High temperature injuries in tomato. V. Fertilization and development of embryo with special reference to the abnormalities caused by high temperature

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1 High temperature injuries in tomato. V. Fertilization and development of embryo with special reference to the abnormalities caused by high temperature SHUICHI IWAHORI Faculty of Agriculture, University of Tokyo, Bunkyoku, Tokyo In the previous paper~6~ it was reported that flowers of one to three days after anthesis, were especially sensitive to high temperature and almost all flowers failed to set fruits. However, the more advanced the stages of flowers at the high temperature treatment, the more resistant to high temperature were the flowers. And the plot treated 5-8 days after anthesis resulted in good fruit set. It is well known that the germination of pollen grains and the elongation of pollen were affected by high temperature~2'10~. Therefore, one of the causes of the poor fruit set may be attributed to the remarkable susceptibilities of pollen germination and pollen tube elongation to high temperature. But it remains unknown whether the development of proembryo and endosperm is affected by high temperature. For the solution of the problems the detailed information of whole processes from pollination to fertilization must be obtained. On the elongation of pollen tubes of tomato there are two reports by Fuji(2) and SMITH and COCHRAN~10~. JUDKINS~7~ reported thatt considerable numbers of pollen grains passed through style within 24 hours after pollination. While, concerning the fertilization and embryogenesis of tomato, there is only one report by SMITH (9) in which he informed that it took about 48 hours from pollination to fertilization. However, the definition of fertilization and the temperature during the experiments which may greatly influence the results~3,4,7~ are not clear in his study. Moreover, there are no inves. tigations on the abnormalities of the developing proembryo and endosperm caused by high temperature. In the present investigation the flowers of the first cluster on the tomato plants grown in the greenhouse maintained 20t constantly, were emasculated and hand-pollinated. With definite intervals the flowers and young fruits were sampled and the fertilization processes andd development of proembryo and endosperm were observed. Another lot of flowers was treated with high temperature at various time intervals after hand-pollination, and the resulted abnormalities of ovules were observed. Materials and Methods In the first experiment tomato plants (cv. Fukuju No. 2), sown on April 1, 1963, transplanted in 18cm clay pots on April 26, were transferred into the greenhouse maintained 20t constantly on May 24, when the first flower of the first cluster just began to open. From May 25 to 30, flowers at anthesis stages* were emasculated and hand-pollinated. By this time the Received for publication April 7, 1966 *In this case the opening of the tip of calyx was defined as anthesiṣ 55

2 380 Jour. Japan. Soc. Hort. Sci. 35. (4) pollination had not yet taken place. The length of styles was about seven millimeters. Flowers were sampled and fixed with FAA at 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 60 and 72 hours after the artificial pollination. Emasculated and unpollinated flowers were also sampled. In the second experiment tomato plants, sown on September 1, 1964, were transferred into the 20t greenhouse. At 18 or 72 hours after hand pollination, the plants with pollinated flowers on them were treated with high temperature of 40t for four hours, then put back and grown in the 20t room. Flowers of each plot were sampled just after and 72 hours after the high temperature treatment. Flowers on the plants left in the 20t room during the high temperature treatment served as control. In the third experiment tomato plants, sown on April 10, 1965, were also transferred into the 20t greenhouse and hand-pollinated. At 24, 48, 72, 96 and 120 hours after pollination, the plants were treated with high temperature of 40t for four hours, then put back and grown in the 20t room. Flowers of each plot were sampled and fixed with FAA immediately after and 72 hours after the treatment. Flowers on the plants left in the 20t room during the treatment served as control. According to the usual paraffin procedure, longitudinal sections of 12,a in thickness were made and stained with DELAFIELD's haematoxylin or 1% acid f uchsin + 1% light green, 8 : 2 by BUCHHOLZ (1). Staining with the latter dye was used especially for the observation of pollen tube elongation and the fertilization processes. The plant materials used in these three experiments were grown in the same greenhouse and kept at 20t constantly after hand-pollination, therefore the temperature condition of these experiments was almost equal. Moreover no significant differences were obtained between the results of these three experiments. 1. Development of normal ovule Observation A very few pollen grains germinated by three hours after pollination. The maximum elongation of pollen tube reached about 0.05 mm (Fig. 1). These results seemed to be in accordance with that of FUJii~2~ who studied the germination of pollen grains in artificial media, and that of SMITH and COCHRAN~10~ who observed the hand-pollinated styles prepared by paraffin methods. The tip of pollen tubes of maximum growth reached the base of the style 15 and 18 hours after pollination (Figs. 4-'5). In the ovules sampled 15 hours after pollination, it was observed that two male nuclei were in contact with egg nucleus and central nucleus, respectively (Figs. 6^ 9). The male nuclei entered into the egg and central nuclei in about ten per cent of the ovules 1821 hours after pollination (Figs. 1012). Although the fusion of male nucleus with egg or with central nucleus could not be observed, 36 hours after pollination, a primary endosperm nucleus was clearly observed in about 30% of the ovules (Fig. 14). Therefore, it seemed reasonable that in the majority of the ovules fertilization took place about 2430 hours after pollination*. In *When flowers are emasculated and hand-pollinated, about O% of ovules appear to fertilize and develop (see IWAHORI et al(s) and VERKERK(12)). 56

3 IWAHORI : High temperature injuries in tomato. V % of 48 hours old ovules two endosperm nuclei were observed which resulted from the first division of the primary endosperm nucleus (Figs. 1516). The mode of endosperm formation of tomato seemed to be cellular type, that is, every nuclear division was accompanied with the cell wall formation. The ovules with four endosperm nuclei were observed most frequently 72 hours after pollination (Figs ), while those with two or eight endosperm nuclei were also observed. By this time fertilized egg, though grew larger in size, did not make division. At 96 hours after pollination, almost all ovules possessed the endosperm consisted of six to eight nuclei (Fig. 19), and in about 10% of ovules the two-celled proembryo was observed (Fig. 20). At 120 hours after pollination, proembryo consisted of four cells (Figs ). Smith~9' stated that with subsequent divisions the top cell gave rise to the cotyledonary portion, the second cell to the hypocotyl, and periblem and plerome of root, the third cell to the root tip and a part of suspensor, and the forth cell to the suspensor. At 144 hours after pollination, the upper two cells divided longitudinally to give to six-celled proembryo (Fig. 23). Proembryo consisted of more than ten cells 192 hours after pollination (Fig. 25). During the course of proembryo development, endosperm continued the growth and division, and filled the ovule. 2. Morphological abnormalities caused by high temperature Treated 18 hours after pollination : It seemed that in almost all ovules fertilization did not take place by this time. Fertilization was not observed in the samples fixed immediately after the treatment. No morphological abnormalities were observed in either egg or central nucleus. However, in the samples fixed three days after the treatment it was found that almost all ovules did not develop but aborted, that might resulted from lack of fertilization (Fig. 26). Treated 24 hours after pollination: In the samples fixed immediately after the treatment a few fertilized ovules and the entry of pollen tubes into the ovules were observed (Fig. 27), though in majority of ovules fertilization did not seem to take place. Degeneration or other abnormalities of egg or central nucleus were scarcely observed, though in a few ovules it was observed that the contents of central nucleus were empty or its nucleolus was abnormally enlarged (Fig. 28). In the samples fixed three days after the treatment developmental stages of the treated ovules were almost equal to those of control. However, 75% of the treated ovules were injured. No morphological abnormalities were observed in proembryo, while in the endosperm were found the empty or collapsed and deeply stained nuclei (Fig. 35). Treated 48 hours after pollination: In the samples fixed immediately after the treatment endosperm consisted of two nuclei, in which the abnormalities of formation of nuclear membrane and empty nucleus were observed (Figs. 29'3O). Proembryo seemed to be hardly injured. Though the control ovules of the same age consisted of four-celled proembryo and endosperm of more than 20 nuclei, in about 20% of the ovules fixed three days after the treatment, the proembryo were two- or one-celled stage, therefore their developmental stages seemed to be retarded. In the ovules of about 40%, degeneration of endosperm was observed, the contents of which were deeply stained, while proembryo appeared to be normal (Fig. 36). 57

4 382 Jour. Japan. Soc. Hort. Sci. 35. ( 4 ) Treated 72 hours after pollination: In about 10% of the ovules sampled immediately after the treatment, irregularly shaped or empty-like nucleus of endosperm was observed (Fig. 31). In 10% of ovules sampled three days after the treatment, progress of the developmental stage of the ovule was retarded. The contents of endosperm cells were deeply stained and degenerated in 30% of the ovules (Fig. 37). Treated 96 hours after pollination: In some ovules fixed immediately after the treatment, some abnormalities were observed. These were the inhibition of nuclear membrane formation, abnormally enlarged nucleolus and empty contents of endosperm (Figs ). In the samples fixed three days after the treatment, about 20% of the ovules were inhibited in its development. In some ovules the development of endosperm was normal, while the proembryo aborted. In 30% of the ovules the deeply stained and degenerated endosperm was observed. The lack of cell wall formation of proembryo was also observed (Fig. 38). Treated 120 hours after pollination: The abnormal ovules or retardation of ovule development were hardly observed in those sampled either just (Fig. 34) or three days after the treatment (Fig. 39). Discussion SMITH and COCHRAN~I0~, in tomato (cv. Bonny Best) grown in the greenhouse maintained 21 C, observed the germination of pollen grains and subsequent tube elongation. One percent of pollen tubes elongated more than 2 mm, 30 hours after pollination, and pollen tubes of more than 4 mm in length were not observed until 42 hours after pollination. JUDKINS~7~, using var. Globe (length of style, 7"-'8 mm), found that when the base of the style was cut 24 hours after pollination, 40 and 30% of the flowers set fruits under the condition of ranging 18-v27 C and 21 C, respectively. He concluded that within 24 hours after pollination a considerable numbers of pollen tubes passed through the base of styles, and that the rate of pollen tube elongation in his experiments was faster than that reported by SMITH and COCHRAN~10~. FUJII~2~ reported that the maximum elongation of pollen tubes reached 3.6mm, 24 hours after placing pollen grains on an artificial medium. The results obtained in the present study seemed to differ from the results of SMITH and COCHRANr101, but rather to be in accordance with that of JUDKINS~7~. SMITH (8'9) reported that fertilization did not take place within 50 hours after pollination in any case, and that two-celled proembryo was not observed until 94 hours after pollination. According to his illustration eight nucleate endosperm was observed 66 hours pollination, that is, 16 hours after fertilization. It is not clear which phase was defined as fertilization by SMITH (9) and how high the temperature was during the experiments, which would influence the duration from pollination to fertilization (3,4,7). In the present study fertilization took place about hours after pollination. Eight nucleate endosperm and two-celled proembryo were observed 96 hours after pollination, that is, 66^J72 hours after fertilization. Therefore, the results of the present investigation differed greatly from that of SMITH'S' as to the time required from pollination to fertilization. As JUD- KINS~7~ by indirect methods reported that within 24 hours after pollination, pollen tubes passed 58

5 IWAHORI : High temperature injuries in tomato. V. 383 Y might through the base of the style in 30-40% of the flowers, the duration required from pollination to fertilization seemed to be shorter than the results of SMITH (9>, and would be 2430 hours as obtained in the present investigation. HOSHIKAWA{5~ stated that "through detailed reexaminations of the fertilization processes in some cereal crop plants recently done by many workers,. it has been found that the time interval between pollination and the completion of fertilization is much shorter than that reported by some previous workers". The results obtained in the present study were nearly in accordance with that of SMITH ~9> concerning the developmental process of proembryo and endosperm. It is well known that the germination of pollen grains and the elongation of pollen tubes are easily affected by high temperature~2~. SMITH and COCHRAN~10) reported that at 38t very few pollen grains germinated on the stigma and that the length of pollen tubes which elongated abnormally, was very short. Although it could not be made clear to what extent the high temperature treatment affected the elongation of pollen tubes in the present study, the fact that almost all ovules which were treated with high temperature 18 hours after pollination aborted, would strongly suggest the elongation of pollen tubes was affected by high temperature so that fertilization did not take place. There are little inf ormations in tomato concerning the abnormalities of proembryo and endosperm resulted from the high temperature treatment. It was revealed in this study that endosperm was more susceptible to high temperature than proembryo, and the cell contents of the endosperm easily became empty or deeply stained and degenerated. However, the endosperm and proembryo treated. at some advanced stages showed the resistance to high temperature. In the present study flowers were hand-pollinated when the calyx just began to open. Natural pollination would take place one or two days later on the flowers grown in field condition. Therefore, the fact that most flowers treated 1'-3 days after anthesis failed to set fruits in the previous investigations, seemed to be attributed mainly to the extreme susceptibiliy of pollen germination and elongation to high temperature. Percentage of fruit set decreased to some extent even in the flowers treated five days after anthesis than control. This be resulted from injury of fertilized ovules, mainly that of endosperm. However, percentage of fruit set hardly decreased in the flowers treated eight days after anthesis. These results were in accordance with the present observations that in the ovules treated five days after pollination, there were hardly any abnormalities. Acknowledgement The auther is indebted to Prof. T. SUGIYAMA for his guidance and invaluable criticism. Thanks are also due to Dr. K. HOSHIKAWA, Laboratory of Crop Science, for his valuable advices. Summary The experiments were conducted to study the fertilization processes and the development of proembryo in the artificially pollinated flowers of the tomato plants grown under the con- 59

6 384 Jour. Japan. Soc. Hort. Sci. 35. (4) dition of 20 C constant, and to observe the abnormalities of ovules caused by high temperature of 40 C for four hours. Pollen grains began to germinate three hours after pollination on the stigma. Fertilization took place 18 hours in the fastest one and hours in the majority of ovules after pollination. Two-, four-, and eight-nucleate endosperm stages were observed 48, 72, and 96 hours after pollination, respectively, in the endosperm which showed the cellular type in the early formative stage. Two-, and four-celled proembryo were observed 96, and 120 hours after pollination, respectively. Proembryo was consisted of more than ten cells 196 hours after pollination. The ovules which had been treated with high temperature 18 hours after pollination aborted. This might be attributed to the retardation or the stoppage of pollen tube elongation by high temperature. In the ovules treated 2496 hours after pollination, degeneration of endosperm was observed. In that case it was found that cytoplasm and nucleus were deeply stained or their contents were empty. In a few ovules the retardation of proembryo development was observed. In the ovules treated 120 hours after pollination, however, the abnormalities of the ovules were hardly observed. Literature 1. BUCHHOLZ, J. Y The dissection, stai- Japanese with English summary). Jour. Jap. fling and mounting of style in the study of Soc. Hort. Sci. 33 : pollen tube distribution. Stain Tech. 6 : 13-24, cited from SMITH and COCHRAN (10). :2. FUJII, T Studies on the flower dropping of fruit vegetables (in Japanese with English summary). Bull. Chiba Coll. Hort. No. 6: HOSHIKAWA, K Cytological studies of double fertilization in wheat (Triticum aestivum L.). Proc. Crop Sci. Soc. Japan 28 : Influence of temperature upon the fertilization of wheat, grown in various levels of nitrogen. Ibid. 28: Studies on the pollen germination and pollen tube growth in relation to the fertilization in wheat. Ibid. 28 : 333-v IWAHORI, S. and K. TAKAHASHI High temperature injuries in tomato. III. Effects of high temperature on flower buds and flowers of different stages of development (in , 10, JUDKINS, W. P Time involved in pollen tube extension through style and rate of fruit growth in tomato. Proc. Amer. Soc. Hort. Sci. 37: SMITH, Relation of temperature to anthesis and blossom drop of the tomato togather with a histological study of pistils. Jour. Agric. Res. 44: Pollination and life-history studies of the tomato(lycopersicon esculentum Mill.). Cornell Univ. Agric. Exp. Sta. Mem.184. and H. COCHRAN Effect of temperature on pollen germination and tube growth in the tomato. Ibid SOUEGES, R Recherches sur l'embryogenie des Solanacees. Soc. Bot. France Bull. 69:163178, , , cited from SMITH and COCHRAN (10). VERKERK, K The pollination of tomatoes. Neth. Jour. Agric. Sci. 5:3754.

7 Figs Normal developmental processes of ovules observed in longitudinal section. Fig. 1. Three hours after pollination. Pollen grains are germinating on the stigma. Fig. 2. Six hours after pollination. Fig. 3. Twelve hours after pollination. Pollen tubes are elongating in the style. Figs Eighteen hours after pollination. Tips of the elongating pollen tubes appear at the base of style. Figs. 6-7, 8~9. Ovule 15 hours after pollination. Male nuclei are in contact with egg nucleus and central nucleus. Fig. 10. Ovule 18 hours after pollination. Male nucleus enters into the egg nucleus. Figs Ovule 21 hours after pollination. Male nuclei enter into the egg nucleus anti central nucleus. Fig. 13. Ovule 24 hours after pollinaton. Fig. 14. Primary endosperm nucleus 36 hours after pollination. Figs. 15-v16. Ovule with two nucleate endosperm 48 hours after pollination. Figs Ovule with four nucleate endosperm 72 hours after pollination. Fig. 19. Ovule with several endosperm nuclei 96 hours after pollination. Fig. 20. Two-celled proembryo 96 hours after pollination. Figs Four-celled proembryo 120 hours after pollination. Fig. 23. Ovule 144 hours after pollination. Fig. 24. Ovule 168 hours after pollination. Fig. 25. Ovule 192 hours after pollination. 61

8 386 Jour. Japan Soc. Hort. Sci. 35 (4) Figs Abnormal development of ovules caused by the high temperature treatment. Fig. 26. Treated 18 hours after pollination, and fixed 72 hours after the treatment. Aborted ovule. Figs Treated 24 hours after pollination, and fixed just after the treatment. Tip of pollen tube appears. Enlarged nucleolus is observed in endosperm nucleus. Figs. 29-v30. Treated 48 hours after pollination, and fixed just after the treatment. Empty nucleus of endosperm (de) is observed. Fig. 31. Treated 72 hours after pollination, and fixed just after the treatment. Irregularly shaped nucleus of endosperm (de) is observed. Figs. 32-v33. Treated 96 hours after pollination, and fixed just after the treatment. Empty nucleus of endosperm (de) is observed. Fig. 34. Treated 120 hours after pollination, and fixed just after the treatment. Only example which shows irregularly shaped nucleus of endosperm. Fig. 35. Treated 24 hours after pollination, and fixed 72 hours after the treatment. Deeplystained and collapsed endosperm is observed. Fig. 36. Treated 48 hours after pollination, and fixed 72 hours after the treatment. Deeplystained and collapsed endosperm is obseved Fig. 37. Treated 72 hours after pollination, and fixed 72 hours after the treatment. Deeplystained and collapsed endosperm is observed. Fig. 38. Treated 96 hours after pollination, and fixed 72 hours after the treatment. Deeplystained and collapsed endosperm, and the lack of cell wall formation of proembryo are observed. Fig. 39. Treated 120 hours after pollination, and fixed 72 hours after the treatment. pt : pollen tube, eg : egg cell, c : central nucleus, m : male nucleus, e : proembryo, en : endosperm de : degenerated endosperm Figs. 1, 2, 5, 15, 17, 19, 2229, 31, 32, x320 Figs. 3, 4. X 100 Figs. 6-14, 16, 18, 20, 21, 30, 33. x400