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lt is well known that the spermatozoids of Ginkgo biloba and Cycas revoluta were discovered in 1896 by S. Hirase and S. Ikeno, respectively, and that it was the first record which proved the existence of motile spermatozoids in gymnosperms. A short sketch of their discovery is given here, as a scene of botanical history in Japan at the end of the l9th century. The detailed description on the structure and development of sexual organs of these plants will not be given as it is not the object of this communication.

Spermatozoids in Ginkgo biloba

Sakugoro Hirase (1856-1925) was a drawing technician and was employed in 1888 at the College of Science, Imperial University, Tokyo, and engaged at the Botanical Laboratory to draw the plant specimens. He became an assistant and learned himself the techniqne of botanical studies and since 1893 began to observe the period of fertilization and embryo formation in Ginkgo biloba (Hirase, 1894a), which was not fully known, though it was investigated by some botanists such as Strasburger (1892). He made microscope preparations of the ovules of Ginkgo (Hirase, 1894b) and found a peculiar radiated structure, attraction sphere, in the pollen tube within the ovule, and further found the existence of two archegonia in the ‘endosperm‘ and canal cells in the archegonium. He could trace the period of fertilization as the middle of September (Hirase, 1895a, b). Much attention was then centered to the peculiar body within the pollen tube, which was ellipsoid in form and provided with a snail-like coiled band, on which numerous cilia were borne. He considered such a body to be a spermatozoid and gave an address on April 25, 1896, at the meeting of the Tokyo Botanical Society, under the title "Spermatozoid of Ginkgo biloba" (Hirase, 1896a). He found such a ciliated body only in microscopic preparations, hut he expected it to be motile. In order to observe the living spermatozoids, he cut numerous ovules and succeeded in finding the motile ones on September 9 of the same year, and gave an address about it on September 26, at the meeting of the Tokyo Botanical Society and published it in the Qctober number of the Botanicai Magazine of the Society (Hirase, 1896b). Further researches on spermatogenesis, fertilization and embryo formation were carried on, and the results were published a little later (Hirase, 1898; Fig. 1). Through his researches, the hfe history of Ginkgo was made clear. At Tokyo, pollination took place between the end of April and early May, and fertilization between the end of September and middle of October.

Fig. 1 — A spermatozoid of Ginkgo biloba, released out of the pollen tube; the spermatozoid body is partly covered by protoplasmic mass; cilia are drawn so finely that they are scarcely visible. x 750 (after Hirase, 1896).

After the discovery of spermatozoids his colleagues of the University discussed and reinvestigated his results, and confirmed them to he mostly correct. Some other workers also observed motile spermatozoids (Fujii, 1898, 1899a, 1899b, 1899c, 1900; Ikeno, 1899a, 1901; Miyake, 1902; Miyoshi, 1896).

Shortly after his work on Ginkgo, Hirase left the University and became a teacher of a middle school at Hikone and then at Kyoto, and died at Kyoto in 1925. lt may be rememhered that he accomplished difficult researches on the life history of Ginkgo biloba by his own endeavour and ingenious experiments, notwithstanding he was not a trained botanist. The Ginkgo tree from which he used to collect his materials was a big female tree growing in the Botanical Garden of the University (Fig.2)., This is still growing there. (note added: see )

Fig. 2 - Ginkgo biloba. A giant female tree in the Botanical Garden of the Imperial University of Tokyo, Koishikawa, Tokyo, from which Hirase discovered the spermatozoids; photographed nearly in 1900.

Spermatozoids in Cycas revoluta

Seiichiro Ikeno (1866-1943) graduated in 1890 in botany from the College of Science, Imperial University at Tokyo, studied botany at the newly established College of Agriculture of the same University, and became an Assistant Professor.

He carried on investigations on Cycas revoluta, since 1895, in order to observe the structure and development of sexual organs, especially the process of fertilization and embryo formation, which was investigated to some extent by others, such as Treub (1881, 1884) and Warming (1877, 1879), in some species of the Cycadaceae. He made numerous microscopic preparations of the ovules of Cycas and observed canal cells in the archegonium which were at that time considered to be absent in the Cycadaceae (Ikeno, 1896a). During these observations he found, in the spring of 1896, the large body with a coiled band and provided with cilia within the pollen tube. lt was just the time when Hirase reported the existence of spermatozoids in Ginkgo, and Ikeno concluded such a body in Cycas also to be the spermatozoid, though he found it only in preparations, and announced it in the November number of the Botanical Magazine of the Tokyo Botanical Society (Ikeno, 1896c).

Further detailed work on the development of pollen tube, spermatogenesis, fertilization and embryo formation was carried on, and the life history of Cycas revoluta was described by him (Ikeno, 1898a; Fig. 3) a little later. He recognized that during spermatogenesis the centrosomal structure on one side of the sperm-body elongated into a blepharoplast, on which numerous cilia were borne (Ikeno, 1898c, 1899b).

Fig. 3A-C - Spermatozoids of Cycas revoluta (cb, centrosome-band (blepharoplast) ; ‘n, nucleus). A. Median section, showing five turns of ciliated spiral band. x 270. B, C. Optical section, showing centrosome-band on which cilia are not shown. B from above; C from oblique side. x 110 (after Ikeno, 1898a).

Cycas revoluta was cultivated in Tokyo, but the seeds were not formed, as Tokyo was a little cold for fertihzation. Therefore, Ikeno and his colleagues collected the material from Kagoshima and Tanegashima, south of Kyusyu. Cycas trees from which Ikeno discovered the spermatozoids (Fig. 4) are still alive in the garden of the Kagoshima Prefectural Museum (formerly the Kogakukan). lt was ascertained by Ikeno that, at Kagoshima, pollination took place during June and July and fertilization during September and October. Kagoshima was far from Tokyo, and he could not collect fresh ovules as he wanted and, therefore, he lost the opportunity of observing motile spermatozoids. lt was Miyake (1905, 1906) who reinvestigated Cycas and observed motile spermatozoids at Naze of Oshima, Oshima Island, Kagoshima and Onezime of Kagoshima, Kagoshima Prefecture, at the end of September, 1899.

After his researches on Cycas Ikeno continued botanical studies as a Professor in the same College, until he retired in 1927. He died in 1943 at Tokyo. The microscopic preparations which he made during his investigations on Cycas are kept at present in the National Science Museum at Tokyo.

Fig. 4 - Cycas revoluta, male and female plants in the garden of the Kogakukan, Kagoshima, from one of which Ikeno discovered spermatozoids; photographed nearly in 1900.

Spermatozoids in Gymnosperms

Through the researches of Hirase, Ikeno and their colleagues the existence of motile spermatozoids in Ginkgo biloba and Cycas revoluta has been proved and, thus, Hirase and Ikeno are recorded in the history of botany as the first discoverers of spermatozoids in gymnosperms. The results of their study were described in some journals of Europe at the request of their editors (Hirase, 1897; Ikeno, 1896b, 1897, 1898h, l898c, 1901; Ikeno & Hirase, 1897), and their works came to be known throughout the world. The Imperial Academy of Japan awarded them prizes, in 1912, for their brilliant investigations.

In 1897 H. J. Webber of the United States of America found in Zamia integrifolia the peculiar structure occurring within the pollen tube and proved it to be a motile spermatozoid (Webber, 1897a, b). His work made the ohservations of Hirase and Ikeno much more authoritative. Webber (1897d, 1901a, 1901b) observed the spermatozoids also in Zamia floridana and Z. pumila. Since then researches on the structure and development of sexnal organs of gymnosperms, especially the Cycadaceae, have been carried on by many botanists and the existence of spermatozoids was reported early in the 20th century by Chamberlain in Dioon edule (1909), Ceratozamia mexicana (1912), Macrozamia moorei (1913) and Stangeria paradoxa (1916); and by Caldwell (1907) in Microcycas calocoma.

The existence of spermatozoids in Ginkgo and Cycadaceae considerably influenced our thinking on the phylogeny of plants. For example, Ginkgo which was considered as a member of the Taxaceae was shifted to a new family Ginkgoaceae (Engler, 1897), order Ginkgoales (Engler, 1898). lt strengthened also the theory of Hofmeister (1851) who maintained that the boundary between pteridophytes and gymnosperms might not be so rigid as was once considered, represented by the surviving links between two groups.

Botany of the l9th Century in Japan

lt may be pointed out that the influence of the discovery of spermatozoids in Ginkgo and Cycas has contributed to the progress of botany in Japan, because the knowledge of modern botany has been introduced just at the last quarter of the l9th century. lt may be said that in Japan the study of plants began since the l7th century, mainly in the study of herbs. Since the l8th century, especially the l9th century, there were famous herbalists who published important herbals. During this period some European natural historians or doctors visited and collected plants in Japan, which were described in Linnean system. Some of the books on these plants reached Japan and stimulated Japanese herbalists. In the 19th century some European books on plant morphology, anatomy and physiology were also introduced and a few of them were translated into Japanese. Some herbalists endeavoured to understand modern botany, and to regulate the classical study of herbs and modern botany. lt was, however, severely forbidden, on account of the policy of seclusionism, to communicate with foreign countries and to introduce the foreign articles, which made the introduction of modern botany very difficult.

Then the Meiji restoration of 1868 took place, and every political and social system was completely revised. The communication with foreign countries and the introduction of foreign articles became free and many Japanese visited European and American countries to learn the modern systems or sciences. In 1877 the Tokyo University¹ was established as the center of modern studies. The university consisted of four colleges, and in the botanical course, which belonged to the College of Science, lectures and experiments were undertaken, at first based on the text books of Europe or America, and professors and students tried to understand modern botany until they were able to undertake original investigations. Thus, in Japan, the last quarter of the l9th century was nothing but a cradle period of modern botany, and it was rather marvellous that important studies by Hirase and Ikeno were accomplished at that time. Their brilliant researches stimulated Japanese botanists so vigorously that they were able to make valuable contributions in the 20th century.

Classical herbalists and senior botanists passed away, but the historic Ginkgo tree at Tokyo and Cycas trees at Kagoshima might have seen the history of Japanese herbal and botany during several centuries, and might have stimulated, and are still inspiring the young students.