Author Institution: Department of Botany and Plant Pathology, The Ohio State University, Columbus 1

Blaydes, Glenn W.

Linck, A. J.

The Ohio Journal of Science

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DEMONSTRATION OF THE CHEMOTROPISM OFPOLLEN TUBES IN VITRO IN FOUR PLANT SPECIES lA. J. LINCK2 AND G. W. BLAYDESDepartment of Botany and Plant Pathology, The Ohio State University, Columbus 10The phenomenon of chemotropism has been reported for pollen tubes (Beckand Joly, 1941; Brink, 1924; Lidforss, 1899; Miyoshi, 1894a; Molisch, 1893; andTsao, 1949) for the spores of certain fungi (Graves, 1916; Miyoshi, 1894b) andfor the organs of higher plants such as roots (Newcombe and Rhodes, 1904).Although numerous species of higher vascular plants have been studied, onlyabout 10 species produced pollen which exhibited chemotropic characteristicstoward some part of the pistil of the same species. Examples of this phenomenonwere first reported at the end of the 19th century (Lidforss, 1899; Molisch, 1893).The literature up to 1924 has been reviewed by Brink. Tsao conducted an ex-tensive study of the chemotropism of pollen tubes and reported her results in 1949with a review of the literature up to that time. The chemical or chemicals re-sponsible for the tropic response have never been isolated.This present study was initiated as a survey of a small number of species ofplants, most of which had never been studied previously, for the possibility ofthe occurrence of the chemotropic characteristic of the pollen tubes toward plantparts of the same species.MATERIALS AND METHODSIn most of the research reported in the literature, a method in vitro was usedto demonstrate chemotropism of pollen tubes. This usually consisted of placinga small segment of some part of the pistil or other floral part on a semisolid mediumand then placing the pollen around this tissue.The method of Tsao (1949) was used in this study with only minor modifica-tions. Pollen was collected from plants grown in the greenhouse. Where pos-sible, flowers just at anthesis or a day or so past anthesis were used in these studies.However, no attempt was made to use pollen of exactly the same age in the tests.Flowers were brought intact into the laboratory and the pistil, other floral organs,or plant parts were cut into small segments. The size of these segments varied,but for segments of the ovulary, for the petals and sepals, pieces about 3-5 mmsquare were used. The style provided small cylinders about 3 mm in length.Ovules, when used in these studies, were excised intact from the ovulary. Themethod consisted of placing about five segments from each floral organ at ap-proximately equally spaced intervals on a semisolid medium in petri dishes.Pollen from the same species was then placed around the floral tissue segment at adistance of about 1 mm with a dissecting needle, under a binocular microscope.Thus, each test or treatment consisted of at least five duplicates in any one experi-ment and each experiment was repeated at least once. Positive chemotropismin a given species was arbitrarily considered as the growth of about 90 percent ormore of the pollen tubes from grains placed 1 mm from the test segment towardthat segment.The medium consisted of 1 percent agar-agar, 10 percent sucrose, and 100ppm Difco Yeast extract. The mixture was autoclaved for 15 minutes at 15pounds pressure. Before the medium had cooled completely it was poured intoContribution from the Department of Botany and Plant Pathology, The Ohio Sate Uni-versity, paper No. 542.2Present address: Department of Plant Pathology and Botany, University of Minnesota,St. Paul 1, Minnesota.THE OHIO JOURNAL OF SCIENCE 60(5): 274, September, 1960.No. 5 CHEMOTROPISM OF POLLEN TUBES 275petri dishes to a depth of about 4 mm. The plant materials were placed on themedium after it had cooled and the dishes were examined under low power atintervals beginning one hour after the pollen was placed on the medium. Forspecies whose pollen did not germinate well, the dishes were examined the follow-ing day and then discarded. For species whose pollen germinated readily on themedium employed here, the results could usually be read in from two to four hours.No attempt was made to adjust the medium in order to obtain good growth of thepollen of all the species studied.RESULTSTen species of plants, representing ten genera and seven families were studied.The results are shown in table 1. Positive chemotropism was demonstrated bypollen of one species of Aloe, one species of Clivia, one species of Gasteria, and onespecies of Haworthia, to segments of one or more floral organs of the same speciesTABLE 1The results of the study of the effect of one or more floral parts onthe direction of growth of pollen of the same species in vitroPositivePlant Family chemotropismAloe confusa Liliaceae + 1Antirrhinum majus Scrophulariaceae —2Clivia nobilis Amaryllidaceae +Crinum sp. Amaryllidaceae —Epiphyllum sp. Cactaceae —Gasteria verrucosa Liliaceae +Haworthia planifolia Liliaceae +Impatiens sultani Balsaminaceae —Kalanchoe sp. Crassulaceae —Nicotiana sp. Solanaceae —190% or more of the pollen tubes grew toward the floral partembedded in the medium.2Growth of the pollen tubes was at random.TABLE 2Plant species in which pollen tube chemotropism was demonstratedTissues toward which pollen tubes ofPlant the same species grewAloe confusa Stigma, style, petal, sepalClivia nobilis Stigma, style, ovulary wall, ovule,leaf, petal, sepalGasteria verrucosa Stigma, ovularyHaworthia planifolia Stigma, style, ovularyrespectively. The pollen of six other species grew at random when placed aroundsegments of floral organs of the same species.In addition to parts of the ovulary such as the stigma, style, ovulary wall,and ovule, other floral parts such as petals and sepals, as well as leaf tissue forcertain species, illicited the chemotropic response to pollen of the same species.These results are given in table 2.Photomicrographs were made of the chemotropic response of the Clivianobilis pollen to parts of the flower and to leaf segments (fig. 1-6).276 A. | . LINCK AND G. \V. BLAYDES Vol. 60Additional studies were carried out on the pollen of Clivia nobilis to learn moreof the nature of the chemotropic factor.With this species it was possible to confirm the results of Tsao (1949) thatthe chemotropic factor is heat stable and readily diffusable into agar. Even afterheating segments of the ovulary in water at 212° F for 15 minutes, it was possibleto obtain the tropic response to this tissue equivalent to the unheated controls.When ovules (6-12) were placed on agar cubes (about 4 mm3 in volume) andallowed to stand for 5-8 hours, it was possible to obtain the chemotropic responseof pollen of Clivia nobilis to these cubes after the ovules had been removed (fig. 7).Again the response was approximately equivalent to controls using excised intactovules on agar plates as previously described. The pollen was placed approxi-rFIGURES 1-6. The demonstration of the growth of pollen tubes of Clivia nobilis toward vegeta-tive and floral parts of the same species. FIGURES 1-3. Growth of the pollentubes toward a segment of the style; toward an entire, excised ovule; and towarda segment of a leaf, respectively. FIGURES 4-6. Growth of the pollen tubestoward the stigma, toward a segment of the sepal, and toward a segment of thepetal, respectively.No. 5 CHEMOTROPISM OF POLLEN TUBES 277mately 1 mm from the agar cubes as in the other experiments with tissue segments.In order to test the possibility that pollen tubes of Clivia nobilis would grow towardany object embedded in the agar, experiments similar to those described werecarried out with the tissue segments replaced by clean glass beads. Growth ofthe tubes of Clivia nobilis pollen grew completely at random around the glassbeads.FIGURE 7. The demonstration of the growth of pollen tubes of Clivia nobilis toward an agarcube containing a diffusate from ovules of the same species.DISCUSSION OF RESULTSThe data presented here in general corroborate the results of Tsao (1949).However, it was not possible to demonstrate chemotropism in the unidentifiedvariety of Antirrhinum majus studied here. The active factor for Clivia nobilismay be chemically similar to the unknown chemical reported by Tsao (1949) forLilium superbum since both are heat stable, water soluble, and diffusable into agar.Tsao (1949) reported that the petals, sepals and leaves of Antirrhinum majusdid not attract the pollen tubes of the same species. In the present study it wasfound that the sepal, petal, and leaf segments of Clivia nobilis did attract the pollentubes of the same species. Thus, it appears that at least in this species the activefactor is distributed throughout the above ground parts of the plant. It wasalso possible to demonstrate chemotropism of the pollen tubes of Aloe confusato segments of the petal and sepal in addition to the tropic response to the stigmaand to segments of the style (table 2).SUMMARY1. Of ten species of plants investigated, it was possible to demonstrate thechemotropism of the pollen tubes of four species to tissue segments of the samespecies. These four species are Aloe confusa, Clivia nobilis, Gasteria verrucosa,and Haworthia planifolia.278 A. J. LINCK AND G. W. BLAYDES Vol. 602. The pollen of these four species exhibited the chemotropic response tovarious segments of the pistils of the respective species, and in addition the pollenof Aloe confusa and Clivia nobilis grew toward petal and sepal segments. Thepollen of Clivia nobilis also demonstrated the chemotropic response to leaf tissuesegments.3. The active factor bringing about the chemotropic growth of the pollentubes of Clivia nobilis was found to be heat stable, water soluble, and diffusableinto agar.LITERATURE CITEDBeck, W. A. and R. A. Joly. 1941. Some growth phenomena in cultured pollen tubes. Trans.Am. Microscop. Soc. 60: 149-162.Brink, R. A. 1924. Physiology of pollen. IV. Chemotropism; effects on growth of groupinggrains; formation and function of callose plugs; summary and conclusions. Am. J. Botany11:417-436.Graves, A. H. 1916. Chemotropism in Rhizopus nigricans. Botan. Gaz. 62: 337-369.Lidforss, B. 1899. Uber den Chemotropismus der Pollenschlauche. Ber. Deut. Botan. Ges.17: 236-242.Miyoshi, M. 1894a. Uber Reizbeiwegungen der Pollenschlauche. Flora 78: 76-93.. 1894b. Uber Chemotropismus der Pilze. Botan. Zeit. 52: 1-28.Molisch, H. 1893. Zur Physiologie des Pollens mit besonderer Riicksicht auf die chemo-tropische Bewegungen der Pollenschlauche. Sitzungsber. Wien. Akad. Wiss. Mathnaturw.Kl. 102: 423-448.Newcombe, F. C. and A. L. Rhodes. 1904. Chemotropism of roots. Botan. Gaz. 37: 23-35.Tsao, Tsung-Hsun. 1949. A study of chemotropism of pollen tubes in vitro. Plant Physiol.24: 494-504.

Demonstration of the Chemotropism of Pollen Tubes in Vitro in Four Plant Species

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Demonstration of the Chemotropism of Pollen Tubes in Vitro in Four Plant Species

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