlation with the fructification of the vegetal body. The knowledge of the way of forming the pollen tube and of factors, which influence this process, results in solving many eco-physiological, genetic, diagnosis aspects, typical of each vegetal taxon [4]. The aim of this paper was the study on the pollen behaviour in the germination process in a widely spread taxon Viola odorata L. Material and Methods The biological material was represented by a population made of a few hundreds of samples with chasmogamous flowers belonging to species Viola odorata, a spontaneous species found in the Botanical Garden of Iassy. For the investigation carried out on sweet violet pollen, we sampled anthers at the stage of anthesis. Pollen grains have been inoculated on agar nutritive mediums, at which sucrose was added at different concentration, from to 3%. The quantity of pollen inoculated on these mediums was the same in all the cases. 1 University of Agricultural Sciences and Veterinary Medicine Ion Ionescu de la Brad Iaşi 6

2 For each experimental variant, we carried out 15 preparations. In order to maintain a wet medium, which was vital for pollen viability, we have used van Tieghem wet chambers [5]. Micromeasurements for determining the dynamics for the extension of pollen tubes were carried out every 3, 24, 48 and 72 hours since the inoculation of pollen grains on nutritive medium. For pointing out the characteristics of pollen tube from this taxon, photographs were taken at Nikon optical microscope and drawings at camera lucida. Results and Discussions After three hours since inoculation, the first pollen tubes appeared on mediums, with glucide concentration ( - 45%) allowed the germination during this interval. The length of these tubes was between 6 and μm, the longest ones being found on 15% sucrose medium, and the shortest ones, on the medium without sucrose (fig. 1). micrometers % sucrose in medium Fig. 1 The average lenght of the pollen tube at Viola odorata L., 3 hours after inoculation After 24 hours since inoculation, pollen tubes have increased in length. Other tubes appeared on other mediums with high until 3% glucide concentration (fig. 2). The longest pollen tubes were formed on 15% sucrose medium ( μm). micrometers % sucrose in medium Fig. 2 The average lenght of the pollen tube at Viola odorata L., 24 hours after inoculation After 48 hours since inoculation, the length of pollen tubes was double, and in some cases, triple. On 15%, 2% and 25% sucrose mediums, pollen tubes were the longest from all experimental variants: μm, 1925 μm and respectively, 175 μm. On the medium without sucrose, the tubes were, on the average, μm long, while on the 61

3 medium with the highest sucrose concentration - 3%, pollen tubes extended until μm (fig. 3) micrometers % sucrose in medium Fig. 3 The average lenght of the pollen tube at Viola odorata L., 48 hours after inoculation After 72 hours since inoculation, we found out that many pollen tubes degenerated. The viable ones either stopped their growth or broke at top, being resorbed (fig. 4). micrometers % sucrose in medium Fig. 4 The average lenght of the pollen tube at Viola odorata L., 72 hours after inoculation The dynamic analysis of length growth for Viola odorata pollen tubes has shown that this parameter was much influenced by glucide concentration of nutritive mediums (fig. 5, 6). In the first 48 hours since inoculation, a significant elongation of pollen tubes took place on all the mediums used in the experiment; then, after 72 hours, a partial resorption of tubes and a general stagnation of the growth process of pollen tubes were noticed. On mediums, which glucide concentration exceeds 11%, the elongation process of pollen tubes was active, even after 72 hours since inoculation, but at minimum height. Different aspects of pollen tubes formed on different nutritive mediums are presented in fig As concerns the structure of Viola odorata L. pollen grains, we found that in this taxon, pollen is bicellular. The generative cell has an elongated shape, being a refringent zone. The germination process starts with the formation of a mammilla. In a few hours, the pollen tube is thus formed. The vegetative cell is quartered on the top of the tube, and the generative cell penetrates the tube when it reaches a mean length of 193 μm. In vitaceae, the generative cell penetrates the tube when it has 1 μm [1]. Therefore, the moment of generative cell penetrating the tube may be considered as a diagnosis character. 62

4 micrometers h 24 h 48 h 72 h time (hours) Fig. 5 Dynamics of average lenght of the pollen tube at Viola odorata L. % sucr. 1% sucr. 5% sucr. 1% sucr. 15% sucr. 2% sucr. 25% sucr. 3% sucr. 35% sucr. 4% sucr. 45% sucr. micrometers h 24 h 48 h 72 h time (hours) Fig. 6 Dynamics of average lenght of the pollen tube at Viola odorata L. 6% sucr. 7% sucr. 8% sucr. 9% sucr. 1% sucr. 11% sucr. 15% sucr. 18% sucr. 2% sucr. 22% sucr. 25% sucr. 3% sucr. During the pollen tube elongation, the generative cell is found at an average distance of 26 μm compared to the base of tube and of 635 μm compared to the top of tube. Viable pollen tubes have a constant diameter on the entire length, of 11 μm, while the tubes broken at the top or near the top have a decreasing diameter to the top. In many variants of the experiment, we found out the formation of pollen tubes with spiral extremity at the base or in the upper third (fig. 13, 14, 15). The spiral forms of these pollen tubes are not conditioned by certain glucide concentrations and cannot be considered abnormalities. During the germination process of Viola odorata pollen, some abnormalities in the shape of pollen tube were found; they do not depend on glucide concentration of medium and on the length of the tube. These abnormalities consist in tubes branched out at the base or near the base of the tube, pollen grains with two tubes, which came from two germinating pores, tubes with dilatations at the top or on the line of the tube (fig. 16, 17). As concerns the length of pollen tubes, we found out that the longest tubes were formed on 1-35% sucrose mediums, when they might have, on the average, 2 μm after h since pollen inoculation on medium. We have also noticed that there was a positive correlation between pollen germination capacity and length of pollen tubes in Viola odorata. After measuring the length of 2 styluses in Viola odorata flowers, which took part of the investigated population, an average of 28 μm was obtained. Therefore, only the pollen tubes with an average length of at least 2 μm could penetrate the entire long stylus of sweet violet flower, in order to reach the ovules. In Viola odorata, a concentration of stigmatic liquid of 1-35% glucide guarantees the success of pollen tubes reaching the ovules during hours. 63

5 Conclusions 1. Viola odorata L. pollen is bicellular. 2. The generative cell penetrates the pollen tube only when it has a length of at least 19 μm. 3. Viable pollen tubes have a constant thickness of 11 micrometers on the entire length, while the broken degenerating tubes have a smaller and smaller diameter to the top. 4. Nutritive mediums with 1-35% glucide concentration lead to the formation of the longest pollen tubes (around 2 micrometers) in hours since inoculation; these tubes can penetrate the long stylus of 28 micrometers of this taxon. 5. There is a positive correlation between pollen germination capacity and length of pollen tubes. 6. During the germination process of Viola odorata pollen, abnormalities in the morphology of pollen tubes may appear (indifferently of the concentration of nutritive layer): branched out tubes, tubes with dilatations placed at different sites, and two pollen tubes of pollen grains. 7. During the germination of sweet violet pollen, spiral pollen tubes may appear at high rates; their presence does not depend on glucide concentration of nutritive medium. This could be a genotypic specific feature. BIBLIOGRAPHY 1. GHERASIM C., 197. Ampelografia R.S.R., vol. 1, Edit. Acad. R.S.R., 189 pp. 2. IVĂNESCU LĂCRĂMIOARA, TOMA IRINA, 23. Embriologie vegetală, Edit. Junimea, Iassy, pp. 3. NEWBIGIN E., ANDERSON M.A., CLARKE A.E., Gametophytic self incompatibility sistem. Plant cell, 5: pp. 4. PĂDUREANU SILVICA 23. The cytological variability of the pollen grain germination in Fetească neagră grspe-vine variety. Anale st. Univ. Al.I. Cuza Iassy, tom XLIX, s.ii a. Biologie vegetală, RAICU P Metode noi în genetică, Edit. Did. Pedag., Bucureşti, PP. 6. RUSSEL S.D., Double fertilization. Intern. Review of Cytology, 14:

6 Fig. 7 Pollen germination on % sucrose medium, 24 hours after inoculation in Viola odorata L. (2X) (Original) Fig. 8 Pollen germination on 1% sucrose medium, 24 hours after inoculation in Viola odorata L. (2X) (Original) 65

7 Fig. 9 Pollen germination on 1% sucrose medium, 24 hours after inoculation in Viola odorata L. (2X) (Original) Fig. 1 Pollen germination on 5% sucrose medium, 48 hours after inoculation in Viola odorata L. (2X) (Original) 66

8 Fig. 11 Pollen germination on 15% sucrose medium, 24 hours after inoculation in Viola odorata L. (2X) (Original) Fig. 12 Pollen germination on 4% sucrose medium, 48 hours after inoculation in Viola odorata L. (2X) (Original) 67

9 Fig. 13. Spiral pollen tubes on 1% sucrose medium, in Viola odorata L. (2X) (Original) Fig. 14. Spiral pollen tubes on 25% sucrose medium,in Viola odorata L. (2X) (Original) 68

10 Fig. 15 Spiral pollen tubes on 15% sucrose medium, in Viola odorata L. (2X) (Original) Fig. 16 Pollen tubes branched and with dilatations, in Viola odorata L.(Original) Fig. 17 Pollen grains with two tubes, in Viola odorata L. (Original) 69