Fertilization and Development of Embryo on Satsuma Orange (Citrus Unshiu MARC.) and Natsudaidai (C

Fertilization and development of embryo on Satsuma orange (Citrus unshiu MARC.) and Natsudaidai (C. natsudaidai HAYATA)

HSU-JENYANG Collegeof Agriculture,University of OsakaPrefecture , Sakai,Osaka

Summary

The mechanism of the Citrus pollen-tube growth 3. The fertilized eggs and nucellar cells began into style and ovary, fertilization and development to divide 40 to 50 days after pollination both in of embryo were studied. The results obtained were selfed Natsudaidai and crossed Satsuma orange with summarized as follows Natsudaidai. 1. The pollen tubes of Natsudaidai grew faster 4. In Natsudaidai, embryo development was very than Satsuma orange in pistil of Natsudaidai or slow and reached the globular stage 58 days, heart- Satsuma orange. shape embryo stage 72 days, and the complete 2. The fertilization took place about 8 days in embryo stage 120 days after self-pollination. selfed Natsudaidai, 12 days in the crossing of 5. The nucellar cells of one to three layers near Satsuma orange with Natsudaidai, and 14 days in the micropylar of embryo sac and the region of self-pollination of Satsuma orange after pollination, the micropylar end to one-third of embryo sac have respectively. the ability to form nucellar embryos.

Introduction

The polyembryony has been found extensively in Citrus, Poncirus and Fortunella plants. This phenomenon was first recognized in orange seed by LEEVWENHOEKin the beginning of the eighteenth century. According to the works of OSAWA(19) ToxopEUs (21) FROST5, FURUSATO(7) and MAHESHWARIand SACHAR~I5~,the extra embryo originate from the nucellar tissue in ovules. It has been considered that pollination is positively necessary for the occurrence of the nucellar embryos (IKEDA(11~,NAGAI and TANIKAWA{17~,FROST~4~, WONG(23)), however, WRIHGT~24~,reported that nucellar embryos occur even without pollination. The fertilization and its mechanism of the initiation in the development of the nucellar embryos have not been studied in detail except for the studies of C. tamurana (MIwA~IS~)and Poncirus trifoliata (OSAWA(19)). In order to obtain a clear understanding of this problem, the author carried out this experiment to study the mechanism of Citrus pollen tube growth into the style and ovary, its fertilization and development of nucellar embryos. Materials and Methods

Materials used in this experiment were 25-year-old Satsuma orange (Citrus unshiu MARL.) and 8-year-old Natsudaidai (C. natsudaidai HAYATA). All these plants are grown in the Citrus Garden of Osaka Agriculture Research Center, Osaka Prefecture. The experiments were conducted during the years from 1964 to 1966. Mature and uniform flower buds for artificial crosses were selected and emasculated . The emasculated flowers were covered with paraffin paper bags. Pollen for desired crosses was

Received for publication November 20, 1967 8 HSU-JEN YANG : Fertilization and development of embryo on Citrus 103

gathered from newly opened flowers which had been previously covered with paraffin paper bags. Pollination was carried out from 10: 30 a.m. to 4: 00 p.m. The pistils were cut off and fixed in chloroform-acetic-alcohol (3: 2 : 2) at the various intervals after pollination. The materials for microtome were processed in ethanol-xylene dehydration series and embeded in paraffin. Sections were cut out 10 to 15 /2 thickness and stained by Haidenhain's iron-alum- haematoxylin procedure.

Results

1. Pollen tube growth The germination of Natsudaidai and Satsuma orange pollens were observed on its own and reciprocal stigmas two hours after pollination (Fig. 1). Pollen grains of Natsudaidai germinated earlier on its own stigmas as well as the stigma of Satsuma orange in comparison to those of Satsuma orange pollen grains. Regarding the growth of pollen tube, the fastest one was Natsudaidai pollen tube in its own style, the second was in the style of Satsuma orange, the third was Satsuma orange pollen tube in its own style, and the slowest was noted in the style of Natsudaidai (Figs. 2 and 3). Natsudaidai pollen tubes reached to its own ovary 4 days after pollination, at that time, Satsuma orange pollen tubes penetrated just only one-half of its own style and one-fourth of Natsudaidai style. All of the Natsudaidai fruits crossed with Satsuma orange dropped off 8 days after pollination (Table 1). 2. Fertilization and development of endosperm Pollen tubes of Natsudaidai reached to its own embryo sac and fertilized 8 days after self- pollination (Fig. 5), and to the Satsuma orange embryo sac 12 days after crossing of Satsuma orange with Natsudaidai. However, Satsuma orange pollen tube penetrated into its own embryo sac 14 days after self-pollination. Then, several endosperm nuclei were observed in the embryo sac 10 days after self-pollination of Natsudaidai (Fig. 6), 14 days after crossing of Satsuma orange with Natsudaidai, and 16 days after self-pollination of Satsuma orange. The endosperm nucleus which had divided succes- sively formed many scattered free nuclei in a thin layer of cytoplasm during 32 days after crossing of Satsuma orange with Natsudaidai (Fig. 7). After then, these free nuclei divided more and more and cell wall was formed surrounding each nuclei and filled the embryo sac up completely. Finally, the endosperm gradually disappeared with the formation of complete embryo (Figs. 15, 16 and 17). 3. Development of embryo

Fig. 4A. Diagram depicting longitudinal section From the observation of this experiment, the of an ovule of Citrus unshiu, showing the oospore remained dormant relatively for a long position of pollen tubes. A. B. C. D and E indicate the position of pollen tubes. a, time after the egg cell has fertilized. The antipodal cell, eg, egg cell. f, f uniculus. 1, oospore did not divide within 40 days after locule. o, ovary, ov, ovule. p, polar nuclei. s, synergid. st, style. vb, vascular bundle. pollination (Fig. 8). Four-celled proembryo was 1.04 Jour. Japan. Soc. Hort. Sci. 37 (2)

Table 1. Length of pollen tubes and the number of endosperm nuclei of C. natsudaidai and C. unshiu, in each of self- and crossing(s)

observed 44 days after self-pollination of Natsudaidai (Fig. 10), and six- and ten-celled proembryo was found 52 and 54 days after crossing of Satsuma orange with Natsudaidai (Figs. 11 and 12). So it may be said in general, the oospore remained dormant for about four to five weeks after fertilization. In Natsudaidai, embryo development was very slow and reached the globular stage 58 days (Fig. 13), heart-shape embryo stage 72 days (Fig. 14), and the complete embryo stage 120 days after self-pollination (Figs. 15 and 16). While, in Satsuma orange the formation of globular embryo was observed 63 days and the embryo which was being differen- tiated and formed the various parts of complete embryo was found 108 days after the crossing (Fig. 17). It was confirmed that when oospore began to divide a few of large nuclei which could be stained with hematoxylin were found in the nucellar tissue of one to three layers near the micropylar of the embryo sac (Fig. 9). Some of these cells began to divide and formed masses of cells and protruded into the embryo sac 84 days after crossing of Satsuma orange with Natsudaidai (Figs. 20 and 21). It was also found that globular nucellar embryo was observed from the region of the micropylar end to one-third of embryo-sac (Figs. 18 and 19). From the above mentioned results, it may be said that not only the nucellar cells near the micropylar end, but also the cells from the micropylar end to one-third of embryo sac have the ability to form nucellar embryos.

Discussion

Fertilization is the fusion of a sperm nucleus with an egg nucleus. The first step of the fertilization is begun with the germination of pollen grains and growth of pollen tube in the stigma tissue. MiwA~16~reported that C. tamurana has not a behavior of self-fruitfulness, owing Hsu-JEN YANG : Fertilization and development of embryo on Citrus 105 to their self-incompatibility. He further reported regarding the growth rate of pollen tube in style that the pollen tube of C. limon grew more quickly when C. tamurana was crossed with C. limon than of self ed C. tamurana. TON and KREZDORNX20) observed that pollen tubes of cross-pollination among Clementin, Minneola and Orlando, with the exception of cross-incom- patibile Orlando x Minneola, and Minneola x Orlando, grew faster than those of the self s. The similar result was obtained from Satsuma orange by the author, i.e., the growth of pollen tube in crossing with Natsudaidai was faster than selfing. However, Natsudaidai showed completely reverse results ; the growth of pollen tube in selfing was more rapidly than the crossing with Satsuma orange. It is also found that many pollen tubes of Natsudaidai were observed in the ovary of self ed Natsudaidai and crossed Satsuma orange with Natsudaidai, but'few pollen tubes of Satsuma orange detected in its self ed ovary, and could not be ascertained any of them in the base of style and ovary of crossed Natsudaidai with Satsuma orange. While, the pollen tube of Satsuma orange grew only one-half of the style of Natsudaidai. Moreover, all of the fruits of Natsudaidai crossed with Satsuma orange dropped off 8 days after pollination. From these results it can be assumed that some kind of inhibitors are formed in the style or ovary of Natsudaidai and these inhibitors suppress the growth of pollen tube of Satsuma orange. It may also be said the cross-incompatibility would be exist when Natsudaidai was crossed with Satsuma orange. As soon as the pollen tube reached the embryo sac by way of the micropyle, a sperm nucleus passed into the embryo sac, and fused with the egg nucleus, thus fertilization took place. TOXOPEUS(21> found that fertilization occurred about three to four weeks after pollination in a Mandarin variety. OSAWA~19~stated that in P. trifoliata its fertilization took place more than two weeks and probably not much less than four weeks after flowering. MIWA~16~found that the fertilization occurred about 7 or 8 days after crossing of C. tamurana with C. limon. The author confirmed that the fertilization occurred about 8 days after the selfing of Natsudaidai, about 12 days after crossing of Satsuma orange with Natsudaidai and about 14 days after selfing of Satsuma orange. Embryonic development in P. trifoliate was studied by OSAWA~19~and by ToxopEUs~21~ in Mandarin. OSAWA(19) found that in Poncirus the first cell division in fertilized egg takes place between two and four weeks after fertilization. However, the author observed that the oospore began to divide about four to five weeks after fertilization in Satsuma orange and Natsudaidai. The super-numerary embryo originating from the nucellar cells were reported by OsAWA~19~, TOXOPEUS~21~,FROST~5~, FURUSATO~7~, MAHESHWARI~15) and SACHAR~15~.These nucellar cells began to divide soon after the fertilized egg have undergone its first division, and formed masses of cells after several divisions. These masses of cells grew into the embryo sac cavity and were completing their further development which consequently formed the nucellar embryos. The nucellar embryo may be distinguished from the sexual embryo by their lateral position, irregular shape and lack of suspensor. The polyembryos derived from the nucellar cells have been found not only in Citrus, Poncirus and Fortunella, but also in Nigritella~15~, Zeuxine~15~, Spiranthes(15), Man gifera (22), Cyanella(15), PachiraOpuntia(14), Mammillaria(15) and Xanthoxylum (3) etc. 11 106 Jour. Japan. Soc. Hort. Sci. 37 (2)

The available evidences on the initiation of the development of the nucellar embryos indicate that pollination is usually necessary but not always. IKEDA~11~, NAGAI and TANIKAWA(17) found on Satsuma orange that prevention of pollination resulted seedless fruits. WRIGHT~24)reported that pollen is not necessary for the production of nucellar embryos in the Marsh grapefruit, FROST(4) stated that nucellar embryony required the stimulation of pollen and did not require the fertilization. WoNG(23) also found in the case of Citrus that four varieties, namely Valencia, Hamlin, Pineapple and Parson Brown, commonly or regularly have seeds if pollinated and produced only seedless fruit when pollen was excluded. Similar test was conducted in the present experiment by using emasculated and bagged flowers and obtained some seedless fruits in Satsuma orange (Table 2). None of the seeds composed with only nucellar embryos were

Table 2. Effect of pollination on fruit setting and seed production in Citrus.

~ X 100 -pollinated artificial. Self-3 Emasculated without pollination. found. It will be concluded from the present study that development of nucellar embryos seemed to syncronize with the first division of the fertilized egg. Therefore, the fertilization is necessary for the initiation of the nucellar embryos development in Citrus.

Acknowledgments

The author wishes to express his deepest gratitude to Drs. J. HIRAI, S. NAKAO, T. YABUNO and S.NAKAGAWA, Department of Horticulture and Agronomy, College of Agriculture, University of Osaka Prefecture, for their valuable guidences throughout the course of this work. The author is also thankful to Mr. J. KIKucHI and Mr. A. NoMVRA of the Osaka Agriculture Research Center, Osaka Prefecture, for providing the necessary materials in this study. HSU-JEN YANG Fertilization and development of embryo on Citrus 107

References

1 BAKER,H.G. 1960. Apomixis and polyembryony The structure and development of the ovule- in Pachira oleaginea (Bombacaceae). Amer. and seed of Opuntia dillenii HAW. Phytomor- Jour. Bot. 47 : 296-302. phology 5 : 112-122. 2. COOPER,W.C., P.C.REECE and J.R.FURR. 1964. 15. and R. C. SACHAR. 1963. Polyem- Citrus breeding in Florida : past, present and bryony (pp. 265-296) in the Recent Advance future. Proc. Fla. Hort. Soci. 75 : 5-13. in the Embryology Angiosperms. MAHESHWARI, 3 DESAI,S.1962. Polyembryony in Xanthoxylum P., Eds. Univ. Delhi. Press. India. MILL. Phytomorphology 12: 184-189. 16. MIWA, T. 1952. Pollination, fertilization and'' 4 FROST,H.B. 1938a. The genetics and cytology fruit drop in Citrus tamurana HORT. Miyazaki of Citrus. Current Sci. 1938 (special no., Univ. Bull. 2 : 1-67. (In Japanese). Genetics) : 24-27. 17. NAGAI, K, and T.TANIKAWA. 1928. On Citrus 5 1938b. Nucellar embryony and juve- pollination. Third Pan-Pacific Sci. Cong. nile characters in clonal varieties of Citrus. (Tokyo, 1926) Proc., 2 : 2023-2029. Jour. Hered. 29 : 423-432. 18. NISHIURA, M., and T. IWASAKI. 1963. Studies:: 6 1948. VIII. Seed reproduction (pp. on the Citrus breeding. I. Variation of seedd 767-815) in The Citrus Industry Vol. 1. formation in Citrus crossing. Jap. Hort. Res. WEBBER,H.J., and L.D.BATCHELOR. Eds. Univ. Sta. Bull B. 2 : 1-13. (In Japanese). Calif. Press. Berkeley and Los Angeles. 19. OSAWA,I. 1912. Cytological and experimental 7. FURUSATO, K. 1952. Studies on polyploidy in studies in Citrus. Jour. Col. Agr. Imp. Univ. Citrus. Jap. Jour. Genet. 27 : 206. (In Japanese). Tokyo 4 : 83-116. 8. 1955. VII. Cytogenetics and breeding 20. TON, L.D., and A.H.KREZDORN. 1967. Growth of Citrus (pp. 229-251). Advanced Biology of pollen tubes in three incompatible varieties: Vol. V. Baif ukan, Tokyo. (In Japanese). of Citrus. Proc. Amer. Hort. Sci. 89 : 211- 9. Y.OHTA and K.ISHIBASHI. 1957. 215. Studies on polyembryony in Citrus. Seiken 21. TOXOPEUS,H. J. 1930. De polyembryonie van. Ziho 8 : 40-48. Citrus en haar beteekenis voor de culture. 10. and .1957. Notes on seedless Cited from The Citrus Industry Vol. 1. (pp.. Citrus species. Ibid. 8 : 100-102. (In Japanese). 767-815). 11. IKEDA,T. 1906. On the parthenocarpy of Citrus. 22. WEBBER, H.J. 1931. The economic importance: Jour. Sci. Agr. Soc. 70 : 2-9. (In Japanese). of apogamy in Citrus and Mangifera. Proc. 12. INOH, S. 1964. VII. Organ Tissue (pp. 387- Amer. Soc. Hort. Sci. 28: 57-61. 564). Plant Histology. Press. Uchida Rokaku- 23. WONG,C.Y. 1939. The influence of pollination hoshinsha, Tokyo. (In Japanese). on seed development in certain varieties of 13. JOHANSEN,D.A. 1950. XVII. Anthophyta Types Citrus. Proc. Amer. Soc. Hort. Sci. 37 : 161- and Variations (pp. 111-125, 200-202), in 164. Plant Embryology. Press. Chronica Botanic 24. WRIGHT,T. 1937. Pollination and the seediness.. Co., U.S.A. of Marsh grapefruit. Trinidad and Tobago 14. MAHESHWARI, P., and R. N. CHOPRA. 1955. Agr. Soc. Proc. 37 : 51-60. 108 Jour. Japan. Soc. Hort. Sci. 37 (2)

温州ミカンおよびナツミカンの受精と胚の発達

楊緒壬 (大阪府立大学農学部)

摘要

本実験はミカソの花柱と子房内における花粉管の伸 3.ナツミカソおよび温州ミカソの受精卵の分裂開始長,および受精と胚発達の機構をより詳細に究明するた時期は,だいたい授粉後40日から50日であつた。めに行なつた。 4.ナツミカソの胚の発達段階において,球形と心臓 1.ナツミカソおよび温州ミカソの正逆授粉の結果, 形の胚になる時期は,それぞれ,授粉後約58日と72日ナツミカソの花粉管の伸長速度は温州ミカンより著しくであつた。胚の各部分が形態的に識別できる時期は授粉速かつた。後120日であつた。 2.授粉後受精までに要する日数はナツミカンの自家 5・珠心胚に分化するある特定の珠心細胞の位置は珠

受精では約8日,温州ミカソにナツミカンを授粉した場孔の周囲,および胚のう壁に沿つて下方へ珠孔から全体合では約12日,温州ミカソの自家受精では約14日での胚のうの長さのほぼ1/3一の所までにおよび,また胚のあつた。う壁に近い1から3層にあつた。

Fig. 1. Two hours after pollination. C. natsudaidai pollen grains are germinating on the stigma of C. unshiu. X 1200. Fig. 2. Six hours after pollination. C. natsudaidai pollen tubes in the stigmatic tissue of C. unshiu. X 400. Fig. 3 Eight days after pollination. C. natsudaidai pollen tube reached the upper part of the ovary of C. uushiu. X 1200. Fig. 4 Showing a mature embryo sac of C. natsudaidai. X 1600. Fig. 5 Eight days after pollination. C.natsudaidai pollen tube reached the embryo sac of C.natsudaidai. ..17nn J\ ir.vv. Fig. 6 Eight days after C.natsudaidai selfing. Fertilization and endosperm nuclei in embryo sac. x800. Fig. 7 Endosperm 32 days after crossing of C.unshiu with C. natsudaidai. X 1200. Fig. 8 Ovule 41 days after C. natsudaidai selfing. Showing the fertilized egg, endosperm and nucellar cells. x800. Fig. 9. Ovule 44 days after the crossing of C.unshiu with C. natsudaidai. Showing four nucellar cells. x1200. Fig. 10. Showing a four-cell proembryo 44 days after C. natsudaidai selfing. X 1000. Fig. 11. Showing a six-cell proembryo 52 days after crossing of C.unshiu with C.natsudaidai. X1200. Fig. 12. Showing a ten-cell proembryo 54 days after crossing of C. unshiu with C. natsudadai. X 1200. Fig. 13. Showing a globular-shape embryo of C. natsudaidai 58 days after pollination. X 1000. Fig. 14. Showing a heart-shape embryo of C. natsudaidai 72 days after pollination. X 1200. Fig. 15. Showing a complete embryo of C. natsudaidai 120 days after pollination. X 100. Fig. 16. Showing the plumule, hypocotyl and radicle. x300. Fig. 17. Showing a later stage of embryo 108 days after crossing of C. unshiu with C. natsudaidai. x300. Fig. 18. Showing a nucellar embryo occurred from the region of the micropylar end to one-third of embryo-sac 54 days after crossing of C. unshiu with C, natsudaidai. X 100. Fig. 19. Magnified the nucellar embryo of Fig. 18. X 1000. Figs. 20. and 21. Lots of many nucellar embryos of C. unshiu 84 days after pollination. X 1000.

a, antipodal cell. c, cotyledon. e, proembryo. eg, egg cell, en, endosperm. es, embryo sac. feg, fertilized ,egg. h, hypocotyl. n, nucellar cell. ne, nucellar embryo. p, polar nucleus. p1, plumule. pt, pollen tube. r, radicle. s, synergid.